Search results for: in vivo dosimetry

Authors: Edmaritz Hernandez-Pagan, Kanjana Laosuntisuk, Alex Harris, Allison Haynes, David Buitrago, Michael Kudenov, Colleen Doherty

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Rare Earth Elements (REEs) are critical metals for modern electronics, green technologies, and defense systems. However, due to their dispersed nature in the Earth’s crust, frequent co-occurrence with radioactive materials, and similar chemical properties, acquiring and purifying REEs is costly and environmentally damaging, restricting access to these metals. Plants could serve as resources for bioengineering REE mining systems. Although there is limited information on how REEs affect plants at a cellular and molecular level, plants with high REE tolerance and hyperaccumulation have been identified. This dissertation aims to develop a plant-based system for harvesting REEs from industrial waste material with a focus on Acid Mine Drainage (AMD), a toxic coal mining product. The objectives are 1) to develop a non-destructive, in vivo detection method for REE detection in Phytolacca plants (REE hyperaccumulator) plants utilizing fluorescence spectroscopy and with a primary focus on dysprosium, 2) to characterize the uptake of REE and Heavy Metals in Phytolacca americana and Phytolacca acinosa (REE hyperaccumulator) in AMD for potential implementation in the plant-based system, 3) to implement the REE detection method to identify REE-binding proteins and peptides for potential enhancement of uptake and selectivity for targeted REEs in the plants implemented in the plant-based system. The candidates are known REE-binding peptides or proteins, orthologs of known metal-binding proteins from REE hyperaccumulator plants, and novel proteins and peptides identified by comparative plant transcriptomics. Lanmodulin, a high-affinity REE-binding protein from methylotrophic bacteria, is used as a benchmark for the REE-protein binding fluorescence assays and expression in A. thaliana to test for changes in REE plant tolerance and uptake.

Keywords: phytomining, agromining, rare earth elements, pokeweed, phytolacca

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Authors: Jeena Gupta

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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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Authors: Prasamsha Panta, Da Yeon Kim, Ja Yong Jang, Min Jae Kim, Jae Ho Kim, Moon Suk Kim

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Introduction: Among the many anticancer drugs used clinically, doxorubicin (Dox), was one of widely used drugs to treat many types of solid tumors such as liver, colon, breast, or lung. Intratumoral injection of chemotherapeutic agents is a potentially more effective alternative to systemic administration because direct delivery of the anticancer drug to the target may improve both the stability and efficacy of anticancer drugs. Injectable in situ-forming gels have attracted considerable attention because they can achieve site specific drug delivery, long term action periods, and improved patient compliance. Objective: Objective of present study is to confirm clinical benefit of intratumoral chemotherapy using injectable in situ-forming poly(ethylene glycol)-b-polycaprolactone diblock copolymer (MP) and Dox with increase in efficacy and reducing the toxicity in patients with cancer diseases. Methods and methodology: We prepared biodegradable MP hydrogel and measured viscosity for the evaluation of thermo-sensitive property. In vivo antitumor activity was performed with normal saline, MP only, single free Dox, repeat free Dox, and Dox-loaded MP gel. The remaining amount of Dox drug was measured using HPLC after the mouse was sacrified. For cytotoxicity studies WST-1 assay was performed. Histological analysis was done with H&E and TUNEL processes respectively. Results: The works in this experiment showed that Dox-loaded MP have biodegradable drug depot property. Dox-loaded MP gels showed remarkable in vitro cytotoxicity activities against cancer cells. Finally, this work indicates that injection of Dox-loaded MP allowed Dox to act effectively in the tumor and induced long-lasting supression of tumor growth. Conclusion: This work has examined the potential clinical utility of intratumorally injected Dox-loaded MP gel, which shows significant effect of higher local Dox retention compared with systemically administered Dox.

Keywords: injectable in-situ forming hydrogel, anticancer, doxorubicin, intratumoral injection

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Authors: Rania Hanafi Mahmoud Said, Rasha Mohamed Taha

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Background: A recent study has explored the potential of an approach to treating infected root canals using a combination of Mineral Trioxide Aggregate (MTA) and Tetracycline-loaded Nanochitosan. Material and methods: Forty dogs were included in the study, with infected periapical areas induced by leaving access openings in their teeth for four months. Bacteriological samples from the infected root canals were collected and managed anaerobically to identify and count the different microorganisms present. The most common microorganisms detected were Prevotella oris, Fusobacterium nucleatum, Streptococcus viridans, Enterococcus faecalis, Clostridium subterminale, Porphyromonas gingivalis, and Peptostreptococcus anaerobius. The dogs were divided into four groups based on the sealant used to treat the infected periapical areas: Group I: Negative control (no treatment) Group II: Positive control (MTA only) Group III: MTA + tetracycline Group IV: MTA + tetracycline loaded on nanochitosan Results: Periapical areas in Group IV showed significantly more bone healing than those in Groups I, II, and III. The newly formed bone was evaluated radiographically, histologically, and immunohistochemically using Osteopontin (OSP) antibodies. Data collected was statistically analysed using SPSS software at a 0.05 significance level. Conclusion: The study concluded that the combined use of Tetracycline-loaded Nanochitosan and MTA presents a promising approach for the treatment of infected root canals. The potent antimicrobial activity of Tetracycline-loaded Nanochitosan, along with the biocompatibility and desirable properties of MTA, may synergistically contribute to improved clinical outcomes in endodontic therapy. This study has important implications for the clinical management of infected root canals. The combination of Tetracycline-loaded Nanochitosan and MTA could provide a more effective and efficient means of treating these challenging cases. Further research is needed to confirm these findings in humans and to optimize the treatment protocol.

Keywords: mineral trioxide aggregate, tetracycline-loaded nanochitosan, periapical infection, osteopontine

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Authors: Amina Aloui, Jalila Ben Salah-Abbès, Abdellah Zinedine, Amar Riba, Noel Durand, Catherine Brabet, Didier Montet, Samir Abbès

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Mycotoxin contamination of food and feed-isa globaconcern, both economically and for public health. Aflatoxin M1 (AFM1) is the principal hydroxylated metabolite of aflatoxin B1. It is frequently found in milk and other dairy products. It is responsible for the development of hepatocellular carcinoma and immunotoxic in humans and animals. The reduction of its bioavailabilitybecomesa great demand in order to protect human and animal health. The use of probiotic bacteria and clay are demonstrated to be able to bind AFM1 in vitro. This study aimed to investigate, in vivo, the activity of two-component mixture: L. rhamnosusGAF06 (LR) and bentonite for reducing the oxidative stress and the histological alterationsinduced by AFM1 in the liver andkidneys. For the experiment, male mice were divided into 7 groups (6 mice/group) and treated, orally, by AFM1, alone or in combination with LR and/or bentonite, for 10 days as follows: group 1 control, group 2 treated with LR alone (2.108 CFU/ml), group 3 treated with bentonite alone (1g/kg), group 4 treated with AFM1 alone (100μg/kg), group 5 co-treated with LR+AFM1, group 6 co-treated with bentonite+AFM1, group 7 co-treated with bentonite+LR+AFM1. At the end of the treatment, the mice were sacrificed, and the livers and kidneys were collected for histological assays. Intracellular antioxidant activities and lipid peroxidation were also studied. The results showed that AFM1causeddamage in liver and kidney tissues, being evidence of hepatotoxicity and nephrotoxicity marked by necrotic cells. It increased the MDA level and decreased the antioxidant enzyme activities (SOD) in both organs. In contrast, the co-treatment with AFM1 plus LR and/or bentonitesignificantly improved the hepatic and renal tissues, regulated kidney, and liver antioxidant enzyme activities. This improvement was more remarkable with the administration of LR-bentonite mixture with AFM1.LR and bentonite alone showed to be safe during the treatment. This mixture can be a promising candidate for future applications in biotechnological processes that aimed to detoxify AFM1in food and feed.

Keywords: aflatoxin M1, bentonite, L. rhamnosus GAF06, oxidative stress, prevention

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Authors: Xiaoquan Huang, Congcong Li, Tingting Wei, Changcun Bai, Na Liu, Meng Tang

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Silver is often doped on nano-titanium dioxide photocatalysts (Ag-TiO₂) by photodeposition method to improve their utilization of visible-light while increasing the toxicity of TiO₂。 However, it is not known what factors influence this toxicity and how to reduce toxicity while maintaining the maximum catalytic activity. In this study, Ag-TiO₂ photocatalysts were synthesized by the photodeposition method with different silver content (AgC) and photodeposition time (PDT). Characterization and catalytic experiments demonstrated that silver was well assembled on TiO₂ with excellent visible-light catalytic activity, and the size of silver increased with PDT. In vitro, the cell viability of lung epithelial cells A549 and BEAS-2B showed that the higher content and smaller size of silver doping caused higher toxicity. In vivo, Ag-TiO₂ catalysts with lower AgC or larger silver particle size obviously caused less pulmonary pro-inflammatory and pro-fibrosis responses. However, the visible light catalytic activity decreased with the increase in silver size. Therefore, in order to optimize the Ag-TiO₂ photocatalyst with the lowest pulmonary toxicity and highest catalytic performance, response surface methodology (RSM) was further performed to optimize the two independent variables of AgC and PDT. Visible-light catalytic activity was evaluated by the degradation rate of Rhodamine B, the antibacterial property was evaluated by killing log value for Escherichia coli, and cytotoxicity was evaluated by IC50 to BEAS-2B cells. As a result, the RSM model showed that AgC and PDT exhibited an interaction effect on catalytic activity in the quadratic model. AgC was positively correlated with antibacterial activity. Cytotoxicity was proportional to AgC while inversely proportional to PDT. Finally, the optimization values were AgC 3.08 w/w% and PDT 28 min. Under this optimal condition, the relatively high silver proportion ensured the visible-light catalytic and antibacterial activity, while the longer PDT effectively reduced the cytotoxicity. This study is of significance for the safe and efficient application of silver-doped TiO₂ photocatalysts.

Keywords: Ag-doped TiO₂, cytotoxicity, inflammtion, fibrosis, response surface methodology

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Authors: Mohammed Y. Elsherbeny, Mohamed Salama, Ahmed Lotfy, Hossam Fareed, Nora Mohammed

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Background: Developmental neurotoxicity (DNT) entails the toxic effects imparted by various chemicals on brain during the early childhood when human brains are vulnerable during this period. DNT study in vivo cannot determine the effect of the neurotoxins, as it is not applicable, so using the neurosphere cells of lab animals as an alternative is applicable and time saving. Methods: Cell culture: Rat neural progenitor cells were isolated from rat embryos’ brain. The cortices were aseptically dissected out and the tissues were triturated. The dispersed tissues were allowed to settle. The supernatant was then transferred to a fresh tube and centrifuged. The pellet was placed in Hank’s balanced salt solution and cultured as free-floating neurospheres in proliferation medium. Differentiation was initiated by growth factor withdrawal in differentiation medium and plating onto a poly-d-lysine/ laminin matrix. Chemical Exposure: Neurospheres were treated for 2 weeks with papaverine in proliferation medium. Proliferation analyses: Spheres were cultured. After 0, 4, 5, 11 and 14 days, sphere size was determined by software analyses (CellProfiler, version 2.1; Broad Institute). Diameter of each neurosphere was measured and exported to excel file further to statistical analysis. Viability test: Trypsin-EDTA solution was added to neurospheres to dissociate neurospheres into single cells suspension, then viability evaluated by the Trypan Blue exclusion test. Result: As regards proliferation analysis and percentage of viable cells of papaverin treated groups: There was no significant change in cells proliferation compared to control at 0, 4, 5, 11 and 14 days with concentrations 1, 5 and 10 µM of papaverine, but there is a significant change in cell viability compared to control after 1 week and 2 weeks with the same concentrations of papaverine. Conclusion: Papaverine has toxic effect on viability of neural cell, not on their proliferation, so it may produce focal neural lesions not growth morphological changes.

Keywords: developmental neurotoxicity, neurotoxin, papaverine, neuroshperes

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Authors: Swati Swati, Yuhang Chen, Robert Reuben

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Coronary stents are devices resembling the shape of a tube which are placed in coronary arteries, to keep the arteries open in the treatment of coronary arterial diseases. Coronary stents are routinely deployed to clear atheromatous plaque. The stent essentially applies an internal pressure to the artery because its structure is cylindrically symmetrical and this may introduce some abnormalities in final arterial shape. The goal of the project is to develop segmented circumferential arterial compliance measuring devices which can be deployed (eventually) in vivo. The segmentation of the device will allow the mechanical asymmetry of any stenosis to be assessed. The purpose will be to assess the quality of arterial tissue for applications in tailored stents and in the assessment of aortic aneurism. Arterial distensibility measurement is of utmost importance to diagnose cardiovascular diseases and for prediction of future cardiac events or coronary artery diseases. In order to arrive at some generic outcomes, a preliminary experimental set-up has been devised to establish the measurement principles for the device at macro-scale. The measurement methodology consists of a strain gauge system monitored by LABVIEW software in a real-time fashion. This virtual instrument employs a balloon within a gelatine model contained in a split cylinder with strain gauges fixed on it. The instrument allows automated measurement of the effect of air-pressure on gelatine and measurement of strain with respect to time and pressure during inflation. Compliance simple creep model has been applied to the results for the purpose of extracting some measures of arterial compliance. The results obtained from the experiments have been used to study the effect of air pressure on strain at varying time intervals. The results clearly demonstrate that with decrease in arterial volume and increase in arterial pressure, arterial strain increases thereby decreasing the arterial compliance. The measurement system could lead to development of portable, inexpensive and small equipment and could prove to be an efficient automated compliance measurement device.

Keywords: arterial compliance, atheromatous plaque, mechanical symmetry, strain measurement

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Authors: Shubhita Mathur, Ritika Kar Bahal, Priyanka Chauhan, Anil K. Tyagi

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Mycobacterium tuberculosis, the causative agent of human tuberculosis, is a major cause of mortality. Bacillus Calmette-Guérin (BCG), the only licensed vaccine available for protection against tuberculosis confers highly variable protection ranging from 0%-80%. Thus, novel vaccine strains need to be evaluated for their potential as a vaccine against tuberculosis. We had previously constructed a triple gene mutant of M. tuberculosis (MtbΔmms), having deletions in genes encoding for phosphatases mptpA, mptpB, and sapM that are involved in host-pathogen interaction. Though vaccination with Mtb∆mms strain induced protection in the lungs of guinea pigs, the mutant strain was not able to control the hematogenous spread of the challenge strain to the spleens. Additionally, inoculation with Mtb∆mms resulted in some pathological damage to the spleens in the early phase of infection. In order to overcome the pathology caused by MtbΔmms in the spleens of guinea pigs and also to control the dissemination of the challenge strain, MtbΔmms was genetically modified by disrupting bioA gene to generate MtbΔmmsb strain. Further, in vivo attenuation of MtbΔmmsb was evaluated, and its protective efficacy was assessed against virulent M. tuberculosis challenge in guinea pigs. Our study demonstrates that Mtb∆mmsb mutant was highly attenuated for growth and virulence in guinea pigs. Vaccination with Mtb∆mmsb mutant generated significant protection in comparison to sham-immunized animals at 4 and 12 weeks post-infection in lungs and spleens of the infected animals. Our findings provide evidence that deletion of genes involved in signal transduction and biotin biosynthesis severely attenuates the pathogen and the single immunization with the auxotroph was able to provide significant protection as compared to sham-immunized animals. The protection imparted by Mtb∆mmsb fell short in comparison to the protection observed in BCG-immunized animals. This study nevertheless indicates the importance of attenuated multiple gene deletion mutants of M. tuberculosis in generating protection against tuberculosis.

Keywords: Mycobacterium tuberculosis, BCG, MtbΔmmsb, bioA, guinea pigs

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Authors: Abdulbaset Mazarzaei

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Natural killer (NK) cells are innate immune effectors that play a pivotal role in combating tumors and infected cells. In recent years, the therapeutic potential of NK cells has gained significant attention due to their remarkable cytotoxic ability. This study focuses on investigating the cytotoxic effect of expanded NK cells enriched with interleukin 12 (IL12) and interleukin 15 (IL15), derived from the leukoreduction filter, on the K562 cell line. Firstly, NK cells were isolated from whole blood samples obtained from healthy volunteers. These cells were subsequently expanded ex vivo using a combination of feeder cells, IL12, and IL15. The expanded NK cells were then harvested and assessed for their cytotoxicity against K562, a well-established human chronic myelogenous leukemia cell line. The cytotoxicity was evaluated using flow cytometry assay. Results demonstrate that the expanded NK cells significantly exhibited enhanced cytotoxicity against K562 cells compared to non-expanded NK cells. Interestingly, the expanded NK cells derived specifically from IL12 and IL15-enriched leukoreduction filters showed a robust cytotoxic effect similar to the whole blood-derived NK cells. These findings suggest that IL12 and IL15 in the leukoreduction filter are crucial in promoting NK cell cytotoxicity. Furthermore, the expanded NK cells displayed relatively similar cytotoxicity profiles to whole blood-derived NK cells, indicating their comparable capability in targeting and eliminating tumor cells. This observation is of significant relevance as expanded NK cells from the leukoreduction filter could potentially serve as a readily accessible and efficient source for adoptive immunotherapy. In conclusion, this study highlights the significant cytotoxic effect of expanded NK cells enriched with IL12 and IL15 obtained from the leukoreduction filter on the K562 cell line. Moreover, it emphasizes that these expanded NK cells exhibit comparable cytotoxicity to whole blood-derived NK cells. These findings reinforce the potential clinical utility of using expanded NK cells from the leukoreduction filter as an effective strategy in adoptive immunotherapy for the treatment of cancer. Further studies are warranted to explore the broader implications of this approach in clinical settings.

Keywords: natural killer (NK) cells, Cytotoxicity, Leukoreduction filter, IL-12 and IL-15 Cytokines

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Authors: Shanker Kalakotla, Krishna Mohan Gottumukkala

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Diabetes is a metabolic disorder characterized by hyperglycemia, carbohydrates, altered lipids and proteins metabolism. In recent research nanotechnology is a blazing field for the researchers; latterly there has been prodigious excitement in the nanomedicine and nano pharmacological area for the study of silver nanoparticles synthesis using natural products. Biological methods have been used to synthesize silver nanoparticles in presence of medicinally active antidiabetic plants, and this intention made us assess the biologically synthesized silver nanoparticles from the seed extract of Psoralea corylfolia using 1 mM silver nitrate solution. The synthesized herbal mediated silver nanoparticles (HMSNP’s) then subjected to various characterization techniques such as XRD, SEM, EDX, TEM, DLS, UV and FT-IR respectively. In current study, the silver nanoparticles tested for in-vitro anti-diabetic activity and possible toxic effects in healthy female albino mice by following OECD guidelines-425. Herbal mediated silver nanoparticles were successfully obtained from bioreduction of silver nitrate using Psoralea corylifolia plant extract. Silver nanoparticles have been appropriately characterized and confirmed using different types of equipment viz., UV-vis spectroscopy, XRD, FTIR, DLS, SEM and EDX analysis. From the behavioral observations of the study, the female albino mice did not show sedation, respiratory arrest, and convulsions. Test compounds did not cause any mortality at the dose level tested (i.e., 2000 mg/kg body weight) doses till the end of 14 days of observation and were considered safe. It may be concluded that LD50 of the HMSNPs was 2000mg/kg body weight. Since LD50 of the HMSNPs was 2000mg/kg body weight, so the preferred dose range for HMSNPs falls between the levels of 200 and 400 mg/kg. Further In-vivo pharmacological models and biochemical investigations will clearly elucidate the mechanism of action and will be helpful in projecting the currently synthesized silver nanoparticles as a therapeutic target in treating chronic ailments.

Keywords: herbal mediated silver nanoparticles, HMSNPs, toxicity of silver nanoparticles, PTP1B in-vitro anti-diabetic assay female albino mice, 425 OECD guidelines

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Authors: Parminder Nain, Jaspreet kaur, Vipin Saini, Sunil Sharma

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Jasminum humile commonly known as yellow Jasmine or Pili chameli, is a medicinal plant used in Ayurveda for treating various diseases, one of which is diabetes mellitus. The current study aimed to establish the antidiabetic and antioxidant properties of aqueous extract of Jasminum humile leaves (AEJHL) in nicotinamide/streptozotocin induced type 2 diabetic rats. Phytochemical screening, HPLC analysis, and acute toxicity study of AEJHL were carried out. Male albino wistar rats (n=42) were divided into seven equal groups. Rats with moderate diabetes having hyperglycemia (blood glucose 250-400 mg/dl) were taken for the experiment. Various concentrations of aqueous extract of Jasminum humile leaves (50, 100, 200 and 300 mg/kg, p.o.), and glibenclamide (1mg/kg, p.o.) were orally administered to diabetic rats for 45 days. The effect of AEJHL on blood glucose, plasma insulin and biochemical parameters such as hemoglobin, total protein, serum creatinine, serum urea, alkaline phosphate, Glutamic-oxalacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT), as well as total cholesterol, triglycerides, and high-density lipoprotein (HDL) were also studied. The antioxidant effect of AEJHL was determined by analyzing hepatic and renal antioxidant markers, like superoxide dismutase (SOD), catalase (CAT), reduced Glutathione (GSH), Glutathione peroxidase (GPx), and lipid peroxidation (LPO) in diabetic rats. After 45-days oral administration of aqueous extract of Jasminum humile leaves significantly (p<0.05) reduced blood sugar and increase plasma insulin level and also reverse all above biochemical parameters and antioxidant enzyme level at dose dependent manner. These findings provide in vivo evidence that the aqueous extract of Jasminum humile leaves possess significant antidiabetic and antioxidant potential in nicotinamide/streptozotocin-induced type-2 diabetes mellitus in rats.

Keywords: antidiabetic, antioxidant, jasminum humile, nicotinamide/streptozotocin, type-2 diabetic

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Authors: Sheraz Gul

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The requirements for progressing a compound to clinical trials is well established and relies on the results from in-vitro and in-vivo animal tests to indicate that it is likely to be safe and efficacious when testing in humans. The typical data package required will include demonstrating compound safety, toxicity, bioavailability, pharmacodynamics (potential effects of the compound on body systems) and pharmacokinetics (how the compound is potentially absorbed, distributed, metabolised and eliminated after dosing in humans). If the desired criteria are met and the compound meets the clinical Candidate criteria and is deemed worthy of further development, a submission to regulatory bodies such as the US Food & Drug Administration for an exploratory Investigational New Drug Study can be made. The purpose of this study is to collect data to establish that the compound will not expose humans to unreasonable risks when used in limited, early-stage clinical studies in patients or normal volunteer subjects (Phase I). These studies are also designed to determine the metabolism and pharmacologic actions of the drug in humans, the side effects associated with increasing doses, and, if possible, to gain early evidence on their effectiveness. In order to reach the above goals, we have developed a pre-clinical high throughput Absorption, Distribution, Metabolism and Excretion–Toxicity (ADME–Toxicity) panel of assays to identify compounds that are likely to meet the Lead and Candidate compound acceptance criteria. This panel includes solubility studies in a range of biological fluids, cell viability studies in cancer and primary cell-lines, mitochondrial toxicity, off-target effects (across the kinase, protease, histone deacetylase, phosphodiesterase and GPCR protein families), CYP450 inhibition (5 different CYP450 enzymes), CYP450 induction, cardio-toxicity (hERG) and gene-toxicity. This panel of assays has been applied to multiple compound series developed in a number of projects delivering Lead and clinical Candidates and examples from these will be presented.

Keywords: absorption, distribution, metabolism and excretion–toxicity , drug discovery, food and drug administration , pharmacodynamics

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Authors: Eric Lacoste

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Laboratory animals are currently widely used as a model of human pathologies in dermatology such as atopic dermatitis (AD). These models provide a better understanding of the pathophysiology of this complex and multifactorial disease, the discovery of potential new therapeutic targets and the testing of the efficacy of new therapeutics. However, confirmation of the correct development of AD is mainly based on histology from skin biopsies requiring invasive surgery or euthanasia of the animals, plus slicing and staining protocols. However, there are currently accessible imaging technologies such as Optical Coherence Tomography (OCT), which allows non-invasive visualization of the main histological structures of the skin (like stratum corneum, epidermis, and dermis) and assessment of the dynamics of the pathology or efficacy of new treatments. Briefly, female immunocompetent hairless mice (SKH1 strain) were sensitized and challenged topically on back and ears for about 4 weeks. Back skin and ears thickness were measured using calliper at 3 occasions per week in complement to a macroscopic evaluation of atopic dermatitis lesions on back: erythema, scaling and excoriations scoring. In addition, OCT was performed on the back and ears of animals. OCT allows a virtual in-depth section (tomography) of the imaged organ to be made using a laser, a camera and image processing software allowing fast, non-contact and non-denaturing acquisitions of the explored tissues. To perform the imaging sessions, the animals were anesthetized with isoflurane, placed on a support under the OCT for a total examination time of 5 to 10 minutes. The results show a good correlation of the OCT technique with classical HES histology for skin lesions structures such as hyperkeratosis, epidermal hyperplasia, and dermis thickness. This OCT imaging technique can, therefore, be used in live animals at different times for longitudinal evaluation by repeated measurements of lesions in the same animals, in addition to the classical histological evaluation. Furthermore, this original imaging technique speeds up research protocols, reduces the number of animals and refines the use of the laboratory animal.

Keywords: atopic dermatitis, mouse model, oxzolone model, histology, imaging

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Authors: Vaibhav D. Bhatt, Anju P. Kunjadia, D. S. Nauriyal, Bhumika J. Joshi, Chaitanya G. Joshi

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Metagenomic analysis of milk samples collected from local cattle breed, kankrej (Bos indicus), Gir (Bos indicus) and Crossbred (Bos indicus X Bos taurus) cattle harbouring subclinical mastitis was carried out by next-generation sequencing (NGS) 454 GS-FLX technology. Around 56 different species including members of Enterobacteriales, Pseudomonadales, Bacillales and Lactobacillales with varying abundance were detected in infected milk. The interesting presence of bacteriophages against Staphylococcus aureus, Escherichia coli, Enterobacter and Yersinia species were observed, especially Enterobacteria and E. coli phages (0∙32%) in Kankrej, Enterobacteria and Staphylococcus phages (1∙05%) in Gir and Staphylococcus phages (2∙32%) in crossbred cattle. NGS findings suggest that phages may be involved in imparting natural resistance of the cattle against pathogens. Further infected milk samples were subjected for bacterial isolation. Fourteen different isolates were identified, and DNA was extracted. Genes (Tet-K, Msr-A, and Mec-A) providing antibiotic resistance to the bacteria were screened by Polymerase Chain Reaction and results were validated with traditional antibiotic assay. Total 3 bacteriophages were isolated from nearby environment of the cattle farm. The efficacy of phages was checked against multi-drug resistant bacteria, identified by PCR. In-vivo study was carried out for phage therapy in mammary glands of female rats “Wister albino”. Mammary glands were infused with MDR isolates for 3 consecutive days. Recovery was observed in infected rats after intramammary infusion of sterile phage suspension. From day 4th onwards, level of C-reactive protein was significant increases up to day 12th . However, significant reduction was observed between days 12th to 18th post treatment. Bacteriophages have significant potential as antibacterial agents and their ability to replicate exponentially within their hosts and their specificity, make them ideal candidates for more sustainable mastitis control.

Keywords: bacteriophages, c-reactive protein, mastitis, metagenomic analysis

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Authors: Endris Yibru Hanurry, Wei-Hsin Hsu, Hsieh-Chih Tsai

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In recent years, the discovery of small interfering RNAs (siRNAs) has got great attention for the treatment of cancer and other diseases. However, the therapeutic efficacy of siRNAs has been faced with many drawbacks because of short half-life in blood circulation, poor membrane penetration, weak endosomal escape and inadequate release into the cytosol. To overcome these drawbacks, we designed a non-viral vector by conjugating polyamidoamine generation 4.5 dendrimer (PDG4.5) with diethylenetriamine (DETA)- and tetraethylenepentamine (TEPA) followed by binding with siRNA to form polyplexes through electrostatic interaction. The result of 1H nuclear magnetic resonance (NMR), 13C NMR, correlation spectroscopy, heteronuclear single–quantum correlation spectroscopy, and Fourier transform infrared spectroscopy confirmed the successful conjugation of DETA and TEPA with PDG4.5. Then, the size, surface charge, morphology, binding ability, stability, release assay, toxicity and cellular internalization were analyzed to explore the physicochemical and biological properties of PDG4.5-DETA and PDG4.5-TEPA polyplexes at specific N/P ratios. The polyplexes (N/P = 8) exhibited spherical nanosized (125 and 85 nm) particles with optimum surface charge (13 and 26 mV), showed strong siRNA binding ability, protected the siRNA against enzyme digestion and accepted biocompatibility to the HeLa cells. Qualitatively, the fluorescence microscopy image revealed the delocalization (Manders’ coefficient 0.63 and 0.53 for PDG4.5-DETA and PDG4.5-TEPA, respectively) of polyplexes and the translocation of the siRNA throughout the cytosol to show a decent cellular internalization and intracellular biodistribution of polyplexes in HeLa cells. Quantitatively, the flow cytometry result indicated that a significant (P < 0.05) amount of siRNA was internalized by cells treated with PDG4.5-DETA (68.5%) and PDG4.5-TEPA (73%) polyplexes. Generally, PDG4.5-DETA and PDG4.5-TEPA were ideal nanocarriers of siRNA in vitro and might be used as promising candidates for in vivo study and future pharmaceutical applications.

Keywords: non-viral carrier, oligoalkylamine, poly(amidoamine) dendrimer, polyplexes, siRNA

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Authors: Fei Ye, Åsa Barrefelt, Manuchehr Abedi-Valugerdi, Khalid M. Abu-Salah, Salman A. Alrokayan, Mamoun Muhammed, Moustapha Hassan

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With appropriate encapsulation in functional nanoparticles drugs are more stable in physiological environment and the kinetics of the drug can be more carefully controlled and monitored. Furthermore, targeted drug delivery can be developed to improve chemotherapy in cancer treatment, not only by enhancing intracellular uptake by target cells but also by reducing the adverse effects in non-target organs. Inorganic imaging agents, delivered together with anti-cancer drugs, enhance the local imaging contrast and provide precise diagnosis as well as evaluation of therapy efficacy. We have developed biodegradable polymeric vesicles as a nanocarrier system for multimodal bio-imaging and anticancer drug delivery. The poly (lactic-co-glycolic acid) PLGA) vesicles were fabricated by encapsulating inorganic imaging agents of superparamagnetic iron oxide nanoparticles (SPION), manganese-doped zinc sulfide (MN:ZnS) quantum dots (QDs) and the anticancer drug busulfan into PLGA nanoparticles via an emulsion-evaporation method. T2-weighted magnetic resonance imaging (MRI) of PLGA-SPION-Mn:ZnS phantoms exhibited enhanced negative contrast with r2 relaxivity of approximately 523 s-1 mM-1 Fe. Murine macrophage (J774A) cellular uptake of PLGA vesicles started fluorescence imaging at 2 h and reached maximum intensity at 24 h incubation. The drug delivery ability PLGA vesicles was demonstrated in vitro by release of busulfan. PLGA vesicles degradation was studied in vitro, showing that approximately 32% was degraded into lactic and glycolic acid over a period of 5 weeks. The biodistribution of PLGA vesicles was investigated in vivo by MRI in a rat model. Change of contrast in the liver could be visualized by MRI after 7 min and maximal signal loss detected after 4 h post-injection of PLGA vesicles. Histological studies showed that the presence of PLGA vesicles in organs was shifted from the lungs to the liver and spleen over time.

Keywords: biodegradable polymers, multifunctional nanoparticles, quantum dots, anticancer drugs

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Authors: T. Northcut, A. Rai, N. Perkins

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Background: The COVID-19 pandemic has had a serious impact on academia in general and social work education in particular, changing permanently the way in which we approach educating students. The new reality of the pandemic coupled with the much-needed focus on racism across the country inspired and required educators to get creative with their teaching styles in order to disrupt the power imbalance in the classroom and attend to the multiple layers of needs of diverse students in precarious sociological and economic circumstances. This paper highlights research examining educators with distinctive positionalities and approaches to classroom instruction who use feminist and antiracist pedagogies while adapting to online teaching during the pandemic. Despite being feminist scholars, whose ideologies developed during different waves of feminism, our commitment to having student-led classrooms, liberation, and equity of all, and striving for social change, unified our feminist teaching pedagogies as well as provided interpersonal support. Methodology: Following a narrative qualitative inquiry methodology, the five authors of this paper came together to discuss our pedagogical styles and underlying values using Zoom in a series of six conversations. Narrative inquiry is an appropriate method to use when researchers are bound by common stories or personal experiences. The use of feminist pedagogy in the classroom before and during the pandemic guided the discussions. After six sessions, we reached the point of data saturation. All data from the dialogic process was recorded and transcribed. We used in vivo, narrative, and descriptive coding for the data analytic process. Results: Analysis of the data revealed several themes, which included (1) the influence of our positionalities as an intersection of race, sexual orientation, gender, and years of teaching experience in the classroom, (2) the meaning and variations between different liberatory pedagogical approaches, (3) the tensions between these approaches and institutional policies and practices, (4) the role of self-reflection in everyday teaching, (5) the distinctions between theory and practice and its utility for students, and (6) the challenges of applying a feminist-centered pedagogical approach during the pandemic while utilizing an online platform. As a collective, we discussed several challenges that limited the use of our feminist pedagogical approaches due to instruction through Zoom.

Keywords: feminist, pedagogy, COVID, zoom

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Authors: Shreya Sara Ittycheria, K. C. Sivakumar, Shijulal Nelson Sathi, Priya Srinivas

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hCG, a heterodimer composed of α and β subunits, is a peptide hormone having numerous biological functions. Although hCG is expressed by placenta during pregnancy, ectopic β-hCG secretion is observed in many non-trophoblastic tumors including that of breast. In-vitro and in-vivo studies done in the lab, have proved that BRCA1 defective cancers express β-hCG and when β-hCG is expressed or supplemented, it promotes tumor progression and exhibits resistance to carboplatin and ABT888, in such cancers but not in BRCA1 wild type cancers. In cancer cells, instead of binding to its regular receptor, LH-CGR, β-hCG binds with Transforming Growth Factor Receptor 2 (TGFβRII) and phosphorylates it resulting in faster tumor progression through the Smad signaling pathway. Targeting β-hCG could be a potential therapeutic strategy for managing BRCA1 defective cancers. Here, molecular docking and dynamic simulation studies were done to identify potential small molecule inhibitors against β-hCG as there are currently no such inhibitors reported. The binding sites of TGFβRII on β-hCG were identified from the top 10 predicted complexes from Z Dock. Virtual screening of selected commercially available small molecules from various libraries such as ZINC, NCI and Life Chemicals amounting to a total of 50,025 molecules were done. Four potential small molecule inhibitors were identified, RgcbPs-1, RgcbPs-2, RgcbPs-3 and RgcbPs-4 with binding affinities -60.778 kcal/mol, -45.447 kcal/mol, -65.2268 kcal/mol and -82.040 kcal/mol respectively. Further, 100ns Molecular Dynamics (MD) simulation showed that these molecules form stable complexes with β-hCG. RgcbPs-1 maintains hydrogen bonds with Q54, L52, Q46, C100, G36, C57, C38 residues, RgcbPs-2 maintains hydrogen bonds with A83 residue, RgcbPs-3 maintains hydrogen bonds with C57, Y58, R94, G101 residues and RgcbPs-4 maintains hydrogen bonds with G36, C38, T40, C57, D99, C100, G101 and L104 residues of β-hCG all of which coincide with the TGFβRII binding site on β-hCG. These results show that these two inhibitors could be used either singly or in combination for inhibiting β-hCG from binding to TGFβRII and thereby directly inhibiting the tumorigenesis pathway.

Keywords: β-hCG, breast cancer, dynamic simulations, molecular docking, small molecule inhibitors, virtual screening.

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Authors: Abdulwahhab Khedr, Tamer Shehata, Hanaa El-Ghamry

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Venlafaxine HCl is a novel antidepressant drug used in the treatment of major depressive disorder, generalized anxiety disorder, social anxiety disorder and panic disorder. Conventional therapeutic regimens with venlafaxine HCl immediate-release dosage forms require frequent dosing due to short elimination half-life of the drug and reduced bioavailability. Hence, this study was carried out to develop sustained-release dosage forms of venlafaxine HCl to reduce its dosing frequency, to improve patient compliance and to reduce side effects of the drug. The polymers used were hydroxypropylmethyl cellulose, xanthan gum, sodium alginate, sodium carboxymethyl cellulose, Carbopol 940 and ethyl cellulose. The physical properties of the prepared tablets including tablet thickness, diameter, weight uniformity, content uniformity, hardness and friability were evaluated. Also, the in-vitro release of venlafaxine HCl from different matrix tablets was studied. Based on physical characters and in-vitro release profiles, certain formulae showing promising sustained-release profiles were subjected to film coating with 15% w/v EC in dichloromethane/ethanol mixture (1:1 ratio) using 1% w/v HPMC as pore former and 30% w/w dibutyl phthalate as plasticizer. The optimized formulations were investigated for drug-excipient compatibility using FTIR and DSC studies. Physical evaluation of the prepared tablets fulfilled the pharmacopoeial requirements for tablet friability test, where the weight loss of the prepared formulae did not exceed 1% of the weight of the tested tablets. Moderate release was obtained from tablets containing HPMC. FTIR and DSC studies for such formulae revealed the absence of any type of chemical interaction between venlafaxine HCl and the used polymers or excipients. Forced swimming test in rats was used to evaluate the antidepressant activity of the selected matrix tablets of venlafaxine HCl. Results showed that formulations significantly decreased the duration of animals’ immobility during the 24 hr-period of the test compared to non-treated group.

Keywords: antidepressant, sustained-release, matrix tablet, venlafaxine hydrochloride

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Authors: Maha Azzam, Mahmoud Gabr, Mahmoud Zakaria, Ayman Refaie, Amani Ismail, Sherry Khater, Sylvia Ashamallah, Mohamed Ghoniem

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Evidence was provided that human bone marrow-derived mesenhymal stem cells (HBM-MSCs) could be differentiated to form insulin-producing cells (IPCs). Transplantation of these cells was able to cure chemically-induced diabetes in nude mice. The efficacy of these cells to control diabetes in large animals was carried out to evaluate the sufficient number of cells needed/Kg body weight and to determine the functional longevity in vivo. Materials/Methods: Ten male mongrel dogs weighing 15-20 Kg were used in this study. Diabetes was chemically-induced in 7 dogs by a mixture of alloxan and streptozotocin. Three non-diabetic served as normal controls. Differentiated HBM-MSCs (5 million/Kg) were encapsulated in theracyte capsules and transplanted beneath the rectus sheath. Each dog received 2 capsules. One dog died 4 days postoperative from inhalation pneumonia. The remaining 6 dogs were followed up for 6-18 months. Results: Four dogs became normoglycemic within 6-8 weeks with normal glucose tolerance curves providing evidence that the transplanted cells were glucose-sensitive and insulin-responsive. In the remaining 2 dogs, fasting blood glucose was reduced but did not reach euglycemic levels. The sera of all transplanted dogs contained human insulin and c-peptide but negligible levels of canine insulin. When the HBM-MSCs loaded capsules were removed, rapid return of diabetic state was noted. The harvested capsules were examined by immunofluorescence. IPCs were seen and co-expression of with c-peptide was confirmed. Furthermore, all the pancreatic endocrine genes were expressed by the transplanted cells. Conclusions: This study provided evidence that theracyte capsules could protect the xenogenic HBM-MSCs from the host immune response. This is an important issue when clinical stem cell therapy is considered for definitive treatment for T1DM.

Keywords: diabetes, mesenchymal stem cells, dogs, Insulin-producing cells

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Authors: Valeria Arcucci, Musarat Ishaq, Steven A. Stacker, Greg J. Goodall, Marc G. Achen

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Metastasis is the lethal aspect of cancer for most patients. Remodelling of lymphatic vessels associated with a tumour is a key initial step in metastasis because it facilitates the entry of cancer cells into the lymphatic vasculature and their spread to lymph nodes and distant organs. Although it is clear that vascular endothelial growth factors (VEGFs), such as VEGF-C and VEGF-D, are key drivers of lymphatic remodelling, the means by which many signaling pathways in endothelial cells are coordinately regulated to drive growth and remodelling of lymphatics in cancer is not understood. We seek to understand the broader molecular mechanisms that control cancer metastasis, and are focusing on microRNAs, which coordinately regulate signaling pathways involved in complex biological responses in health and disease. Here, using small RNA sequencing, we found that a specific microRNA, miR-132, is upregulated in expression in lymphatic endothelial cells (LECs) in response to the lymphangiogenic growth factors. Interestingly, ectopic expression of miR-132 in LECs in vitro stimulated proliferation and tube formation of these cells. Moreover, miR-132 is expressed in lymphatic vessels of a subset of human breast tumours which were previously found to express high levels of VEGF-D by immunohistochemical analysis on tumour tissue microarrays. In order to dissect the complexity of regulation by miR-132 in lymphatic biology, we performed Argonaute HITS-CLIP, which led us to identify the miR-132-mRNA interactome in LECs. We found that this microRNA in LECs is involved in the control of many different pathways mainly involved in cell proliferation and regulation of the extracellular matrix and cell-cell junctions. We are now exploring the functional significance of miR-132 targets in the biology of LECs using biochemical techniques, functional in vitro cell assays and in vivo lymphangiogenesis assays. This project will ultimately define the molecular regulation of lymphatic remodelling by miR-132, and thereby identify potential therapeutic targets for drugs designed to restrict the growth and remodelling of tumour lymphatics resulting in metastatic spread.

Keywords: argonaute HITS-CLIP, cancer, lymphatic remodelling, miR-132, VEGF

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Authors: Tanu Khanuja, Harikrishnan N. Unni

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Head is the most vulnerable part of human body and may cause severe life threatening injuries. As the in vivo brain response cannot be recorded during injury, computational investigation of the head model could be really helpful to understand the injury mechanism. Majority of the physical damage to living tissues are caused by relative motion within the tissue due to tensile and shearing structural failures. The present Finite Element study focuses on investigating intracranial pressure and stress/strain distributions resulting from impact loads on various sites of human head. This is performed by the development of the 3D model of a human head with major segments like cerebrum, cerebellum, brain stem, CSF (cerebrospinal fluid), and skull from patient specific MRI (magnetic resonance imaging). The semi-automatic segmentation of head is performed using AMIRA software to extract finer grooves of the brain. To maintain the accuracy high number of mesh elements are required followed by high computational time. Therefore, the mesh optimization has also been performed using tetrahedral elements. In addition, model validation with experimental literature is performed as well. Hard tissues like skull is modeled as elastic whereas soft tissues like brain is modeled with viscoelastic prony series material model. This paper intends to obtain insights into the severity of brain injury by analyzing impacts on frontal, top, back, and temporal sites of the head. Yield stress (based on von Mises stress criterion for tissues) and intracranial pressure distribution due to impact on different sites (frontal, parietal, etc.) are compared and the extent of damage to cerebral tissues is discussed in detail. This paper finds that how the back impact is more injurious to overall head than the other. The present work would be helpful to understand the injury mechanism of traumatic brain injury more effectively.

Keywords: dynamic impact analysis, finite element analysis, intracranial pressure, MRI, traumatic brain injury, von Misses stress

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Authors: Liaqat Ali, Hubert E. Blum, Türkân Sakinc

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Enterococcus faecium is an emerging multidrug-resistant nosocomial pathogen increased dramatically worldwide and causing bacteremia, endocarditis, urinary tract and surgical site infections in immunocomprised patients. The capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system are also involved in hindering leukocyte killing of enterococci. The gene cluster (enterococcal polysaccharide antigen) of E. faecalis encoding homologues of many genes involved in polysaccharide biosynthesis. We identified two putative loci with 22 kb and 19 kb which contained 11 genes encoding for glycosyltransferases (GTFs); this was confirmed by using genome comparison of already sequenced strains that has no homology to known capsule genes and the epa-locus. The polysaccharide-conjugate vaccines have rapidly emerged as a suitable strategy to combat different pathogenic bacteria, therefore, we investigated a polysaccharide biosynthesis CapD protein in E. faecium contains 336 amino acids and had putative function for N-linked glycosylation. The deletion/knock-out capD mutant was constructed and complemented by homologues recombination method and confirmed by using PCR and sequencing. For further characterization and functional analysis, in-vitro cell culture and in-vivo a mouse infection models were used. Our ΔcapD mutant shows a strong hydrophobicity and all strains exhibited biofilm production. Subsequently, the opsonic activity was tested in an opsonophagocytic assay which shows increased in mutant compared complemented and wild type strains but more than two fold decreased in colonization and adherence was seen on surface of uroepithelial cells. However, a significant higher bacterial colonialization was observed in capD mutant during animal bacteremia infection. Unlike other polysaccharides biosynthesis proteins, CapD does not seems to be a major virulence factor in enterococci but further experiments and attention is needed to clarify its function, exact mechanism and involvement in pathogenesis of enteroccocal nosocomial infections eventually to develop a vaccine/ or targeted therapy.

Keywords: E. faecium, pathogenesis, polysaccharides, biofilm formation

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Authors: Manuela Amorim, Cláudia S. Marques, Maria Conceição Calhau, Hélder J. Pinheiro, Maria Manuela Pintado

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Recently it has been recognized peptides from different food sources with biological activities. However, no relevant study has proven the potential of brewer yeast peptides in the modulation of gut microbiota. The importance of human intestinal microbiota in maintaining host health is well known. Probiotics, prebiotics and the combination of these two components, can contribute to support an adequate balance of the bacterial population in the human large intestine. The survival of many bacterial species inhabiting the large bowel depends essentially on the substrates made available to them, most of which come directly from the diet. Some of these substrates can be selectively considered as prebiotics, which are food ingredients that can stimulate beneficial bacteria such as Lactobacilli or Bifidobacteria growth in the colon. Moreover, conventional food can be used as vehicle to intake bioactive compounds that provide those health benefits and increase people well-being. In this way, the main objective of this work was to study the potential prebiotic activity of brewer yeast peptide extract (BYP) obtained via hydrolysis of yeast proteins by cardosins present in Cynara cardunculus extract for possible use as a functional ingredient. To evaluate the effect of BYP on the modulation of gut microbiota in diet-induced obesity model, Wistar rats were fed either with a standard or a high-fat diet. Quantified via 16S ribosomal RNA (rRNA) expression by quantitative PCR (qPCR), genera of beneficial bacteria (Lactobacillus spp. and Bifidobacterium spp.) and three main phyla (Firmicutes, Bacteroidetes and Actinobacteria) were assessed. Results showed relative abundance of Lactobacillus spp., Bifidobacterium spp. and Bacteroidetes was significantly increased (P < 0.05) by BYP. Consequently, the potential health-promoting effects of WPE through modulation of gut microbiota were demonstrated in vivo. Altogether, these findings highlight the possible intervention of BYP as gut microbiota enhancer, promoting healthy life style, and the incorporation in new food products, leads them bringing associated benefits endorsing a new trend in the improvement of new value-added food products.

Keywords: functional ingredients, gut microbiota, prebiotics, brewer yeast peptide extract

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Authors: Ananya Gupta, Sangeeta Bhaskar

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Mycobacterium indicus pranii (MIP) is a saprophytic mycobacterium. It is a predecessor of M. avium complex (MAC). Whole genome analysis and growth kinetics studies have placed MIP in between pathogenic and non-pathogenic species. It shares significant antigenic repertoire with M. tuberculosis and have unique immunomodulatory properties. MIP provides better protection than BCG against pulmonary tuberculosis in animal models. Immunization with MIP by aerosol route provides significantly higher protection as compared to immunization by subcutaneous (s.c.) route. However, mechanism behind differential protection has not been studied. In this study, using mice model we have evaluated and compared the M.tb specific immune response in lung compartments (airway lumen / lung interstitium) as well as spleen following MIP immunization via nasal (i.n.) and s.c. route. MIP i.n. vaccination resulted in increased seeding of memory T cells (CD4+ and CD8+ T-cells) in the airway lumen. Frequency of CD4+ T cells expressing Th1 migratory marker (CXCR3) and activation marker (CD69) were also high in airway lumen of MIP i.n. group. Significantly high ex vivo secretion of cytokines- IFN-, IL-12, IL-17 and TNF- from cells of airway luminal spaces provides evidence of antigen-specific lung immune response, besides generating systemic immunity comparable to MIP s.c. group. Analysis of T cell response on per cell basis revealed that antigen specific T-cells of MIP i.n. group were functionally superior as higher percentage of these cells simultaneously secreted IFN-gamma, IL-2 and TNF-alpha cytokines as compared to MIP s.c. group. T-cells secreting more than one of the cytokines simultaneously are believed to have robust effector response and crucial for protection, compared with single cytokine secreting T-cells. Adoptive transfer of airway luminal T-cells from MIP i.n. group into trachea of naive B6 mice revealed that MIP induced CD8 T-cells play crucial role in providing long term protection. Thus the study demonstrates that MIP intranasal vaccination induces M.tb specific memory T-cells in the airway lumen that results in an early and robust recall response against M.tb infection.

Keywords: airway lumen, Mycobacterium indicus pranii, Th1 migratory markers, vaccination

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Authors: Cheng-Kuang Hsu, Chih-Hsiang Chang, Chi-Chih Wang

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Black soybeans contain high amount of antioxidants including polyphenols, anthocyanins and flavones. The protective function of black soybean against CCl4 (a strong oxidant) induced acute and chronic liver damage was investigated in vivo using SD rats or ICR mouse. The evaluation of CCl4 induced oxidative stress in the liver tissues included the measurements of the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the concentration of thiobarbituric acid reactive substances (TBARS), the activities of antioxidant enzymes (superoxide dismutase SOD, catalase, and glutathione peroxidase GPx), as well as the level of histological lesion in the liver tissues. For chronic experiment, a decoction at the concentration of 100 or 1000 mg/kg of body weight, produced by baking black soybean at 130°C for 5 min and followed by immerging in 100°C hot water for 20 min, showed the inhibitory effect against CCl4 induced liver damage in SD rats. Hot-water extract (80 °C for 30 min) from un-preheated black soybean at the concentration of 200 mg/kg of body weight could not reduce ALT and AST levels in CCl4 treated SD rats, but the hot-water extract from preheated black soybean did enhance antioxidant enzymes activities, decline ALT and AST levels. Specially, the hot-water extract from the seed cost of black soybean had the highest liver protective function since it can reduce vacuolization and necrosis in the liver tissues. For acute experiment, the hot-water extracts from black soybean and the seed coat, as well as pure cyanidin-3-glucoside (C3G) could reduce ALT and AST levels of CCl4 induced ICR mouse. The decoction and hot-water extract from the seed coat of black soybean had higher total polyphenols, anthocyanins and flavones contents than those extracts from whole black soybean. Such results agreed with high liver protective function in the decoction and hot-water from the seed coat of black soybean. Black soybean showed protective function only after preheating process (baking at 130°C for 5 to 10 min) because preheating treatment damaged the cell wall and made the extraction of the antioxidants more effectively.

Keywords: black soybean, liver protective function, antioxidant, antioxidative stress

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Authors: Hadiza Abdullahi

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Sialic Acids and sialidases have been implicated in many disease states particularly bacterial and viral infections which are common opportunist infections of HIV disease. Their role in HIV/AIDS is contemplated. A study was carried out to determine Sialic Acid profile and Sialidase Activity in HIV infected and Apparently Healthy individuals, and also determine the relationship between the sialic acid levels and sialidase activity. Blood samples were collected from 200 subjects (150 HIV infected individuals and 50 apparently healthy individuals divided into four groups- HIV ART Naïve, HIV Stable (on ART but have been stable with no clinical episodes), HIV-OI (on ART with opportunistic infections), and Apparently Healthy). Complete Blood Count, Erythrocyte Surface Sialic Acid (ESSA), Free Serum Sialic Acid (FSSA) concentrations and Sialidase activity were determined for all 200 subjects. Analysis of variance (ANOVA) was used to compare the results of the different groups of HIV infected individuals as well as controls. The mean haemoglobin (HGB), Packed Cell Volume (PCV) and Red Blood Cells (RBC) concentrations were significantly lower (P ≤ 0.05) in the HIV groups compared with the apparently healthy groups. Anaemia and neutropaenia were the most common heamatological abnormalities observed in this study with highest prevalence of anaemia found in the ART naive group. The mean FSSA was 0.4±0.4mg/ml. There was a significant difference (p ≤ 0.05) between some groups. The highest levels of FSSA was observed in the HIV ART naïve (0.65±0.5mg/ml). The mean ESSA value for the study population was 0.54±0.35mg/ml with no significant difference (p ≤ 0.05) between groups. The mean sialidase activity values were 0.52±0.1 µmol/min/µl, 0.40±0.1 µmol/min/µl, 0.45±0.1 µmol/min/µl and 0.41±0.1 µmol/min/µl for the HIV ART naïve, HIV stable, HIV/OIs and apparently healthy groups respectively. No significant difference (p ≤ 0.05) was found between groups and also in gender and age. The finding in this study of higher mean sialidase activity and FSSA levels in the ART naïve HIV group compared with other groups indicate that the virus and other opportunistic pathogens may be sialidase producers in vivo which cleave off sialic acids from erythrocytes surface, leading to high levels of FSSA, anaemia and neutropaenia seen in this group. The higher ESSA concentration found in the HIV stable group along with lowest FSSA concentration in the group suggests the presence of sialyltransferases.

Keywords: erythrocyte surface sialic acid, free serum sialic acid, HIV, sialidase

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Authors: Vaishali Patil, Neeraj Masand

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In older people, Alzheimer’s disease (AD) is turning out to be a lethal disease. According to the amyloid hypothesis, aggregation of the amyloid β–protein (Aβ), particularly its 42-residue variant (Aβ42), plays direct role in the pathogenesis of AD. Aβ is generated through sequential cleavage of amyloid precursor protein (APP) by β–secretase (BACE) and γ–secretase (GS). Thus in the treatment of AD, γ-secretase modulators (GSMs) are potential disease-modifying as they selectively lower pathogenic Aβ42 levels by shifting the enzyme cleavage sites without inhibiting γ–secretase activity. This possibly avoids known adverse effects observed with complete inhibition of the enzyme complex. Virtual screening, via drug-like ADMET filter, QSAR and molecular docking analyses, has been utilized to identify novel γ–secretase modulators with sulphonamide nucleus. Based on QSAR analyses and docking score, some novel analogs have been synthesized. The results obtained by in silico studies have been validated by performing in vivo analysis. In the first step, behavioral assessment has been carried out using Scopolamine induced amnesia methodology. Later the same series has been evaluated for neuroprotective potential against the oxidative stress induced by Scopolamine. Biochemical estimation was performed to evaluate the changes in biochemical markers of Alzheimer’s disease such as lipid peroxidation (LPO), Glutathione reductase (GSH), and Catalase. The Scopolamine induced amnesia model has shown increased Acetylcholinesterase (AChE) levels and the inhibitory effect of test compounds in the brain AChE levels have been evaluated. In all the studies Donapezil (Dose: 50µg/kg) has been used as reference drug. The reduced AChE activity is shown by compounds 3f, 3c, and 3e. In the later stage, the most potent compounds have been evaluated for Aβ42 inhibitory profile. It can be hypothesized that this series of alkyl-aryl sulphonamides exhibit anti-AD activity by inhibition of Acetylcholinesterase (AChE) enzyme as well as inhibition of plaque formation on prolong dosage along with neuroprotection from oxidative stress.

Keywords: gamma-secretase inhibitors, Alzzheimer's disease, sulphonamides, QSAR

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Authors: Asif Bilal, Fouzia Tanvir, Sibtain Ahmad

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Estrogen receptor alpha, also known as estrogen receptor-1, is highly involved in risk of mammary carcinoma. The objectives of this study were to identify non-synonymous SNPs of estrogen receptor and their association with breast cancer and to identify the chemotherapeutic responses of phytochemicals against it via in-silico study design. For this purpose, different online tools. to identify pathogenic SNPs the tools were SIFT, Polyphen, Polyphen-2, fuNTRp, SNAP2, for finding disease associated SNPs the tools SNP&GO, PhD-SNP, PredictSNP, MAPP, SNAP, MetaSNP, PANTHER, and to check protein stability Mu-Pro, I-Mutant, and CONSURF were used. Post-translational modifications (PTMs) were detected by Musitedeep, Protein secondary structure by SOPMA, protein to protein interaction by STRING, molecular docking by PyRx. Seven SNPs having rsIDs (rs760766066, rs779180038, rs956399300, rs773683317, rs397509428, rs755020320, and rs1131692059) showing mutations on I229T, R243C, Y246H, P336R, Q375H, R394S, and R394H, respectively found to be completely deleterious. The PTMs found were 96 times Glycosylation; 30 times Ubiquitination, a single time Acetylation; and no Hydroxylation and Phosphorylation were found. The protein secondary structure consisted of Alpha helix (Hh) is (28%), Extended strand (Ee) is (21%), Beta turn (Tt) is 7.89% and Random coil (Cc) is (44.11%). Protein-protein interaction analysis revealed that it has strong interaction with Myeloperoxidase, Xanthine dehydrogenase, carboxylesterase 1, Glutathione S-transferase Mu 1, and with estrogen receptors. For molecular docking we used Asiaticoside, Ilekudinuside, Robustoflavone, Irinoticane, Withanolides, and 9-amin0-5 as ligands that extract from phytochemicals and docked with this protein. We found that there was great interaction (from -8.6 to -9.7) of these ligands of phytochemicals at ESR1 wild and two mutants (I229T and R394S). It is concluded that these SNPs found in ESR1 are involved in breast cancer and given phytochemicals are highly helpful against breast cancer as chemotherapeutic agents. Further in vitro and in vivo analysis should be performed to conduct these interactions.

Keywords: breast cancer, ESR1, phytochemicals, molecular docking

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