Search results for: NTRK gene fusion

Authors: Neeraj Neeraj, Soumen Basu, Banibrata Maity

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Glutathione (GSH) is one of the most important biomolecules having small molecular weight, which helps in various cellular functions like regulation of gene, xenobiotic metabolism, preservation of intracellular redox activities, signal transduction, etc. Therefore, the detection of GSH requires huge attention by using extremely selective and sensitive techniques. Herein, a rapid fluorometric nanosensor is designed by combining carbon dots (Cdots) and MnO₂ nanoparticles of different sizes for the detection of GSH. The bottom-up approach, i.e., microwave method, was used for the preparation of the water soluble and greatly fluorescent Cdots by using ascorbic acid as a precursor. MnO₂ nanospheres of different sizes (large, medium, and small) were prepared by varying the ratio of concentration of methionine and KMnO₄ at room temperature, which was confirmed by HRTEM analysis. The successive addition of MnO₂ nanospheres in Cdots results fluorescence quenching. From the fluorescence intensity data, Stern-Volmer quenching constant values (KS-V) were evaluated. From the fluorescence intensity and lifetime analysis, it was found that the degree of fluorescence quenching of Cdots followed the order: large > medium > small. Moreover, fluorescence recovery studies were also performed in the presence of GSH. Fluorescence restoration studies also show the order of turn on follows the same order, i.e., large > medium > small, which was also confirmed by quantum yield and lifetime studies. The limits of detection (LOD) of GSH in presence of Cdots@different sized MnO₂ nanospheres were also evaluated. It was observed thatLOD values were in μM region and lowest in case of large MnO₂ nanospheres. The separation distance (d) between Cdots and the surface of different MnO₂ nanospheres was determined. The d values increase with increase in the size of the MnO₂ nanospheres. In summary, the synthesized Cdots@MnO₂ nanocomposites acted as a rapid, simple, economical as well as environmental-friendly nanosensor for the detection of GSH.

Keywords: carbon dots, fluorescence, glutathione, MnO₂ nanospheres, turn off-on

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Authors: Patrícia Severino, Luciana Nalone, Daniele Martins, Marco Chaud, Classius Ferreira, Cristiane Bani, Ricardo Albuquerque

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Hydrogels produced with polymers have been used in the development of dressings for wound treatment and tissue revitalization. Our study on polymer nanocomposites containing silver nanoparticles shows antimicrobial activity and applications in wound healing. The effects are linked with the slow oxidation and Ag⁺ liberation to the biological environment. Furthermore, bacterial cell membrane penetration and metabolic disruption through cell cycle disarrangement also contribute to microbial cell death. The silver antimicrobial activity has been known for many years, and previous reports show that low silver concentrations are safe for human use. This work aims to develop a hydrogel using natural polymers (sodium alginate and gelatin) combined with silver nanoparticles for wound healing and with antimicrobial properties in cutaneous lesions. The hydrogel development utilized different sodium alginate and gelatin proportions (20:80, 50:50 and 80:20). The silver nanoparticles incorporation was evaluated at the concentrations of 1.0, 2.0 and 4.0 mM. The physico-chemical properties of the formulation were evaluated using ultraviolet-visible (UV-Vis) absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and thermogravimetric (TG) analysis. The morphological characterization was made using transmission electron microscopy (TEM). Human fibroblast (L2929) viability assay was performed with a minimum inhibitory concentration (MIC) assessment as well as an in vivo cicatrizant test. The results suggested that sodium alginate and gelatin in the (80:20) proportion with 4 mM of AgNO₃ in the (UV-Vis) exhibited a better hydrogel formulation. The nanoparticle absorption spectra of this analysis showed a maximum band around 430 - 450 nm, which suggests a spheroidal form. The TG curve exhibited two weight loss events. DSC indicated one endothermic peak at 230-250 °C, due to sample fusion. The polymers acted as stabilizers of a nanoparticle, defining their size and shape. Human fibroblast viability assay L929 gave 105 % cell viability with a negative control, while gelatin presented 96% viability, alginate: gelatin (80:20) 96.66 %, and alginate 100.33 % viability. The sodium alginate:gelatin (80:20) exhibited significant antimicrobial activity, with minimal bacterial growth at a ratio of 1.06 mg.mL⁻¹ in Pseudomonas aeruginosa and 0.53 mg.mL⁻¹ in Staphylococcus aureus. The in vivo results showed a significant reduction in wound surface area. On the seventh day, the hydrogel-nanoparticle formulation reduced the total area of injury by 81.14 %, while control reached a 45.66 % reduction. The results suggest that silver-hydrogel nanoformulation exhibits potential for wound dressing therapeutics.

Keywords: nanocomposite, wound healing, hydrogel, silver nanoparticle

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Authors: Lee A. Browne, K. Rumbold

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The first fluorinating enzyme, 5'-fluoro-5'-deoxyadenosine synthase (fluorinase) was isolated from the soil bacterium Streptomyces cattleya. Such an enzyme, with the ability to catalyze a C-F bond, presents great potential as a biocatalyst. Naturally fluorinated compounds are extremely rare in nature. As a result, the number of fluorinases identified remains relatively few. The field of fluorination is almost completely synthetic. However, with the increasing demand for fluorinated organic compounds of commercial value in the agrochemical, pharmaceutical and materials industries, it has become necessary to utilize biologically based methods such as biocatalysts. A key step in this crucial process is the large-scale production of the fluorinase enzyme in considerable quantities for industrial applications. Thus, this study aimed to optimize expression of the fluorinase enzyme in both prokaryotic and eukaryotic expression systems in order to obtain high protein yields. The fluorinase gene was cloned into the pET 41b(+) and pPinkα-HC vectors and used to transform the expression hosts, E.coli BL21(DE3) and Pichia pastoris (PichiaPink™ strains) respectively. Expression trials were conducted to select optimal conditions for expression in both expression systems. Fluorinase catalyses a reaction between S-adenosyl-L-Methionine (SAM) and fluoride ion to produce 5'-fluorodeoxyadenosine (5'FDA) and L-Methionine. The activity of the enzyme was determined using HPLC by measuring the product of the reaction 5'FDA. A gradient mobile phase of 95:5 v/v 50mM potassium phosphate buffer to a final mobile phase containing 80:20 v/v 50mM potassium phosphate buffer and acetonitrile were used. This resulted in the complete separation of SAM and 5’-FDA which eluted at 1.3 minutes and 3.4 minutes respectively. This proved that the fluorinase enzyme was active. Optimising expression of the fluorinase enzyme was successful in both E.coli and PichiaPink™ where high expression levels in both expression systems were achieved. Protein production will be scaled up in PichiaPink™ using fermentation to achieve large-scale protein production. High level expression of protein is essential in biocatalysis for the availability of enzymes for industrial applications.

Keywords: biocatalyst, expression, fluorinase, PichiaPink™

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Authors: Reihane Mehrparvar

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Human age could exceed further by altering gene expression through food intaking, although as a consequence of recent century eating patterns, human life-span getting shorter by emerging irregulating in autophagy mechanism, insulin, leptin, gut microbiota which are important etiological factors of type-2 diabetes, obesity, infertility, cancer, metabolic and autoimmune diseases. However, restricted calorie intake and vigorous exercise might be beneficial for losing weight and metabolic regulation in a short period but could not be implementable in the long term as a way of life. Therefore, the lack of a dietary program that is compatible with the genes of the body is essential. Sweet and high-glycemic-index (HGI) foods were associated with type-2 diabetes and cancer morbidity. The neuropsychological perspective characterizes the inclination of sweet and HGI-food consumption as addictive behavior; hence this process engages preference of gut microbiota, neural node, and dopaminergic functions. Moreover, meal composition is not the only factor that affects body hemostasis. In this narrative review, it is believed to attempt to investigate how the body responded to different food intakes and represent an accurate model based on current evidence. Eating frequently and ingesting unassimilable protein and carbohydrates may not be compatible with human genes and could cause impairments in the self-renovation mechanism. This trajectory indicates our body is more adapted to starvation and eating animal meat and marrow. Here has been recommended a model that takes into account three important factors: frequent eating, meal composition, and circadian rhythm, which may offer a promising intervention for obesity, inflammation, cardiovascular, autoimmune disorder, type-2 diabetes, insulin resistance, infertility, and cancer through intensifying autophagy-mechanism and eliminate medical costs.

Keywords: metabolic disease, anti-aging, type-2 diabetes, autophagy

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Authors: Cristian Soitu, Alexander Feuerborn, Cyril Deroy, Alfonso Castrejon-Pita, Peter R. Cook, Edmond J. Walsh

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Microfluidics now stands as an academically mature technology after a quarter of a century research activities have delivered a vast array of proof of concepts for many biological workflows. However, translation to industry remains poor, with only a handful of notable exceptions – e.g. digital PCR, DNA sequencing – mainly because of biocompatibility issues, limited range of readouts supported or complex operation required. This technology exploits the domination of interfacial forces over gravitational ones at the microscale, replacing solid walls with fluid ones as building blocks for cell micro-environments. By employing only materials used by biologists for decades, the system is shown to be biocompatible, and easy to manufacture and operate. The method consists in displacing a continuous fluid layer into a pattern of isolated chambers overlaid with an immiscible liquid to prevent evaporation. The resulting fluid arrangements can be arrays of micro-chambers with rectangular footprint, which use the maximum surface area available, or structures with irregular patterns. Pliant, self-healing fluid walls confine volumes as small as 1 nl. Such fluidic structures can be reconfigured during the assays, giving the platform an unprecedented level of flexibility. Common workflows in cell biology are demonstrated – e.g. cell growth and retrieval, cloning, cryopreservation, fixation and immunolabeling, CRISPR-Cas9 gene editing, and proof-of-concept drug tests. This fluid-shaping technology is shown to have potential for high-throughput cell- and organism-based assays. The ability to make and reconfigure on-demand microfluidic circuits on standard Petri dishes should find many applications in biology, and yield more relevant phenotypic and genotypic responses when compared to standard microfluidic assays.

Keywords: fluid walls, micro-chambers, reconfigurable, freestyle

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Authors: S. A. El-Marasy, S. A. El Awdan, R. M. Abd-Elsalam

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This study aimed to investigate the possible neuroprotective effect of chrysin on thioacetamide (TAA)-induced hepatic encephalopathy in rats. Also, the effect of chrysin on motor impairment, cognitive deficits, oxidative stress, neuroinflammation, apoptosis and histopathological damage was assessed. Male Wistar rats were randomly allocated into five groups. The first group received the vehicle (distilled water) for 21 days and is considered as normal group. While the second one received intraperitoneal dose of TAA (200 mg/kg) at three alternative days during the third week of the experiment to induce HE and is considered as control group. The other three groups were orally administered chrysin for 21 days (25, 50, 100 mg/kg) and starting from day 17; rats received intraperitoneal dose of TAA (200 mg/kg) at three alternative days. Then behavioral, biochemical, histopathological and immunohistochemical analyses were assessed. Then behavioral, biochemical, histopathological and immunohistochemical analyses were assessed. Chrysin reversed TAA-induced motor coordination in rotarod test, cognitive deficits in object recognition test (ORT) and attenuated serum ammonia, hepatic liver enzymes, reduced malondialdehyde (MDA), elevated reduced glutathione (GSH), reduced nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) brain contents. Chrysin administration also reduced Toll-4 receptor (TLR-4) gene expression, caspase-3 protein expression, hepatic necrosis and astrocyte swelling. This study depicts that chrysin exerted neuroprotective effect in TAA-induced HE rats, evidenced by improvement of cognitive deficits, motor incoordination and histopathological changes such as astrocyte swelling and vacuolization; hallmarks in HE, via reducing hyperammonemia, ameliorating hepatic function, in addition to its anti-oxidant, inactivation of TLR-4/NF-κB inflammatory pathway, and anti-apoptotic effects.

Keywords: chrysin, hepatic encephalopathy, oxidative stress, rats, thioacetamide, TLR4/NF-κB pathway

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Authors: Mia Bahar, Özlem Bozkurt

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Obsessive-compulsive disorder (OCD) is a typical, persistent, and long-lasting mental health condition in which a person experiences uncontrollable, recurrent thoughts (or "obsessions") and/or activities (or "compulsions") that they feel compelled to engage in repeatedly. Obsessive-compulsive disorder is both underdiagnosed and undertreated. It frequently manifests in a variety of medical settings and is persistent, expensive, and burdensome. Obsessive-compulsive neurosis was long believed to be a condition that offered valuable insight into the inner workings of the unconscious mind. Obsessive-compulsive disorder is now recognized as a prime example of a neuropsychiatric condition susceptible to particular pharmacotherapeutic and psychotherapy therapies and mediated by pathology in particular neural circuits. An obsessive-compulsive disorder which is called OCD, usually has two components, one cognitive and the other behavioral, although either can occur alone. Obsessions are often repetitive and intrusive thoughts that invade consciousness. These obsessions are incredibly hard to control or dismiss. People who have OCD often engage in rituals to reduce anxiety associated with intrusive thoughts. Once the ritual is formed, the person may feel extreme relief and be free from anxiety until the thoughts of contamination intrude once again. These thoughts are strengthened through a manifestation of negative reinforcement because they allow the person to avoid anxiety and obscurity. These thoughts are described as autogenous, meaning they most likely come from nowhere. These unwelcome thoughts are related to actions which we can describe as Thought Action Fusion. The thought becomes equated with an action, such as if they refuse to perform the ritual, something bad might happen, and so people perform the ritual to escape the intrusive thought. In almost all cases of OCD, the person's life gets extremely disturbed by compulsions and obsessions. Studies show OCD is an estimated 1.1% prevalence, making it a challenging issue with high co-morbidities with other issues like depressive episodes, panic disorders, and specific phobias. The first to reveal brain anomalies in OCD were numerous CT investigations, although the results were inconsistent. A few studies have focused on the orbitofrontal cortex (OFC), anterior cingulate gyrus (AC), and thalamus, structures also implicated in the pathophysiology of OCD by functional neuroimaging studies, but few have found consistent results. However, some studies have found abnormalities in the basal ganglion. There have also been some discussions that OCD might be genetic. OCD has been linked to families in studies of family aggregation, and findings from twin studies show that this relationship is somewhat influenced by genetic variables. Some Research has shown that OCD is a heritable, polygenic condition that can result from de novo harmful mutations as well as common and unusual variants. Numerous studies have also presented solid evidence in favor of a significant additive genetic component to OCD risk, with distinct OCD symptom dimensions showing both common and individual genetic risks.

Keywords: compulsions, obsessions, neuropsychiatric, genetic

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Authors: Debabrata Biswas

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The current trend in the development of antibiotic resistance by multiple bacterial pathogens has resulted in a troubling loss of effective antibiotic options for human. The emergence of multi-drug-resistant pathogens has necessitated higher dosages and combinations of multiple antibiotics, further exacerbating the problem of antibiotic resistance. Zoonotic bacterial pathogens, such as Salmonella, Campylobacter, Shiga toxin-producing Escherichia coli (such as enterohaemorrhagic E. coli or EHEC), and Listeria are the most common and predominant foodborne enteric infectious agents. It was observed that these pathogens gained/developed their ability to survive in the presence of antibiotics either in farm animal gut or farm environment and researchers believe that therapeutic and sub-therapeutic antibiotic use in farm animal production might play an important role in it. The mechanism of action of antimicrobial components used in farm animal production in genomic interplay in the gut and farm environment, has not been fully characterized. Even the risk of promoting the exchange of mobile genetic elements between microbes specifically pathogens needs to be evaluated in depth, to ensure sustainable farm animal production, safety of our food and to mitigate/limit the enteric infection with multiple antibiotic resistant bacterial pathogens. Due to the consumer’s demand and considering the current emerging situation, many countries are in process to withdraw antibiotic use in farm animal production. Before withdrawing use of the sub-therapeutic antibiotic or restricting the use of therapeutic antibiotics in farm animal production, it is essential to find alternative natural antimicrobials for promoting the growth of farm animal and/or treating animal diseases. Further, it is also necessary to consider whether that compound(s) has the potential to trigger the acquisition or loss of genetic materials in zoonotic and any other bacterial pathogens. Development of alternative therapeutic and sub-therapeutic antimicrobials for farm animal production and food processing and preservation and their effective implementation for sustainable strategies for farm animal production as well as the possible risk for horizontal gene transfer in major enteric pathogens will be focus in the study.

Keywords: food safety, natural antimicrobial, sustainable farming, antibiotic resistance

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Authors: Pei Chi Fang, Wei Chen Lin

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Trichomonas tenax is a microaerophilic oral protozoan found in patients with poor oral hygiene. It participates in the inflammatory process of periodontal disease and can potentially be aspirated into the lungs, giving rise to pulmonary trichomoniasis. However, the precise roles of T. tenax in the pulmonary system remain largely unexplored and warrant comprehensive epidemiological investigation. To assess the prevalence of T. tenax infection, we collected bronchoalveolar lavage fluid (BALF) samples from hospitalized patients with lung diseases. A specific nested PCR approach was employed to determine prevalence rates, yielding 21 positive cases out of 61 samples from Ditmanson Medical Foundation Chia-Yi Christian Hospital, and 11 positive cases out of 55 samples from National Cheng Kung University Hospital. Furthermore, there is a critical need for comprehensive data regarding the presence of T. tenax in environmental surface watersheds. In this context, we present findings from investigations in the Yanshuei and Donggang river basins in southern Taiwan, which are crucial sources for public drinking water in the region. In order to elucidate potential implications on pulmonary virus infections, we conducted an analysis of gene expression level changes in H292 cell line after exposure to T. tenax. Our findings revealed significant regulation of multiple virus-related genes, including IFI44L and IFITM3. Ongoing research endeavors are focused on identifying the key components within T. tenax responsible for these observed effects. Crucially, this study lays the groundwork for a preliminary understanding of T. tenax prevalence in patients with pulmonary diseases. It also seeks to establish a meaningful correlation between lung infections and oral hygiene practices, with the ultimate aim of informing distinct treatment and prevention strategies.

Keywords: parasitology, genes, virus, human health, infection, lung

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Authors: Petra Borilova Linhartova, Michaela Ruckova, Sabina Sevcikova, Natalie Mlcuchova, Jan Bohm, Katerina Zukalova, Monika Vlachova, Jiri Dolina, Lumir Kunovsky, Radek Kroupa, Zdenek Pavlovsky, Zdenek Danek, Tereza Deissova, Lydie Izakovicova Holla, Ondrej Slaby, Zdenek Kala

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Esophageal adenocarcinoma (EAC) is a highly aggressive malignancy that frequently develops from Barrett's esophagus (BE), a premalignant pathologic change occurring in the lower end of the esophagus. Specific microRNAs (miRNAs), small non-coding RNAs that function as posttranscriptional regulators of gene expression, were repeatedly proved to play key roles in the pathogenesis of these diseases. This pilot study aimed to analyze four selected miRNAs in esophageal tissues from healthy controls (HC) and patients with reflux esophagitis (RE)/BE/EAC, as well as to compare expression at the site of Barrett's mucosa/adenocarcinoma and healthy esophageal tissue outside the area of the main pathology in patients with BE/EAC. In this pilot study, 22 individuals (3 HC, 8 RE, 5 BE, 6 EAC) were included and endoscopically examined. RNA was isolated from the fresh-frozen esophageal tissue (stored in the RNAlater™ Stabilization Solution −70°C) using the AllPrep DNA/RNA/miRNA Universal Kit. Subsequent RT-qPCR analysis was performed using selected TaqMan MicroRNA Assays for miR-21, miR-34a, miR-196a, miR-196b, and endogenous control (RNU44). While the expression of miR-21 in the esophageal tissue with the main pathology was decreased in BE and EAC patients in comparison to the group of HC and RE patients (p=0.01), the expression of miR-196a was increased in the BE and EAC patients (p<0.01). Correlations between those miRNAs expression in tissue and severity of diagnosis were observed (p<0.05). In addition, miR-196a was significantly more expressed at the site with the main pathology than in paired adjacent esophageal tissue in BE and EAC patients (p<0.01). In conclusion, our pilot results showed that miR-196a, which regulates the proliferation, invasion, and migration (and was previously associated with esophageal squamous cell carcinoma and marked as a potential therapeutic target), could be a diagnostic tissue biomarker for BE and EAC as well.

Keywords: microRNA, barrett´s esophagus, esophageal adenocarcinoma, biomarker

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Authors: Tanakrit Doltanakarn

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Introduction: These days, cannabis is being accepted in many countries due to the fact that cannabis could be use in medical. The medical cannabis is used to treat and reduce the pain many diseases. For example, neuropathic pain, Parkinson, autism disorders, cancer pain reduce the adverse effect of chemotherapy, diabetes, and migraine. Active ingredients in cannabis that modulate patients' perceptions of their conditions include Δ9‐tetrahydrocannabinol (THC), cannabidiol (CBD), flavonoids, and terpenes. However, there is an adverse effect of cannabis, cardiovascular effects, psychosis, schizophrenia, mood disorder, and cognitive alternation. These effects are from the THC and CBD ingredients in the cannabis. The metabolize processes of delta-9 THC to 11-OH-delta 9 -THC (inactive form), THC were cause of adverse effects. Interestingly, the distributions of CYP2C9 gene (CYP2C9*2 and CYP2C9*3, poor metabolizer) that might affect incidences of adverse effects in patients who treated with medical cannabis. Objective: The aim of this study we want to investigate the association between genetic polymorphism of CYP2C9 frequency and Thai patients who treated with medical cannabis. Materials and Methods:We recruited sixty-five unrelated Thai patients from the College of Pharmacy, Rangsit University. DNA were extracted using Genomic DNA Mini Kit. Genotyping of CYP2C9*2 (430C>T, rs1799853) and CYP2C9*3 (1075A>C, rs1057910) were genotyped by the TaqMan Real-time PCR assay. Results: Among these 31 medicals cannabis-induced ADRs patients, they were diagnosed with 22 (33.85%) tachycardia and 3 (4.62%) arrhythmia. There were 34 (52.31%) medical cannabis-tolerant controls who were included in this study.40 (61.53%) Thai patients were female, and 25 (38.46%) were male, with median age of 57 (range 27 – 87) years. In this study, we found none of the medical cannabis-induced ADRs carried CYP2C9*2 variant along with medical cannabis-tolerant control group. CYP2C9*3 variant (intermediate metabolizer, IM) was found just only one of thirty-one (3.23%) in the medical cannabis-induced ADRs and two of thirty-fourth (5.88%) in the tolerant controls. Conclusions: Thus, the distribution of CYP2C9 alleles offer a comprehensive view of pharmacogenomics marker in Thai population that could be used as a reference for worldwide to investigate the pharmacogenomics application.

Keywords: medical cannabis, adverse effect, CYP2C9, thai patients

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Authors: Shawn P. Daly

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Communities heavily engaged in the tourism industry discover their values intersect, meld, and conflict with those of visitors. Maintaining cultural integrity in the face of powerful external pressures causes stress among society members. This effect represents a less studied aspect of sustainable tourism. The present paper brings a perspective unique to the tourism literature: biculturalism. The grounded theories, coherent hypotheses, and validated constructs and indicators of biculturalism represent a sound base from which to consider sociocultural issues in sustainable tourism. Five models describe the psychological state of individuals operating at cultural crossroads: assimilation (joining the new culture), acculturation (grasping the new culture but remaining of the original culture), alternation (varying behavior to cultural context), multicultural (maintaining distinct cultures), and fusion (blending cultures). These five processes divide into two units of analysis (individual and society), permitting research questions at levels important for considering sociocultural sustainability. Acculturation modelling has morphed into dual processes of acculturation (new culture adaptation) and enculturation (original culture adaptation). This dichotomy divides sustainability research questions into human impacts from assimilation (acquiring new culture, throwing away original), separation (rejecting new culture, keeping original), integration (acquiring new culture, keeping original), and marginalization (rejecting new culture, throwing away original). Biculturalism is often cast in terms of its emotional, behavioral, and cognitive dimensions. Required cultural adjustments and varying levels of cultural competence lead to physical, psychological, and emotional outcomes, including depression, lowered life satisfaction and self-esteem, headaches, and back pain—or enhanced career success, social skills, and life styles. Numerous studies provide empirical scales and research hypotheses for sustainability research into tourism’s causality and effect on local well-being. One key issue in applying biculturalism to sustainability scholarship concerns identification and specification of the alternative new culture contacting local culture. Evidence exists for tourism industry, universal tourist, and location/event-specific tourist culture. The biculturalism paradigm holds promise for researchers examining evolving cultural identity and integrity in response to mass tourism. In particular, confirmed constructs and scales simplify operationalization of tourism sustainability studies in terms of human impact and adjustment.

Keywords: biculturalism, cultural integrity, psychological and sociocultural adjustment, tourist culture

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Authors: Leila Rashki Ghaleno, Mohamad Amin Hajari, Leila Montazeri, Abdolhossein Shahverdi, Mojtaba Rezazadeh Valojerdi

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Background: Spermatogonia stem cells (SSCs) exhibit pluripotency, enabling them to undergo differentiation into many cell lineages, including neurons, glia, endothelial cells, and hepatocytes when cultured in vitro. Although the specific mechanisms are not yet fully understood, it has been observed that biopolymer agents, such as hyaluronic acid (HA) and alginate (Alg), have the potential to induce transdifferentiation of SSCs. The current work aimed to examine the process of in vitro spermatogenesis and the conversion of mouse testicular cells into hepatocytes and erythrocyte-like cells utilizing the HA-Alg hydrogel. Method: After being extracted from the testes of a 5-day postpartum mouse (5 DPP), the testicular cells were separated into two enzymatic stages and then put into a composite hydrogel containing 0.5% HA and 1% alginate. On days 14 and 28 of culture, the colonies' growth, the cells' viability, and their histology were assessed. Result: Despite observing significant cell proliferation on day 14 and the development of circular-shaped organoids on day 28, it was noted that the organoids generated in the HA-Alg medium tended to maintain their circular morphology on day 28. Notably, the testicular cells underwent transdifferentiation into cell types resembling erythrocytes and hepatocytes. The hepatocyte-like cells exhibited the presence of glycogen and lipid deposits, indicating their hepatocyte-like characteristics. Interestingly, immunostaining analysis revealed the secretion of albumin and the presence of VEGFR on day 14. However, on day 28, albumin expression was not detected, while the expression of Sox9 (a marker for hepatocytes), Vegf, CD34, and C-kit (markers for erythrocytes) showed increased levels in the gene expression evaluation. Conclusion: The present findings indicated that HA-Alg could be a potent and effective agent for the transdifferentiation of testis cells into erythrocyte and hepatocyte-like cells, as recent studies have confirmed the transformation of SSCs into hepatocyte cells during in vitro culture.

Keywords: 3D culture, mouse testicular cell, hyaluronic acid, liver organoids

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Authors: Marzieh Khedmati, Shannon L. Bartelt-Hunt

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Vegetative Filter Strips (VFS) are used for controlling the volume of runoff and decreasing contaminant concentrations in runoff before entering water bodies. Many studies have investigated the role of VFS in sediment and nutrient removal, but little is known about their efficiency for the removal of emerging contaminants such as antimicrobial resistance genes (ARGs). Vegetative Filter Strip Modeling System (VFSMOD) was used to simulate the efficiency of VFS in this regard. Several studies demonstrated the ability of VFSMOD to predict reductions in runoff volume and sediment concentration moving through the filters. The objectives of this study were to calibrate the VFSMOD with experimental data and assess the efficiency of the model in simulating the filter behavior in removing ARGs (ermB) and tylosin. The experimental data were obtained from a prior study conducted at the University of Nebraska (UNL) Rogers Memorial Farm. Three treatment factors were tested in the experiments, including manure amendment, narrow grass hedges and rainfall events. Sediment Delivery Ratio (SDR) was defined as the filter efficiency and the related experimental and model values were compared to each other. The VFS Model generally agreed with the experimental results and as a result, the model was used for predicting filter efficiencies when the runoff data are not available. Narrow Grass Hedges (NGH) were shown to be effective in reducing tylosin and ARGs concentration. The simulation showed that the filter efficiency in removing ARGs is different for different soil types and filter lengths. There is an optimum length for the filter strip that produces minimum runoff volume. Based on the model results increasing the length of the filter by 1-meter leads to higher efficiency but widening beyond that decreases the efficiency. The VFSMOD, which was proved to work well in estimation of VFS trapping efficiency, showed confirming results for ARG removal.

Keywords: antimicrobial resistance genes, emerging contaminants, narrow grass hedges, vegetative filter strips, vegetative filter strip modeling system

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Authors: Kirsten Wilson, Patrick M. Brauer, Sandra Babic, Diana Golubeva, Jessica Van Eyk, Tinya Wang, Avanti Karkhanis, Tim A. Le Fevre, Andy I. Kokaji, Allen C. Eaves, Sharon A. Louis, , Nooshin Tabatabaei-Zavareh

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Long-lived plasma cells are rare, non-proliferative B cells generated from antibody-secreting cells (ASCs) following an immune response to protect the host against pathogen re-exposure. Despite their therapeutic potential, the lack of in vitro protocols in the field makes it challenging to use B cells as a cellular therapeutic tool. As a result, there is a need to establish robust and reproducible methods for the generation of B cells. To address this, we have developed a culture system for generating B cells from hematopoietic stem and/or progenitor cells (HSPCs) derived from human umbilical cord blood (CB) or pluripotent stem cells (PSCs). HSPCs isolated from CB were cultured using the StemSpan™ B Cell Generation Kit and produced CD19+ B cells at a frequency of 23.2 ± 1.5% and 59.6 ± 2.3%, with a yield of 91 ± 11 and 196 ± 37 CD19+ cells per input CD34+ cell on culture days 28 and 35, respectively (n = 50 - 59). CD19+IgM+ cells were detected at a frequency of 31.2 ± 2.6% and were produced at a yield of 113 ± 26 cells per input CD34+ cell on culture day 35 (n = 50 - 59). The B cell receptor loci of CB-derived B cells were sequenced to confirm V(D)J gene rearrangement. ELISpot analysis revealed that ASCs were generated at a frequency of 570 ± 57 per 10,000 day 35 cells, with an average IgM+ ASC yield of 16 ± 2 cells per input CD34+ cell (n = 33 - 42). PSC-derived HSPCs were generated using the STEMdiff™ Hematopoietic - EB reagents and differentiated to CD10+CD19+ B cells with a frequency of 4 ± 0.8% after 28 days of culture (n = 37, 1 embryonic and 3 induced pluripotent stem cell lines tested). Subsequent culture of PSC-derived HSPCs increased CD19+ frequency and generated ASCs from 1 - 2 iPSC lines. This method is the first report of a serum- and feeder-free system for the generation of B cells from CB and PSCs, enabling further B lineage-specific research for potential future clinical applications.

Keywords: stem cells, B cells, immunology, hematopoiesis, PSC, differentiation

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Authors: Mona Alharbi

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Melioidosis has emerged as a lethal disease. Unfortunately, the molecular mechanisms of virulence and pathogenicity of Burkholderia pseudomallei remain unknown. However, proteomics research has selected putative targets in B. pseudomallei that might play roles in the B. pseudomallei virulence. BPSL 2418 putative protein has been predicted as a free methionine sulfoxide reductase and interestingly there is a link between the level of the methionine sulfoxide in pathogen tissues and its virulence. Therefore in this work, we describe the cloning expression, purification, and crystallization of BPSL 2418 and the solution of its 3D structure using X-ray crystallography. Also, we aimed to identify the substrate binding and reduced forms of the enzyme to understand the role of BPSL 2418. The gene encoding BPSL2418 from B. pseudomallei was amplified by PCR and reclone in pETBlue-1 vector and transformed into E. coli Tuner DE3 pLacI. BPSL2418 was overexpressed using E. coli Tuner DE3 pLacI and induced by 300μM IPTG for 4h at 37°C. Then BPS2418 purified to better than 95% purity. The pure BPSL2418 was crystallized with PEG 4000 and PEG 6000 as precipitants in several conditions. Diffraction data were collected to 1.2Å resolution. The crystals belonged to space group P2 21 21 with unit-cell parameters a = 42.24Å, b = 53.48Å, c = 60.54Å, α=γ=β= 90Å. The BPSL2418 binding MES was solved by molecular replacement with the known structure 3ksf using PHASER program. The structure is composed of six antiparallel β-strands and four α-helices and two loops. BPSL2418 shows high homology with the GAF domain fRMsrs enzymes which suggest that BPSL2418 might act as methionine sulfoxide reductase. The amino acids alignment between the fRmsrs including BPSL 2418 shows that the three cysteines that thought to catalyze the reduction are fully conserved. BPSL 2418 contains the three conserved cysteines (Cys⁷⁵, Cys⁸⁵ and Cys¹⁰⁹). The active site contains the six antiparallel β-strands and two loops where the disulfide bond formed between Cys⁷⁵ and Cys¹⁰⁹. X-ray structure of free methionine sulfoxide binding and native forms of BPSL2418 were solved to increase the understanding of the BPSL2418 catalytic mechanism.

Keywords: X-Ray Crystallography, BPSL2418, Burkholderia pseudomallei, Melioidosis

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Authors: Marinna Madrid, Nabiha Saklayen, Marinus Huber, Nicolas Vogel, Christos Boutopoulos, Michel Meunier, Eric Mazur

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Delivering material into cells is important for a diverse range of biological applications, including gene therapy, cellular engineering and imaging. We present a plasmonic substrate for delivering membrane-impermeable material into cells at high throughput and high efficiency while maintaining cell viability. The substrate fabrication is based on an affordable and fast colloidal self-assembly process. When illuminated with a femtosecond laser, the light interacts with the electrons at the surface of the metal substrate, creating localized surface plasmons that form bubbles via energy dissipation in the surrounding medium. These bubbles come into close contact with the cell membrane to form transient pores and enable entry of membrane-impermeable material via diffusion. We use fluorescence microscopy and flow cytometry to verify delivery of membrane-impermeable material into HeLa CCL-2 cells. We show delivery efficiency and cell viability data for a range of membrane-impermeable cargo, including dyes and biologically relevant material such as siRNA. We estimate the effective pore size by determining delivery efficiency for hard fluorescent spheres with diameters ranging from 20 nm to 2 um. To provide insight to the cell poration mechanism, we relate the poration data to pump-probe measurements of micro- and nano-bubble formation on the plasmonic substrate. Finally, we investigate substrate stability and reusability by using scanning electron microscopy (SEM) to inspect for damage on the substrate after laser treatment. SEM images show no visible damage. Our findings indicate that self-assembled plasmonic substrates are an affordable tool for high-throughput, high-efficiency delivery of material into mammalian cells.

Keywords: femtosecond laser, intracellular delivery, plasmonic, self-assembly

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Authors: Denis Mustafov, Emmanouil Karteris, Maria Braoudaki

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Glioblastoma multiform (GBM) is a heterogenous primary brain tumour that kills most affected patients. To the authors best knowledge, despite all research efforts there is no early diagnostic biomarker for GBM. MicroRNAs (miRNAs) are short non-coding RNA molecules which are deregulated in many cancers. The aim of this research was to determine miRNAs with a diagnostic impact and to potentially identify promising therapeutic targets for glioblastoma multiform. In silico analysis was performed to identify deregulated miRNAs with diagnostic relevance for glioblastoma. The expression profiles of the chosen miRNAs were then validated in vitro in the human glioblastoma cell lines A172 and U-87MG. Briefly, RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed using the mirVANA miRNA isolation kit. Quantitative Real-Time Polymerase Chain Reaction was performed to verify their expression. The presence of five target proteins within the A172 cell line was evaluated by Western blotting. The expression of the CORO1C protein within 32 GBM cases was examined via immunohistochemistry. The miRNAs identified in silico included miR-21-5p, miR-34a and miR-128a. These miRNAs were shown to target deregulated GBM genes, such as CDK6, E2F3, BMI1, JAG1, and CORO1C. miR-34a and miR-128a showed low expression profiles in comparison to a control miR-RNU-44 in both GBM cell lines suggesting tumour suppressor properties. Opposing, miR-21-5p demonstrated greater expression indicating that it could potentially function as an oncomiR. Western blotting revealed expression of all five proteins within the A172 cell line. In silico analysis also suggested that CORO1C is a target of miR-128a and miR-34a. Immunohistochemistry demonstrated that 75% of the GBM cases showed moderate to high expression of CORO1C protein. Greater understanding of the deregulated expression of miR-128a and the upregulation of CORO1C in GBM could potentially lead to the identification of a promising diagnostic biomarker signature for glioblastomas.

Keywords: non-coding RNAs, gene expression, brain tumours, immunohistochemistry

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Authors: Konstantin Yu. Moiseev, Antonina F. Budnik, Andrey I. Emanuilov, Petr M. Masliukov

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The vast majority of sympathetic ganglionic neurons are catecholaminergic. Some sympathetic neurons lack catecholamines and mostly use acetylcholine as their main neurotransmitter. Some cholinergic postganglionic neurons also express neuronal nitric oxide synthase (nNOS). Preganglionic sympathetic neurons are cholinergic and most of them are also nNOS-immunoreactive (IR). The purpose of this study was to gain further insight into the neuroplasticity of sympathetic neurons during postnatal ontogenesis by comparing the development of pre- and postganglionic neurons expressing nNOS in different mammals. nNOS was investigated by immunohistochemistry in the sympathetic superior cervical ganglion (SCG), stellate ganglion (SG), celiac ganglion (CG) and spinal cord from rats, mice and cats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 2-month-old and 2-year-old). In rats and mice, nNOS-positive neurons were not found in sympathetic ganglia from birth onwards. In cats, non-catecholaminergic nNOS-IR sympathetic ganglionic neurons are present from the moment of birth. In all studied age groups, substantial populations of nNOS-IR cells (up to 8.3%) was found in the SG, with a much smaller population found in the SCG (<1%) and only few cells observed in the CG. The percentage of nNOS-IR neurons in the CG and SCG did not significantly change during development. The proportion of nNOS-IR neuron profiles in the SG increased in first 20 days of life from 2.3±0.15% to 8.3±0.56%. In the SG, percentages of nNOS-IR sympathetic neurons colocalizing vasoactive intestinal peptide increased in the first 20 days of life. Choline acetyltransferase (ChAT)-IR and calcitonin gene-related peptide-IR neurons were not observed in the sympathetic ganglia of newborn animals and did not appear until 10 days after birth. In the SG of newborn and 10-day-old kittens, the majority of NOS-IR neurons were calbindin (CB)-IR, whereas in the SCG and CG of cats of all age groups and in the SG of 30-day-old and older kittens, the vast majority of NOS-IR neurons lacked CB. In newborn mammals, the most of sympathetic preganglionic neurons in the nucleus intermediolateralis thoracolumbalis pars principalis (nucl.ILp) were nNOS-IR. The percentage of nNOS-IR neurons decreased and the same parameter of ChAT-IR neurons increased during the development. We conclude that the development of nNOS-IR preganglionic and ganglionic sympathetic neurons in different mammals has time and species differences.

Keywords: sympathetic neuron, nitric oxide synthase, immunohistochemistry, development

Procedia PDF Downloads 194

Authors: Rita Alfattal, Maryam Alfarhan, Adeeb M. Algaith, Buthaina Albash, Reem M. Elshafie, Asma Alshammari, Ahmad Alahmad, Fatima Dashti, Rasha Alsafi, Hind Alsharhan

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Defects of respiratory chain complex III (CIII) result in characteristic but rare mitochondrial disorders associated with distinct neuroradiological findings. The underlying molecular defects affecting mitochondrial CIII assembly factors are few and yet to be identified. LYRM7 assembly factor is required for proper CIII assembly where it acts as a chaperone for the Rieske iron‐sulfur (UQCRFS1) protein in the mitochondrial matrix and stabilizing it. We present here the seventeenth individual with LYRM7-associated mitochondrial leukoencephalopathy harboring a previously reported rare pathogenic homozygous LYRM 7 variant, c.2T>C, (p.Met1?). Like previously reported individuals, our 4-year-old male proband presented with recurrent metabolic and lactic acidosis, encephalopathy, and myopathy. Further, he has additional, previously unreported features, including an acute stroke like episode with bilateral central blindness and optic neuropathy, recurrent hyperglycemia and hypertension associated with metabolic crisis. However, he has no signs of psychomotor regression. He has been stable clinically with residual left-sided reduced visual acuity and amblyopia, and no more metabolic crises for 2-year-period while on the mitochondrial cocktail. Although the reported brain MRI findings in other affected individuals are homogenous, it is slightly different in our index, revealing evidence of bilateral almost symmetric multifocal periventricular T2 hyperintensities with hyperintensities of the optic nerves, optic chiasm, and corona radiata but with no cavitation or cystic changes. This report describes new clinical and radiological findings of LYRM7-associated disease. The report also summarizes the clinical and molecular data of previously reported individuals describing the full phenotypic spectrum.

Keywords: LYRM7 gene defect, mitochondrial disease, , lactic acidosis, , genetic disorder

Procedia PDF Downloads 49

Authors: C. Miranda, R. Soares, S. Cunha, L. Ferreira, G. Igrejas, P. Poeta

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Different animal species, including ornamental animals, are reported as potential reservoirs of antibiotic resistance genes. Consequently, these resistances can be disseminated in the environment and transferred to humans. Moreover, multidrug-resistant bacteria reduce the efficacy of antibiotics, as the case of vancomycin-resistant enterococci. Enterococcus faecalis and E. faecium are described as the main nosocomial pathogens. In this line, the aim of this study was to characterize resistance and virulence genes of enterococci species isolated from samples of food supplied to ornamental animals during 2020. The 29 enterococci isolates (10 E. faecalis and 19 E. faecium) were tested for the presence of the resistance genes for the following antibiotics: erythromicyn (ermA, ermB and ermC), tetracycline (tetL, tetM, tetK and tetO), quinupristin/dalfopristin (vatD and vatE), gentamicin (aac(6’)-aph(2’’)-Ia), chloramphenicol (catA), streptomycin (ant(6)-Ia) and vancomycin (vanA and vanB). The same isolates were also tested for 10 virulence factors genes (esp, ace, gelE, agg, fsr, cpd, cylA, cylB, cylM and cylLL). The resistance and virulence genes were performed by PCR, using specific primers and conditions. Negative and positive controls were used in all PCR assays. The most prevalent resistance genes detected in both enterococci species were ermB (n=15, 52%), ermC (n=7, 24%), tetK (n=8, 28%) and vatE (n=4, 14%). Resistance genes for vancomycin were found in ten (34%) E. faecalis and ten (34%) E. faecium isolates. Only E. faecium isolates showed the presence of ermA (n=2, 7%), tetL (n=13, 45%) and ant(6)-Ia gene (n=4, 14%). A total of nine (31%) enterococci isolates were classified as multidrug-resistant bacteria (3 E. faecalis and 6 E. faecium). In three E. faecalis and one E. faecium were not detected resistance genes. The virulence genes detected in both species were agg (n=6, 21%) and cylLL (n=11, 38%). In general, each isolate showed only one of these virulence genes. Five E. faecalis and eleven E. faecium isolates were negative for all analyzed virulence genes. These preliminary results showed the presence of multidrug-resistant enterococci in food supplied to ornamental animals, in particular vancomycin-resistant enterococci. This genotypic characterization reinforces the relevance to public health in the control of antibiotic-resistant bacteria.

Keywords: antibiotic resistance, enterococci, feed, ornamental animals

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Authors: Hala Raslan, Noha Eltaweel, Hanaa Rasmi, Solaf Kamel, May Magdy, Sherif Ismail, Khalda Amr

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Introduction: Rheumatoid arthritis (RA) is an inflammatory, autoimmune disorder with progressive joint damage. Osteoarthritis (OA) is a degenerative disease of the articular cartilage that shows multiple clinical manifestations or symptoms resembling those of RA. Genetic predisposition is believed to be a principal etiological factor for RA and OA. In this study, we aimed to measure the expression of the top deregulated miRNAs that might be the cause of pathogenesis in both diseases, according to our latest NGS analysis. Six of the deregulated miRNAs were selected as they had multiple target genes in the RA pathway, so they are more likely to affect the RA pathogenesis.Methods: Eighty cases were recruited in this study; 45 rheumatoid arthiritis (RA), 30 osteoarthiritis (OA) patients, as well as 20 healthy controls. The selection of the miRNAs from our latest NGS study was done using miRwalk according to the number of their target genes that are members in the KEGG RA pathway. Total RNA was isolated from plasma of all recruited cases. The cDNA was generated by the miRcury RT Kit then used as a template for real-time PCR with miRcury Primer Assays and the miRcury SYBR Green PCR Kit. Fold changes were calculated from CT values using the ΔΔCT method of relative quantification. Results were compared RA vs Controls and OA vs Controls. Target gene prediction and functional annotation of the deregulated miRNAs was done using Mienturnet. Results: Six miRNAs were selected. They were miR-15b-3p, -128-3p, -194-3p, -328-3p, -542-3p and -3180-5p. In RA samples, three of the measured miRNAs were upregulated (miR-194, -542, and -3180; mean Rq= 2.6, 3.8 and 8.05; P-value= 0.07, 0.05 and 0.01; respectively) while the remaining 3 were downregulated (miR-15b, -128 and -328; mean Rq= 0.21, 0.39 and 0.6; P-value= <0.0001, <0.0001 and 0.02; respectively) all with high statistical significance except miR-194. While in OA samples, two of the measured miRNAs were upregulated (miR-194 and -3180; mean Rq= 2.6 and 7.7; P-value= 0.1 and 0.03; respectively) while the remaining 4 were downregulated (miR-15b, -128, -328 and -542; mean Rq= 0.5, 0.03, 0.08 and 0.5; P-value= 0.0008, 0.003, 0.006 and 0.4; respectively) with statistical significance compared to controls except miR-194 and miR-542. The functional enrichment of the selected top deregulated miRNAs revealed the highly enriched KEGG pathways and GO terms. Conclusion: Five of the studied miRNAs were greatly deregulated in RA and OA, they might be highly involved in the disease pathogenesis and so might be future therapeutic targets. Further functional studies are crucial to assess their roles and actual target genes.

Keywords: MiRNAs, expression, rheumatoid arthritis, osteoarthritis

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Authors: Mohamed Trigui, Fatma Masmoudi, Imen Zouari

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Massive utilizations of chemical fertilizer and chemical pesticides in agriculture sector to improve the farming productivity have created increasing environmental damages. Then, agriculture must become sustainable, focusing on production systems that respect the environment and help to reduce climate change. Isolation and microbial identification of new bacterial strains from naturally saline habitats and compost extracts could be a prominent way in pest management and crop production under saline conditions. In this study, potential mechanisms involved in plant growth promotion and suppressive activity against fungal diseases of a compost extract produced from poultry manure/olive husk compost and halotolerant and halophilic bacterial strains under saline stress were investigated. On the basis of the antimicrobial tests, different strains isolated from Sfax solar saltern (Tunisia) and from compost extracts were selected and tested for their plant growth promoting traits, such as siderophores production, nitrogen fixation, phosphate solubilization and the production of extracellular hydrolytic enzymes (protease and lipase) under in-vitro conditions. Among 450 isolated bacterial strains, 16 isolates showed potent antifungal activity against the tested plant pathogenic fungi. Their identification based on 16S rRNA gene sequence revealed they belonged to different species. Some of these strains were also characterized for their plant growth promoting capacities. Obtained results showed the ability of four strains belonging to Bacillus genesis to ameliorate germination rate and root elongation compared to the untreated positive controls. Combinatorial capacity of halotolerant bacteria with antimicrobial activity and plant growth promoting traits could be promising sources of interesting bioactive substances under saline stress.

Keywords: abiotic stress, biofertilizer, biotic stress, compost extract, halobacteria, plant growth promoting (PGP), soil fertility

Procedia PDF Downloads 62

Authors: Eitan Yefenof

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Glucocorticoid (GC) hormones, e.g. Dexamethasone and Prednisone, are widely used in the therapy of leukemia and lymphoma owing to their apoptogenic effect on lymphoid cells. However, the emergence of GC resistant cells during therapy is a major cause for treatment failure, urging the need for novel strategies that maintain leukemia sensitivity to the pro-apoptotic activity of GCs. GCs act by binding to the GC receptor (GR), which, in its inactive state, is sequestered in the cytosol by a multi-subunit complex of heat shock proteins. Upon ligand binding, the complex dissociates, allowing GR activation and translocation to the nucleus, where it regulates transcription of multiple genes. We demonstrated that in addition to gene expression, GR also regulates microRNA (miR) expression. Deep-sequencing analysis revealed 14 miRs that are regulated in GC-sensitive but resistant leukemias upon treatment with GC. GC up-regulates miR-103, miR-15~16 and miR-30e/d, while down-regulates miR-17, mir-18a, miR-19a, miR-19b, miR-20a and miR-92a (members of the miR-17∼92a multi-cistron). Upon transfection, miR-103 confers GC apoptotic sensitivity to otherwise GC-resistant cell. Furthermore, knocking down miR-103 expression reduces the GC apoptotic response of sensitive cells. miR-103 abrogates c-Myc expression, an oncogenic transcription factor which is deregulated in many cancers. In addition, miR-103 up-regulates Bim, a pro-apoptotic protein crucial for GC-induced death. Activated glycogen synthase kinase 3 (GSK3) is also crucial for GC-induced apoptosis. GSK3 is active in GC-sensitive but not in GC-resistant cells. We found that GSK3 associates with the GR multi-subunit complex. Upon GC exposure, it dissociates from the GR and interacts with Bim to enable activation of the mitochondrial apoptosis pathway. miR-103 mediated c-Myc ablation is followed by down-regulation of the multi-cistron miR-17~92a, in particular miR-18a and miR-20a. miR-18a targets GR for degradation whereas miR-20a targets Bim degradation. Hence, miR-103 acts, in concert with Bim and GR, as a "tumor suppressor" that leads to reduced proliferation, cell-cycle arrest and cell death. We suggest that miR-103 can provide a diagnostic tool that predicts the sensitivity of leukemia to GC based therapy. Furthermore, exosomal delivery of miR-103 or up-regulation of the endogenous miR-103 could confer apoptotic sensitivity to resistant cells at the outset, thus becoming a useful therapeutic tool combined with GCs.

Keywords: apoptosis, leukemia, micro-RNA, steroids

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Authors: Yuda Purwana Roswanjaya, Wouter Kohlen, Rene Geurts

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The Trema genus represents a group of fast-growing tropical tree species within the Cannabaceae. Interestingly, five species nested in this lineage -known as Parasponia- can establish rhizobium nitrogen-fixing root nodules, similar to those found in legumes. Parasponia and legumes use a conserved genetic network to control root nodule formation, among which are genes also essential for mycorrhizal symbiosis (the so-called common symbiotic pathway). However, Trema species lost several genes that function exclusively in nodulation, suggesting a loss-of the nodulation trait in Trema. Strikingly, in a Trema orientalis population found in Malaysian Borneo we identified a truncated SYMBIOSIS RECEPTOR KINASE (SYMRK) mutant allele lacking a large portion of the c-terminal kinase domain. In legumes this gene is essential for nodulation and mycorrhization. This raises the question whether Trema orientalis can still be mycorrhized. To answer this question, we established quantitative mycorrhization assay for Parasponia andersonii and Trema orientalis. Plants were grown in closed pots on half strength Hoagland medium containing 20 µM potassium phosphate in sterilized sand and inoculated with 125 spores of Rhizopagus irregularis (Agronutrion-DAOM197198). Mycorrhization efficiency was determined by analyzing the frequency of mycorrhiza (%F), the intensity of the mycorrhizal colonization (%M) and the arbuscule abundance (%A) in the root system. Trema orientalis RG33 can be mycorrhized, though with lower efficiency compared to Parasponia andersonii. From this we conclude that a functional SYMRK kinase domain is not essential for Trema orientalis mycorrhization. In ongoing experiments, we aim to investigate the role of SYMRK in Parasponia andersonii mycorrhization and nodulation. For this two Parasponia andersonii symrk CRISPR-Cas9 mutant alleles were created. One mimicking the TorSYMRKRG33 allele by deletion of exon 13-15, and a full Parasponia andersonii SYMRK knockout.

Keywords: endomycorrhization, Parasponia andersonii, symbiosis receptor kinase (SYMRK), Trema orientalis

Procedia PDF Downloads 136

Authors: Benlaifa Meriem, Berredjem Hajira, Bouasla Radia, Berredjem Malika, Djebar Med Reda

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Toxoplasmosis is a cosmopolitan infection due to Toxoplasma gondii (T.gondii). It is a significant cause of congenital disease and an important opportunistic pathogen which has become a worldwide increasing problem due to the AIDS epidemic. Current available drugs do not give satisfactory results and often have only a static and several adverse side effects as it is the case of pyrimethamine. So, the need to develop and evaluate new drugs is critical. The purpose of this study is to investigate the in vitro and in vivo effects of the new chiral N-acylsulfonamide bis-oxazolidin-2-ones on T.gondii. In this study, anti-T.gondii RH strain activities, of two new chiral N-acylsulfonamide bis-oxazolidin-2-ones were evaluated in vitro, using a MRC-5 fibroblast tissue cultures to determine the concentration that inhibit parasite multiplication by 50% (IC50) of each drug and in vivo, by PCR detection of the tachyzoites in mice ascites after new molecules treatment, using the 35-fold repetitive B1 gene of T.gondii. The in vitro results demonstrated that the treatment with the tested molecules decreased the amount of tachyzoites in cell culture in a dose-dependent manner. The inhibition was complete for concentrations over 4 mg/ml. The IC50 of Mol 1 and Mol 2 were 1.5 and 3 mg/ml, respectively, and were quite similar to the control one (2 mg/ml). The Mol 1 was highly active against T.gondii in cell cultures than Mol 2; these results were similar to those of sulfadiazine-treated group (p < 0.05). Toxoplasma-specific DNA was demonstrated in all ascites samples from infected mice of the different tested groups. Mol 1 showed better effect than Mol 2, but it did not completely inhibit the parasite proliferation. The intensity of amplification products increased when the treatment started late after infection. These findings suggest continuous parasite replication despite the treatment. In conclusion, our results showed a promising treatment effect of the tested molecules and suggest that in vitro, the Mol 1, and Mol 2 have a dose-dependent effect and a high cytotoxicity on the studied cells. The present study revealed that concentration and duration of tested molecules treatment are major factors that influence the course of Toxoplasma infection in infected mice.

Keywords: cytotoxicity, PCR, sulfonamide, Toxoplasma gondii

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Authors: Marjan Moradi Fard, Hossein Rassi, Masoud Houshmand

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Aim: Breast cancer is one of the most common cancers affecting the morbidity and mortality of Iranian women. This disease is a result of collective alterations of oncogenes and tumor suppressor genes. Studies have produced conflicting results concerning the role of p53 codon 72 polymorphism (G>C) and miR-146a rs2910164 polymorphism (G>C) on the risk of several cancers; therefore, a research was performed to estimate the association between the p53 codon 72 polymorphism and miR-146a rs2910164 polymorphism in breast cancer. Methods and Materials: A total of 45 archival breast cancer samples from khatam hospital and 40 healthy samples were collected. Verification of each cancer reported in a relative was sought through the pathology reports of the hospital records. Then, DNA extracted from all samples by standard methods and p53 codon 72 polymorphism genotypes and miR-146a rs2910164 polymorphism genotypes were analyzed using multiplex PCR. The tubules, mitotic activity, necrosis, polymorphism and grade of breast cancer were staged by Nottingham histological grading and immunohistochemical staining of the sections from the paraffin wax embedded tissues for the expression of ER, PR and p53 was carried out using a standard method. Finally, data analysis was performed using the 7 version of the Epi Info(TM) 2012 software and test chi-square(x2) for trend. Results: Successful DNA extraction was assessed by PCR amplification of b-actin gene (99 bp). According to the results, p53 GG genotype and miR-146a rs2910164 CC genotype was significantly associated with increased risk of breast cancer in the study population. In this study, we established that tumors of p53 GG genotype and miR-146a rs2910164 CC genotype exhibited higher mitotic activity, higher polymorphism, lower necrosis, lower tubules, higher ER- and PR-negatives and lower TP53-positives than the other genotypes. Conclusion: The present study provided preliminary evidence that a p53 GG genotype may effect breast cancer risk in the study population, interacting synergistically with miR-146a rs2910164 CC genotype. Our results demonstrate that the testing of p53 codon 72 polymorphism genotypes and miR-146a rs2910164 polymorphism genotypes in combination with clinical parameters can serve as major risk factors in the early identification of breast cancers.

Keywords: breast cancer, p53 codon 72 polymorphism, miR-146a rs2910164 polymorphism, genotypes

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Authors: Muhammad Qasim, Dolors Puigjaner, Josep Maria López, Joan Herrero, Carme Olivé, Gerard Fortuny

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Computational modeling of the human pelvis using the finite element (FE) method has become extremely important to understand the mechanics of prostate motion and deformation when transrectal ultrasound (TRUS) guided biopsy is performed. The number of reliable and validated hyperelastic constitutive FE models of the male pelvis region is limited, and given models did not precisely describe the anatomical behavior of pelvis organs, mainly of the prostate and its neoplasms location. The motion and deformation of the prostate during TRUS-guided biopsy makes it difficult to know the location of potential lesions in advance. When using this procedure, practitioners can only provide roughly estimations for the lesions locations. Consequently, multiple biopsy samples are required to target one single lesion. In this study, the whole pelvis model (comprised of the rectum, bladder, pelvic muscles, prostate transitional zone (TZ), and peripheral zone (PZ)) is used for the simulation results. An isotropic hyperelastic approach (Signorini model) was used for all the soft tissues except the vesical muscles. The vesical muscles are assumed to have a linear elastic behavior due to the lack of experimental data to determine the constants involved in hyperelastic models. The tissues and organ geometry is taken from the existing literature for 3D meshes. Then the biomechanical parameters were obtained under different testing techniques described in the literature. The acquired parametric values for uniaxial stress/strain data are used in the Signorini model to see the anatomical behavior of the pelvis model. The five mesh nodes in terms of small prostate lesions are selected prior to biopsy and each lesion’s final position is targeted when TRUS probe force of 30 N is applied at the inside rectum wall. Code_Aster open-source software is used for numerical simulations. Moreover, the overall effects of pelvis organ deformation were demonstrated when TRUS–guided biopsy is induced. The deformation of the prostate and neoplasms displacement showed that the appropriate material properties to organs altered the resulting lesion's migration parametrically. As a result, the distance traveled by these lesions ranged between 3.77 and 9.42 mm. The lesion displacement and organ deformation are compared and analyzed with our previous study in which we used linear elastic properties for all pelvic organs. Furthermore, the visual comparison of axial and sagittal slices are also compared, which is taken for Magnetic Resource Imaging (MRI) and TRUS images with our preliminary study.

Keywords: code-aster, magnetic resonance imaging, neoplasms, transrectal ultrasound, TRUS-guided biopsy

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Authors: Monica Jong, Antonios Banos, Tom Scott, Chris Webster, David Fletcher

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Hydrogen, as a clean alternative to methane, is relatively easy to make, either from water using electrolysis or from methane using steam reformation. However, hydrogen is much trickier to store than methane, and without effective storage, it simply won’t pass muster as a suitable methane substitute. Physical storage of hydrogen is quite inefficient. Storing hydrogen as a compressed gas at pressures up to 900 times atmospheric is volumetrically inefficient and carries safety implications, whilst storing it as a liquid requires costly and constant cryogenic cooling to minus 253°C. This is where DU steps in as a possible solution. Across the periodic table, there are many different metallic elements that will react with hydrogen to form a chemical compound known as a hydride (or metal hydride). From a chemical perspective, the ‘king’ of the hydride forming metals is palladium because it offers the highest hydrogen storage volumetric capacity. However, this material is simply too expensive and scarce to be used in a scaled-up bulk hydrogen storage solution. Depleted Uranium is the second most volumetrically efficient hydride-forming metal after palladium. The UK has accrued a significant amount of DU because of manufacturing nuclear fuel for many decades, and that is currently without real commercial use. Uranium trihydride (UH3) contains three hydrogen atoms for every uranium atom and can chemically store hydrogen at ambient pressure and temperature at more than twice the density of pure liquid hydrogen for the same volume. To release the hydrogen from the hydride, all you do is heat it up. At temperatures above 250°C, the hydride starts to thermally decompose, releasing hydrogen as a gas and leaving the Uranium as a metal again. The reversible nature of this reaction allows the hydride to be formed and unformed again and again, enabling its use as a high-density hydrogen storage material which is already available in large quantities because of its stockpiling as a ‘waste’ by-product. Whilst the tritium storage credentials of Uranium have been rigorously proven at the laboratory scale and at the fusion demonstrator JET for over 30 years, there is a need to prove the concept for depleted uranium hydrogen storage (HyDUS) at scales towards that which is needed to flexibly supply our national power grid with energy. This is exactly the purpose of the HyDUS project, a collaborative venture involving EDF as the interested energy vendor, Urenco as the owner of the waste DU, and the University of Bristol with the UKAEA as the architects of the technology. The team will embark on building and proving the world’s first pilot scale demonstrator of bulk chemical hydrogen storage using depleted Uranium. Within 24 months, the team will attempt to prove both the technical and commercial viability of this technology as a longer duration energy storage solution for the UK. The HyDUS project seeks to enable a true by-product to wonder material story for depleted Uranium, demonstrating that we can think sustainably about unlocking the potential value trapped inside nuclear waste materials.

Keywords: hydrogen, long duration storage, storage, depleted uranium, HyDUS

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Authors: Jeena Gupta

Abstract:

In the today’s world of pharmaceutical products, one should not forget the healing properties of inexpensive food materials especially spices. They are known to possess hidden pharmaceutical ingredients, imparting them the qualities of being anti-microbial, anti-oxidant, anti-inflammatory and anti-carcinogenic. Further aberrant epigenetic regulatory mechanisms like DNA methylation, histone modifications or altered microRNA expression patterns, which regulates gene expression without changing DNA sequence, contribute significantly in the development of various diseases. Changing lifestyles and diets exert their effect by influencing these epigenetic mechanisms which are thus the target of dietary phytochemicals. Bioactive components of plants have been in use since ages but their potential to reverse epigenetic alterations and prevention against diseases is yet to be explored. Spices being rich repositories of many bioactive constituents are responsible for providing them unique aroma and taste. Some spices like curcuma and garlic have been well evaluated for their epigenetic regulatory potential, but for others, it is largely unknown. We have evaluated the biological activity of phyto-active components of Fennel, Cardamom and Fenugreek by in silico molecular modeling, in vitro and in vivo studies. Ligand-based similarity studies were conducted to identify structurally similar compounds to understand their biological phenomenon. The database searching has been done by using Fenchone from fennel, Sabinene from cardamom and protodioscin from fenugreek as a query molecule in the different small molecule databases. Moreover, the results of the database searching exhibited that these compounds are having potential binding with the different targets found in the Protein Data Bank. Further in addition to being epigenetic modifiers, in vitro study had demonstrated the antimicrobial, antifungal, antioxidant and cytotoxicity protective effects of Fenchone, Sabinene and Protodioscin. To best of our knowledge, such type of studies facilitate the target fishing as well as making the roadmap in drug design and discovery process for identification of novel therapeutics.

Keywords: epigenetics, spices, phytochemicals, fenchone

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