Search results for: peptide inhibitors

Authors: Priyanka Jain, Ashish K. Sharma, Esha Shukla, K. J. Mukherjee

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We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.

Keywords: cellular stress response, GFP, knock-outs, up-regulated genes

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Authors: Saule Balmagambetova, Zhenisgul Tlegenova, Saule Madinova

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Cardiotoxicity associated with anticancer treatment, now defined as cancer therapy-related cardiac dysfunction (CTRCD), accompanies cancer patients and negatively impacts their survivorship. Currently, a cardio-oncological service is being created in Kazakhstan based on the provisions of the European Society of Cardio-oncology (ESC) Guidelines. In the frames of a pilot project, a cohort study on CTRCD conditions was initiated at the Aktobe Cancer center. One hundred twenty-eight newly diagnosed breast cancer patients started on doxorubicin and/or trastuzumab were recruited. Echocardiography with global longitudinal strain (GLS) assessment, biomarkers panel (cardiac troponin (cTnI), brain natriuretic peptide (BNP), myeloperoxidase (MPO), galectin-3 (Gal-3), D-dimers, C-reactive protein (CRP)), and other tests were performed at baseline and every three months. Patients were stratified by the cardiovascular risks according to the ESC recommendations and allocated into the risk groups during the pre-treatment visit. Of them, 10 (7.8%) patients were assigned to the high-risk group, 48 (37.5%) to the medium-risk group, and 70 (54.7%) to the low-risk group, respectively. High-risk patients have been receiving their cardioprotective treatment from the outset. Patients were also divided by treatment - in the anthracycline-based 83 (64.8%), in trastuzumab- only 13 (10.2%), and in the mixed anthracycline/trastuzumab group 32 individuals (25%), respectively. Mild symptomatic CTRCD was revealed and treated in 2 (1.6%) participants, and a mild asymptomatic variant in 26 (20.5%). Mild asymptomatic conditions are defined as left ventricular ejection fraction (LVEF) ≥50% and further relative reduction in GLS by >15% from baseline and/or a further rise in cardiac biomarkers. The listed biomarkers were assessed longitudinally in repeated-measures linear regression models during 12 months of observation. The associations between changes in biomarkers and CTRCD and between changes in biomarkers and LVEF were evaluated. Analysis by risk groups revealed statistically significant differences in baseline LVEF scores (p 0.001), BNP (p 0.0075), and Gal-3 (p 0.0073). Treatment groups found no statistically significant differences at baseline. After 12 months of follow-up, only LVEF values showed a statistically significant difference by risk groups (p 0.0011). When assessing the temporal changes in the studied parameters for all treatment groups, there were statistically significant changes from visit to visit for LVEF (p 0.003); GLS (p 0.0001); BNP (p<0.00001); MPO (p<0.0001); and Gal-3 (p<0.0001). No moderate or strong correlations were found between the biomarkers values and LVEF, between biomarkers and GLS. Between the biomarkers themselves, a moderate, close to strong correlation was established between cTnI and D-dimer (r 0.65, p<0.05). The dose-dependent effect of anthracyclines has been confirmed: the summary dose has a moderate negative impact on GLS values: -r 0.31 for all treatment groups (p<0.05). The present study found myeloperoxidase as a promising biomarker of cardiac dysfunction in the mixed anthracycline/trastuzumab treatment group. The hazard of CTRCD increased by 24% (HR 1.21; 95% CI 1.01;1.73) per doubling in baseline MPO value (p 0.041). Increases in BNP were also associated with CTRCD (HR per doubling, 1.22; 95% CI 1.12;1.69). No cases of chemotherapy discontinuation due to cardiotoxic complications have been recorded. Further observations are needed to gain insight into the ability of biomarkers to predict CTRCD onset.

Keywords: breast cancer, chemotherapy, cardiotoxicity, Kazakhstan

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Authors: Lana Burmistrova

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Introduction: The influence of visual impairments on several cognitive processes used in the music learning process is an increasingly important area in special education and cognitive musicology. Many children have several visual impairments due to the refractive errors and irreversible inhibitors. However, based on the compensatory neuroplasticity and functional reorganization, congenitally blind (CB) and early blind (EB) individuals use several areas of the occipital lobe to perceive and process auditory and tactile information. CB individuals have greater memory capacity, memory reliability, and less false memory mechanisms are used while executing several tasks, they have better working memory (WM) and short-term memory (STM). Blind individuals use several strategies while executing tactile and working memory n-back tasks: verbalization strategy (mental recall), tactile strategy (tactile recall) and combined strategies. Methods and design: The aim of the pilot study was to substantiate similar tendencies while executing attention, memory and combined auditory tasks in blind and sighted individuals constructed for this study, and to investigate attention, memory and combined mechanisms used in the music learning process. For this study eight (n=8) blind and eight (n=8) sighted individuals aged 13-20 were chosen. All respondents had more than five years music performance and music learning experience. In the attention task, all respondents had to identify pitch changes in tonal and randomized melodic pairs. The memory task was based on the mismatch negativity (MMN) proportion theory: 80 percent standard (not changed) and 20 percent deviant (changed) stimuli (sequences). Every sequence was named (na-na, ra-ra, za-za) and several items (pencil, spoon, tealight) were assigned for each sequence. Respondents had to recall the sequences, to associate them with the item and to detect possible changes. While executing the combined task, all respondents had to focus attention on the pitch changes and had to detect and describe these during the recall. Results and conclusion: The results support specific features in CB and EB, and similarities between late blind (LB) and sighted individuals. While executing attention and memory tasks, it was possible to observe the tendency in CB and EB by using more precise execution tactics and usage of more advanced periodic memory, while focusing on auditory and tactile stimuli. While executing memory and combined tasks, CB and EB individuals used passive working memory to recall standard sequences, active working memory to recall deviant sequences and combined strategies. Based on the observation results, assessment of blind respondents and recording specifics, following attention and memory correlations were identified: reflective attention and STM, reflective attention and periodic memory, auditory attention and WM, tactile attention and WM, auditory tactile attention and STM. The results and the summary of findings highlight the attention and memory features used in the music learning process in the context of blindness, and the tendency of the several attention and memory types correlated based on the task, strategy and individual features.

Keywords: attention, blindness, memory, music learning, strategy

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Authors: Zhuoran Li, Guan Qin

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A robust and efficient compositional equilibrium characterization model for hydrate-containing systems is required, especially for time-critical simulations such as subsea pipeline flow assurance analysis, compositional simulation in hydrate reservoirs etc. A multiphase flash calculation framework, which combines Gibbs energy minimization function and cubic plus association (CPA) EoS, is developed to describe the highly non-ideal phase behavior of hydrate-containing systems. A non-iterative eigenvalue problem-solving approach for the trust-region sub-problem is selected to guarantee efficiency. The developed flash model is based on the state-of-the-art objective function proposed by Michelsen to minimize the Gibbs energy of the multiphase system. It is conceivable that a hydrate-containing system always contains polar components (such as water and hydrate inhibitors), introducing hydrogen bonds to influence phase behavior. Thus, the cubic plus associating (CPA) EoS is utilized to compute the thermodynamic parameters. The solid solution theory proposed by van der Waals and Platteeuw is applied to represent hydrate phase parameters. The trust-region method combined with the trust-region sub-problem non-iterative eigenvalue problem-solving approach is utilized to ensure fast convergence. The developed multiphase flash model's accuracy performance is validated by three available models (one published and two commercial models). Hundreds of published hydrate-containing system equilibrium experimental data are collected to act as the standard group for the accuracy test. The accuracy comparing results show that our model has superior performances over two models and comparable calculation accuracy to CSMGem. Efficiency performance test also has been carried out. Because the trust-region method can determine the optimization step's direction and size simultaneously, fast solution progress can be obtained. The comparison results show that less iteration number is needed to optimize the objective function by utilizing trust-region methods than applying line search methods. The non-iterative eigenvalue problem approach also performs faster computation speed than the conventional iterative solving algorithm for the trust-region sub-problem, further improving the calculation efficiency. A new thermodynamic framework of the multiphase flash model for the hydrate-containing system has been constructed in this work. Sensitive analysis and numerical experiments have been carried out to prove the accuracy and efficiency of this model. Furthermore, based on the current thermodynamic model in the oil and gas industry, implementing this model is simple.

Keywords: equation of state, hydrates, multiphase equilibrium, trust-region method

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Authors: S. D. N. K. Bathige, G. I. Godahewa, Navaneethaiyer Umasuthan, Jehee Lee

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Kunitz-type serine protease inhibitors (KTIs) are identified in various organisms including animals, plants and microbes. These proteins shared single or multiple Kunitz inhibitory domains link together or associated with other types of domains. Characteristic Kunitz type domain composed of around 60 amino acid residues with six conserved cysteine residues to stabilize by three disulfide bridges. KTIs are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis and inflammation. In this study, two Kunitz-type domain containing protein was identified from rock bream database and designated as RbKunitz. The coding sequence of RbKunitz encoded for 507 amino acids with 56.2 kDa theoretical molecular mass and 5.7 isoelectric point (pI). There are several functional domains including MANEC superfamily domain, PKD superfamily domain, and LDLa domain were predicted in addition to the two characteristic Kunitz domain. Moreover, trypsin interaction sites were also identified in Kunitz domain. Homology analysis revealed that RbKunitz shared highest identity (77.6%) with Takifugu rubripes. Completely conserved 28 cysteine residues were recognized, when comparison of RbKunitz with other orthologs from different taxonomical groups. These structural evidences indicate the rigidity of RbKunitz folding structure to achieve the proper function. The phylogenetic tree was constructed using neighbor-joining method and exhibited that the KTIs from fish and non-fish has been evolved in separately. Rock bream was clustered with Takifugu rubripes. The SYBR Green qPCR was performed to quantify the RbKunitz transcripts in different tissues and challenged tissues. The mRNA transcripts of RbKunitz were detected in all tissues (muscle, spleen, head kidney, blood, heart, skin, liver, intestine, kidney and gills) analyzed and highest transcripts level was detected in gill tissues. Temporal transcription profile of RbKunitz in rock bream blood tissues was analyzed upon LPS (lipopolysaccharide), Poly I:C (Polyinosinic:polycytidylic acid) and Edwardsiella tarda challenge to understand the immune responses of this gene. Compare to the unchallenged control RbKunitz exhibited strong up-regulation at 24 h post injection (p.i.) after LPS and E. tarda injection. Comparatively robust expression of RbKunits was observed at 3 h p.i. upon Poly I:C challenge. Taken together all these data indicate that RbKunitz may involve into to immune responses upon pathogenic stress, in order to protect the rock bream.

Keywords: Kunitz-type, rock bream, immune response, serine protease inhibitor

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Authors: Alireza Barari, Saeed Shirali, Ahmad Abdi

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Abstract: Introduction: Physical activity may be related to male reproductive function by affecting on thehypothalamic-pituitary-gonadal(HPG) axis. Our aim was to determine the effects of 6 weeks resistance exercise on reproductive hormones, HPG axis. The hypothalamic-pituitary-gonadal (HPG) axis refers tothe effects of endocrine glands in three-level including (i) the hypothalamic releasing hormone GnRH, which is synthesized in in a small heterogenous neuronal population and released in a pulsatile fashion, (ii) the anterior pituitary hormones, follicle-stimulating hormone(FSH) and luteinizing hormone (LH) and (iii) the gonadal hormones, which include both steroid such as testosterone (T), estradiol and progesterone and peptide hormones (such as inhibin). Hormonal changes that create a more anabolic environment have been suggested to contribute to the adaptation to strength exercise. Physical activity has an extensive impact on male reproductive function depending upon the intensity and duration of the exercise and the fitness level of the individual. However, strenuous exercise represents a physical stress and inflammation changed that challenges homeostasis. Materials and methods: Sixteen male volunteered were included in a 6-week control period followed by 6 weeks of resistance training (leg press, lat pull, chest press, squat, seatedrow, abdominal crunch, shoulder press, biceps curl and triceps press down) four times per week. intensity of training loading was 60%-75% of one maximum repetition. Participants performed 3 sets of 10 repetitions. Rest periods were two min between exercises and sets. Start with warm up exercises include: The muscles relax and stretch the body, which was for 10 minutes. Body composition, VO2max and the circulating level of free testosterone (fT), luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex hormone binding globulin (SHBG) and inhibin B measured prior and post 6-week intervention. The hormonal levels of each serum sample were measured using commercially available ELISA kits. Analysis of anthropometrical data and hormonal level were compared using the independent samples t- test in both groups and using SPSS (version 19). P ≤ 0.05 was considered statistically significant. Results: For muscle strength, both lower- and upper-body strength were increased significantly. Aerobic fitness level improved in trained participant from 39.4 ± 5.6 to 41.9 ± 5.3 (P = 0.002). fT concentration rise progressively in the trained group and was significantly greater than those in the control group (P = 0.000). By the end of the 6-week resistance training, serum SHBG significantly increased in the trained group compared with the control group (P = 0.013). In response to resistance training, LH, FSH and inhibin B were not significantly changed. Discussion: According to our finfings, 6 weeks of resistance training induce fat loss without any changes in body weight and BMI. A decline of 25.3% in percentage of body fat with statiscally same weight was due to increase in muscle mass that happened during resistance exercise periods . Six weeks of resistance training resulted in significant improvement in BF%, VO2max and increasing strength and the level of fT and SHBG.

Keywords: resistance, hypothalamic, pituitary, gonadal axis

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Authors: Javier Enciso-Benavides, Fredy Fabian, Carlos Castaneda, Luis Alfaro, Alex Choque, Aparicio Aguilar, Javier Enciso

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Background: The use of biomarkers in breast cancer diagnosis, therapy, and prognosis has gained increasing interest. Cancer stem cells (CSCs) are a subpopulation of tumor cells that can drive tumor initiation and may cause relapse. Therefore, due to the importance of diagnosis, therapy, and prognosis, several biomarkers that characterize CSCs have been identified; however, in treatment-naïve triple-negative breast tumors, there is an urgent need to identify new biomarkers and therapeutic targets. According to this, the aim of this study was to identify serum proteins associated with cancer stem cells and pluripotency in women with triple-negative breast tumors in order to subsequently identify a biomarker for this type of breast tumor. Material and Methods: Whole blood samples from 12 women with histopathologically diagnosed triple-negative breast tumors were used after obtaining informed consent from the patient. Blood serum was obtained by conventional procedure and frozen at -80ºC. Identification of cancer stem cell-associated proteins was performed by matrix-assisted laser desorption/ionisation-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS), protein analysis was obtained using the AB Sciex TOF/TOF™ 5800 system (AB Sciex, USA). Sequences not aligned by ProteinPilot™ software were analyzed by Protein BLAST. Results: The following proteins related to pluripotency and cancer stem cells were identified by MALDI TOF/TOF mass spectrometry: A-chain, Serpin A12 [Homo sapiens], AIEBP [Homo sapiens], Alpha-one antitrypsin, AT {internal fragment} [human, partial peptide, 20 aa] [Homo sapiens], collagen alpha 1 chain precursor variant [Homo sapiens], retinoblastoma-associated protein variant [Homo sapiens], insulin receptor, CRA_c isoform [Homo sapiens], Hydroxyisourate hydrolase [Streptomyces scopuliridis], MUCIN-6 [Macaca mulatta], Alpha-actinin-3 [Chrysochloris asiatica], Polyprotein M, CRA_d isoform, partial [Homo sapiens], Transcription factor SOX-12 [Homo sapiens]. Recommendations: The serum proteins identified in this study should be investigated in the exosome of triple-negative breast cancer stem cells and in the blood serum of women without breast cancer. Subsequently, proteins found only in the blood serum of women with triple-negative breast cancer should be identified in situ in triple-negative breast cancer tissue in order to identify a biomarker to study the evolution of this type of cancer, or that could be a therapeutic target. Conclusions: Eleven cancer stem cell-related serum proteins were identified in 12 women with triple-negative breast cancer, of which MUCIN-6, retinoblastoma-associated protein variant, transcription factor SOX-12, and collagen alpha 1 chain are the most representative and have not been studied so far in this type of breast tumor. Acknowledgement: This work was supported by Proyecto CONCYTEC–Banco Mundial “Mejoramiento y Ampliacion de los Servicios del Sistema Nacional de Ciencia Tecnología e Innovacion Tecnologica” 8682-PE (104-2018-FONDECYT-BM-IADT-AV).

Keywords: triple-negative breast cancer, MALDI TOF/TOF MS, serum proteins, cancer stem cells

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Authors: Ekaterina S. Marchenko, Gulsharat A. Baigonakova, Kirill M. Dubovikov, Igor A. Khlusov

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NiTi alloys combine biomechanical and biochemical properties. This makes them a perfect candidate for medical applications. However, there is a serious problem with these alloys, such as the release of Ni from the matrix. Ni ions are known to be toxic to living tissues and leach from the matrix into the surrounding implant tissues due to corrosion after prolonged use. To prevent the release of Ni ions, corrosive strong coatings are usually used. Titanium nitride-based coatings are perfect corrosion inhibitors and also have good bioactive properties. However, there is an opportunity to improve the biochemical compatibility of the surface by depositing another layer. This layer can consist of elements such as calcium and phosphorus. The Ca and P ions form different calcium phosphate phases, which are present in the mineral part of human bones. We therefore believe that these elements must promote osteogenesis and osteointegration. In view of the above, the aim of this study is to investigate the effect of crystallinity on the biocompatibility of a two-layer coating deposited on NiTi substrate by sputtering. The first step of the research, apart from the NiTi polishing, is the layer-by-layer deposition of Ti-Ni-Ti by magnetron sputtering and the subsequent synthesis of this composite in an N atmosphere at 900 °C. The total thickness of the corrosion resistant layer is 150 nm. Plasma assisted RF sputtering was then used to deposit a bioactive film on the titanium nitride layer. A Ca-P powder target was used to obtain such a film. We deposited three types of Ca-P layers with different crystallinity and compared them in terms of cytotoxicity. One group of samples had no Ca-P coating and was used as a control. We obtained different crystallinity by varying the sputtering parameters such as bias voltage, plasma source current and pressure. XRD analysis showed that all coatings are calcium phosphate, but the sample obtained at maximum bias and plasma source current and minimum pressure has the most intense peaks from the coating phase. SEM and EDS showed that all three coatings have a homogeneous and dense structure without cracks and consist of calcium, phosphorus and oxygen. Cytotoxic tests carried out on three types of samples with Ca-P coatings and a control group showed that the control sample and the sample with Ca-P coating obtained at maximum bias voltage and plasma source current and minimum pressure had the lowest number of dead cells on the surface, around 11 ± 4%. Two other types of samples with Ca-P coating have 40 ± 9% and 21 ± 7% dead cells on the surface. It can therefore be concluded that these two sputtering modes have a negative effect on the corrosion resistance of the whole samples. The third sputtering mode does not affect the corrosion resistance and has the same level of cytotoxicity as the control. It can be concluded that the most suitable sputtering mode is the third with maximum bias voltage and plasma source current and minimum pressure.

Keywords: calcium phosphate coating, cytotoxicity, NiTi alloy, two-layer coating

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Authors: Priyanka Sharma, Ranjita Das, Mohan C. Kalita, Debajit Thakur

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Background: Actinomycetes are the richest source of novel bioactive secondary metabolites such as antibiotics, enzymes and other therapeutically useful metabolites with diverse biological activities. The present study aims at the antimicrobial potential and genetic diversity of culturable Actinomycetes isolated from protected forest ecosystems of Assam which includes Kaziranga National Park (26°30˝-26°45˝N and 93°08˝-93°36˝E), Pobitora Wildlife Sanctuary (26º12˝-26º16˝N and 91º58˝-92º05˝E) and Gibbon Wildlife Sanctuary (26˚40˝-26˚45˝N and 94˚20˝-94˚25˝E) which are located in the North-eastern part of India. Northeast India is a part of the Indo-Burma mega biodiversity hotspot and most of the protected forests of this region are still unexplored for the isolation of effective antibiotic-producing Actinomycetes. Thus, there is tremendous possibility that these virgin forests could be a potential storehouse of novel microorganisms, particularly Actinomycetes, exhibiting diverse biological properties. Methodology: Soil samples were collected from different ecological niches of the protected forest ecosystems of Assam and Actinomycetes were isolated by serial dilution spread plate technique using five selective isolation media. Preliminary screening of Actinomycetes for an antimicrobial activity was done by spot inoculation method and the secondary screening by disc diffusion method against several test pathogens, including multidrug resistant Staphylococcus aureus (MRSA). The strains were further screened for the presence of antibiotic synthetic genes such as type I polyketide synthases (PKS-I), type II polyketide synthases (PKS-II) and non-ribosomal peptide synthetases (NRPS) genes. Genetic diversity of the Actinomycetes producing antimicrobial metabolites was analyzed through 16S rDNA-RFLP using Hinf1 restriction endonuclease. Results: Based on the phenotypic characterization, a total of 172 morphologically distinct Actinomycetes were isolated and screened for antimicrobial activity by spot inoculation method on agar medium. Among the strains tested, 102 (59.3%) strains showed activity against Gram-positive bacteria, 98 (56.97%) against Gram-negative bacteria, 92 (53.48%) against Candida albicans MTCC 227 and 130 (75.58%) strains showed activity against at least one of the test pathogens. Twelve Actinomycetes exhibited broad spectrum antimicrobial activity in the secondary screening. The taxonomic identification of these twelve strains by 16S rDNA sequencing revealed that Streptomyces was found to be the predominant genus. The PKS-I, PKS-II and NRPS genes detection indicated diverse bioactive products of these twelve Actinomycetes. Genetic diversity by 16S rDNA-RFLP indicated that Streptomyces was the dominant genus amongst the antimicrobial metabolite producing Actinomycetes. Conclusion: These findings imply that Actinomycetes from the protected forest ecosystems of Assam, India, are a potential source of bioactive secondary metabolites. These areas are as yet poorly studied and represent diverse and largely unscreened ecosystem for the isolation of potent Actinomycetes producing antimicrobial secondary metabolites. Detailed characterization of the bioactive Actinomycetes as well as purification and structure elucidation of the bioactive compounds from the potent Actinomycetes is the subject of ongoing investigation. Thus, to exploit Actinomycetes from such unexplored forest ecosystems is a way to develop bioactive products.

Keywords: Actinomycetes, antimicrobial activity, forest ecosystems, RFLP

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Authors: Preethi Kumari P., Shetty Prakasha, Rao Suma A.

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Mild steel has been widely employed as construction materials for pipe work in the oil and gas production such as down hole tubular, flow lines and transmission pipelines, in chemical and allied industries for handling acids, alkalis and salt solutions due to its excellent mechanical property and low cost. Acid solutions are widely used for removal of undesirable scale and rust in many industrial processes. Among the commercially available acids hydrochloric acid is widely used for pickling, cleaning, de-scaling and acidization of oil process. Mild steel exhibits poor corrosion resistance in presence of hydrochloric acid. The high reactivity of mild steel in presence of hydrochloric acid is due to the soluble nature of ferrous chloride formed and the cementite phase (Fe3C) normally present in the steel is also readily soluble in hydrochloric acid. Pitting attack is also reported to be a major form of corrosion in mild steel in the presence of high concentrations of acids and thereby causing the complete destruction of metal. Hydrogen from acid reacts with the metal surface and makes it brittle and causes cracks, which leads to pitting type of corrosion. The use of chemical inhibitor to minimize the rate of corrosion has been considered to be the first line of defense against corrosion. In spite of long history of corrosion inhibition, a highly efficient and durable inhibitor that can completely protect mild steel in aggressive environment is yet to be realized. It is clear from the literature review that there is ample scope for the development of new organic inhibitors, which can be conveniently synthesized from relatively cheap raw materials and provide good inhibition efficiency with least risk of environmental pollution. The aim of the present work is to evaluate the electrochemical parameters for the corrosion inhibition behavior of an aromatic hydrazide derivative, 4-hydroxy- N '-[(E)-1H-indole-2-ylmethylidene)] benzohydrazide (HIBH) on mild steel in 2M hydrochloric acid using Tafel polarization and electrochemical impedance spectroscopy (EIS) techniques at 30-60 °C. The results showed that inhibition efficiency increased with increase in inhibitor concentration and decreased marginally with increase in temperature. HIBH showed a maximum inhibition efficiency of 95 % at 8×10-4 M concentration at 30 °C. Polarization curves showed that HIBH act as a mixed-type inhibitor. The adsorption of HIBH on mild steel surface obeys the Langmuir adsorption isotherm. The adsorption process of HIBH at the mild steel/hydrochloric acid solution interface followed mixed adsorption with predominantly physisorption at lower temperature and chemisorption at higher temperature. Thermodynamic parameters for the adsorption process and kinetic parameters for the metal dissolution reaction were determined.

Keywords: electrochemical parameters, EIS, mild steel, tafel polarization

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Authors: Suikinai Nobre Santos, Ranko Gacesa, Paul F. Long, Itamar Soares de Melo

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Extremophilic microorganism are adapted to biotopes combining several stress factors (temperature, pressure, radiation, salinity and pH), which indicate the richness valuable resource for the exploitation of novel biotechnological processes and constitute unique models for investigations their biomolecules (1, 2). The above information encourages us investigate bioprospecting synthesized compounds by a noval actinomycete, designated thermotolerant Streptomyces caatingaensis CMAA 1322, isolated from sample soil tropical dry forest (Caatinga) in the Brazilian semiarid region (3-17°S and 35-45°W). This set of constrating physical and climatic factores provide the unique conditions and a diversity of well adapted species, interesting site for biotechnological purposes. Preliminary studies have shown the great potential in the production of cytotoxic, pesticidal and antimicrobial molecules (3). Thus, to extend knowledge of the genes clusters responsible for producing biosynthetic pathways of natural products in strain CMAA1322, whole-genome shotgun (WGS) DNA sequencing was performed using paired-end long sequencing with PacBio RS (Pacific Biosciences). Genomic DNA was extracted from a pure culture grown overnight on LB medium using the PureLink genomic DNA kit (Life Technologies). An approximately 3- to 20-kb-insert PacBio library was constructed and sequenced on an 8 single-molecule real-time (SMRT) cell, yielding 116,269 reads (average length, 7,446 bp), which were allocated into 18 contigs, with 142.11x coverage and N50 value of 20.548 bp (BioProject number PRJNA288757). The assembled data were analyzed by Rapid Annotations using Subsystems Technology (RAST) (4) the genome size was found to be 7.055.077 bp, comprising 6167 open reading frames (ORFs) and 413 subsystems. The G+C content was estimated to be 72 mol%. The closest-neighbors tool, available in RAST through functional comparison of the genome, revealed that strain CMAA1322 is more closely related to Streptomyces hygroscopicus ATCC 53653 (similarity score value, 537), S. violaceusniger Tu 4113 (score value, 483), S. avermitilis MA-4680 (score value, 475), S. albus J1074 (score value, 447). The Streptomyces sp. CMAA1322 genome contains 98 tRNA genes and 135 genes copies related to stress response, mainly osmotic stress (14), heat shock (16), oxidative stress (49). Functional annotation by antiSMASH version 3.0 (5) identified 41 clusters for secondary metabolites (including two clusters for lanthipeptides, ten clusters for nonribosomal peptide synthetases [NRPS], three clusters for siderophores, fourteen for polyketide synthetase [PKS], six clusters encoding a terpene, two clusters encoding a bacteriocin, and one cluster encoding a phenazine). Our work provide in comparative analyse of genome and extract produced (data no published) by lineage CMAA1322, revealing the potential of microorganisms accessed from extreme environments as Caatinga” to produce a wide range of biotechnological relevant compounds.

Keywords: caatinga, streptomyces, environmental stresses, biosynthetic pathways

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Authors: Melissa K. Hord, Stephen P. Whiteside

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With lifetime prevalence rates ranging from 6% to 15%, anxiety disorders are among the most common childhood mental health diagnoses. Anxiety disorders diagnosed in childhood generally show an unremitting course, lead to additional psychopathology and interfere with social, emotional, and academic development. Effective evidence-based treatments include cognitive-behavioral therapy (CBT) and selective serotonin reuptake inhibitors (SSRI’s). However, if anxious children receive any treatment, it is usually through primary care, typically consists of medication, and very rarely includes evidence-based psychotherapy. Despite the high prevalence of anxiety disorders, there have only been two independent research labs that have investigated long-term results for CBT treatment for all childhood anxiety disorders and two for specific anxiety disorders. Generally, the studies indicate that the majority of youth maintain gains up to 7.4 years after treatment. These studies have not been replicated. In addition, little is known about the additional mental health care received by these patients in the intervening years after anxiety treatment, which seems likely to influence maintenance of gains for anxiety symptoms as well as the development of additional psychopathology during the subsequent years. The original sample consisted of 335 children ages 7 to 17 years (mean 13.09, 53% female) diagnosed with an anxiety disorder in 2010. Medical record review included provider billing records for mental health appointments during the five years after anxiety treatment. The subsample for this study was classified into three groups: 64 children who received CBT in an anxiety disorders clinic, 56 who received treatment from a psychiatrist, and 10 who were seen in a primary care setting. Chi-square analyses resulted in significant differences in mental health care utilization across the five years after treatment. Youth receiving treatment in primary care averaged less than one appointment each year and the appointments continued at the same rate across time. Children treated by a psychiatrist averaged approximately 3 appointments in the first two years and 2 in the subsequent three years. Importantly, youth treated in the anxiety clinic demonstrated a gradual decrease in mental health appointments across time. The nuanced differences will be presented in greater detail. The results of the current study have important implications for developing dissemination materials to help guide parents when they are selecting treatment for their children. By including all mental health appointments, this study recognizes that anxiety is often comorbid with additional diagnoses and that receiving evidence-based treatment may have long-term benefits that are associated with improvements in broader mental health. One important caveat might be that the acuity of mental health influenced the level of care sought by patients included in this study; however, taking this possibility into account, it seems those seeking care in a primary care setting continued to require similar care at the end of the study, indicating little improvement in symptoms was experienced.

Keywords: anxiety, children, mental health, outcomes

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Authors: Milu T. Cherian, Sergio C. Chai, Morgan A. Casal, Taosheng Chen

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This study aims to use CINPA1, a recently discovered small-molecule inhibitor of the xenobiotic receptor CAR (constitutive androstane receptor) for understanding the binding modes of CAR and to guide CAR-mediated gene expression profiling studies in human primary hepatocytes. CAR and PXR are xenobiotic sensors that respond to drugs and endobiotics by modulating the expression of metabolic genes that enhance detoxification and elimination. Elevated levels of drug metabolizing enzymes and efflux transporters resulting from CAR activation promote the elimination of chemotherapeutic agents leading to reduced therapeutic effectiveness. Multidrug resistance in tumors after chemotherapy could be associated with errant CAR activity, as shown in the case of neuroblastoma. CAR inhibitors used in combination with existing chemotherapeutics could be utilized to attenuate multidrug resistance and resensitize chemo-resistant cancer cells. CAR and PXR have many overlapping modulating ligands as well as many overlapping target genes which confounded attempts to understand and regulate receptor-specific activity. Through a directed screening approach we previously identified a new CAR inhibitor, CINPA1, which is novel in its ability to inhibit CAR function without activating PXR. The cellular mechanisms by which CINPA1 inhibits CAR function were also extensively examined along with its pharmacokinetic properties. CINPA1 binding was shown to change CAR-coregulator interactions as well as modify CAR recruitment at DNA response elements of regulated genes. CINPA1 was shown to be broken down in the liver to form two, mostly inactive, metabolites. The structure-activity differences of CINPA1 and its metabolites were used to guide computational modeling using the CAR-LBD structure. To rationalize how ligand binding may lead to different CAR pharmacology, an analysis of the docked poses of human CAR bound to CITCO (a CAR activator) vs. CINPA1 or the metabolites was conducted. From our modeling, strong hydrogen bonding of CINPA1 with N165 and H203 in the CAR-LBD was predicted. These residues were validated to be important for CINPA1 binding using single amino-acid CAR mutants in a CAR-mediated functional reporter assay. Also predicted were residues making key hydrophobic interactions with CINPA1 but not the inactive metabolites. Some of these hydrophobic amino acids were also identified and additionally, the differential coregulator interactions of these mutants were determined in mammalian two-hybrid systems. CINPA1 represents an excellent starting point for future optimization into highly relevant probe molecules to study the function of the CAR receptor in normal- and pathophysiology, and possible development of therapeutics (for e.g. use for resensitizing chemoresistant neuroblastoma cells).

Keywords: antagonist, chemoresistance, constitutive androstane receptor (CAR), multi-drug resistance, structure activity relationship (SAR), xenobiotic resistance

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Authors: Salwa Karboune, Amanda Waglay

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Recent studies indicate that health-promoting functional ingredients and nutraceuticals can help support and improve the overall public health, which is timely given the aging of the population and the increasing cost of health care. The development of novel ‘natural’ functional ingredients is increasingly challenging. Biocatalysis offers powerful approaches to achieve this goal. Our recent research has been focusing on the development of innovative biocatalytic approaches towards the isolation of protein isolates from potato by-products and the generation of peptides. Potato is a vegetable whose high-quality proteins are underestimated. In addition to their high proportion in the essential amino acids, potato proteins possess angiotensin-converting enzyme-inhibitory potency, an ability to reduce plasma triglycerides associated with a reduced risk of atherosclerosis, and stimulate the release of the appetite regulating hormone CCK. Potato proteins have long been considered not economically feasible due to the low protein content (27% dry matter) found in tuber (Solanum tuberosum). However, potatoes rank the second largest protein supplying crop grown per hectare following wheat. Potato proteins include patatin (40-45 kDa), protease inhibitors (5-25 kDa), and various high MW proteins. Non-destructive techniques for the extraction of proteins from potato pulp and for the generation of peptides are needed in order to minimize functional losses and enhance quality. A promising approach for isolating the potato proteins was developed, which involves the use of multi-enzymatic systems containing selected glycosyl hydrolase enzymes that synergistically work to open the plant cell wall network. This enzymatic approach is advantageous due to: (1) the use of milder reaction conditions, (2) the high selectivity and specificity of enzymes, (3) the low cost and (4) the ability to market natural ingredients. Another major benefit to this enzymatic approach is the elimination of a costly purification step; indeed, these multi-enzymatic systems have the ability to isolate proteins, while fractionating them due to their specificity and selectivity with minimal proteolytic activities. The isolated proteins were used for the enzymatic generation of active peptides. In addition, they were applied into a reduced gluten cookie formulation as consumers are putting a high demand for easy ready to eat snack foods, with high nutritional quality and limited to no gluten incorporation. The addition of potato protein significantly improved the textural hardness of reduced gluten cookies, more comparable to wheat flour alone. The presentation will focus on our recent ‘proof-of principle’ results illustrating the feasibility and the efficiency of new biocatalytic processes for the production of innovative functional food ingredients, from potato by-products, whose potential health benefits are increasingly being recognized.

Keywords: biocatalytic approaches, functional ingredients, potato proteins, peptides

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Authors: Mattias Bergstrom

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Significance of the Study: This research will provide new insights into the risks with digital embedded infrastructure. Through this research, we will analyze each risk and its potential negation strategies, especially for AI and autonomous automation. Moreover, the analysis that is presented in this paper will convey valuable information for future research that can create more stable, secure, and efficient autonomous systems. To learn and understand the risks, a large IoT system was envisioned, and risks with hardware, tampering, and cyberattacks were collected, researched, and evaluated to create a comprehensive understanding of the potential risks. Potential solutions have then been evaluated on an open source IoT hardware setup. This list shows the identified passive and active risks evaluated in the research. Passive Risks: (1) Hardware failures- Critical Systems relying on high rate data and data quality are growing; SCADA systems for infrastructure are good examples of such systems. (2) Hardware delivers erroneous data- Sensors break, and when they do so, they don’t always go silent; they can keep going, just that the data they deliver is garbage, and if that data is not filtered out, it becomes disruptive noise in the system. (3) Bad Hardware injection- Erroneous generated sensor data can be pumped into a system by malicious actors with the intent to create disruptive noise in critical systems. (4) Data gravity- The weight of the data collected will affect Data-Mobility. (5) Cost inhibitors- Running services that need huge centralized computing is cost inhibiting. Large complex AI can be extremely expensive to run. Active Risks: Denial of Service- It is one of the most simple attacks, where an attacker just overloads the system with bogus requests so that valid requests disappear in the noise. Malware- Malware can be anything from simple viruses to complex botnets created with specific goals, where the creator is stealing computer power and bandwidth from you to attack someone else. Ransomware- It is a kind of malware, but it is so different in its implementation that it is worth its own mention. The goal with these pieces of software is to encrypt your system so that it can only be unlocked with a key that is held for ransom. DNS spoofing- By spoofing DNS calls, valid requests and data dumps can be sent to bad destinations, where the data can be extracted for extortion or to corrupt and re-inject into a running system creating a data echo noise loop. After testing multiple potential solutions. We found that the most prominent solution to these risks was to use a Peer 2 Peer consensus algorithm over a blockchain to validate the data and behavior of the devices (sensors, storage, and computing) in the system. By the devices autonomously policing themselves for deviant behavior, all risks listed above can be negated. In conclusion, an Internet middleware that provides these features would be an easy and secure solution to any future autonomous IoT deployments. As it provides separation from the open Internet, at the same time, it is accessible over the blockchain keys.

Keywords: IoT, security, infrastructure, SCADA, blockchain, AI

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Authors: K. K. Balavenkatraman, B. Bertschi, K. Bigot, A. Grevot, A. Doelemeyer, S. D. Chibout, A. Wolf, F. Pognan, N. Manevski, O. Kretz, P. Swart, K. Litherland, J. Ashton-Chess, B. Ling, R. Wettstein, D. J. Schaefer

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Skin toxicity is poorly detected during preclinical studies, and drug-induced side effects in humans such as rashes, hyperplasia or more serious events like bullous pemphigus or toxic epidermal necrolysis represent an important hurdle for clinical development. In vitro keratinocyte-based epidermal skin models are suitable for the detection of chemical-induced irritancy, but do not recapitulate the biological complexity of full skin and fail to detect potential serious side-effects. Normal healthy skin explants may represent a valuable complementary tool, having the advantage of retaining the full skin architecture and the resident immune cell diversity. This study investigated several conditions for the maintenance of good morphological structure after several days of culture and the retention of phase II metabolism for 24 hours in skin explants in vitro. Human skin samples were collected with informed consent from patients undergoing plastic surgery and immediately transferred and processed in our laboratory by removing the underlying dermal fat. Punch biopsies of 4 mm diameter were cultured in an air-liquid interface using transwell filters. Different cultural conditions such as the effect of calcium, temperature and cultivation media were tested for a period of 14 days and explants were histologically examined after Hematoxylin and Eosin staining. Our results demonstrated that the use of Williams E Medium at 32°C maintained the physiological integrity of the skin for approximately one week. Upon prolonged incubation, the upper layers of the epidermis become thickened and some dead cells are present. Interestingly, these effects were prevented by addition of EGFR inhibitors such as Afatinib or Erlotinib. Phase II metabolism of the skin such as glucuronidation (4-methyl umbeliferone), sulfation (minoxidil), N-acetyltransferase (p-toluidene), catechol methylation (2,3-dehydroxy naphthalene), and glutathione conjugation (chlorodinitro benzene) were analyzed by using LCMS. Our results demonstrated that the human skin explants possess metabolic activity for a period of at least 24 hours for all the substrates tested. A time course for glucuronidation with 4-methyl umbeliferone was performed and a linear correlation was obtained over a period of 24 hours. Longer-term culture studies will indicate the possible evolution of such metabolic activities. In summary, these results demonstrate that human skin explants maintain a normal structure for several days in vitro and are metabolically active for at least the first 24 hours. Hence, with further characterisation, this model may be suitable for the study of drug-induced toxicity.

Keywords: human skin explant, phase II metabolism, epidermal growth factor receptor, toxicity

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Authors: Thanthrige Thiunuwan Priyathilaka, Don Anushka Sandaruwan Elvitigala, Bong-Soo Lim, Hyung-Bok Jeong, Jehee Lee

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Toll like receptors (TLRs) are a phylogeneticaly conserved family of pattern recognition receptors, which participates in the host immune responses against various pathogens and pathogen derived mitogen. TLR21, a non-mammalian type, is almost restricted to the fish species even though those can be identified rarely in avians and amphibians. Herein, this study was carried out to identify and characterize TLR21 from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at transcriptional and genomic level. In this study, the full length cDNA and genomic sequence of RbTLR21 was identified using previously constructed cDNA sequence database and BAC library, respectively. Identified RbTLR21 sequence was characterized using several bioinformatics tools. The quantitative real time PCR (qPCR) experiment was conducted to determine tissue specific expressional distribution of RbTLR21. Further, transcriptional modulation of RbTLR21 upon the stimulation with Streptococcus iniae (S. iniae), rock bream iridovirus (RBIV) and Edwardsiella tarda (E. tarda) was analyzed in spleen tissues. The complete coding sequence of RbTLR21 was 2919 bp in length which can encode a protein consisting of 973 amino acid residues with molecular mass of 112 kDa and theoretical isoelectric point of 8.6. The anticipated protein sequence resembled a typical TLR domain architecture including C-terminal ectodomain with 16 leucine rich repeats, a transmembrane domain, cytoplasmic TIR domain and signal peptide with 23 amino acid residues. Moreover, protein folding pattern prediction of RbTLR21 exhibited well-structured and folded ectodomain, transmembrane domain and cytoplasmc TIR domain. According to the pair wise sequence analysis data, RbTLR21 showed closest homology with orange-spotted grouper (Epinephelus coioides) TLR21with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 shows a close evolutionary relationship with its ortholog from Danio rerio. Genomic structure of RbTLR21 consisted of single exon similar to its ortholog of zebra fish. Sevaral putative transcription factor binding sites were also identified in 5ʹ flanking region of RbTLR21. The RBTLR 21 was ubiquitously expressed in all the tissues we tested. Relatively, high expression levels were found in spleen, liver and blood tissues. Upon induction with rock bream iridovirus, RbTLR21 expression was upregulated at the early phase of post induction period even though RbTLR21 expression level was fluctuated at the latter phase of post induction period. Post Edwardsiella tarda injection, RbTLR transcripts were upregulated throughout the experiment. Similarly, Streptococcus iniae induction exhibited significant upregulations of RbTLR21 mRNA expression in the spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed a homolog of TLR21 family members and RbTLR21 may be involved in host immune responses against bacterial and DNA viral infections.

Keywords: rock bream, toll like receptor 21 (TLR21), pattern recognition receptor, genomic characterization

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Authors: Jong H. Kim, Kathleen L. Chan, Luisa W. Cheng

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Control of fungal pathogens, such as foodborne mycotoxin producers, is problematic as effective antimycotic agents are often very limited. Mycotoxin contamination significantly interferes with the safe production of foods or crops worldwide. Moreover, expansion of fungal resistance to commercial drugs or fungicides is a global human health concern. Therefore, there is a persistent need to enhance the efficacy of commercial antimycotic agents or to develop new intervention strategies. Disruption of the cellular antioxidant system should be an effective method for pathogen control. Such disruption can be achieved with safe, redox-active compounds. Natural phenolic derivatives are potent redox cyclers that inhibit fungal growth through destabilization of the cellular antioxidant system. The goal of this study is to identify novel, redox-active compounds that disrupt the fungal antioxidant system. The identified compounds could also function as sensitizing agents to conventional antimycotics (i.e., chemosensitization) to improve antifungal efficacy. Various benzo derivatives were tested against fungal pathogens. Gene deletion mutants of the yeast Saccharomyces cerevisiae were used as model systems for identifying molecular targets of benzo analogs. The efficacy of identified compounds as potent antifungal agents or as chemosensitizing agents to commercial drugs or fungicides was examined with methods outlined by the Clinical Laboratory Standards Institute or the European Committee on Antimicrobial Susceptibility Testing. Selected benzo derivatives possessed potent antifungal or antimycotoxigenic activity. Molecular analyses by using S. cerevisiae mutants indicated antifungal activity of benzo derivatives was through disruption of cellular antioxidant or cell wall integrity system. Certain benzo analogs screened overcame tolerance of Aspergillus signaling mutants, namely mitogen-activated protein kinase mutants, to fludioxonil fungicide. Synergistic antifungal chemosensitization greatly lowered minimum inhibitory or fungicidal concentrations of test compounds, including inhibitors of mitochondrial respiration. Of note, salicylaldehyde is a potent antimycotic volatile that has some practical application as a fumigant. Altogether, benzo derivatives targeting cellular antioxidant system of fungi (along with cell wall integrity system) effectively suppress fungal growth. Candidate compounds possess the antifungal, antimycotoxigenic or chemosensitizing capacity to augment the efficacy of commercial antifungals. Therefore, chemogenetic approaches can lead to the development of novel antifungal intervention strategies, which enhance the efficacy of established microbe intervention practices and overcome drug/fungicide resistance. Chemosensitization further reduces costs and alleviates negative side effects associated with current antifungal treatments.

Keywords: antifungals, antioxidant system, benzo derivatives, chemosensitization

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Authors: Wei Xiao, Gangzhi Cai, Xingliang Qin, Hongyan Ren, Zaidong Hua, Zhe Zhu, Hongwei Xiao, Ximin Zheng, Jie Yao, Yanzhen Bi

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Myostatin (MSTN) is the master regulator of double muscling phenotype with overgrown muscle and decreased fatness in animals, but its action mode to regulate fat deposition remains to be elucidated. In this study a swin-specific pathway through which MSTN acts to regulate the fat deposition was deciphered. Deep sequenincing of the mRNA and miRNA of fat tissues of MSTN knockout (KO) and wildtype (WT) pigs discovered the positive correlation of myocyte enhancer factor 2C (MEF2C) and fat-inhibiting miR222 expression, and the inverse correlation of miR222 and stearoyl-CoA desaturase 5 (SCD5) expression. SCD5 is rodent-absent and expressed only in pig, sheep and cattle. Fatty acid spectrum of fat tissues revealed a lower percentage of oleoyl-CoA (18:1) and palmitoleyl CoA (16:1) in MSTN KO pigs, which are the catalyzing products of SCD5-mediated desaturation of steroyl CoA (18:0) and palmitoyl CoA (16:0). Blood metrics demonstrated a 45% decline of triglyceride (TG) content in MSTN KO pigs. In light of these observations we hypothesized that MSTN might act through MEF2C/miR222/SCD5 pathway to regulate desaturation of fatty acid as well as triglyceride synthesis in pigs. To this end, real-time PCR and Western blotting were carried out to detect the expression of the three genes stated above. These experiments showed that MEF2C expression was up-regulated by nearly 2-fold, miR222 up-regulated by nearly 3-fold and SCD5 down-regulated by nearly 50% in MSTN KO pigs. These data were consistent with the expression change in deep sequencing analysis. Dual luciferase reporter was then used to confirm the regulation of MEF2C upon the promoter of miR222. Ecotopic expression of MEF2C in preadipocyte cells enhanced miR222 expression by 3.48-fold. CHIP-PCR identified a putative binding site of MEF2C on -2077 to -2066 region of miR222 promoter. Electrophoretic mobility shift assay (EMSA) demonstrated the interaction of MEF2C and miR222 promoter in vitro. These data indicated that MEF2C transcriptionally regulates the expression of miR222. Next, the regulation of miR222 on SCD5 mRNA as well as its physiological consequences were examined. Dual luciferase reporter testing revealed the translational inhibition of miR222 upon the 3´ UTR (untranslated region) of SCD5 in preadipocyte cells. Transfection of miR222 mimics and inhibitors resulted in the down-regulation and up-regulation of SCD5 in preadipocyte cells respectively, consistent with the results from reporter testing. RNA interference of SCD5 in preadipocyte cells caused 26.2% reduction of TG, in agreement with the results of TG content in MSTN KO pigs. In summary, the results above supported the existence of a molecular pathway that MSTN signals through MEF2C/miR222/SCD5 to regulate the fat deposition in pigs. This swine-specific pathway offers potential molecular markers for the development and breeding of a new pig line with optimised fatty acid composition. This would benefit human health by decreasing the takeup of saturated fatty acid.

Keywords: fat deposition, MEF2C, miR222, myostatin, SCD5, pig

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Authors: Fernanda Gonçalves, Karla Alcântara, Vanessa Moura, Patrícia Nolasco, Jorge Kalil, Maristela Hernandez

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Introduction: Atherosclerosis is a chronic disease where two striking features are observed: retention of lipids and inflammation. Understanding the interaction between immune cells and lipoproteins involved in atherogenesis are urgent challenges, since cardiovascular diseases are the leading cause of death worldwide. Macrophages are critical to the development of atherosclerotic plaques and in the perpetuation of inflammation in these lesions. These cells are also directly involved in unstable plaque rupture. Recently different populations of macrophages are being identified in atherosclerotic lesions. Although the presence of M2 macrophages (macrophages activated by the alternative pathway, eg. The IL-4) has been identified, the function of these cells in atherosclerosis is not yet defined. M2 macrophages have a high endocytic capacity, they promote remodeling of tissues and to have anti-inflammatory activity. However, in atherosclerosis, especially unstable plaques, severe inflammatory reaction, accumulation of cellular debris and intense degradation of the tissue is observed. Thus, it is possible that the M2 macrophages have altered function (phenotype) in atherosclerosis. Objective: Our aim is to evaluate if the presence of oxidized LDL alters the phenotype and function of M2 macrophages in vitro. Methods: For this, we will evaluate whether the addition of lipoprotein in M2 macrophages differentiated in vitro with IL -4 induces 1) a reduction in the secretion of anti-inflammatory cytokines (CBA and ELISA), 2) secretion of inflammatory cytokines (CBA and ELISA), 3) expression of cell activation markers (Flow cytometry), 4) alteration in gene expression of molecules adhesion and extracellular matrix (Real-Time PCR) and 5) Matrix degradation (confocal microscopy). Results: In oxLDL stimulated M2 macrophages cultures we did not find any differences in the expression of the cell surface markers tested, including: HLA-DR, CD80, CD86, CD206, CD163 and CD36. Also, cultures stimulated with oxLDL had similar phagocytic capacity when compared to unstimulated cells. However, in the supernatant of these cultures an increase in the secretion of the pro-inflammatory cytokine IL-8 was detected. No significant changes where observed in IL-6, IL-10, IL-12 and IL-1b levels. The culture supernatant also induced massive extracellular matrix (produced by mouse embryo fibroblast) filaments degradation. When evaluating the expression of 84 extracellular matrix and adhesion molecules genes, we observed that the stimulation of oxLDL in M2 macrophages decreased 47% of the genes and increased the expression of only 3% of the genes. In particular we noted that oxLDL inhibit the expression of 60% of the genes constituents of extracellular matrix and collagen expressed by these cells, including fibronectin1 and collagen VI. We also observed a decrease in the expression of matrix protease inhibitors, such as TIMP 2. On the opposite, the matricellular protein thrombospondin had a 12 fold increase in gene expression. In the presence of native LDL 90% of the genes had no altered expression. Conclusion: M2 macrophages stimulated with oxLDL secrete the pro-inflammatory cytokine IL-8, have an altered extracellular matrix constituents gene expression, and promote the degradation of extracellular matrix. M2 macrophages may contribute to the perpetuation of inflammation in atherosclerosis and to plaque rupture.

Keywords: atherosclerosis, LDL, macrophages, m2

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Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

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The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

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Authors: Robin Anderson, David Nisbet

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Nitrate, 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH) are biologically active chemicals that can accumulate naturally in rangeland grasses forages consumed by grazing cattle, sheep and goats. While toxic to livestock if accumulations and amounts consumed are high enough, particularly in animals having no recent exposure to the forages, these chemicals are known to be potent inhibitors of methane-producing bacteria inhabiting the rumen. Consequently, there is interest in examining their potential use as anti-methanogenic compounds to decrease methane emissions by grazing ruminants. Presently, rumen microbes, collected freshly from a cannulated Holstein cow maintained on 50:50 corn based concentrate:alfalfa diet were mixed (10 mL fluid) in 18 x 150 mm crimp top tubes with 0.5 of high nitrate-containing barley (Hordeum vulgare; containing 272 µmol nitrate per g forage dry matter), and NPA- or NPOH- containing milkvetch forages (Astragalus canadensis and Astragalus miser containing 80 and 174 soluble µmol NPA or NPOH/g forage dry matter respectively). Incubations containing 0.5 g alfalfa (Medicago sativa) were used as controls. Tubes (3 per each respective forage) were capped and incubated anaerobically (using oxygen free carbon dioxide) for 24 h at 39oC after which time amounts of total gas produced were measured via volume displacement and headspace samples were analyzed by gas chromatography to determine concentrations of hydrogen and methane. Fluid samples were analyzed by gas chromatography to measure accumulations of fermentation acids. A completely randomized analysis of variance revealed that the nitrate-containing barley and both the NPA- and the NPOH-containing milkvetches significantly decreased methane production, by > 50%, when compared to methane produced by populations incubated similarly with alfalfa (70.4 ± 3.6 µmol/ml incubation fluid). Accumulations of hydrogen, which are typically increased when methane production is inhibited, by incubations with the nitrate-containing barley and the NPA- and NPOH-containing milkvetches did not differ from accumulations observed in the alfalfa controls (0.09 ± 0.04 µmol/mL incubation fluid). Accumulations of fermentation acids produced in the incubations containing the high-nitrate barley and the NPA- and NPOH-containing milkvetches likewise did not differ from accumulations observed in incubations containing alfalfa (123.5 ± 10.8, 36.0 ± 3.0, 17.1 ± 1.5, 3.5 ± 0.3, 2.3 ± 0.2, 2.2 ± 0.2 µmol/mL incubation fluid for acetate, propionate, butyrate, valerate, isobutyrate, and isovalerate, respectively). This finding indicates the microbial populations did not compensate for the decreased methane production via compensatory changes in production of fermentative acids. Stoichiometric estimation of fermentation balance revealed that > 77% of reducing equivalents generated during fermentation of the forages were recovered in fermentation products and the recoveries did not differ between the alfalfa incubations and those with the high-nitrate barley or the NPA- or NPOH-containing milkvetches. Stoichiometric estimates of amounts of hexose fermented similarly did not differ between the nitrate-, NPA and NPOH-containing incubations and those with the alfalfa, averaging 99.6 ± 37.2 µmol hexose consumed/mL of incubation fluid. These results suggest that forages containing nitrate, NPA or NPOH may be useful to reduce methane emissions of grazing ruminants provided risks of toxicity can be effectively managed.

Keywords: nitrate, nitropropanol, nitropropionic acid, rumen methane emissions

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Authors: K. Karapetyan, F. Tkhruni, A. Aghajanyan, T. S. Balabekyan, L. Arstamyan

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Introduction: Globalization of the food supply has created the conditions favorable for the emergence and spread of food-borne and especially dangerous pathogens (EDP) in developing countries. The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the processed foods product. Antimicrobial compounds of lactic acid bacteria (LAB) possess bactericidal or bacteriostatic activity against intestinal pathogens, spoilage organisms and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus and Salmonella. Endemic strains of LAB were isolated. The strains, showing broad spectrum of antimicrobial activity against food spoiling microorganisms, were selected. The genotyping by 16S rRNA sequencing, GS-PCR, RAPD PCR methods showed that they were presented by Lactobacillus rhamnosus109, L.plantarum 65, L.plantarum 66 and Enterococcus faecium 64 species. LAB are deposited in "Microbial Depository Center" (MDC) SPC "Armbiotechnology". Methods: LAB strains were isolated from different dairy products from rural households from the highland regions of Armenia. Serially diluted samples were spread on MRS (Merck, Germany) and hydrolyzed milk agar (1,2 % w/v). Single colonies from each LAB were individually inoculated in liquid MRS medium and incubated at 37oC for 24 hours. Culture broth with biomass was centrifuged at 10,000 g during 20 min for obtaining of cell free culture broth (CFC). The antimicrobial substances from CFC broth were purified by the combination of adsorption-desorption and ion-exchange chromatography methods. Separation of bacteriocins was performed using a HPLC method on "Avex ODS" C18 column. Mass analysis of peptides recorded on the device API 4000 in the electron ionization mode. The spot-on-lawn method on the test culture plated in the solid medium was applied. The antimicrobial activity is expressed in arbitrary units (AU/ml). Results. Purification of CFC broth of LAB allowed to obtain partially purified antimicrobial preparations which contains bacteriocins with broad spectrum of antimicrobial activity. Investigation of their main biochemical properties shown, that inhibitory activity of preparations is partially reduced after treatment with proteinase K, trypsin, pepsin, suggesting a proteinaceous nature of bacteriocin-like substances containing in CFC broth. Preparations preserved their activity after heat treatment (50-121 oC, 20 min) and were stable in the pH range 3–8. The results of SDS PAAG electrophoresis show that L.plantarum 66 and Ent.faecium 64 strains have one bacteriocin (BCN) with maximal antimicrobial activity with approximate molecular weight 2.0-3.0 kDa. From L.rhamnosus 109 two BCNs were obtained. Mass spectral analysis indicates that these bacteriocins have peptide bonds and molecular weight of BCN 1 and BCN 2 are approximately 1.5 kDa and 700 Da. Discussion: Thus, our experimental data shown, that isolated endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms of different taxonomic group, such as Salmonella sp., Esherichia coli, Bacillus sp., L.monocytogenes, Proteus mirabilis, Staph. aureus, Ps. aeruginosa. Obtained results proved the perspectives for use of endemic strains in the preservation of foodstuffs. Acknowledgments: This work was realized with financial support of the Project Global Initiatives for Preliferation Prevention (GIPP) T2- 298, ISTC A-1866.

Keywords: antimicrobial activity, bacteriocins, endemic strains, food safety

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Authors: Cristina Diaz, Lucia Perez, Simone Visigalli, Giuseppe Di Florio, Gonzalo Fuentes, Roberto Canziani, Paolo Gronchi

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During the last 50 years, the selection of materials is one of the main decisions in machine design for different industrial applications. It is due to numerous physical, chemical, mechanical and technological factors to consider in it. Corrosion effects are related with all of these factors and impact in the life cycle, machine incidences and the costs for the life of the machine. Corrosion affects the deterioration or destruction of metals due to the reaction with the environment, generally wet. In food industry, dewatering industry, concrete industry, paper industry, etc. corrosion is an unsolved problem and it might introduce some alterations of some characteristics in the final product. Nowadays, depending on the selected metal, its surface and its environment of work, corrosion prevention might be a change of metal, use a coating, cathodic protection, use of corrosion inhibitors, etc. In the vast majority of the situations, use of a corrosion resistant material or in its defect, a corrosion protection coating is the solution. Stainless steels are widely used in machine design, because of their strength, easily cleaned capacity, corrosion resistance and appearance. Typical used are AISI 304 and AISI 316. However, their benefits don’t fit every application, and some coatings are required against corrosion such as some paintings, galvanizing, chrome plating, SiO₂, TiO₂ or ZrO₂ coatings, etc. In this work, some coatings based in a bilayer made of Titanium-Tantalum, Titanium-Niobium, Titanium-Hafnium or Titanium-Zirconium, have been developed used magnetron sputtering configuration by PVD (Physical Vapor Deposition) technology, for trying to reduce corrosion effects on AISI 304, AISI 316 and comparing it with Titanium alloy substrates. Ti alloy display exceptional corrosion resistance to chlorides, sour and oxidising acidic media and seawater. In this study, Ti alloy (99%) has been included for comparison with coated AISI 304 and AISI 316 stainless steel. Corrosion tests were conducted by a Gamry Instrument under ASTM G5-94 standard, using different electrolytes such as tomato salsa, wine, olive oil, wet compost, a mix of sand and concrete with water and NaCl for testing corrosion in different industrial environments. In general, in all tested environments, the results showed an improvement of corrosion resistance of all coated AISI 304 and AISI 316 stainless steel substrates when they were compared to uncoated stainless steel substrates. After that, comparing these results with corrosion studies on uncoated Ti alloy substrate, it was observed that in some cases, coated stainless steel substrates, reached similar current density that uncoated Ti alloy. Moreover, Titanium-Zirconium and Titanium-Tantalum coatings showed for all substrates in study including coated Ti alloy substrates, a reduction in current density more than two order in magnitude. As conclusion, Ti-Ta, Ti-Zr, Ti-Nb and Ti-Hf coatings have been developed for improving corrosion resistance of AISI 304 and AISI 316 materials. After corrosion tests in several industry environments, substrates have shown improvements on corrosion resistance. Similar processes have been carried out in Ti alloy (99%) substrates. Coated AISI 304 and AISI 316 stainless steel, might reach similar corrosion protection on the surface than uncoated Ti alloy (99%). Moreover, coated Ti Alloy (99%) might increase its corrosion resistance using these coatings.

Keywords: coatings, corrosion, PVD, stainless steel

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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: Yung-Chih Kuo, Che-Yu Lin, Jay-Shake Li, Yung-I Lou

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Curcumin (CRM) and nerve growth factor (NGF) were entrapped in liposomes (LIP) with cardiolipin (CL) to downregulate the phosphorylation of mitogen-activated protein kinases for Alzheimer’s disease (AD) management. AD belongs to neurodegenerative disorder with a gradual loss of memory, yielding irreversible dementia. CL-conjugated LIP loaded with CRM (CRM-CL/LIP) and that with NGF (NGF-CL/LIP) were applied to AD models of SK-N-MC cells and Wistar rats with an insult of β-amyloid peptide (Aβ). Lipids comprising 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (Avanti Polar Lipids, Alabaster, AL), 1',3'-bis[1,2- dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (CL; Avanti Polar Lipids), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (Avanti Polar Lipids), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (Avanti Polar Lipids) and CRM (Sigma–Aldrich, St. Louis, MO) were dissolved in chloroform (J. T. Baker, Phillipsburg, NJ) and condensed using a rotary evaporator (Panchum, Kaohsiung, Taiwan). Human β-NGF (Alomone Lab, Jerusalem, Israel) was added in the aqueous phase. Wheat germ agglutinin (WGA; Medicago AB, Uppsala, Sweden) was grafted on LIP loaded with CRM for (WGA-CRM-LIP) and CL-conjugated LIP loaded with CRM (WGA-CRM-CL/LIP) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma–Aldrich) and N-hydroxysuccinimide (Alfa Aesar, Ward Hill, MA). The protein samples of SK-N-MC cells (American Type Tissue Collection, Rockville, MD) were used for sodium dodecyl sulfate (Sigma–Aldrich) polyacrylamide gel (Sigma–Aldrich) electrophoresis. In animal study, the LIP formulations were administered by intravenous injection via a tail vein of male Wistar rats (250–280 g, 8 weeks, BioLasco, Taipei, Taiwan), which were housed in the Animal Laboratory of National Chung Cheng University in accordance with the institutional guidelines and the guidelines of Animal Protection Committee under the Council of Agriculture of the Republic of China. We found that CRM-CL/LIP could inhibit the expressions of phosphorylated p38 (p-p38), p-Jun N-terminal kinase (p-JNK), and p-tau protein at serine 202 (p-Ser202) to retard the neuronal apoptosis. Free CRM and released CRM from CRM-LIP and CRM-CL/LIP were not in a straightforward manner to effectively inhibit the expression of p-p38 and p-JNK in the cytoplasm. In addition, NGF-CL/LIP enhanced the quantities of p-neurotrophic tyrosine kinase receptor type 1 (p-TrkA) and p-extracellular-signal-regulated kinase 5 (p-ERK5), preventing the Aβ-induced degeneration of neurons. The membrane fusion of NGF-LIP activated the ERK5 pathway and the targeting capacity of NGF-CL/LIP enhanced the possibility of released NGF to affect the TrkA level. Moreover, WGA-CRM-LIP improved the permeation of CRM across the blood–brain barrier (BBB) and significantly reduced the Aβ plaque deposition and malondialdehyde level and increased the percentage of normal neurons and cholinergic function in the hippocampus of AD rats. This was mainly because the encapsulated CRM was protected by LIP against a rapid degradation in the blood. Furthermore, WGA on LIP could target N-acetylglucosamine on endothelia and increased the quantity of CRM transported across the BBB. In addition, WGA-CRM-CL/LIP could be effective in suppressing the synthesis of acetylcholinesterase and reduced the decomposition of acetylcholine for better neurotransmission. Based on the in vitro and in vivo evidences, WGA-CRM-CL/LIP can rescue neurons from apoptosis in the brain and can be a promising drug delivery system for clinical AD therapy.

Keywords: Alzheimer’s disease, β-amyloid, liposome, mitogen-activated protein kinase

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Authors: D. Malouch, M. Berchel, C. Dreanno, S. Stachowski-Haberkorn, P-A. Jaffres

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Biofouling is a complex natural phenomenon that involves biological, physical and chemical properties related to the environment, the submerged surface and the living organisms involved. Bio-colonization of artificial structures can cause various economic and environmental impacts. The increase in costs associated with the over-consumption of fuel from biocolonized vessels has been widely studied. Measurement drifts from submerged sensors, as well as obstructions in heat exchangers, and deterioration of offshore structures are major difficulties that industries are dealing with. Therefore, surfaces that inhibit biocolonization are required in different areas (water treatment, marine paints, etc.) and many efforts have been devoted to produce efficient and eco-compatible antifouling agents. The different steps of surface fouling are widely described in literature. Studying the biofilm and its stages provides a better understanding of how to elaborate more efficient antifouling strategies. Several approaches are currently applied, such as the use of biocide anti-fouling paint (mainly with copper derivatives) and super-hydrophobic coatings. While these two processes are proving to be the most effective, they are not entirely satisfactory, especially in a context of a changing legislation. Nowadays, the challenge is to prevent biofouling with non-biocide compounds, offering a cost effective solution, but with no toxic effects on marine organisms. Since the micro-fouling phase plays an important role in the regulation of the following steps of biofilm formation, it is desired to reduce or delate biofouling of a given surface by inhibiting the micro-fouling at its early stages. In our recent works, we reported that some amphiphilic compounds exhibited bacteriostatic or bactericidal properties at a concentration that did not affect mammalian eukaryotic cells. These remarkable properties invited us to assess this type of bio-inspired phospholipids to prevent the colonization of surfaces by marine bacteria. Of note, other studies reported that amphiphilic compounds interacted with bacteria leading to a reduction of their development. An amphiphilic compound is a molecule consisting of a hydrophobic domain and a polar head (ionic or non-ionic). These compounds appear to have interesting antifouling properties: some ionic compounds have shown antimicrobial activity, and zwitterions can reduce nonspecific adsorption of proteins. Herein, we investigate the potential of amphiphilic compounds as inhibitors of bacterial growth and marine biofilm formation. The aim of this study is to compare the efficacy of four synthetic phospholipids that features a cationic charge or a zwitterionic polar-head group to prevent microfouling with marine bacteria. Toxicity of these compounds was also studied in order to identify the most promising compounds that inhibit biofilm development and show low cytotoxicity on two links representative of coastal marine food webs: phytoplankton and oyster larvae.

Keywords: amphiphilic phospholipids, biofilm, marine fouling, non-toxique assays

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Authors: Esther Friedlander, Philip Friedlander

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The use of immunotherapy that inhibits programmed death-1 (PD-1) or its ligand PD-L1 confers survival benefits in patients with non-small cell lung cancer (NSCLC). However, approximately 45% of patients experience primary treatment resistance, necessitating the development of strategies to improve efficacy. While the renin-angiotensin system (RAS) has systemic hemodynamic effects, tissue-specific regulation exists along with modulation of immune activity in part through regulation of myeloid cell activity, leading to the hypothesis that RAS inhibition may improve anti-PD-1/L-1 efficacy. A retrospective analysis was conducted that included 173 advanced solid tumor cancer patients treated at Valley Hospital, a community Hospital in New Jersey, USA, who were treated with a PD-1/L-1 inhibitor in a defined time period showing a statistically significant relationship between RAS pathway inhibition (RASi through concomitant treatment with an ACE inhibitor or angiotensin receptor blocker) and positive efficacy to the immunotherapy that was independent of age, gender and cancer type. Subset analysis revealed strong numerical benefit for efficacy in both patients with squamous and nonsquamous NSCLC as determined by documented clinician assessment of efficacy and by duration of therapy. A PUBMED literature search was now conducted to identify studies assessing the effect of RAS pathway inhibition on anti-PD-1/L1 efficacy in advanced solid tumor patients and compare these findings to those seen in the Valley Hospital retrospective study with a focus on NSCLC specifically. A total of 11 articles were identified assessing the effects of RAS pathway inhibition on the efficacy of checkpoint inhibitor immunotherapy in advanced cancer patients. Of the 11 studies, 10 assessed the effect on survival of RASi in the context of treatment with anti-PD-1/PD-L1, while one assessed the effect on CTLA-4 inhibition. Eight of the studies included patients with NSCLC, while the remaining 2 were specific to genitourinary malignancies. Of the 8 studies, two were specific to NSCLC patients, with the remaining 6 studies including a range of cancer types, of which NSCLC was one. Of these 6 studies, only 2 reported specific survival data for the NSCLC subpopulation. Patient characteristics, multivariate analysis data and efficacy data seen in the 2 NSLCLC specific studies and in the 2 basket studies, which provided data on the NSCLC subpopulation, were compared to that seen in the Valley Hospital retrospective study supporting a broader effect of RASi on anti-PD-1/L1 efficacy in advanced NSLCLC with the majority of studies showing statistically significant benefit or strong statistical trends but with one study demonstrating worsened outcomes. This comparison of studies extends published findings to the community hospital setting and supports prospective assessment through randomized clinical trials of efficacy in NSCLC patients with pharmacodynamic components to determine the effect on immune cell activity in tumors and on the composition of the tumor microenvironment.

Keywords: immunotherapy, cancer, angiotensin, efficacy, PD-1, lung cancer, NSCLC

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Authors: Binbin Chen, Dennis Y. C. Leung

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Aluminium (Al) -air cell holds a high volumetric capacity density of 8.05 Ah cm-3, benefit from the trivalence of Al ions. Additional benefits of Al-air cell are low price and environmental friendliness. Furthermore, the Al energy conversion process is characterized of 100% recyclability in theory. Along with a large base of raw material reserve, Al attracts considerable attentions as a promising material to be integrated within the global energy system. However, despite the early successful applications in military services, several problems exist that prevent the Al-air cells from widely civilian use. The most serious issue is the parasitic corrosion of Al when contacts with electrolyte. To overcome this problem, super-pure Al alloyed with various traces of metal elements are used to increase the corrosion resistance. Nevertheless, high-purity Al alloys are costly and require high energy consumption during production process. An alternative approach is to add inexpensive inhibitors directly into the electrolyte. However, such additives would increase the internal ohmic resistance and hamper the cell performance. So far these methods have not provided satisfactory solutions for the problem within Al-air cells. For the operation of alkaline Al-air cell, there are still other minor problems. One of them is the formation of aluminium hydroxide in the electrolyte. This process decreases ionic conductivity of electrolyte. Another one is the carbonation process within the gas diffusion layer of cathode, blocking the porosity of gas diffusion. Both these would hinder the performance of cells. The present work optimizes the above problems by building an Al-air cell operation system, consisting of four components. A top electrolyte tank containing fresh electrolyte is located at a high level, so that it can drive the electrolyte flow by gravity force. A mechanical rechargeable Al-air cell is fabricated with low-cost materials including low grade Al, carbon paper, and PMMA plates. An electrolyte waste tank with elaborate channel is designed to separate the hydrogen generated from the corrosion, which would be collected by gas collection device. In the first section of the research work, we investigated the performance of the mechanical rechargeable Al-air cell with a constant flow rate of electrolyte, to ensure the repeatability experiments. Then the whole system was assembled together and the feasibility of operating was demonstrated. During experiment, pure hydrogen is collected by collection device, which holds potential for various applications. By collecting this by-product, high utilization efficiency of aluminum is achieved. Considering both electricity and hydrogen generated, an overall utilization efficiency of around 90 % or even higher under different working voltages are achieved. Fluidic electrolyte could remove aluminum hydroxide precipitate and solve the electrolyte deterioration problem. This operation system provides a low-cost strategy for harvesting energy from the abundant secondary Al. The system could also be applied into other metal-air cells and is suitable for emergency power supply, power plant and other applications. The low cost feature implies great potential for commercialization. Further optimization, such as scaling up and optimization of fabrication, will help to refine the technology into practical market offerings.

Keywords: aluminium-air cell, high efficiency, hydrogen, mechanical recharge

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Authors: A. Abdi, A. Abbasi Daloee, A. Barari

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Aim: The adaptations that occur in human body after doing exercises training are a factor to help healthy people stay away from certain diseases. One of the main adaptations is a change in blood circulation, especially in vessels. The increase of capillary density is dependent on the balance between angiogenic and angiostatic factors. Most studies show that the changes made to angiogenic developmental factors resulted from physical exercises indicate the low level of stimulators compared with inhibitors. It is believed that the plasma level of VEGF-A, the important angiogenic factor, is reduced after physical exercise. Findings indicate that the extract of crataegus plant reduces the platelet-derived growth factor receptor (PDGFR) autophosphorylation in human's fibroblast. More importantly, crataegus (1 to 100 mg in liter) clearly leads to the inhibition of PDGFR autophosphorylation in vascular smooth muscle cells (VSMCs). Angiogenesis is a process that can be classified into physiological and pathophysiological forms. collagen XVIII is a part of extracellular protein and heparan sulfate proteoglycans in vascular epithelial and endothelial basement membrane cause the release of endostatin from noncollagenous collagen XVIII. Endostatin inhibits the growth of endothelial cells, inhibits angiogenesis, weakens different types of cancer, and the growth of tumors. The purpose of the current study was to investigate the effect of intensive aerobic running with or without aqueous extraction of black Crataegus elbursensis on Collagen XVIII in male rats. Design: Thirty-two Wistar male rats (4-6 weeks old, 125-135 gr weight) were acquired from the Pasteur's Institute (Amol, Mazandaran), and randomly assigned into control (n = 16) and training (n = 16) groups. Rats were further divided into saline-control (SC) (n=8), saline-training (ST) (n=8), crataegus pentaegyna extraction -control (CPEC) (n=8), and crataegus pentaegyna extraction - training (CPET) (n=8). The control (SC and CPEC) groups remained sedentary; whereas the training groups underwent a high running exercise program. plasma were excised and immediately frozen in liquid nitrogen. Statistical analysis was performed using a one way analysis of variance and Tukey test. Significance was accepted at P = 0.05. Results: The results show that aerobic exercise group had the highest concentration collagen XVIII compared to other groups and then respectively black crataegus, training-crataegus and control groups. Conclusion: In general, researchers in this study concluded that the increase of collagen XVIII (albeit insignificant) as a result of physical activity and consumption of black crataegus extract could possibly serve as a regional inhibitor of angiogenesis and another evidence for the anti-cancer effects of physical activities. Since the research has not managed in this study to measure the amount of plasma endostatin, it is suggested that both indices are measured with important angiogenic factors so that we can have a more accurate interpretation of changes to angiogenic and angiostatic factors resulted from physical exercises.

Keywords: aerobic running, Crataegus elbursensis, Collagen XVIII

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