Search results for: mRNA marker

Authors: A. Bortkiewcz, A. Dudarewicz, P. Małecki, M. Kłaczyński, T. Wszołek, Małgorzata Pawlaczyk-Łuszczyńska

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Low-frequency noise (LFN) has been recognized as a special environmental pollutant. It is usually considered a broadband noise with the dominant content of low frequencies from 10 Hz to 250 Hz. A growing body of data shows that LFN differs in nature from other environmental noises, which are at comparable levels but not dominated by low-frequency components. The primary and most frequent adverse effect of LFN exposure is annoyance. Moreover, some recent investigations showed that LFN at relatively low A-weighted sound pressure levels (40−45 dB) occurring in office-like areas could adversely affect the mental performance, especially of high-sensitive subjects. It is well documented that high-frequency noise disturbs various types of human functions; however, there is very little data on the impact of LFN on well-being and health, including the cardiovascular system. Heart rate variability (HRV) is a sensitive marker of autonomic regulation of the circulatory system. Walker and co-workers found that LFN has a significantly more negative impact on cardiovascular response than exposure to high-frequency noise and that changes in HRV parameters resulting from LFN exposure tend to persist over time. The negative reactions of the cardiovascular system in response to LFN generated by wind turbines (20-200 Hz) were confirmed by Chiu. The scientific aim of the study is to assess the relationship between the spectral-temporal characteristics of LFN and the activity of the autonomic nervous system, considering the subjective assessment of annoyance, sensitivity to this type of noise, and cognitive and general health status. The study will be conducted in 20 male students in a special, acoustically prepared, constantly supervised room. Each person will be tested 4 times (4 sessions), under conditions of non-exposure (sham) and exposure to noise of wind turbines recorded at a distance of 250 meters from the turbine with different frequencies and frequency ranges: acoustic band 20 Hz-20 kHz, infrasound band 5-20 Hz, acoustic band + infrasound band. The order of sessions of the experiment will be randomly selected. Each session will last 1 h. There will be a 2-3 days break between sessions to exclude the possibility of the earlier session influencing the results of the next one. Before the first exposure, a questionnaire will be conducted on noise sensitivity, general health status using the GHQ questionnaire, hearing organ status and sociodemographic data. Before each of the 4 exposures, subjects will complete a brief questionnaire on their mood and sleep quality the night before the test. After the test, the subjects will be asked about any discomfort and subjective symptoms during the exposure. Before the test begins, Holter ECG monitoring equipment will be installed. HRV will be analyzed from the ECG recordings, including time and frequency domain parameters. The tests will always be performed in the morning (9-12) to avoid the influence of diurnal rhythm on HRV results. Students will perform psychological tests 15 minutes before the end of the test (Vienna Test System).

Keywords: neurovegetative control, heart rate variability (HRV), cognitive processes, low frequency noise

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Authors: Rajesh Kumar Gautam, Debabrata Seth

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Thioflavin-T (ThT) play a key role of an important biologically active fluorescent sensor for amyloid fibrils. ThT molecule has been developed a method to detect the analysis of different type of diseases such as neurodegenerative disorders, Alzheimer’s, Parkinson’s, and type II diabetes. ThT was used as a fluorescent marker to detect the formation of amyloid fibril. In the presence of amyloid fibril, ThT becomes highly fluorescent. ThT undergoes twisting motion around C-C bonds of the two adjacent benzothiazole and dimethylaniline aromatic rings, which is predominantly affected by the micro-viscosity of the local environment. The present study articulates photophysics and torsional dynamics of biologically active molecule ThT in the presence of deep-eutectic solvents (DESs). DESs are environment-friendly, low cost and biodegradable alternatives to the ionic liquids. DES resembles ionic liquids, but the constituents of a DES include a hydrogen bond donor and acceptor species, in addition to ions. Due to the presence of the H-bonding network within a DES, it exhibits structural heterogeneity. Herein, we have prepared two different DESs by mixing urea with choline chloride and N, N-diethyl ethanol ammonium chloride at ~ 340 K. It was reported that deep eutectic mixture of choline chloride with urea gave a liquid with a freezing point of 12°C. We have experimented by taking two different concentrations of ThT. It was observed that at higher concentration of ThT (50 µM) it forms aggregates in DES. The photophysics of ThT as a function of temperature have been explored by using steady-state, and picoseconds time-resolved fluorescence emission spectroscopic techniques. From the spectroscopic analysis, we have observed that with rising temperature the fluorescence quantum yields and lifetime values of ThT molecule gradually decreases; this is the cumulative effect of thermal quenching and increase in the rate of the torsional rate constant. The fluorescence quantum yield and fluorescence lifetime decay values were always higher for DES-II (urea & N, N-diethyl ethanol ammonium chloride) than those for DES-I (urea & choline chloride). This was mainly due to the presence of structural heterogeneity of the medium. This was further confirmed by comparison with the activation energy of viscous flow with the activation energy of non-radiative decay. ThT molecule in less viscous media undergoes a very fast twisting process and leads to deactivation from the photoexcited state. In this system, the torsional motion increases with increasing temperature. We have concluded that beside bulk viscosity of the media, structural heterogeneity of the medium play crucial role to guide the photophysics of ThT in DESs. The analysis of the experimental data was carried out in the temperature range 288 ≤ T = 333K. The present articulate is to obtain an insight into the DESs as media for studying various photophysical processes of amyloid fibrils sensing molecule of ThT.

Keywords: deep eutectic solvent, photophysics, Thioflavin T, the torsional rate constant

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Authors: Tomas Makaras, Julija Razumienė, Vidutė Gurevičienė, Gintarė Sauliutė, Milda Stankevičiūtė

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Stress responses represent animal’s natural reactions to various challenging conditions and could be used as a welfare indicator. Regardless of the wide use of glucose measurements in stress evaluation, there are some inconsistencies in its acceptance as a stress marker, especially when it comes to comparison with non-invasive cortisol measurements in the fish challenging stress. To meet the challenge and to test the reliability and applicability of glucose measurement in practice, in this study, different environmental/anthropogenic exposure scenarios were simulated to provoke chemical-induced stress in fish (14-days exposure to landfill leachate) followed by a 14-days stress recovery period and under the cumulative effect of leachate fish subsequently exposed to pathogenic oomycetes (Saprolegnia parasitica) to represent a possible infection in fish. It is endemic to all freshwater habitats worldwide and is partly responsible for the decline of natural freshwater fish populations. Brown trout (Salmo trutta fario) and sea trout (Salmo trutta trutta) juveniles were chosen because of a large amount of literature on physiological stress responses in these species was known. Glucose content in fish by applying invasive and non-invasive glucose measurement procedures in different test mediums such as fish blood, gill tissues and fish-holding water were analysed. The results indicated that the quantity of glucose released in the holding water of stressed fish increased considerably (approx. 3.5- to 8-fold) and remained substantially higher (approx. 2- to 4-fold) throughout the stress recovery period than the control level suggesting that fish did not recover from chemical-induced stress. The circulating levels of glucose in blood and gills decreased over time in fish exposed to different stressors. However, the gill glucose level in fish showed a decrease similar to the control levels measured at the same time points, which was found to be insignificant. The data analysis showed that concentrations of β-D glucose measured in gills of fish treated with S. parasitica differed significantly from the control recovery, but did not differ from the leachate recovery group showing that S. parasitica presence in water had no additive effects. In contrast, a positive correlation between blood and gills glucose were determined. Parallel trends in blood and water glucose changes suggest that water glucose measurement has much potency in predicting stress. This study demonstrated that measuring β-D-glucose in fish-holding water is not stressful as it involves no handling and manipulation of an organism and has critical technical advantages concerning current (invasive) methods, mainly using blood samples or specific tissues. The quantification of glucose could be essential for studies examining the stress physiology/aquaculture studies interested in the assessment or long-term monitoring of fish health.

Keywords: brown trout, landfill leachate, sea trout, pathogenic oomycetes, β-D-glucose

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Authors: Ting Lu, Ya-Ru Diao, Fei Fan, Ye Xue, Lei Shi, Xian-e Tang, Meng-jun Zhan, Zhen-hua Deng

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Accurate adult age estimation (AAE) is a significant and challenging task in forensic and archeology fields. Attempts have been made to explore optimal adult age metrics, and the rib is considered a potential age marker. The traditional way is to extract age-related features designed by experts from macroscopic or radiological images followed by classification or regression analysis. Those results still have not met the high-level requirements for practice, and the limitation of using feature design and manual extraction methods is loss of information since the features are likely not designed explicitly for extracting information relevant to age. Deep learning (DL) has recently garnered much interest in imaging learning and computer vision. It enables learning features that are important without a prior bias or hypothesis and could be supportive of AAE. This study aimed to develop DL models for AAE based on CT images and compare their performance to the manual visual scoring method. Chest CT data were reconstructed using volume rendering (VR). Retrospective data of 2500 patients aged 20.00-69.99 years were obtained between December 2019 and September 2021. Five-fold cross-validation was performed, and datasets were randomly split into training and validation sets in a 4:1 ratio for each fold. Before feeding the inputs into networks, all images were augmented with random rotation and vertical flip, normalized, and resized to 224×224 pixels. ResNeXt was chosen as the DL baseline due to its advantages of higher efficiency and accuracy in image classification. Mean absolute error (MAE) was the primary parameter. Independent data from 100 patients acquired between March and April 2022 were used as a test set. The manual method completely followed the prior study, which reported the lowest MAEs (5.31 in males and 6.72 in females) among similar studies. CT data and VR images were used. The radiation density of the first costal cartilage was recorded using CT data on the workstation. The osseous and calcified projections of the 1 to 7 costal cartilages were scored based on VR images using an eight-stage staging technique. According to the results of the prior study, the optimal models were the decision tree regression model in males and the stepwise multiple linear regression equation in females. Predicted ages of the test set were calculated separately using different models by sex. A total of 2600 patients (training and validation sets, mean age=45.19 years±14.20 [SD]; test set, mean age=46.57±9.66) were evaluated in this study. Of ResNeXt model training, MAEs were obtained with 3.95 in males and 3.65 in females. Based on the test set, DL achieved MAEs of 4.05 in males and 4.54 in females, which were far better than the MAEs of 8.90 and 6.42 respectively, for the manual method. Those results showed that the DL of the ResNeXt model outperformed the manual method in AAE based on CT reconstruction of the costal cartilage and the developed system may be a supportive tool for AAE.

Keywords: forensic anthropology, age determination by the skeleton, costal cartilage, CT, deep learning

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Authors: Olga Kruzhkova, Irina Vorobyeva, Roman Porozov

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Vandalism is a common phenomenon, but its definition is still not clearly defined. In the public sense, vandalism is the blatant cases of pogroms in cemeteries, destruction of public places (regardless of whether these actions are authorized), damage to significant objects of culture and history (monuments, religious buildings). From a legal point of view, only such an act can be called vandalism, which is aimed at 'desecrating buildings or other structures, damaging property on public transport or in other public places'. The key here is the notion of public property that is being damaged. In addition, the principal is the semantics of messages, expressed in a kind of sign system (drawing, inscription, symbol), which initially threatens public order, the calmness of citizens, public morality. Because of this, the legal qualification of vandalism doesn’t include a sufficiently wide layer of environmental destructions that are common in modern urban space (graffiti and other damage to private property, broken shop windows, damage to entrances and elevator cabins), which in ordinary consciousness are seen as obvious facts of vandalism. At the same time, the understanding of vandalism from the position of psychology implies an appeal to the question of the limits of the activity of the subject of vandalism and his motivational basis. Also recently, the discourse on the positive meaning of some forms of vandalism (graffiti, street-art, etc.) has been activated. But there is no discussion of the role and significance of vandalism in public and individual life, although, like any socio-cultural and socio-psychological phenomenon, vandalism is not groundless and meaningless. Our aim of the study was to identify and describe the functions of vandalism as a socio-cultural and socio-psychological phenomenon of the life of the urban community, as well as personal determinants of its manifestations. The study was conducted in the spatial environment of the Russian megalopolis (Ekaterinburg) by photographing visual results of vandal acts (6217 photos) with subsequent trace-assessment and image content analysis, as well as diagnostics of personal characteristics and motivational basis of vandal activity of possible subjects of vandalism among youth. The results of the study allowed to identify the functions of vandalism at the socio-environmental and individual-subjective levels. The socio-environmental functions of vandalism include the signaling function, the function of preparing of social changes, the constructing function, and the function of managing public moods. The demonstrative-protest function, the response function, the refund function, and the self-expression function are assigned to the individual-subjective functions of vandalism. A two-dimensional model of vandal functions has been formed, where functions are distributed in the spaces 'construction reconstruction', 'emotional regulation/moral regulation'. It is noted that any function of vandal activity at the individual level becomes a kind of marker of 'points of tension' at the social and environmental level. Acknowledgment: The research was supported financially by Russian Science Foundation, (Project No. 17-18-01278).

Keywords: destruction, urban environment, vandal behavior, vandalism, vandalism functions

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Authors: Amelia J. McFarland, Andrew K. Davey, Shailendra Anoopkumar-Dukie

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Microglia are the resident macrophage population of the central nervous system (CNS), contributing to both innate and adaptive immune response, and brain homeostasis. Activation of microglia occurs in response to a multitude of pathogenic stimuli in their microenvironment; this induces morphological and functional changes, resulting in a state of acute neuroinflammation which facilitates injury resolution. Adequate microglial function is essential for the health of the neuroparenchyma, with microglial dysfunction implicated in numerous CNS pathologies. Given the critical role that these macrophage-derived cells play in CNS homeostasis, there is a high demand for microglial models suitable for use in neuroscience research. The isolation of primary human microglia, however, is both difficult and costly, with microglial activation an unwanted but inevitable result of the extraction process. Consequently, there is a need for the development of alternative experimental models which exhibit morphological, biochemical and functional characteristics of human microglia without the difficulties associated with primary cell lines. In this study, our aim was to evaluate whether THP-1 human peripheral blood monocytes would display microglial-like qualities following an induced differentiation, and, therefore, be suitable for use as surrogate microglia. To achieve this aim, THP-1 human peripheral blood monocytes from acute monocytic leukaemia were differentiated with a range of phorbol 12-myristate 13-acetate (PMA) concentrations (50-200 nM) using two different protocols: a 5-day continuous PMA exposure or a 3-day continuous PMA exposure followed by a 5-day rest in normal media. In each protocol and at each PMA concentration, microglial-like cell morphology was assessed through crystal violet staining and the presence of CD-14 microglial / macrophage cell surface marker. Lipopolysaccharide (LPS) from Escherichia coli (055: B5) was then added at a range of concentrations from 0-10 mcg/mL to activate the PMA-differentiated THP-1 cells. Functional microglial-like behavior was evaluated by quantifying the release of prostaglandin (PG)-E2 and pro-inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α using mediator-specific ELISAs. Furthermore, production of global reactive oxygen species (ROS) and nitric oxide (NO) were determined fluorometrically using dichlorodihydrofluorescein diacetate (DCFH-DA) and diaminofluorescein diacetate (DAF-2-DA) respectively. Following PMA-treatment, it was observed both differentiation protocols resulted in cells displaying distinct microglial morphology from 10 nM PMA. Activation of differentiated cells using LPS significantly augmented IL-1β, TNF-α and PGE2 release at all LPS concentrations under both differentiation protocols. Similarly, a significant increase in DCFH-DA and DAF-2-DA fluorescence was observed, indicative of increases in ROS and NO production. For all endpoints, the 5-day continuous PMA treatment protocol yielded significantly higher mediator levels than the 3-day treatment and 5-day rest protocol. Our data, therefore, suggests that the differentiation of THP-1 human monocyte cells with PMA yields a homogenous microglial-like population which, following stimulation with LPS, undergo activation to release a range of pro-inflammatory mediators associated with microglial activation. Thus, the use of PMA-differentiated THP-1 cells represents a suitable microglial model for in vitro research.

Keywords: differentiation, lipopolysaccharide, microglia, monocyte, neuroscience, THP-1

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Authors: Andrew N. Saylor, James R. Peters

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Scoliosis is a complex 3D deformity of the thoracic and lumbar spines, clinically diagnosed by measurement of a Cobb angle of 10 degrees or more on a coronal X-ray. The Cobb angle is the angle made by the lines drawn along the proximal and distal endplates of the respective proximal and distal vertebrae comprising the curve. Traditionally, Cobb angles are measured manually using either a marker, straight edge, and protractor or image measurement software. The task of measuring the Cobb angle can also be represented by a function taking the spine geometry rendered using X-ray imaging as input and returning the approximate angle. Although the form of such a function may be unknown, it can be approximated using artificial neural networks (ANNs). The performance of ANNs is affected by many factors, including the choice of activation function and network architecture; however, the effects of these parameters on the accuracy of scoliotic deformity measurements are poorly understood. Therefore, the objective of this study was to systematically investigate the effect of ANN architecture and activation function on Cobb angle measurement from the coronal X-rays of scoliotic subjects. The data set for this study consisted of 609 coronal chest X-rays of scoliotic subjects divided into 481 training images and 128 test images. These data, which included labeled Cobb angle measurements, were obtained from the SpineWeb online database. In order to normalize the input data, each image was resized using bi-linear interpolation to a size of 500 × 187 pixels, and the pixel intensities were scaled to be between 0 and 1. A fully connected (dense) ANN with a fixed cost function (mean squared error), batch size (10), and learning rate (0.01) was developed using Python Version 3.7.3 and TensorFlow 1.13.1. The activation functions (sigmoid, hyperbolic tangent [tanh], or rectified linear units [ReLU]), number of hidden layers (1, 3, 5, or 10), and number of neurons per layer (10, 100, or 1000) were varied systematically to generate a total of 36 network conditions. Stochastic gradient descent with early stopping was used to train each network. Three trials were run per condition, and the final mean squared errors and mean absolute errors were averaged to quantify the network response for each condition. The network that performed the best used ReLU neurons had three hidden layers, and 100 neurons per layer. The average mean squared error of this network was 222.28 ± 30 degrees2, and the average mean absolute error was 11.96 ± 0.64 degrees. It is also notable that while most of the networks performed similarly, the networks using ReLU neurons, 10 hidden layers, and 1000 neurons per layer, and those using Tanh neurons, one hidden layer, and 10 neurons per layer performed markedly worse with average mean squared errors greater than 400 degrees2 and average mean absolute errors greater than 16 degrees. From the results of this study, it can be seen that the choice of ANN architecture and activation function has a clear impact on Cobb angle inference from coronal X-rays of scoliotic subjects.

Keywords: scoliosis, artificial neural networks, cobb angle, medical imaging

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Authors: S. Najafi, E. Bertini, M. Pezzotti, G.B. Tornielli, S. Zenoni

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Grapevine is an important agricultural fruit crop plant consumed worldwide and with a key role in the global economy. Grapevine is strongly affected by both biotic and abiotic stresses, which impact grape growth at different stages, such as during plant and berry development and pre- and post-harvest, consequently causing significant economic losses. Recently global warming has propelled the anticipation of the onset of berry ripening, determining the reduction of a grape color and increased volatilization of aroma compounds. Climate change could negatively alter the physiological characteristics of the grape and affect the berry and wine quality. Modern plant breeding can provide tools such as genome editing for improving grape resilience traits while maintaining intact the viticultural and oenological quality characteristics of the genotype. This study aims at developing a platform for genome editing application in grapevine plants with the final goal to improve berry quality, biotic, and abiotic resilience traits. We chose to directly deliver ribonucleoproteins (RNP, preassembled Cas protein and guide RNA) into plant protoplasts, and, from these cell structures, regenerate grapevine plants edited in specific selected genes controlling traits of interest. Edited plants regenerated by somatic embryogenesis from protoplasts will then be sequenced and molecularly characterized. Embryogenic calli of Sultana and Shiraz cultivars were initiated from unopened leaves of in-vitro shoot tip cultures and from stamens, respectively. Leaves were placed on NB2 medium while stamens on callus initiation medium (PIV) medium and incubated in the dark at 28 °C for three months. Viable protoplasts, tested by FDA staining, isolated from embryogenic calli were cultured by disc method at 1*105 protoplasts/ml. Mature well-shaped somatic embryos developed directly in the protoplast culture medium two months later and were transferred in the light into to shooting medium for further growth. Regenerated plants were then transferred to the greenhouse; no phenotypic alterations were observed when compared to non in-vitro cultured plants. The performed experiments allowed to established an efficient protocol of embryogenic calli production, protoplast isolation, and regeneration of the whole plant through somatic embryogenesis in both Sultana and Shiraz. Regenerated plants, through direct somatic embryogenesis deriving from a single cell, avoid the risk of chimerism during the regeneration process, therefore improving the genome editing process. As pre-requisite of genome editing, an efficient method for transfection of protoplast by yellow fluorescent protein (YFP) marker genes was also established and experiments of direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) in protoplasts to achieve efficient DNA-free targeted mutations are in progress.

Keywords: CRISPR-cas9, plant regeneration, protoplast isolation, Vitis vinifera

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Authors: Parul Kulshreshtha, Subrata Sinha, Rakesh Bhatnagar

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This study was conducted by taking B. anthracis as a model pathogen. On infecting a host, B. anthracis secretes three proteins, namely, protective antigen (PA, 83kDa), edema factor (EF, 89 kDa) and lethal factor (LF, 90 kDa). These three proteins are the components of two anthrax toxins. PA binds to the cell surface receptors, namely, tumor endothelial marker (TEM) 8 and capillary morphogenesis protein (CMG) 2. TEM8 and CMG2 interact with LDL-receptor related protein (LRP) 6 for endocytosis of EF and LF. On entering the cell, EF acts as a calmodulin-dependent adenylate cyclase that causes a prolonged increase of cytosolic cyclic adenosine monophosphate (cAMP). LF is a metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MAPKK/MEK) close to their N-terminus. By secreting these two toxins, B.anthracis ascertains death of the host. Once the systemic levels of the toxins rise, antibiotics alone cannot save the host. Therefore, toxin-specific inhibitors have to be developed. In this wake, monoclonal antibodies have been developed for the neutralization of toxic effects of anthrax toxins. We created hybridomas by using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor of B. anthracis) to obtain anti-toxin antibodies. Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immunized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies from all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H8 and H10) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). The protective efficacy of H7, H8, H10 and H11 was investigated. H7, H8 and H10 were found to be protective. H11 was found to have disease enhancing characteristics in-vitro and in mouse model of challenge with B. anthracis. In this study the disease enhancing character of H11 monoclonal antibody and anti-rLFn polyclonal sera was investigated. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature both in-vitro and in-vivo. But combination of H11 with LETscFv (an scFv with VH and VL identical to H10 but lacking Fc region) could not abrogate the disease-enhancing character of H11 mAb. Therefore it was concluded that for suppression of disease enhancement, Fc portion was absolutely essential for interaction of H10 with H11. Our study indicates that the protective potential of an antibody depends equally on its idiotype/ antigen specificity and its isotype. A number of monoclonal and engineered antibodies are being explored as immunotherapeutics but it is absolutely essential to characterize each one for their individual and combined protective potential. Although new in the sphere of toxin-based diseases, it is extremely important to characterize the disease-enhancing nature of polyclonal as well as monoclonal antibodies. This is because several anti-viral therapeutics and vaccines have failed in the face of this phenomenon. The passive –immunotherapy thus needs to be well formulated to avoid any contraindications.

Keywords: immunotherapy, polyclonal, monoclonal, antibody-dependent disease enhancement

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Authors: Anna Höfer, Johannes Herrmann, Patrick Meybohm, Christopher Lotz

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Mitochondria are pivotal for energy supply and regulation of cellular functions. Deficiencies of mitochondrial metabolism have been implicated in diverse stressful conditions including infections. Platelets are key mediators for thrombo-inflammation during development and resolution of acute respiratory distress syndrome (ARDS). Previous data point to an exhausted platelet phenotype in critically-ill patients with coronavirus 19 disease (COVID-19) impacting the course of disease. The objective of this work was to characterize platelet mitochondrial metabolism in patients suffering from COVID-19 ARDSA longitudinal analysis of platelet mitochondrial metabolism in 24 patients with COVID-19 induced ARDS compared to 35 healthy controls (ctrl) was performed. Blood samples were analyzed at two time points (t1=day 1; t2=day 5-7 after study inclusion). The activity of mitochondrial citrate synthase was photometrically measured. The impact of oxidative stress on mitochondrial permeability was assessed by a photometric calcium-induced swelling assay and the activity of superoxide dismutase (SOD) by a SOD assay kit. The amount of protein carbonylation and the activity of mitochondria complexes I-IV were photometrically determined. Levels of interleukins (IL)-1α, IL-1β and tumor necrosis factor (TNF-) α were measured by a Multiplex assay kit. Median age was 54 years, 63 % were male and BMI was 29.8 kg/m2. SOFA (12; IQR: 10-15) and APACHE II (27; IQR: 24-30) indicated critical illness. Median Murray Score was 3.4 (IQR: 2.8-3.4), 21/24 (88%) required mechanical ventilation and V-V ECMO support in 14/24 (58%). Platelet counts in ARDS did not change during ICU stay (t1: 212 vs. t2: 209 x109/L). However, mean platelet volume (MPV) significantly increased (t1: 10.6 vs. t2: 11.9 fL; p<0.0001). Citrate synthase activity showed no significant differences between ctrl and ARDS patients. Calcium induced swelling was more pronounced in patients at t1 compared to t2 and to ctrl (50µM; t1: 0.006 vs. ctrl: 0.016 ΔOD; p=0.001). The amount of protein carbonylation as marker for irreversible proteomic modification constantly increased during ICU stay and compared to ctrl., without reaching significance. In parallel, superoxid dismutase activity gradually declined during ICU treatment vs. ctrl (t2: - 29 vs. ctrl.: - 17 %; p=0.0464). Complex I analysis revealed significantly stronger activity in ARDS vs. ctrl. (t1: 0.633 vs. ctrl.: 0.415 ΔOD; p=0.0086). There were no significant differences in complex II, III or IV activity in platelets from ARDS patients compared to ctrl. IL-18 constantly increased during the observation period without reaching significance. IL-1α and TNF-α did not differ from ctrl. However, IL-1β levels were significantly elevated in ARDS (t1: 16.8; t2: 16.6 vs. ctrl.: 12.4 pg/mL; p1=0.0335, p2=0.0032). This study reveals new insights in platelet mitochondrial metabolism during COVID-19 caused ARDS. it data point towards enhanced platelet activity with a pronounced turnover rate. We found increased activity of mitochondria complex I and evidence for enhanced oxidative stress. In parallel, protective mechanisms against oxidative stress were narrowed with elevated levels of IL-1β likely causing a pro-apoptotic environment. These mechanisms may contribute to platelet exhaustion in ARDS.

Keywords: acute respiratory distress syndrome (ARDS), coronavirus 19 disease (COVID-19), oxidative stress, platelet mitochondrial metabolism

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Authors: Esra Sengul, R. G. Aktas, M. E. Sitar, H. Isan

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Hepatocellular carcinoma (HCC) is a hepatocellular tumor commonly found on the surface of the chronic liver. HepG2 is the most commonly used cell type in HCC studies. The main proteins remaining in the blood serum after separation of plasma fibrinogen are albumin and globulin. The fact that the albumin showed hepatocellular damage and reflect the synthesis capacity of the liver was the main reason for our use. Alpha-Fetoprotein (AFP) is an albumin-like structural embryonic globulin found in the embryonic cortex, cord blood, and fetal liver. It has been used as a marker in the follow-up of tumor growth in various malign tumors and in the efficacy of surgical-medical treatments, so it is a good protein to look at with albumins. We have seen the morphological changes of dimethyl sulfoxide (DMSO) on HepG2 and decided to investigate its biochemical effects. We examined the effects of DMSO, which is used in cell cultures, on albumin, AFP and total protein at low doses. Material Method: Cell Culture: Medium was prepared in cell culture using Dulbecco's Modified Eagle Media (DMEM), Fetal Bovine Serum Dulbecco's (FBS), Phosphate Buffered Saline and trypsin maintained at -20 ° C. Fixation of Cells: HepG2 cells, which have been appropriately developed at the end of the first week, were fixed with acetone. We stored our cells in PBS at + 4 ° C until the fixation was completed. Area Calculation: The areas of the cells are calculated in the ImageJ (IJ). Microscope examination: The examination was performed with a Zeiss Inverted Microscope. Daytime photographs were taken at 40x, 100x 200x and 400x. Biochemical Tests: Protein (Total): Serum sample was analyzed by a spectrophotometric method in autoanalyzer. Albumin: Serum sample was analyzed by a spectrophotometric method in autoanalyzer. Alpha-fetoprotein: Serum sample was analyzed by ECLIA method. Results: When liver cancer cells were cultured in medium with 1% DMSO for 4 weeks, a significant difference was observed when compared with the control group. As a result, we have seen that DMSO can be used as an important agent in the treatment of liver cancer. Cell areas were reduced in the DMSO group compared to the control group and the confluency ratio increased. The ability to form spheroids was also significantly higher in the DMSO group. Alpha-fetoprotein was lower than the values of an ordinary liver cancer patient and the total protein amount increased to the reference range of the normal individual. Because the albumin sample was below the specimen value, the numerical results could not be obtained on biochemical examinations. We interpret all these results as making DMSO a caretaking aid. Since each one was not enough alone we used 3 parameters and the results were positive when we refer to the values of a normal healthy individual in parallel. We hope to extend the study further by adding new parameters and genetic analyzes, by increasing the number of samples, and by using DMSO as an adjunct agent in the treatment of liver cancer.

Keywords: hepatocellular carcinoma, HepG2, dimethyl sulfoxide, cell culture, ELISA

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Authors: B. Sudhakara Reddy

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This study provides a snapshot of the sustainability of selected Indian cities by employing 70 indicators in four dimensions to develop an overall city sustainability index. In recent years, the concept of ‘urban sustainability’ has become prominent due to its complexity. Urban areas propel growth and at the same time poses a lot of ecological, social and infrastructural problems and risks. In case of developing countries, the high population density of and the continuous in-migration run the highest risk in natural and man-made disasters. These issues combined with the inability of policy makers in providing basic services makes the cities unsustainable. To assess whether any given policy is moving towards or against urban sustainability it is necessary to consider the relationships among its various dimensions. Hence, in recent years, while preparing the sustainability index, an integral approach involving indicators of different dimensions such as ‘economic’, ‘environmental’ and 'social' is being used. It is also important for urban planners, social analysts and other related institutions to identify and understand the relationships in this complex system. The objective of the paper is to develop a city performance index (CPI) to measure and evaluate the urban regions in terms of sustainable performances. The objectives include: i) Objective assessment of a city’s performance, ii) setting achievable goals iii) prioritise relevant indicators for improvement, iv) learning from leaders, iv) assessment of the effectiveness of programmes that results in achieving high indicator values, v) Strengthening of stakeholder participation. Using the benchmark approach, a conceptual framework is developed for evaluating 25 Indian cities. We develop City Sustainability index (CSI) in order to rank cities according to their level of sustainability. The CSI is composed of four dimensions: Economic, Environment, Social, and Institutional. Each dimension is further composed of multiple indicators: (1) Economic that considers growth, access to electricity, and telephone availability; (2) environmental that includes waste water treatment, carbon emissions, (3) social that includes, equity, infant mortality, and 4) institutional that includes, voting share of population, urban regeneration policies. The CSI, consisting of four dimensions disaggregate into 12 categories and ultimately into 70 indicators. The data are obtained from public and non-governmental organizations, and also from city officials and experts. By ranking a sample of diverse cities on a set of specific dimensions the study can serve as a baseline of current conditions and a marker for referencing future results. The benchmarks and indices presented in the study provide a unique resource for the government and the city authorities to learn about the positive and negative attributes of a city and prepare plans for a sustainable urban development. As a result of our conceptual framework, the set of criteria we suggest is somewhat different to any already in the literature. The scope of our analysis is intended to be broad. Although illustrated with specific examples, it should be apparent that the principles identified are relevant to any monitoring that is used to inform decisions involving decision variables. These indicators are policy-relevant and, hence they are useful tool for decision-makers and researchers.

Keywords: benchmark, city, indicator, performance, sustainability

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Authors: Colin S. Chen, Chien-Jung Tien, Hsin-Jan Huang

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For weathering studies, the change of chemical constituents by biodegradation effect in diesel-contaminated soils are important factors to be considered, especially when there is a prolonged period of weathering processes. The objective was to evaluate biodegradation effects onto hydrocarbon fingerprinting and distribution patterns of diesel fuels, fuel source screening and differentiation, source-specific marker compounds, and diagnostic ratios of diesel fuel constituents by laboratory and field studies. Biodegradation processes of diesel contaminated soils were evaluated by experiments lasting for 15 and 12 months, respectively. The degradation of diesel fuel in top soils was affected by organic carbon content and biomass of microorganisms in soils. Higher depletion of total petroleum hydrocarbon (TPH), n-alkanes, and polynuclear aromatic hydrocarbons (PAHs) and their alkyl homologues was observed in soils containing higher organic carbon content and biomass. Decreased ratio of selected isoprenoids (i.e., pristane (Pr) and phytane (Ph)) including n-C17/pristane and n-C18/phytane was observed. The ratio of pristane/phytane was remained consistent for a longer period of time. At the end of the experimental period, a decrease of pristane/phytane was observed. Biomarker compounds of bicyclic sesquiterpanes (BS) were less susceptible to the effects of biodegradation. The ratios of characteristic factors such as C15 sesquiterpane/ 8β(H)-drimane (BS3/BS5), C15 sesquiterpane/ 8β(H)-drimane (BS4/BS5), 8β(H)-drimane/8β(H)-homodrimane (BS5/BS10), and C15 sesquiterpane/8β(H)-homodrimane (BS3/BS10) could be adopted for source identification of diesel fuels in top soil. However, for biodegradation processes lasted for six months but shorter than nine months, only BS3/BS5 and BS3/BS10 could be distinguished in two diesel fuels. In subsoil experiments (contaminated soil located 50 cm below), the ratios of characteristic factors including BS3/BS5, BS4/BS5, and BS5/BS10 were valid for source identification of two diesel fuels for nine month biodegradation. At the early stage of contamination, biomass of soil decreased significantly. However, 6 and 7 dominant species were found in soils in top soil experiments, respectively. With less oxygen and nutrients in subsoil, less biomass of microorganisms was observed in subsoils. Only 2 and 4 diesel-degrading species of microorganisms were identified in two soils, respectively. Parameters of double ratio such as fluorene/C1-fluorene: C2-phenanthrene/C3-phenanthrene (C0F/C1F:C2P/C3P) in both top and subsoil, C2-naphthalene/C2-phenanthrene: C1-phenanthrene/C3-phenanthrene (C2N/C2P:C1P/C3P), and C1-phenanthrene/C1-fluorene: C3-naphthalene/C3-phenanthrene (C1P/C1F:C3N/C3P) in subsoil could serve as forensic indicators in diesel contaminated sites. BS3/BS10:BS4/BS5 could be used in 6 to 9 months of biodegradation processes. Results of principal component analysis (PCA) indicated that source identification of diesel fuels in top soil could only be perofrmed for weathering process less than 6 months. For subsoil, identification can be conducted for weathering process less than 9 months. Ratio of isoprenoids (pristane and phytane) and PAHs might be affected by biodegradation in spilled sites. The ratios of bicyclic sesquiterpanes could serve as forensic indicators in diesel-contaminated soils. Finally, source identification was attemped for samples collected from different fuel contaminated sites by using the unique pattern of sesquiterpanes. It was anticipated that the information generated from this study would be adopted by decision makers to evaluate the liability of cleanup in diesel contaminated sites.

Keywords: biodegradation, diagnostic ratio, diesel fuel, environmental forensics

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Authors: Truong H. Le, Ngoc M. To, Quang N. Tran, Luu T. Cao, Chi V. Le

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Introduction: In Vietnam, the current monitoring and treatment for ordinary diabetic patient mostly based on glucose monitoring with HbA1c test for every three months (recommended goal is HbA1c < 6.5%~7%). For diabetes in pregnant women or Gestational diabetes mellitus (GDM), glycemic control until the time of delivery is extremly important because it could reduce significantly medical implications for both the mother and the child. Besides, GDM requires continuos glucose monitoring at least every two weeks and therefore an alternative marker of glycemia for short-term control is considering a potential tool for the healthcare providers. There are published studies have indicated that the glycosylated serum protein is a better indicator than glycosylated hemoglobin in GDM monitoring. Based on the actual practice in Vietnam, this study was designed to evaluate the role of serum fructosamine as a monitoring tool in GDM treament and its correlations with fasting blood glucose (G0), 2-hour postprandial glucose (G2) and glycosylated hemoglobin (HbA1c). Methods: A cohort study on pregnant women diagnosed with GDM by the 75-gram oralglucose tolerance test was conducted at Endocrinology Department, Cho Ray hospital, Vietnam from June 2014 to March 2015. Cho Ray hospital is the final destination for GDM patient in the southern of Vietnam, the study population has many sources from other pronvinces and therefore researchers belive that this demographic characteristic can help to provide the study result as a reflection for the whole area. In this study, diabetic patients received a continuos glucose monitoring method which consists of bi-weekly on-site visit every 2 weeks with glycosylated serum protein test, fasting blood glucose test and 2-hour postprandial glucose test; HbA1c test for every 3 months; and nutritious consultance for daily diet program. The subjects still received routine treatment at the hospital, with tight follow-up from their healthcare providers. Researchers recorded bi-weekly health conditions, serum fructosamine level and delivery outcome from the pregnant women, using Stata 13 programme for the analysis. Results: A total of 500 pregnant women was enrolled and follow-up in this study. Serum fructosamine level was found to have a light correlation with G0 ( r=0.3458, p < 0.001) and HbA1c ( r=0.3544, p < 0.001), and moderately correlated with G2 ( r=0.4379, p < 0.001). During study timeline, the delivery outcome of 287 women were recorded with the average age of 38.5 ± 1.5 weeks, 9% of them have macrosomia, 2.8% have premature birth before week 35th and 9.8% have premature birth before week 37th; 64.8% of cesarean section and none of them have perinatal or neonatal mortality. The study provides a reference interval of serum fructosamine for GDM patient was 112.9 ± 20.7 μmol/dL. Conclusion: The present results suggests that serum fructosamine is as effective as HbA1c as a reflection of blood glucose control in GDM patient, with a positive result in delivery outcome (0% perinatal or neonatal mortality). The reference value of serum fructosamine measurement provided a potential monitoring utility in GDM treatment for hospitals in Vietnam. Healthcare providers in Cho Ray hospital is considering to conduct more studies to test this reference as a target value in their GDM treatment and monitoring.

Keywords: gestational diabetes mellitus, monitoring tool, serum fructosamine, Vietnam

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Authors: Sonia Boscenco, Zihan Wang, Euclides José de Mendoça Filho, João Paulo Hoppe, Irina Pokhvisneva, Geoffrey B.C. Hall, Michael J. Meaney, Patricia Pelufo Silveira

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Entropy can be described as a measure of the number of states of a system, and when used in the context of physiological time-based signals, it serves as a measure of complexity. In functional connectivity data, entropy can account for the moment-to-moment variability that is neglected in traditional functional magnetic resonance imaging (fMRI) analyses. While brain fMRI resting state entropy has been associated with some pathological conditions like schizophrenia, no investigations have explored the association between brain entropy measures and individual differences in child behavior in healthy children. We describe a novel exploratory approach to evaluate brain fMRI resting state data in two child cohorts, and MAVAN (N=54, 4.5 years, 48% males) and GUSTO (N = 206, 4.5 years, 48% males) and its associations to child behavior, that can be used in future research in the context of child exposures and long-term health. Following rs-fMRI data pre-processing and Shannon entropy calculation across 32 network regions of interest to acquire 496 unique functional connections, partial correlation coefficient analysis adjusted for sex was performed to identify associations between entropy data and Strengths and Difficulties questionnaire in MAVAN and Child Behavior Checklist domains in GUSTO. Significance was set at p < 0.01, and we found eight significant associations in GUSTO. Negative associations were found between two frontoparietal regions and cerebellar posterior and oppositional defiant problems, (r = -0.212, p = 0.006) and (r = -0.200, p = 0.009). Positive associations were identified between somatic complaints and four default mode connections: salience insula (r = 0.202, p < 0.01), dorsal attention intraparietal sulcus (r = 0.231, p = 0.003), language inferior frontal gyrus (r = 0.207, p = 0.008) and language posterior superior temporal gyrus (r = 0.210, p = 0.008). Positive associations were also found between insula and frontoparietal connection and attention deficit / hyperactivity problems (r = 0.200, p < 0.01), and insula – default mode connection and pervasive developmental problems (r = 0.210, p = 0.007). In MAVAN, ten significant associations were identified. Two positive associations were found = with prosocial scores: the salience prefrontal cortex and dorsal attention connection (r = 0.474, p = 0.005) and the salience supramarginal gyrus and dorsal attention intraparietal sulcus (r = 0.447, p = 0.008). The insula and prefrontal connection were negatively associated with peer problems (r = -0.437, p < 0.01). Conduct problems were negatively associated with six separate connections, the left salience insula and right salience insula (r = -0.449, p = 0.008), left salience insula and right salience supramarginal gyrus (r = -0.512, p = 0.002), the default mode and visual network (r = -0.444, p = 0.009), dorsal attention and language network (r = -0.490, p = 0.003), and default mode and posterior parietal cortex (r = -0.546, p = 0.001). Entropy measures of resting state functional connectivity can be used to identify individual differences in brain function that are correlated with variation in behavioral problems in healthy children. Further studies applying this marker into the context of environmental exposures are warranted.

Keywords: child behaviour, functional connectivity, imaging, Shannon entropy

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Authors: Maryam Hajrezaie, Mahmood Ameen Abdulla, Nazia Abdul Majid, Hapipa Mohd Ali, Pouya Hassandarvish, Maryam Zahedi Fard

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Background: Colon cancer is one of the most prevalent cancers in the world and is the third leading cause of death among cancers in both males and females. The incidence of colon cancer is ranked fourth among all cancers but varies in different parts of the world. Cancer chemoprevention is defined as the use of natural or synthetic compounds capable of inducing biological mechanisms necessary to preserve genomic fidelity. Andrographolide is the major labdane diterpenoidal constituent of the plant Andrographis paniculata (family Acanthaceae), used extensively in the traditional medicine. Extracts of the plant and their constituents are reported to exhibit a wide spectrum of biological activities of therapeutic importance. Laboratory animal model studies have provided evidence that Andrographolide play a role in inhibiting the risk of certain cancers. Objective: Our aim was to evaluate the chemopreventive efficacy of the Andrographolide in the AOM induced rat model. Methods: To evaluate inhibitory properties of andrographolide on colonic aberrant crypt foci (ACF), five groups of 7-week-old male rats were used. Group 1 (control group) were fed with 10% Tween 20 once a day, Group 2 (cancer control) rats were intra-peritoneally injected with 15 mg/kg Azoxymethan, Gropu 3 (drug control) rats were injected with 15 mg/kg azoxymethan and 5-Flourouracil, Group 4 and 5 (experimental groups) were fed with 10 and 20 mg/kg andrographolide each once a day. After 1 week, the treatment group rats received subcutaneous injections of azoxymethane, 15 mg/kg body weight, once weekly for 2 weeks. Control rats were continued on Tween 20 feeding once a day and experimental groups 10 and 20 mg/kg andrographolide feeding once a day for 8 weeks. All rats were sacrificed 8 weeks after the azoxymethane treatment. Colons were evaluated grossly and histopathologically for ACF. Results: Administration of 10 mg/kg and 20 mg/kg andrographolide were found to be effectively chemoprotective, as evidenced microscopily and biochemically. Andrographolide suppressed total colonic ACF formation up to 40% to 60%, respectively, when compared with control group. Pre-treatment with andrographolide, significantly reduced the impact of AOM toxicity on plasma protein and urea levels as well as on plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GGT) activities. Grossly, colorectal specimens revealed that andrographolide treatments decreased the mean score of number of crypts in AOM-treated rats. Importantly, rats fed andrographolide showed 75% inhibition of foci containing four or more aberrant crypts. The results also showed a significant increase in glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), and Prostaglandin E2 (PGE2) activities and a decrease in malondialdehyde (MDA) level. Histologically all treatment groups showed a significant decrease of dysplasia as compared to control group. Immunohistochemical staining showed up-regulation of Hsp70 and down-regulation of Bax proteins. Conclusion: The current study demonstrated that Andrographolide reduce the number of ACF. According to these data, Andrographolide might be a promising chemoprotective activity, in a model of AOM-induced in ACF.

Keywords: chemopreventive, andrographolide, colon cancer, aberrant crypt foci (ACF)

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Authors: Fernanda Piculo, Giovana Vesentini, Gabriela Marini, Debora Cristina Damasceno, Angelica Mercia Pascon Barbosa, Marilza Vieira Cunha Rudge

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Introduction: Women with previous gestational diabetes mellitus (GDM) were significantly more likely to have urinary incontinence (UI) and pelvic floor muscle dysfunction compared to non-diabetic women two years after a cesarean section. Additional results demonstrated that induced diabetes causes detrimental effects on pregnant rat urethral muscle. These results indicate the need for exploration of the mechanistic role of a recovery factor in female UI. Chemokine ligand 7 (CCL7) was significantly over expressed in rat serum, urethral and vaginal tissues immediately following induction of stress UI in a rat model simulating birth trauma. CCL7 over expression has shown potency for stimulating targeted stem cell migration and provide a translational link (clinical measurement) which further provide opportunities for treatment. The aim of this study was to investigate the CCL7 levels profile in diabetic pregnant women with urinary incontinence during pregnancy over the first year postpartum. Methods: This study was conducted in the Perinatal Diabetes Research Center of the Botucatu Medical School/UNESP, and was approved by the Research Ethics Committee of the Institution (CAAE: 20639813.0.0000.5411). The diagnosis of GDM was established between 24th and 28th gestational weeks, by the 75 g-OGTT test according to ADA’s criteria. Urinary incontinence was defined according to the International Continence Society and the CCL7 levels was measured by ELISA (R&D Systems, Catalog Number DCC700). Two hundred twelve women were classified into four study groups: normoglycemic continent (NC), normoglycemic incontinent (NI), diabetic continent (DC) and diabetic incontinent (DI). They were evaluated at six-time-points: 12-18, 24-28 and 34-38 gestational weeks, 24-48 hours, 6 weeks and 6-12 months postpartum. Results: At 12-18 weeks, it was possible to consider only two groups, continent and incontinent, because at this early gestational period has not yet been the diagnosis of GDM. The group with GDM and UI (DI group) showed lower levels of CCL7 in all time points during pregnancy and postpartum, compared to normoglycemic groups (NC and NI), indicating that these women have not recovered from child birth induced UI during the 6-12 months postpartum compared to their controls, and that the progression of UI and/or lack of recovery throughout the first postpartum year can be related with lower levels of CCL7. Instead, serum CCL7 was significantly increased in the NC group. Taken together, these findings of overexpression of CCL7 in the NC group and decreased levels in the DI group, could confirm that diabetes delays the recovery from child birth induced UI, and that CCL7 could potentially be used as a serum marker of injury. Conclusion: This study demonstrates lower levels of CCL7 in the DI group during pregnancy and postpartum and suggests that the progression of UI in diabetic women and/or lack of recovery throughout the first postpartum year can be related with low levels of CCL7. This provides a translational potential where CCL7 measurement could be used as a surrogate for injury after delivery. Successful controlled CCL7 mediated stem cell homing to the lower urinary tract could one day introduce the potential for non-operative treatment or prevention of stress urinary incontinence.

Keywords: CCL7, gestational diabetes, pregnancy, urinary incontinence

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Authors: Prajna Paramita Naik, Sujit Kumar Bhutia

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Background: Regardless of the development multimodal treatment strategies, oral squamous cell carcinoma (OSCC) is often associated with a high rate of recurrence, metastasis and chemo- and radio- resistance. The present study inspected the relevance of CD44, ABCB1 and ADAM17 expression as a putative stem cell compartment in oral squamous cell carcinoma (OSCC) and deciphered the role of autophagy in regulating the expression of aforementioned proteins, stemness and chemoresistance. Methods: A retrospective analysis of CD44, ABCB1 and ADAM17 expression with respect to the various clinicopathological factors of sixty OSCC patients were determined via immunohistochemistry. The correlation among CD44, ABCB1 and ADAM17 expression was established. Sphere formation assay, flow cytometry and fluorescence microscopy were conducted to elucidate the stemness and chemoresistance nature of established cisplatin-resistant oral cancer cells (FaDu). The pattern of expression of CD44, ABCB1 and ADAM17 in parental (FaDu-P) and resistant FaDu cells (FaDu-CDDP-R) were investigated through fluorescence microscopy. Western blot analysis of autophagy marker proteins was performed to compare the status of autophagy in parental and resistant FaDu cell. To investigate the role of autophagy in chemoresistance and stemness, sphere formation assay, immunofluorescence and Western blot analysis was performed post transfection with siATG14 and the level of expression of autophagic proteins, mitochondrial protein and stemness-associated proteins were analyzed. The statistical analysis was performed by GraphPad Prism 4.0 software. p-value was defined as follows: not significant (n.s.): p > 0.05;*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001 were considered statistically significant. Results: In OSCC, high CD44, ABCB1 and ADAM17 expression were significantly correlated with higher tumor grades and poor differentiation. However, the expression of these proteins was not related to the age and sex of OSCC patients. Moreover, the expression of CD44, ABCB1 and ADAM17 were positively correlated with each other. In vitro and OSCC tissue double labeling experiment data showed that CD44+ cells were highly associated with ABCB1 and ADAM17 expression. Further, FaDu-CDDP-R cells showed higher sphere forming capacity along with increased fraction of the CD44+ population and β-catenin expression FaDu-CDDP-R cells also showed accelerated expression of CD44, ABCB1 and ADAM17. A comparatively higher autophagic flux was observed in FaDu-CDDP-R against FaDu-P cells. The expression of mitochondrial proteins was noticeably reduced in resistant cells as compared to parental cells indicating the occurrence of autophagy-mediated mitochondrial degradation in oral cancer. Moreover, inhibition of autophagy was coupled with the decreased formation of orospheres suggesting autophagy-mediated stemness in oral cancer. Blockade of autophagy was also found to induce the restoration of mitochondrial proteins in FaDu-CDDP-R cells indicating the involvement of mitophagy in chemoresistance. Furthermore, a reduced expression of CD44, ABCB1 and ADAM17 was also observed in ATG14 deficient cells FaDu-P and FaDu-CDDP-R cells. Conclusion: The CD44+ ⁄ABCB1+ ⁄ADAM17+ expression in OSCC might be associated with chemoresistance and a putative CSC compartment. Further, the present study highlights the contribution of mitophagy in chemoresistance and confirms the potential involvement of autophagic regulation in acquisition of stem-like characteristics in OSCC.

Keywords: ABCB1, ADAM17, autophagy, CD44, chemoresistance, mitophagy, OSCC, stemness

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Authors: Thiago E. Goto, Carla C. Lopes, Helena B. Nader, Anielle C. A. Silva, Noelio O. Dantas, José R. Siqueira Jr., Luciano Caseli

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Quantum dots (QD) are semiconductor nanocrystals that can be employed in biological research as a tool for fluorescence imagings, having the potential to expand in vivo and in vitro analysis as cancerous cell biomarkers. Particularly, cadmium selenide (CdSe) magic-sized quantum dots (MSQDs) exhibit stable luminescence that is feasible for biological applications, especially for imaging of tumor cells. For these facts, it is interesting to know the mechanisms of action of how such QDs mark biological cells. For that, simplified models are a suitable strategy. Among these models, Langmuir films of lipids formed at the air-water interface seem to be adequate since they can mimic half a membrane. They are monomolecular films formed at liquid-gas interfaces that can spontaneously form when organic solutions of amphiphilic compounds are spread on the liquid-gas interface. After solvent evaporation, the monomolecular film is formed, and a variety of techniques, including tensiometric, spectroscopic and optic can be applied. When the monolayer is formed by membrane lipids at the air-water interface, a model for half a membrane can be inferred where the aqueous subphase serve as a model for external or internal compartment of the cell. These films can be transferred to solid supports forming the so-called Langmuir-Blodgett (LB) films, and an ampler variety of techniques can be additionally used to characterize the film, allowing for the formation of devices and sensors. With these ideas in mind, the objective of this work was to investigate the specific interactions of CdSe MSQDs with tumorigenic and non-tumorigenic cells using Langmuir monolayers and LB films of lipids and specific cell extracts as membrane models for diagnosis of cancerous cells. Surface pressure-area isotherms and polarization modulation reflection-absorption spectroscopy (PM-IRRAS) showed an intrinsic interaction between the quantum dots, inserted in the aqueous subphase, and Langmuir monolayers, constructed either of selected lipids or of non-tumorigenic and tumorigenic cells extracts. The quantum dots expanded the monolayers and changed the PM-IRRAS spectra for the lipid monolayers. The mixed films were then compressed to high surface pressures and transferred from the floating monolayer to solid supports by using the LB technique. Images of the films were then obtained with atomic force microscopy (AFM) and confocal microscopy, which provided information about the morphology of the films. Similarities and differences between films with different composition representing cell membranes, with or without CdSe MSQDs, was analyzed. The results indicated that the interaction of quantum dots with the bioinspired films is modulated by the lipid composition. The properties of the normal cell monolayer were not significantly altered, whereas for the tumorigenic cell monolayer models, the films presented significant alteration. The images therefore exhibited a stronger effect of CdSe MSQDs on the models representing cancerous cells. As important implication of these findings, one may envisage for new bioinspired surfaces based on molecular recognition for biomedical applications.

Keywords: biomembrane, langmuir monolayers, quantum dots, surfaces

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Authors: Khelladi Hadj Mostefa, Krouf Djamil, Taleb-Dida Nawel

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Background: Obesity results from a prolonged imbalance between energy intake and energy expenditure, as depending on basal metabolic rate. Oils and proteins from sea have important therapeutic (such as obesity and hypercholesterolemia) and antioxidant effects. Sardine are a widely consumed fish in the Mediterranean region. Its consumption provides humans with various nutrients such as oils (rich in omega 3 plyunsaturated fatty acids)) and proteins. Methods: Sardine oil (SO) and sardine proteins (SP) were extracted and purified. Mixture of vegetable oils (olive-walnut-sunflower) were prepared from oils produced in Algeria. Eighteen wistar rats are fed a high fat diet enriched with 1% cholesterol for 30 days to induce obesity and hypercholesterolemia. The rats are divided into 3 groups. The first group consumes 20% sardine protein combined with 5% sardine oil (38% SFA (saturated fatty acids), 31% MIFA (monounsaturated fatty acids) and 31% PIFA (polyunsaturated fatty acids)) (SPso). The second group consumes 20% sardine protein combined with 5% of a mixture of vegetable oils (VO) containing 13% SFA, 58% MIFA and 29% PIFA (PSvo), and the third group consuming 20% casein combined with 5% of the mixture of vegetable oils and serves as a semi-synthetic reference (CASvo). Body weights and glycaemia are measured weekly After 28 days of experimentation, the rats are sacrificed, the blood and the liver removed. Serum assays of total cholesterol (TC) and triglycerides (TG) were performed by enzymatic colorimetric methods. Evaluation of lipid peroxidation was performed by assaying thiobarbituric acid reactive species (TBARS) and hydroperoxides values. The protein oxidation was performed by assaying carbonyl derivatives values. Finally, evaluation of antioxidant defense is made by measuring the activity of antioxidant enzymes, the superoxide dismutase (SOD) and the catalase (CAT).Results: After 28 days, the body weight (BW) of the rats increased significantly in SPso and SPvo groups compared to CAS group, by +11% and 7%, respectively. Cholesterolemia (TC) increased significantly in the SPso and SPvo groups compared to the CAS group (P<0.01), while triglyceridemia (TG) decreased significantly in the SPso group compared to SPvo and CAS groups (P<0.01). Albumin (marker of inflammation) increased in the PSs group compared to SPvo and CAS groups by +35% and +13%, respectively. The serum TBARS levels are -40% lower in SPso group compared to SPvo group, and they are -80% and -76% lower in SPso compared to SPvo and CAS groups, respectively. The level of carbonyls derivatives in the serum and liver are significantly reduced in the SPso group compared to the SPvo and CAS groups. Superoxide dismutase (SOD) activity decreased in liver of SPso group compared to SPvo group (P<0.01). While that of CAT is increased in liver tissue of SPso group compared to SPvo group (P<0.01). Conclusion: Sardine oil combined with sardine protein has a hypotriglyceridemic effect, reduces body weight, attenuates inflammation and seems to protect against lipid peroxidation and protein oxidation and increases antioxidant defense in hypercholesterolemic and obese rats. This could be in favor of a protective effect against obesity and cardiovascular diseases.

Keywords: rat, obesity, hypercholesterolemia, sardine protein, sardine oil, vegetable oils mixture, lipid peroxidation, protein oxidation, antioxidant defense

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Authors: Sarwan Dhir, Suma Basak, Dipika Parajulee

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Alfalfa is renowned for its nutritional and biopharmaceutical value as a perennial forage legume. However, establishing a rapid plant regeneration protocol using somatic embryogenesis and efficient transformation frequency are the crucial prerequisites for gene editing in alfalfa. This study was undertaken to establish and improve the protocol for somatic embryogenesis and subsequent plant regeneration. The experiments were conducted in response to natural sensitivity using various antibiotics such as cefotaxime, carbenicillin, gentamycin, hygromycin, and kanamycin. Using 3-week-old leaf tissue, somatic embryogenesis was initiated on Gamborg’s B5 basal (B5H) medium supplemented with 3% maltose, 0.9µM Kinetin, and 4.5µM 2,4-D. Embryogenic callus (EC) obtained from the B5H medium exhibited a high rate of somatic embryo formation (97.9%) after 3 weeks when the cultures were placed in the dark. Different developmental stages of somatic embryos and cotyledonary stages were then transferred to Murashige and Skoog’s (MS) basal medium under light, resulting in a 94% regeneration rate of plantlets. Our results indicate that leaf segments can grow (tolerate) up to 450 mg/L of cefotaxime and 400 mg/L of carbenicillin in the culture medium. However, the survival threshold for hygromycin at 12.5 mg/L, kanamycin at 250 mg/L, gentamycin at 50 mg/L, and timentin (300 mg/L). The experiment to improve the protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector was also conducted. The vector contains two reporter genes such as β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene (HPT), all driven under the CaMV 35s promoter. Various transformation parameters were optimized using 3-week-old in vitro-grown plantlets. The different parameters such as types of explant, leaf ages, preculture days, segment sizes, wounding types, bacterial concentrations, infection periods, co-cultivation periods, different concentrations of acetosyringone, silver nitrate, and calcium chloride were optimized for transient gene expression. The transient gene expression was confirmed via histochemical GUS and GFP visualization under fluorescent microscopy. The data were analyzed based on the semi-quantitative observation of the percentage and number of blue GUS spots on different days of agro-infection. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old leaf segments wounded using a scalpel blade of 11 size- after 3 days of post-incubation at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of bacterial-leaf segment co-cultivation, with the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. Our results suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The stable gene expression in the putative transgenic tissue was confirmed using PCR amplification of both marker genes, indicating that gene expression in explants was not solely due to Agrobacterium, but also from transformed cells. The improved protocol could be used for generating transgenic alfalfa plants using genome editing techniques such as CRISPR/Cas9.

Keywords: Medicago sativa l. (Alfalfa), agrobacterium tumefaciens, β-glucuronidase, green fluorescent protein, transient gene

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Authors: Majed Al Otaibi, Melissa Lessard-Beaudoin, Amel Loudghi, Raphael Chouinard-Watkins, Melanie Plourde, Frederic Calon, C. Alexandre Castellano, Stephen Cunnane, Helene Payette, Pierrette Gaudreau, Denis Gris, Rona K. Graham

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Introduction: Alzheimer disease (AD) is a chronic disorder that affects millions of individuals worldwide. Symptoms include memory dysfunction, and also alterations in attention, planning, language and overall cognitive function. Olfactory dysfunction is a common symptom of several neurological disorders including AD. Studying the mechanisms underlying the olfactory dysfunction may therefore lead to the discovery of potential biomarkers and/or treatments for neurodegenerative diseases. Objectives: To determine if olfactory dysfunction predicts future cognitive impairment in the aging population and to characterize the olfactory system in a murine model expressing a genetic factor of AD. Method: For the human study, quantitative olfactory tests (UPSIT and OMT) have been done on 93 subjects (aged 80 to 94 years) from the Quebec Longitudinal Study on Nutrition and Successful Aging (NuAge) cohort accepting to participate in the ORCA secondary study. The telephone Modified Mini Mental State examination (t-MMSE) was used to assess cognition levels, and an olfactory self-report was also collected. In a separate cohort, olfactory cortical volume was calculated using MRI results from healthy old adults (n=25) and patients with AD (n=18) using the AAL single-subject atlas and performed with the PNEURO tool (PMOD 3.7). For the murine study, we are using Western blotting, RT-PCR and immunohistochemistry. Result: Human Study: Based on the self-report, 81% of the participants claimed to not suffer from any problem with olfaction. However, based on the UPSIT, 94% of those subjects showed a poor olfactory performance and different forms of microsmia. Moreover, the results confirm that olfactory function declines with age. We also detected a significant decrease in olfactory cortical volume in AD individuals compared to controls. Murine study: Preliminary data demonstrate there is a significant decrease in expression levels of the proform of caspase-3 and the caspase substrate STK3, in the olfactory bulb of mice expressing human APOE4 compared with controls. In addition, there is a significant decrease in the expression level of the caspase-9 proform and caspase-8 active fragment. Analysis of the mature neuron marker, NeuN, shows decreased expression levels of both isoforms. The data also suggest that Iba-1 immunostaining is increased in the olfactory bulb of APOE4 mice compared to wild type mice. Conclusions: The activation of caspase-3 may be the cause of the decreased levels of STK3 through caspase cleavage and may play role in the inflammation observed. In the clinical study, our results suggest that seniors are unaware of their olfactory function status and therefore it is not sufficient to measure olfaction using the self-report in the elderly. Studying olfactory function and cognitive performance in the aging population will help to discover biomarkers in the early stage of the AD.

Keywords: Alzheimer's disease, APOE4, cognition, caspase, brain atrophy, neurodegenerative, olfactory dysfunction

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Authors: Vincent Oghenekevbe Olughor, Olusoji Mayowa Ige

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Except for a preexisting liver disease and malnutrition, there are no predilections for low serum albumin (SA) levels in humans. At normal reference levels (4.0-6.0g/dl) SA is a universal marker for mortality and morbidity risks assessments where depletion by 1.0g/dl increases mortality risk by 137% and morbidity by 89%.It has 40 known functions contributing significantly to the sustenance of human life. A depletion in SA to <2.2g/dl, in most clinical settings worldwide, leads to loss of oncotic pressure of blood causing clinical manifestations of bipedal Oedema, in which the patients remain conscious. SA also contributes significantly to buffering of blood to a life-sustaining pH of 7.35-7.45. A drop in blood pH to <6.9 will lead to instant coma and death, which can occur after SA continues to deplete after manifestations of bipedal Oedema. In an intervention study conducted in 2014 following the discovery that “SA is depleted during malaria fever”, a Nutraceutical formulated for use as treatment adjunct to prevent SA depletions during malaria to <2.4g/dl after Efficacy testing was found to be satisfactory. There are five known types of Malaria caused by Apicomplexan parasites, Plasmodium: the most lethal being that caused by Plasmodium falciparum causing malignant tertian malaria, in which the fever was occurring every 48 hours coincides with the dumping of malaria-toxins (Hemozoin) into blood, causing contamination: blood must remain sterile. Other Apicomplexan parasites, Toxoplasma and Cryptosporidium, are opportunistic infections of HIV. Separate studies showed SA depletions in MDRTB (multidrug resistant TB), and MDRTB-HIV patients by the same mechanism discovered with malaria and such depletions will be further complicated whenever Apicomplexan parasitic infections co-exist. Both Apicomplexan parasites and the TB parasite belong to the Obligate-group of Parasites, which are parasites that replicate only inside its host; and most of them have capacities to over-consume host nutrients during parasitaemia. In MDRTB patients the body attempts repeatedly to prevent depletions in SA to critical levels in the presence of adequate nutrients and only for a while in MDRTB-HIV patients. These groups of patients will, therefore, benefit from the already tested Nutraceutical in malaria patients. The Nutraceutical bio-Powder was formulated (to BP 1988 specification) from twelve nature-based food-grade nutrients containing all dedicated nutrients for ensuring improved synthesis of Albumin by the liver. The Nutraceutical was administered daily for 38±2days in 23 children, in a prospective phase-2 clinical trial, and its impact on body weight and core blood parameters were documented at the start and end of efficacy testing period. Sixteen children who did not experience malaria-induced depletions of SA had significant SA increase; seven children who experienced malaria-induced depletions of SA had insignificant SA decrease. The Packed Cell Volume Percentage (PCV %), a measure of the Oxygen carrying capacity of blood and the amount of nutrients the body can absorb, increased in both groups. The total serum proteins (SA+ Globulins) increased or decreased within the continuum of normal. In conclusion, MDRTB and MDRTB-HIV patients will benefit from a variant of this Nutraceutical when used as treatment adjunct.

Keywords: antitrypsin-free Nutraceutical, apicomplexan parasites, no predilections for low serum albumin, toxoplasmosis

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Authors: Kiran Datta, Daniella Holland-Hart, Anthony Byrne

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Introduction: Oesophago-gastric (OG) cancers are the fifth commonest in the UK, accounting for over 12,000 deaths each year. Most patients will present at later stages of the disease, with only 21% of patients with stage 4 disease surviving longer than a year. As a result, many patients are unsuitable for curative surgery and instead receive palliative treatment to improve prognosis and symptom burden. However, palliative chemotherapy can result in significant toxicity: almost half of the patients are unable to complete their chemotherapy regimen, with this proportion rising significantly in older and frailer patients. In addition, clinical trials often exclude older and frailer patients due to strict inclusion criteria, meaning there is limited evidence to guide which patients are most likely to benefit from palliative chemotherapy. Inappropriate chemotherapy administration is at odds with the goals of palliative treatment and care, which are to improve quality of life, and this also represents a significant resource expenditure. This literature review aimed to examine and appraise evidence regarding treatment resilience in order to guide clinicians in identifying the most suitable candidates for palliative chemotherapy. Factors influencing treatment resilience were assessed, as measured by completion rates, dose reductions, and toxicities. Methods: This literature review was conducted using rapid review methodology, utilising modified systematic methods. A literature search was performed across the MEDLINE, EMBASE, and Cochrane Library databases, with results limited to papers within the last 15 years and available in English. Key inclusion criteria included: 1) participants with either oesophageal, gastro-oesophageal junction, or gastric cancers; 2) patients treated with palliative chemotherapy; 3) available data evaluating the association between baseline participant characteristics and treatment resilience. Results: Of the 2326 papers returned, 11 reports of 10 studies were included in this review after excluding duplicates and irrelevant papers. Treatment resilience factors that were assessed included: age, performance status, frailty, inflammatory markers, and sarcopenia. Age was generally a poor predictor for how well patients would tolerate chemotherapy, while poor performance status was a better indicator of the need for dose reduction and treatment non-completion. Frailty was assessed across one cohort using multiple screening tools and was an effective marker of the risk of toxicity and the requirement for dose reduction. Inflammatory markers included lymphopenia and the Glasgow Prognostic Score, which assessed inflammation and hypoalbuminaemia. Although quick to obtain and interpret, these findings appeared less reliable due to the inclusion of patients treated with palliative radiotherapy. Sarcopenia and body composition were often associated with chemotherapy toxicity but not the rate of regimen completion. Conclusion: This review demonstrates that there are numerous measures that can estimate the ability of patients with oesophago-gastric cancer to tolerate palliative chemotherapy, and these should be incorporated into clinical assessments to promote personalised decision-making around treatment. Age should not be a barrier to receiving chemotherapy and older and frailer patients should be included in future clinical trials to better represent typical patients with oesophago-gastric cancers. Decisions regarding palliative treatment should be guided by these factors identified as well as patient preference.

Keywords: frailty, oesophago-gastric cancer, palliative chemotherapy, treatment resilience

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Authors: Suman Chaudhary, Rupinder K. Kanwar, Jagat R. Kanwar

Abstract:

Azadirachta indica, also known as Neem belonging to the mahogany family is actively gaining interest in the era of modern day medicine due to its extensive applications in homeopathic medicine such as Ayurveda and Unani. More than 140 phytochemicals have been extracted from neem leaves, seed, bark and flowers for agro-medicinal applications. Among the various components, neem leaf protein (NLP) is currently the most investigated active ingredient, due to its immunomodulatory activities against tumor growth. However, these therapeutic ingredients of neem are susceptible to degradation and cannot withstand the drastic pH changes under physiological environment, and therefore, there is an urgent need of an alternative strategy such as a nano-delivery system to exploit its medicinal benefits. This study hypothesizes that guar gum (GG) derived biodegradable nano-carrier based encapsulation of NLP will improve its stability, specificity and sensitivity, thus facilitating targeted anti-cancer therapeutics. GG is a galactomannan derived from the endosperm of the guar beans seeds. Synthesis of guar nanocapsules (NCs) was performed using nanoprecipitation technique where the GG was encapsulated with NLP. Preliminary experiments conducted to characterize the NCs confirmed spherical morphology with a narrow size distribution of 30-40 nm. Differential scanning colorimetric analysis (DSC) validated the stability of these NCs even at a temperature range of 50-60°C which was well within the physiological and storage conditions. Thermogravimetric (TGA) analysis indicated high decomposition temperature of these NCs ranging upto 350°C. Additionally, Fourier Transform Infrared spectroscopy (FTIR) and the SDS-PAGE data acquired confirmed the successful encapsulation of NLP in the NCs. The anti-cancerous therapeutic property of this NC was tested on colon cancer cells (caco-2) as they are one of the most prevalent form of cancer. These NCs (both NLP loaded and void) were also tested on human intestinal epithelial cells (FHs 74) cells to evaluate their effect on normal cells. Cytotoxicity evaluation of the NCs in the cell lines confirmed that the IC50 for NLP in FHs 74 cells was ~2 fold higher than in caco-2 cells, indicating that this nanoformulation system possessed biocompatible anti-cancerous properties Immunoconfocal microscopy analysis confirmed the time dependent internalization of the NCs within 6h. Recent findings performed using Annexin V and PI staining indicated a significant increase (p ≤ 0.001) in the early and late apoptotic cell population when treated with the NCs signifying the role of NLP in inducing apoptosis in caco-2 cells. This was further validated using Western blot, Polymerase chain reaction (PCR) and Fluorescence activated cell sorter (FACS) aided protein expressional analysis which presented a downregulation of survivin, an anti-apoptotic cell marker and upregulation of Bax/Bcl-2 ratio (pro-apoptotic indicator). Further, both the NLP NC and unencapsulated NLP treatment destabilized the mitochondrial membrane potential subsequently facilitating the release of the pro-apoptotic caspase cascade initiator, cytochrome-c. Future studies will be focused towards granting specificity to these NCs towards cancer cells, along with a comprehensive analysis of the anti-cancer potential of this naturally occurring compound in different cancer and in vivo animal models, will validate the clinical application of this unprecedented protein therapeutic.

Keywords: anti-tumor, guar gum, nanocapsules, neem leaf protein

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Authors: Gloria M. Ntuba-Jua, Afui M. Mih, Eneke E. T. Bechem

Abstract:

Prunus africana (Hook. F.) Kalkman, commonly known as Pygeum or African cherry belongs to the Rosaceae family. It is a medium to large, evergreen tree with a spreading crown of 10 to 20 m. It is used by the traditional medical practitioners for the treatment of over 45ailments in Cameroon and sub-Sahara Africa. In modern medicine, it is used in the treatment of benign prostrate hyperplasia (BPH), prostate gland hypertrophy (enlarged prostate glands). This is possible because of its ability to produce some secondary metabolites which are believed to have bioactivity against these ailments. The ready international market for the sale of Prunus bark, uncontrolled exploitation, illegal harvesting using inappropriate techniques and poor timing of harvesting have contributed enormously to making the plant endangered. It is known to harbor a large number of endophytic fungi with the potential to produce similar secondary metabolites as the parent plant. Alternative sourcing of medicinal principles through endophytic fungi requires succinct knowledge of the endophytic fungi. This will serve as a conservation measure for Prunus africana by reducing dependence on Prunus bark for such metabolites. This work thus sought to compare the production of some major secondary metabolites produced by P. africana and some of its associated endophytic fungi. The leaves and stem bark of the plant from different provenances were soaked in methanol for 72 hrs to yield the methanolic crude extract. The phytochemical screening of the methanolic crude extracts using different standard procedures revealed the presence of tannins, flavonoids, terpenoids, saponins, phenolics and steroids. Pure cultures of some predominantly isolated endophyte species from the difference Prunus provenances such as Curvularia sp, and Morphospecies P001 were also grown in Potato Dextrose Broth (PDB) for 21 days and later extracted with Methylene dichloride (MDC) solvent after 24hrs to produce crude culture extracts. Qualitative assessment of crude culture extracts showed the presence of tannins, terpenoids, phenolics and steroids particularly β-Sitosterol, (a major bioactive metabolite) as did the plant tissues. Qualitative analysis by thin layer chromatography (TLC) was done to confirm and compare the production of β-Sitosterol (as marker compounds) in the crude extracts of the plant and endophyte. Samples were loaded on TLC silica gel aluminium barked plate (Kieselgel 60 F254, 0.2 mm, Merck) using acetone/hexane, (3.0:7.0) solvent system. They were visualized under an ultra violet lamp (UV254 and UV360). TLC revealed that leaves had a higher concentration of β-sitosterol in terms of band intensity than stem barks from the different provenances. The intensity of β-sitosterol bands in the culture extracts of endophytes was comparable to the plant extracts except for Curvularia sp (very minute) whose band was very faint. The ability of these fungi to make β-sitosterol was confirmed by TLC analysis with the compound having chromatographic properties (retention factor) similar to those of β-sitosterol standard. The ability of these major endophytes to produce secondary metabolites similar to the host has therefore been demonstrated. There is, therefore, the potential of developing the in vitro production system of Prunus secondary metabolites thereby enhancing its conservation.

Keywords: Caneroon, endophytic fungi, Prunus africana, secondary metabolite

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Authors: Neelofar, Khursheed Alam, Jamal Ahmad

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Protein modification in diabetes mellitus may lead to early glycation products (EGPs) or amadori product as well as advanced glycation end products (AGEs). Early glycation involves the reaction of glucose with N-terminal and lysyl side chain amino groups to form Schiff’s base which undergoes rearrangements to form more stable early glycation product known as Amadori product. After Amadori, the reactions become more complicated leading to the formation of advanced glycation end products (AGEs) that interact with various AGE receptors, thereby playing an important role in the long-term complications of diabetes. Millard reaction or nonenzymatic glycation reaction accelerate in diabetes due to hyperglycation and alter serum protein’s structure, their normal functions that lead micro and macro vascular complications in diabetic patients. In this study, Human Serum Albumin (HSA) with a constant concentration was incubated with different concentrations of glucose at 370C for a week. At 4th day, Amadori product was formed that was confirmed by colorimetric method NBT assay and TBA assay which both are authenticate early glycation product. Conformational changes in native as well as all samples of Amadori albumin with different concentrations of glucose were investigated by various biophysical and biochemical techniques. Main biophysical techniques hyperchromacity, quenching of fluorescence intensity, FTIR, CD and SDS-PAGE were used. Further conformational changes were observed by biochemical assays mainly HMF formation, fructoseamine, reduction of fructoseamine with NaBH4, carbonyl content estimation, lysine and arginine residues estimation, ANS binding property and thiol group estimation. This study find structural and biochemical changes in Amadori modified HSA with normal to hyperchronic range of glucose with respect to native HSA. When glucose concentration was increased from normal to chronic range biochemical and structural changes also increased. Highest alteration in secondary and tertiary structure and conformation in glycated HSA was observed at the hyperchronic concentration (75mM) of glucose. Although it has been found that Amadori modified proteins is also involved in secondary complications of diabetes as AGEs but very few studies have been done to analyze the conformational changes in Amadori modified proteins due to early glycation. Most of the studies were found on the structural changes in Amadori protein at a particular glucose concentration but no study was found to compare the biophysical and biochemical changes in HSA due to early glycation with a range of glucose concentration at a constant incubation time. So this study provide the information about the biochemical and biophysical changes occur in Amadori modified albumin at a range of glucose normal to chronic in diabetes. Although many implicates currently in use i.e. glycaemic control, insulin treatment and other chemical therapies that can control many aspects of diabetes. However, even with intensive use of current antidiabetic agents more than 50 % of diabetic patient’s type 2 suffers poor glycaemic control and 18 % develop serious complications within six years of diagnosis. Experimental evidence related to diabetes suggests that preventing the nonenzymatic glycation of relevant proteins or blocking their biological effects might beneficially influence the evolution of vascular complications in diabetic patients or quantization of amadori adduct of HSA by authentic antibodies against HSA-EGPs can be used as marker for early detection of the initiation/progression of secondary complications of diabetes. So this research work may be helpful for the same.

Keywords: diabetes mellitus, glycation, albumin, amadori, biophysical and biochemical techniques

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Authors: Maryline Vere, Wilma ten Ham-Baloyi, Lucy Ochola, Opeoluwa Oyedele, Lindsey Beyleveld, Siphokazi Tili, Takafira Mduluza, Paula E. Melariri

Abstract:

Schistosomiasis, caused by Schistosoma (S.) haematobium and Schistosoma (S.) mansoni parasites poses a significant public health challenge in low-income regions. Diagnosis typically relies on identifying specific urine biomarkers such as haematuria, protein, and leukocytes for S. haematobium, while the Point-of-Care Circulating Cathodic Antigen (POC-CCA) assay is employed for detecting S. mansoni. Urinalysis and the POC-CCA assay are favoured for their rapid, non-invasive nature and cost-effectiveness. However, traditional diagnostic methods such as Kato-Katz and urine filtration lack sensitivity in low-transmission areas, which can lead to underreporting of cases and hinder effective disease control efforts. Therefore, in this study, urinalysis and the POC-CCA assay was utilised to diagnose schistosomiasis effectively among school-going children in Nelson Mandela Bay Municipality. This was a cross-sectional study with a total of 759 children, aged 5 to 14 years, who provided urine samples. Urinalysis was performed using urinary dipstick tests, which measure multiple parameters, including haematuria, protein, leukocytes, bilirubin, urobilinogen, ketones, pH, specific gravity and other biomarkers. Urinalysis was performed by dipping the strip into the urine sample and observing colour changes on specific reagent pads. The POC-CCA test was conducted by applying a drop of urine onto a cassette containing CCA-specific antibodies, and the presence of a visible test line indicated a positive result for S. mansoni infection. Descriptive statistics were used to summarize urine parameters, and Pearson correlation coefficients (r) were calculated to analyze associations among urine parameters using R software (version 4.3.1). Among the 759 children, the prevalence of S. haematobium using haematuria as a diagnostic marker was 33.6%. Additionally, leukocytes were detected in 21.3% of the samples, and protein was present in 15%. The prevalence of positive POC-CCA test results for S. mansoni was 3.7%. Urine parameters exhibited low to moderate associations, suggesting complex interrelationships. For instance, specific gravity and pH showed a negative correlation (r = -0.37), indicating that higher specific gravity was associated with lower pH. Weak correlations were observed between haematuria and pH (r = -0.10), bilirubin and ketones (r = 0.14), protein and bilirubin (r = 0.13), and urobilinogen and pH (r = 0.12). A mild positive correlation was found between leukocytes and blood (r = 0.23), reflecting some association between these inflammation markers. In conclusion, the study identified a significant prevalence of schistosomiasis among school-going children in Nelson Mandela Bay Municipality, with S. haematobium detected through haematuria and S. mansoni identified using the POC-CCA assay. The detection of leukocytes and protein in urine samples serves as critical biomarkers for schistosomiasis infections, reinforcing the presence of schistosomiasis in the study area when considered alongside haematuria. These urine parameters are indicative of inflammatory responses associated with schistosomiasis, underscoring the necessity for effective diagnostic methodologies. Such findings highlight the importance of comprehensive diagnostic assessments to accurately identify and monitor schistosomiasis prevalence and its associated health impacts. The significant burden of schistosomiasis in this population highlights the urgent need to develop targeted control interventions to effectively reduce its prevalence in the study area.

Keywords: schistosomiasis, urinalysis, haematuria, POC-CCA

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Authors: Jan F. Talts, Amit Saxena, Kåre Engkilde

Abstract:

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most frequent side effects caused by anti-neoplastic agents, with a prevalence from 19 % to 85 %. Clinically, CIPN is a mostly sensory neuropathy leading to pain and to motor and autonomic changes. Due to its high prevalence among cancer patients, CIPN constitutes a major problem for both cancer patients and survivors, especially because currently, there is no single effective method of preventing CIPN. Hearing loss is the most common form of sensory impairment in humans and can be caused by ototoxic chemical compounds such as chemotherapy (platinum-based antineoplastic agents).In rodents, single or repeated cisplatin injections induce peripheral neuropathy and hearing impairment mimicking human disorder, allowing studying the efficacy of new pharmacological candidates in chemotherapy-induced hearing loss and peripheral neuropathy. RNA sequencing data from full term amniotic fluid (TAF) mesenchymal stemcell (MSC) clones was used to identify neural-specific markers present on TAF-MSC. Several prospective neural markers were tested by flow cytometry on cultured TAF-MSC. One of these markers was used for cell-sorting using Tyto MACSQuant cell sorter, and the neural marker positive cell population was expanded for several passages to the final therapeutic product stage. Peripheral neuropathy and hearing loss was induced in mice by administration of cisplatin in three week-long cycles. The efficacy of neural-specific TAF-MSC in treating hearing loss and pain perception was evaluated by administration of three injections of 3 million cells/kg by intravenous route or three injections of 3 million cells/kg by intra-arterial route after each cisplatin cycle treatment. Auditory brainstem responses (ABR) are electric potentials recorded from scalp electrodes, and the first ABR wave represents the summed activity of the auditory nerve fibers contacting the inner hair cells. For ABR studies, mice were anesthetized, then earphones were placed in the left ear of each mouse, an active electrode was placed in the vertex of the skull, a reference electrode under the skin of the mastoid bone, and a ground electrode in the neck skin. The stimuli consisted of tone pips of five frequencies (2, 4, 6, 12, 16, and 24 kHz) at various sound levels (from 0 to 90 dB) ranging to cover the mouse auditory frequency range. The von Frey test was used to assess the onset and maintenance of mechanical allodynia over time. Mice were placed in clear plexiglass cages on an elevated mesh floor and tested after 30 min of habituation. Mechanical paw withdrawal threshold was examined using an electronic von Frey anesthesiometer. Cisplatin groups treated with three injections of 3 million cells/kg by intravenous route and three injections of 3 million cells/kg by intra-arterial route after each cisplatin cycle treatment presented, a significant increase of hearing acuity characterized by a decrease of ABR threshold and a decrease of neuropathic pain characterized by an increase of von Frey paw withdrawal threshold compared to controls only receiving cisplatin. This study shows that treatment with MSCselected for neural specificity presents significant positive efficacy on the chemotherapy-induced neuropathic pain and the chemotherapy-induced hearing loss.

Keywords: mesenchymal stem cell, peripheral neuropathy, amniotic fluid, regenerative medicine

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Authors: Irina Alexandra Paun, Mona Mihailescu, Marian Zamfirescu, Catalin Romeo Luculescu, Adriana Maria Acasandrei, Cosmin Catalin Mustaciosu, Roxana Cristina Popescu, Maria Dinescu

Abstract:

A major concern in bone tissue engineering is to develop complex 3D architectures that mimic the natural cells environment, facilitate the cells growth in a defined manner and allow the flow transport of nutrients and metabolic waste. In particular, porous structures of controlled pore size and positioning are indispensable for growing human-like bone structures. Another concern is to monitor both the structures and the seeded cells with high spatial resolution and without interfering with the cells natural environment. The present approach relies on laser-based technologies employed for fabricating porous biomimetic structures that support the growth of osteoblast-like cells and for their non-invasive 3D imaging. Specifically, the porous structures were built by two photon polymerization –direct writing (2PP_DW) of the commercially available photoresists IL-L780, using the Photonic Professional 3D lithography system. The structures consist of vertical tubes with micrometer-sized heights and diameters, in a honeycomb-like spatial arrangement. These were fabricated by irradiating the IP-L780 photoresist with focused laser pulses with wavelength centered at 780 nm, 120 fs pulse duration and 80 MHz repetition rate. The samples were precisely scanned in 3D by piezo stages. The coarse positioning was done by XY motorized stages. The scanning path was programmed through a writing language (GWL) script developed by Nanoscribe. Following laser irradiation, the unexposed regions of the photoresist were washed out by immersing the samples in the Propylene Glycol Monomethyl Ether Acetate (PGMEA). The porous structures were seeded with osteoblast like MG-63 cells and their osteogenic potential was tested in vitro. The cell-seeded structures were analyzed in 3D using the digital holographic microscopy technique (DHM). DHM is a marker free and high spatial resolution imaging tool, where the hologram acquisition is performed non-invasively i.e. without interfering with the cells natural environment. Following hologram recording, a digital algorithm provided a 3D image of the sample, as well as information about its refractive index, which is correlated with the intracellular content. The axial resolution of the images went down to the nanoscale, while the temporal scales ranged from milliseconds up to hours. The hologram did not involve sample scanning and the whole image was available in one frame recorded going over 200μm field of view. The digital holograms processing provided 3D quantitative information on the porous structures and allowed a quantitative analysis of the cellular response in respect to the porous architectures. The cellular shape and dimensions were found to be influenced by the underlying micro relief. Furthermore, the intracellular content gave evidence on the beneficial role of the porous structures in promoting osteoblast differentiation. In all, the proposed laser-based protocol emerges as a promising tool for the fabrication and non-invasive imaging of porous constructs for bone tissue engineering. Acknowledgments: This work was supported by a grant of the Romanian Authority for Scientific Research and Innovation, CNCS-UEFISCDI, project PN-II-RU-TE-2014-4-2534 (contract 97 from 01/10/2015) and by UEFISCDI PN-II-PT-PCCA no. 6/2012. A part of this work was performed in the CETAL laser facility, supported by the National Program PN 16 47 - LAPLAS IV.

Keywords: biomimetic, holography, laser, osteoblast, two photon polymerization

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