Search results for: beta 2 toxin gene

Authors: Sajewicz-Krukowska Joanna, Domańska-Blicharz Katarzyna, Tarasiuk Karolina, Marzec-Kotarska Barbara

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Astroviral infections pose a significant problem in the poultry industry, leading to multiple adverse effects such as decreased egg production, breeding disorders, poor weight gain, and even increased mortality. Commonly observed chicken astrovirus (CAstV) was recently reported to be responsible for "white chicks syndrome" associated with increased embryo/chick mortality. The CAstV-mediated pathogenesis in chicken occurs due to complex interactions between the infectious pathogen and the immune system. Many aspects of CAstV-chicken interactions remain unclear, and there is no information available regarding gene expression changes in the chicken's spleen in response to CAstV infection. We aimed to investigate the molecular background triggered by CAstV infection. Ten 21-day-old SPF White Leghorn chickens were divided into two groups of 5 birds each. One group was inoculated with CAstV, and the other was used as the negative control. On 4th dpi, spleen samples were collected and immediately frozen at -70°C for RNA isolation. We analysed transcriptional profiles of the chickens' spleens at the 4th day following infection using RNA-seq to establish differentially expressed genes (DEGs). The RNA-seq findings were verified by quantitative real-time PCR (qRT-PCR). A total of 31959 transcripts were identified in response to CAstV infection. Eventually 45 DEGs (p-value<0.05; Log2Foldchange>1)were recognized in the spleen after CAstV infection (26 upregulated DEGs and 19 downregulated DEGs). qRT-PCR performed on 4 genes (IFIT5, OASL, RASD1, DDX60) confirmed RNAseq results. Top differentially expressed genes belonged to novel putative IFN-induced CAstV restriction factors. Most of the DEGs were associated with RIG-I–like signalling pathway or, more generally, with an innate antiviral response(upregulated: BLEC3, CMPK2, IFIT5, OASL, DDX60, IFI6, and downregulated: SPIK5, SELENOP, HSPA2, TMEM158, RASD1, YWHAB). The study provided a global analysis of host transcriptional changes that occur during CAstV infection in vivo and proved the cell cycle in the spleen and immune signalling in chickens were predominantly affected upon CAstV infection.

Keywords: chicken astrovirus, CastV, RNA-seq, transcriptome, spleen

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Authors: Emiru Birhane, Tesfay Gidey, Haftu Abrha, Abrha Brhan, Amanuel Zenebe, Girmay Gebresamuel, Florent Noulèkoun

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Dracaena ombet and Dobera glabra are two of the most rare and endangered tree species in dryland areas. Unfortunately, their sustainability is being compromised by different anthropogenic and natural factors. However, the impacts of ongoing land use and climate change on the population structure and distribution of the species are less explored. This study was carried out in the grazing lands and hillside areas of the Desa'a dry Afromontane forest, northern Ethiopia, to characterize the population structure of the species and predict the impact of climate change on their potential distributions. In each land-use type, abundance, diameter at breast height, and height of the trees were collected using 70 sampling plots distributed over seven transects spaced one km apart. The geographic coordinates of each individual tree were also recorded. The results showed that the species populations were characterized by low abundance and unstable population structure. The latter was evinced by a lack of seedlings and mature trees. The study also revealed that the total abundance and dendrometric traits of the trees were significantly different between the two land uses. The hillside areas had a denser abundance of bigger and taller trees than the grazing lands. Climate change predictions using the MaxEnt model highlighted that future temperature increases coupled with reduced precipitation would lead to significant reductions in the suitable habitats of the species in northern Ethiopia. The species' suitable habitats were predicted to decline by 48–83% for D. ombet and 35–87% for D. glabra. Hence, to sustain the species populations, different strategies should be adopted, namely the introduction of alternative livelihoods (e.g., gathering NTFP) to reduce the overexploitation of the species for subsistence income and the protection of the current habitats that will remain suitable in the future using community-based exclosures. Additionally, the preservation of the species' seeds in gene banks is crucial to ensure their long-term conservation.

Keywords: grazing lands, hillside areas, land-use change, MaxEnt, range limitation, rare and endangered tree species

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Authors: Farida Azzouz-Olden, Arthur G. Hunt, Gloria Degrandi-Hoffman

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Bees are confronting several environmental challenges, including the intermingled effects of malnutrition and disease. Intuitively, pollen is the healthiest nutritional choice; however, commercial substitutes, such as BeePro and MegaBee, are widely used. Herein we examined how feeding natural and artificial diets shapes transcription in the abdomen of the honey bee, and how transcription shifts in combination with Nosema parasitism. Gene ontology enrichment revealed that, compared with poor diet (carbohydrates (C)), bees fed pollen (P > C), BeePro (B > C), and MegaBee (M > C) showed a broad upregulation of metabolic processes, especially lipids; however, pollen feeding promoted more functions and superior proteolysis. The superiority of the pollen diet was also evident through the remarkable overexpression of vitellogenin in bees fed pollen instead of MegaBee or BeePro. Upregulation of bioprocesses under carbohydrates feeding compared to pollen (C > P) provided a clear poor nutritional status, uncovering stark expression changes that were slight or absent relatively to BeePro (C > B) or MegaBee (C > M). Poor diet feeding (C > P) induced starvation response genes and hippo signaling pathway, while it repressed growth through different mechanisms. Carbohydrate feeding (C > P) also elicited ‘adult behavior’, and developmental processes suggesting transition to foraging. Finally, it altered the ‘circadian rhythm’, reflecting the role of this mechanism in the adaptation to nutritional stress in mammals. Nosema-infected bees fed pollen compared to carbohydrates (PN > CN) upheld certain bioprocesses of uninfected bees (P > C). Poor nutritional status was more apparent against pollen (CN > PN) than BeePro (CN > BN) or MegaBee (CN > MN). Nosema accentuated the effects of malnutrition since more starvation-response genes and stress response mechanisms were upregulated in CN > PN compared to C > P. The bioprocess ‘Macromolecular complex assembly’ was also enriched in CN > PN, and involved genes associated with human HIV and/or influenza, thus providing potential candidates for bee-Nosema interactions. Finally, the enzyme Duox emerged as essential for guts defense in bees, similarly to Drosophila. These results provide evidence of the superior nutritional status of bees fed pollen instead of artificial substitutes in terms of overall health, even in the presence of a pathogen.

Keywords: honeybee, immunity, Nosema, nutrition, RNA-seq

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Authors: Atitallah Sofien, Bouyahia Olfa, Attar Souleima, Missaoui Nada, Ben Rabeh Rania, Yahyaoui Salem, Mazigh Sonia, Boukthir Samir

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Introduction: Ciliopathies are a spectrum of diseases that have in common a defect in the synthesis of ciliary proteins. It is a rare cause of neonatal cholestasis. Clinical presentation varies extremely, and the main affected organs are the kidneys, liver, and pancreas. Methodology: This is a descriptive case report of a newborn who was admitted for exploration of neonatal cholestasis in the Paediatric Department C at the Children’s Hospital of Tunis, where the investigations concluded with a rare genetic mutation. Results: This is the case of a newborn male with no family history of hepatic and renal diseases, born to consanguineous parents, and from a well-monitored uneventful pregnancy. He developed jaundice on the second day of life, for which he received conventional phototherapy in the neonatal intensive care unit. He was admitted at 15 days for mild bronchiolitis. On clinical examination, intense jaundice was noted with normal stool and urine colour. Initial blood work showed an elevation in conjugated bilirubin and a high gamma-glutamyl transferase level. Transaminases and prothrombin time were normal. Abdominal sonography revealed hepatomegaly, splenomegaly, and undifferentiated renal cortex with bilateral medullar micro-cysts. Kidney function tests were normal. The infant received ursodeoxycholic acid and vitamin therapy. Genetic testing showed a homozygous mutation in the DCDC2 gene that hadn’t been documented before confirming the diagnosis of renal-hepatic ciliopathy. The patient has regular follow-ups, and his conjugated bilirubin and gamma-glutamyl transferase levels have been decreasing. Conclusion: Genetic testing has revolutionized the approach to etiological diagnosis in pediatric cholestasis. It enables personalised treatment strategies to better enhance the quality of life of patients and prevent potential complications following adequate long-term monitoring.

Keywords: cholestasis, newborn, ciliopathy, DCDC2, genetic

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Authors: Aleya Ferdausi, Meriel Jones, Anthony Halls

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The Amaryllidaceae genus Narcissus contains secondary metabolites, which are important sources of bioactive compounds such as pharmaceuticals indicating that their biological activity extends from the native plant to humans. Transcriptome analysis (RNA-seq) is an effective platform for the identification and functional characterization of candidate genes as well as to identify genes encoding uncharacterized enzymes. The biotechnological production of secondary metabolites in plant cell or organ cultures has become a tempting alternative to the extraction of whole plant material. The biochemical pathways for the production of secondary metabolites require primary metabolites to undergo a series of modifications catalyzed by enzymes such as cytochrome P450s, methyltransferases, glycosyltransferases, and acyltransferases. Differential gene expression analysis of Narcissus was obtained from two conditions, i.e. field and in vitro callus. Callus was obtained from modified MS (Murashige and Skoog) media supplemented with growth regulators and twin-scale explants from Narcissus cv. Carlton bulb. A total of 2153 differentially expressed transcripts were detected in Narcissus bulb and in vitro callus, and 78.95% of those were annotated. It showed the expression of genes involved in the biosynthesis of alkaloids were present in both conditions i.e. cytochrome P450s, O-methyltransferase (OMTs), NADP/NADPH dehydrogenases or reductases, SAM-synthetases or decarboxylases, 3-ketoacyl-CoA, acyl-CoA, cinnamoyl-CoA, cinnamate 4-hydroxylase, alcohol dehydrogenase, caffeic acid, N-methyltransferase, and NADPH-cytochrome P450s. However, cytochrome P450s and OMTs involved in the later stage of Amaryllidaceae alkaloids biosynthesis were mainly up-regulated in field samples. Whereas, the enzymes involved in initial biosynthetic pathways i.e. fructose biphosphate adolase, aminotransferases, dehydrogenases, hydroxyl methyl glutarate and glutamate synthase leading to the biosynthesis of precursors; tyrosine, phenylalanine and tryptophan for secondary metabolites were up-regulated in callus. The knowledge of probable genes involved in secondary metabolism and their regulation in different tissues will provide insight into the Narcissus plant biology related to alkaloid production.

Keywords: narcissus, callus, transcriptomics, secondary metabolites

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Authors: Fatna Benmessaoud, Mohammed Cheikh, Vencent Velay, Vanessa Vedal, Farhad Rezai-Aria, Christine Boher

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The global mechanical behavior of materials is strongly linked to their microstructure, especially their crystallographic texture and their grains morphology. These material aspects determine the mechanical fields character (heterogeneous or homogeneous), thus, they give to the global behavior a degree of anisotropy according the initial microstructure. For these reasons, the prediction of global behavior of materials in relationship with the microstructure must be performed with a multi-scale approach. Therefore, multi-scale modeling in the context of crystal plasticity is widely used. In this present contribution, a phenomenological elasto-viscoplastic model developed in the crystal plasticity context and finite element method are used to investigate the effects of crystallographic texture and grains sizes on global behavior of a polycrystalline equiaxed Ti-6Al-4V alloy. The constitutive equations of this model are written on local scale for each slip system within each grain while the strain and stress mechanical fields are investigated at the global scale via finite element scale transition. The beta phase of Ti-6Al-4V alloy modeled is negligible; its percent is less than 10%. Three families of slip systems of alpha phase are considered: basal and prismatic families with a burgers vector and pyramidal family with a burgers vector. The twinning mechanism of plastic strain is not observed in Ti-6Al-4V, therefore, it is not considered in the present modeling. Nine representative elementary volumes (REV) are generated with Voronoi tessellations. For each individual equiaxed grain, the own crystallographic orientation vis-à-vis the loading is taken into account. The meshing strategy is optimized in a way to eliminate the meshing effects and at the same time to allow calculating the individual grain size. The stress and strain fields are determined in each Gauss point of the mesh element. A post-treatment is used to calculate the local behavior (in each grain) and then by appropriate homogenization, the macroscopic behavior is calculated. The developed model is validated by comparing the numerical simulation results with an experimental data reported in the literature. It is observed that the present model is able to predict the global mechanical behavior of Ti-6Al-4V alloy and investigate the microstructural parameters' effects. According to the simulations performed on the generated volumes (REV), the macroscopic mechanical behavior of Ti-6Al-4V is strongly linked to the active slip systems family (prismatic, basal or pyramidal). The crystallographic texture determines which family of slip systems can be activated; therefore it gives to the plastic strain a heterogeneous character thus an anisotropic macroscopic mechanical behavior. The average grains size influences also the Ti-6Al-4V mechanical proprieties, especially the yield stress; by decreasing of the average grains size, the yield strength increases according to Hall-Petch relationship. The grains sizes' distribution gives to the strain fields considerable heterogeneity. By increasing grain sizes, the scattering in the localization of plastic strain is observed, thus, in certain areas the stress concentrations are stronger than other regions.

Keywords: microstructural parameters, multi-scale modeling, crystal plasticity, Ti-6Al-4V alloy

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Authors: Anuschka Curran, Sandra Barnard

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Coral reefs are of the most diverse and productive ecosystems on the planet, but due to the impact of climate change, these infrastructures are dying off primarily through coral bleaching. Coral bleaching can be described as the process by which zooxanthellae (algal endosymbionts) are expelled from the gastrodermal cavity of the respective coral host, causing increased coral whitening. The general consensus is that mass coral bleaching is due to the dysfunction of photosynthetic processes in the zooxanthellae as a result of the combined action of elevated temperature and light-stress. The question then is, do zooxanthellae have the potential to play a key role in the future of coral reef restoration through genetic engineering? The aim of this study is firstly to review the different zooxanthellae taxa and their traits with respect to environmental stress, and secondly, to review the information available on the protective mechanisms present in zooxanthellae cells when experiencing temperature fluctuations, specifically concentrating on heat shock proteins and the antioxidant stress response of zooxanthellae. The eight clades (A-H) previously recognized were redefined into seven genera. Different zooxanthellae taxa exhibit different traits, such as their photosynthetic stress responses to light and temperature. Zooxanthellae have the ability to determine the amount and type of heat shock proteins (hsps) present during a heat response. The zooxanthellae can regulate both the host’s respective hsps as well as their own. Hsps, generally found in genotype C3 zooxanthellae, such as Hsp70 and Hsp90, contribute to the thermal stress response of the respective coral host. Antioxidant activity found both within exposed coral tissue, and the zooxanthellae cells can prevent coral hosts from expelling their endosymbionts. The up-regulation of gene expression, which may mitigate thermal stress induction of any of the physiological aspects discussed, can ensure stable coral-zooxanthellae symbiosis in the future. It presents a viable alternative strategy to preserve reefs amidst climate change. In conclusion, despite their unusual molecular design, genetic engineering poses as a useful tool in understanding and manipulating variables and systems within zooxanthellae and therefore presents a solution that can ensure stable coral-zooxanthellae symbiosis in the future.

Keywords: antioxidant enzymes, genetic engineering, heat-shock proteins, Symbiodinium

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Authors: Siwimol Phoomniyom, Pathom Karaipoom, Rossaphorn Kittyaowaman

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Background: C. trachomatis is the most common bacterial sexually transmitted infections. Although infection with C. trachomatis can be treated with antibiotic, it is frequently asymptomatic, especially in extragenital sites. Hence, if screening tests are not performed, undetected and untreated is a crucial problem for C. trachomatis infection, especially in Thailand, which is less well studied. We sought to assess the prevalence of C. trachomatis infection in multiple anatomical sites among patients attending Bangrak STIs Center. Methods: We examined laboratory results of all patients at baseline visit from 3 January 2018 to 27 December 2019. These results were tested by a validated in-house real time PCR specify for the cryptic plasmid gene of C. trachomatis. The prevalence of C. trachomatis was analyzed by anatomical sites, sexes, and ages. Urogenital samples were obtained from urethral swab of men and cervical swab of women. The median ages of the patients were 32 years (range 13-89 years). Chi-square test by IBM SPSS statistic version 20 was used to assess difference in the distribution of variables between groups. Results: Among 3,789 patients, the prevalence for C. trachomatis infection was the highest in rectal (16.1%), followed by urogenital (11.2%) and pharyngeal (3.5%) sites. Rectal and urogenital infection in men was higher than in women, with the highest prevalence of 16.6% in rectal site. Both rectal and urogenital sites also showed statistically significant differences between sexes (P<0.001). Meanwhile, pharyngeal C. trachomatis infection rate was higher in women than men. Interestingly, the chlamydia prevalence was the highest in age 13-19 years of all three sites (18.5%, urogenital; 17.7%, rectal; 6.5%, pharyngeal), with statistically significant difference between age groups (P<0.001). Total of 45 C. trachomatis infections, 20.0%, 51.1%, and 6.7% were isolated from urogenital, rectal, and pharyngeal sites. In total, 75.6%, 26.7%, and 80.0% of chlamydia infections would have been missed, if only urogenital, rectal, or pharyngeal screening was performed. Conclusions: The highest source of C. trachomatis infection was the rectal site. While, the highest prevalence in men was at rectal site, that in women was at urogenital site. The highest chlamydia prevalence was found in adolescent age group, indicating that the pediatric population was a high-risk group. This finding also elucidated that a high proportion of C. trachomatis infection would be missed, if only single anatomical site screening was performed, especially in extragenital sites. Hence, extragenital screening is also required for the extensive C. trachomatis detection.

Keywords: chlamydia trachomatis, anatomical sites, sexes, ages

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Authors: Shveta Bathla, Preeti Rawat, Sudarshan Kumar, Rubina Baithalu, Jogender Singh Rana, Tushar Kumar Mohanty, Ashok Kumar Mohanty

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Pregnancy is a complex process which includes series of events such as fertilization, formation of blastocyst, implantation of embryo, placental formation and development of fetus. The success of these events depends on various interactions which are synchronized by endocrine interaction between a receptive dam and competent embryo. These interactions lead to change in expression of hormones and proteins. But till date no protein biomarker is available which can be used to detect successful completion of these events. We employed quantitative proteomics approach to develop putative serological biomarker which has diagnostic applicability for early detection of pregnancy in cows. For this study, sera were collected from control (non-pregnant, n=6) and pregnant animals on successive days of pregnancy (7, 19, 45, n=6). The sera were subjected to depletion for removal of albumin using Norgen depletion kit. The tryptic peptides were labeled with iTRAQ. The peptides were pooled and fractionated using bRPLC over 80 min gradient. Then 12 fractions were injected to nLC for identification and quantitation in DDA mode using ESI. Identification using Mascot search revealed 2056 proteins out of which 352 proteins were differentially expressed. Twenty proteins were upregulated and twelve proteins were down-regulated with fold change > 1.5 and < 0.6 respectively (p < 0.05). The gene ontology studies of DEPs using Panther software revealed that majority of proteins are actively involved in catalytic activities, binding and enzyme regulatory activities. The DEP'S such as NF2, MAPK, GRIPI, UGT1A1, PARP, CD68 were further subjected to pathway analysis using KEGG and Cytoscape plugin Cluego that showed involvement of proteins in successful implantation, maintenance of pluripotency, regulation of luteal function, differentiation of endometrial macrophages, protection from oxidative stress and developmental pathways such as Hippo. Further efforts are continuing for targeted proteomics, western blot to validate potential biomarkers and development of diagnostic kit for early pregnancy diagnosis in cows.

Keywords: bRPLC, Cluego, ESI, iTRAQ, KEGG, Panther

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Authors: Abu S. Mustafa, Mohammad W. Khan, Faraz Shaheed Khan, Nazima Habibi

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Brucellosis is a zoonotic disease of worldwide prevalence. There are at least four species and several strains of Brucella that cause human disease. Brucella genomes have very limited variation across strains, which hinder strain identification using classical molecular techniques, including PCR and 16 S rDNA sequencing. The aim of this study was to perform whole genome sequencing of clinical isolates of Brucella and perform bioinformatics and comparative genomics analyses to determine the existence of genetic differences across the isolates of a single Brucella species and strain. The draft sequence data were generated from 15 clinical isolates of Brucella melitensis (biovar 2 strain 63/9) using MiSeq next generation sequencing platform. The generated reads were used for further assembly and analysis. All the analysis was performed using Bioinformatics work station (8 core i7 processor, 8GB RAM with Bio-Linux operating system). FastQC was used to determine the quality of reads and low quality reads were trimmed or eliminated using Fastx_trimmer. Assembly was done by using Velvet and ABySS softwares. The ordering of assembled contigs was performed by Mauve. An online server RAST was employed to annotate the contigs assembly. Annotated genomes were compared using Mauve and ACT tools. The QC score for DNA sequence data, generated by MiSeq, was higher than 30 for 80% of reads with more than 100x coverage, which suggested that data could be utilized for further analysis. However when analyzed by FastQC, quality of four reads was not good enough for creating a complete genome draft so remaining 11 samples were used for further analysis. The comparative genome analyses showed that despite sharing same gene sets, single nucleotide polymorphisms and insertions/deletions existed across different genomes, which provided a variable extent of diversity to these bacteria. In conclusion, the next generation sequencing, bioinformatics, and comparative genome analysis can be utilized to find variations (point mutations, insertions and deletions) across different genomes of Brucella within a single strain. This information could be useful in surveillance and epidemiological studies supported by Kuwait University Research Sector grants MI04/15 and SRUL02/13.

Keywords: brucella, bioinformatics, comparative genomics, whole genome sequencing

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Authors: Virve Pääkkönen, Heidi M. Cuffaro, Leo Tjäderhane

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Objectives: Odontoblasts are the outermost cell layer of dental pulp and form the dentin. Importance of bacterial products, e.g. lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria and lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, have been indicated in the pathogenesis of pulpitis. Gram-positive bacteria are more prevalent in superficial carious lesion while the amount gram-negative is higher in the deep lesions. Objective of this study was to investigate the effect of these bacterial products on inflammatory response of pulp tissue. Interleukins (IL) were of special interest. Various ILs have been observed in the dentin-pulp complex of carious tooth in vivo. Methods: Tissue culture method was used for testing the effect of LTA and LPS on human odontoblasts. Enzymatic isolation technique was used to extract living odontoblasts for cell cultures. DNA microarray and quantitative PCR (qPCR) were used to characterize the changes in the expression profile of the tissue cultured odontoblasts. Laser microdissection was used to cut healthy and affected dentin and odontoblast layer directly under carious lesion for experiments. Cytokine array detecting 80 inflammatory cytokines was used to analyze the protein content of conditioned culture media as well as dentin and odontoblasts from the carious teeth. Results: LPS caused increased gene expression IL-1α, and -8 and decrease of IL-1β, 12 , -15 and -16 after 1h treatment, while after 24h treatment decrease of IL-8, -11 and 23 mRNAs was observed. LTA treatment caused cell death in the tissue cultured odontoblasts but in in the cell culture but not in cell culture. Cytokine array revealed at least 2-fold down-regulation of IL-1β, -10 and -12 in response to LPS treatment. Cytokine array of odontoblasts of carious teeth, as well as LPS-treated tissue-cultured odontoblasts, revealed increased protein amounts of IL-16, epidermal growth factor (EGF), angiogenin and IGFBP-1 as well as decreased amount of fractalkine. In carious dentin, increased amount of IL-1β, EGF and fractalkine was observed, as well as decreased level of GRO-1 and HGF. Conclusion: LPS caused marked changes in the expression of inflammatory cytokines in odontoblasts. Similar changes were observed in the odontoblasts cut directly under the carious lesion. These results help to shed light on the inflammatory processes happening during caries.

Keywords: inflammation, interleukin, lipoteichoic acid, odontoblasts

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Authors: Moin Uddin, M. Masroor A. Khan, Faisal Rasheed, Tariq Ahmad Dar, Akbar Ali, Lalit Varshney

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Oligomers, obtained by exposing the natural polysaccharides (alginate, carrageenan, chitosan, etc.) to cobalt-60 generated gamma radiation may prove as potent plant growth promoters when applied as foliar sprays to the plants. They function as endogenous growth elicitors, triggering the synthesis of different enzymes and modulating various plant responses by exploiting the gene expression. Exogenous application of Jasmonic acid or of its methyl ester, methyl jasmonate (MeJ) has been reported to increase the secondary metabolites production in medicinal and aromatic plants. Keeping this in mind, three pot experiments were conducted to test whether the foliar application of irradiated-chitosan (IC) and MeJ, applied alone or in combination, could augment the active constituents as well as growth, physiological and yield attributes of Catharanthus roseus, which carries anticancer alkaloids, viz. vincristine and vinblastine, in its leaves in addition to various other useful alkaloids. Totally, 5 spray treatments, comprising various aqueous solutions of IC [20, 40, 80 and 160 mg L-1 (Experiment 1)], MeJ (10, 20, 30 and 40 mg L-1 (Experiment 2)] and those of IC+MeJ [40+20, 40+30, 80+20, 80+30, 160+20 and 160+30 mg L-1 (Experiment 3)], were applied at seven days interval. Total leaf-alkaloids content as well as growth, physiological and yield parameters, evaluated at 120 days after sowing, were significantly enhanced by IC application. IC application could not increase the leaf-content of vincristine and vinblastine; nonetheless, it significantly augmented the yield of these alkaloids owing to enhancing the dry mass of leaves per plant. MeJ application, particularly at 30 mg L-1, increased both content (17%) and yield (48%) of total leaf-alkaloids as well as the content and yield of vincristine ( 29 and 63%, respectively) and vinblastine (14 and 44%, respectively) alkaloids, though it significantly decreased most other parameters studied, particularly at higher concentrations (30 and 40 mg L-1 of MeJ). As compared to the control (water-spray treatment), collective application of IC (80 mg L-1) and MeJ (20 mg L-1) resulted in the highest values of most of the parameters studied. However, 80 mg L-1 of IC applied with 30 mg L-1 of MeJ gave the best results for the content and yield of total as well as anticancer leaf-alkaloids (vincristine and vinblastine). Comparing the control, it increased the content and yield of total leaf-alkaloids (37 and 118%, respectively) and those of vincristine (65 and 163%, respectively) and vinblastine (31 and 107%, respectively). Conclusively, the applied technique significantly enhanced the production of total as well as anticancer alkaloids of Catharanthus roseus.

Keywords: anticancer alkaloids (vincristine and vinblastine), catharanthus roseus, irradiated chitosan, methyl jasmonate

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Authors: Shaimaa Eltanani, Thangal Yumnamcha, Ahmed S. Ibrahim

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Purpose: Mitochondria dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to dissect the role of different mitochondrial components, specifically, those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs. Methods: Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real-time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components; the capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: Rotenone for complex I; Oligomycin for ATP synthase; and FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: cell-cell interactions (Rb), cell-matrix interactions (α), and cell membrane permeability (Cm). Results: Rotenone (1 µM) produced the greatest reduction in the Z, followed by FCCP (1 µM), whereas no reduction in the Z was observed after the treatment with Oligomycin (1 µM). Following this further, we deconvoluted the effect of these inhibitors on Rb, α, and Cm. Firstly, rotenone (1 µM) completely abolished the resistance contribution of Rb, as the Rb became zero immediately after the treatment. Secondly, FCCP (1 µM) eliminated the resistance contribution of Rb only after 2.5 hours and increased Cm without considerable effect on α. Lastly, Oligomycin had the lowest impact among these inhibitors on Rb, which became similar to the control group at the end of the experiment without noticeable effects on Cm or α. Conclusion: These results demonstrate differential roles for complex I, complex V, and coupling of OxPhos in maintaining the barrier functionality of HRECs, in which complex I being the most important component in regulating the barrier functionality and the spreading behavior of HRECs. Such differences can be used in investigating gene expression as well as for screening selective agents that improve the functionality of complex I to be used in the therapeutic approach for treating REC-related retinal diseases.

Keywords: human retinal endothelial cells (hrecs), rotenone, oligomycin, fccp, oxidative phosphorylation, oxphos, capacitance, impedance, ecis modeling, rb resistance, α resistance, and barrier integrity

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Authors: Yelin Zhao, Xinxiu Li, Martin Smelik, Oleg Sysoev, Firoj Mahmud, Dina Mansour Aly, Mikael Benson

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Background: Early diagnosis of pancreatic cancer is clinically challenging due to vague, or no symptoms, and lack of biomarkers. Polygenic risk score (PRS) scores may provide a valuable tool to assess increased or decreased risk of PC. This study aimed to develop such PRS by filtering genetic variants identified by GWAS using transcriptional programs identified by single-cell RNA sequencing (scRNA-seq). Methods: ScRNA-seq data from 24 pancreatic ductal adenocarcinoma (PDAC) tumor samples and 11 normal pancreases were analyzed to identify differentially expressed genes (DEGs) in in tumor and microenvironment cell types compared to healthy tissues. Pathway analysis showed that the DEGs were enriched for hundreds of significant pathways. These were clustered into 40 “programs” based on gene similarity, using the Jaccard index. Published genetic variants associated with PDAC were mapped to each program to generate program PRSs (pPRSs). These pPRSs, along with five previously published PRSs (PGS000083, PGS000725, PGS000663, PGS000159, and PGS002264), were evaluated in a European-origin population from the UK Biobank, consisting of 1,310 PDAC participants and 407,473 non-pancreatic cancer participants. Stepwise Cox regression analysis was performed to determine associations between pPRSs with the development of PC, with adjustments of sex and principal components of genetic ancestry. Results: The PDAC genetic variants were mapped to 23 programs and were used to generate pPRSs for these programs. Four distinct pPRSs (P1, P6, P11, and P16) and two published PRSs (PGS000663 and PGS002264) were significantly associated with an increased risk of developing PC. Among these, P6 exhibited the greatest hazard ratio (adjusted HR[95% CI] = 1.67[1.14-2.45], p = 0.008). In contrast, P10 and P4 were associated with lower risk of developing PC (adjusted HR[95% CI] = 0.58[0.42-0.81], p = 0.001, and adjusted HR[95% CI] = 0.75[0.59-0.96], p = 0.019). By comparison, two of the five published PRS exhibited an association with PDAC onset with HR (PGS000663: adjusted HR[95% CI] = 1.24[1.14-1.35], p < 0.001 and PGS002264: adjusted HR[95% CI] = 1.14[1.07-1.22], p < 0.001). Conclusion: Compared to published PRSs, scRNA-seq-based pPRSs may be used not only to assess increased but also decreased risk of PDAC.

Keywords: cox regression, pancreatic cancer, polygenic risk score, scRNA-seq, UK biobank

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Authors: Daria Reczynska, Justyna Jarczak, Michal Czopowicz, Danuta Sloniewska, Karina Horbanczuk, Wieslaw Jarmuz, Jaroslaw Kaba, Emilia Bagnicka

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Since people, animals and plants are constantly exposed to pathogens they have developed very complex systems of defense. Among ca. 1000 antimicrobial peptides from different families so far identified, approximately 30 belonging to cathelicidin family can be found in mammals. Cathelicidins probably constitute the first line of defense because they can act at a physiological salt concentration which is present in healthy tissues. Moreover, the low salt concentration which is present in infected tissues inhibits their activity. In goat bactenecin 7.5 (BAC7.5), bactenecin 5 (BAC5), myeloid antimicrobial peptide 28 (MAP28), myeloid antimicrobial peptide 34 (MAP34 A and B), goat bactenecin3.4 (ChBac3.4) were identified. Caprine arthritis-encephalitis (CAE) caused by small ruminant lentivirus (SRLV) is economic problem. The main CAE symptoms are weight loss, arthritis, pneumonia and mastitis (significant elevation of the somatic cell count and deterioration of some technological parameters). The study was conducted on 24 dairy goats. The animals were divided into two groups: experimental (SRLV-infected) and control (non-infected). The blood samples were collected five times: on the 1st, 7th, 30th, 90th and 150thday of lactation. The levels of transcripts of BAC7.5, BAC5, MAP28 and MAP34 genes in blood leucocytes were measured using qPCR method. There were no differences in mRNA levels of studied genes between stages of lactation. The differences were observed in expressions of BAC5, MAP28 and MAP34 genes with lower levels in the experimental group. There was no difference in BAC7.5 expression between groups. The decreased levels of transcripts of cathelicidin genes in blood leucocytes of SRLV-infected goats may indicate the disturbances of homeostasis in organisms. It can be concluded that SRLV infection seems to inhibit expression of cathelicidin genes. The study was financed by a grant from the National Scientific Center No. UMO-2013/09/B/NZ/03514.

Keywords: goat, CAEV, cathelicidins, blood leukocytes, gene expression

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Authors: Rijnbout-St.James Willem, Lindner Volkhard, Scholand Mary Beth, Ashton M. Tillett, Di Gennaro Michael Jude, Smith Silvia Enrica

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Introduction: Fibrosing lung diseases are characterized by changes in the lung interstitium and are classified based on etiology: 1) environmental/exposure-related, 2) autoimmune-related, 3) sarcoidosis, 4) interstitial pneumonia, and 4) idiopathic. Among interstitial lung diseases (ILD) idiopathic forms, idiopathic pulmonary fibrosis (IPF) is the most severe. Pathogenesis of IPF is characterized by an increased presence of proinflammatory mediators, resulting in alveolar injury, where injury to alveolar epithelium precipitates an increase in collagen deposition, subsequently thickening the alveolar septum and decreasing gas exchange. Identifying biomarkers implicated in the pathogenesis of lung fibrosis is key to developing new therapies and improving the efficacy of existing therapies. The transforming growth factor-beta (TGF-B1), a mediator of tissue repair associated with WNT5A signaling, is partially responsible for fibroblast proliferation in ILD and is the target of Pirfenidone, one of the antifibrotic therapies used for patients with IPF. Canonical TGF-B signaling is mediated by the proteins SMAD 2/3, which are, in turn, indirectly regulated by Collagen Triple Helix Repeat Containing 1 (CTHRC1). In this study, we tested the following hypotheses: 1) CTHRC1 is more elevated in the ILD cohort compared to unaffected controls, and 2) CTHRC1 is differently expressed among ILD types. Material and Methods: CTHRC1 levels were measured by ELISA in 171 plasma samples from the deidentified University of Utah ILD cohort. Data represent a cohort of 131 ILD-affected participants and 40 unaffected controls. CTHRC1 samples were categorized by a pulmonologist based on affectation status and disease subtypes: IPF (n = 45), sarcoidosis (4), nonspecific interstitial pneumonia (16), hypersensitivity pneumonitis (n = 7), interstitial pneumonia (n=13), autoimmune (n = 15), other ILD - a category that includes undifferentiated ILD diagnoses (n = 31), and unaffected controls (n = 40). We conducted a single-factor ANOVA of plasma CTHRC1 levels to test whether CTHRC1 variance among affected and non-affected participants is statistically significantly different. In-silico analysis was performed with Ingenuity Pathway Analysis® to characterize the role of CTHRC1 in the pathway of lung fibrosis. Results: Statistical analyses of CTHRC1 in plasma samples indicate that the average CTHRC1 level is significantly higher in ILD-affected participants than controls, with the autoimmune ILD being higher than other ILD types, thus supporting our hypotheses. In-silico analyses show that CTHRC1 indirectly activates and phosphorylates SMAD3, which in turn cross-regulates TGF-B1. CTHRC1 also may regulate the expression and transcription of TGFB-1 via WNT5A and its regulatory relationship with CTNNB1. Conclusion: In-silico pathway analyses demonstrate that CTHRC1 may be an important biomarker in ILD. Analysis of plasma samples indicates that CTHRC1 expression is positively associated with ILD affectation, with autoimmune ILD having the highest average CTHRC1 values. While characterizing CTHRC1 levels in plasma can help to differentiate among ILD types and predict response to Pirfenidone, the extent to which plasma CTHRC1 level is a function of ILD severity or chronicity is unknown.

Keywords: interstitial lung disease, CTHRC1, idiopathic pulmonary fibrosis, pathway analyses

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Authors: N. N. Pandey, Raghvendra Singh, Biju S. Kamlam, Bipin K. Vishwakarma, Preetam Kala

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The rainbow trout (Oncorhynchus mykiss) is an exotic salmonid fish, well known for its fast growth, tremendous ability to thrive in diverse conditions, delicious flesh and hard fighting nature in Europe and other countries. Rainbow trout farming has a great potential for its contribution to the mainstream economy of Himalayan states in India and other temperate countries. These characteristics establish them as one of the most widely introduced and cultured fish across the globe, and its farming is also prominent in the cold water regions of India. Nevertheless, genetic fatigue, slow growth, early maturity, and low productivity are limiting the expansion of trout production. Moreover, farms adjacent to natural streams or other water sources are subject to escape of domesticated rainbow trout into the wild, which is a serious environmental concern as the escaped fish is subject to contaminate and disrupt the receiving ecosystem. A decline in production traits due to early maturity prolongs the culture duration and affects the profit margin of rainbow trout farms in India. A viable strategy that could overcome these farming constraints in large scale operation is the production of triploid fish that are sterile and more heterozygous. For better triploidy induction rate (TR), heat shock at 28°C for 10 minutes and pressure shock 9500 psi pressure for 5 minutes is applied to green eggs with 90-100% of triploidy success and 72-80% survival upto swim-up fry stage. There is 20% better growth in aquaculture with triploids rainbow trout over diploids. As compared to wild diploid fish, larger sized and fitter triploid rainbow trout in natural waters attract to trout anglers, and support the development of recreational fisheries by state fisheries departments without the risk of contaminating existing gene pools and disrupting local fish diversity. Overall, enhancement of productivity in rainbow trout farms and trout production in coldwater regions, development of lucrative trout angling and better ecological management is feasible with triploid rainbow trout.

Keywords: rainbow trout, triploids fish, heat shock, pressure shock, trout angling

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Authors: Basma Hassabo, Sarah Ahmed, Aisha Hameed

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Aims and Objectives: To determine the incidence of maternal mortality amongst pregnant women with sickle cell disease (SCD) in the United Kingdom and to determine exact cause of death in these women. Background: SCD is caused by the ‘sickle’ gene and is characterized by episodes of severe bone pain and other complications like acute chest syndrome, chronic pulmonary hypertension, stroke, retinopathy, chronic renal failure, hepato-splenic crises, avascular bone necrosis, sepsis and leg ulcers. SCD is a continual cause of maternal mortality and fetal complications, and it comprises 1.5% of all Direct and Indirect deaths in the UK. Sepsis following premature rupture of membranes with ascending infection, post-partum infection and pre-labour overwhelming septic shock is one of its leading causes of death. Over the last fifty years of maternal mortality reports in UK, between 1 to 4 pregnant women died in each triennium. Material and Method: This is a retrospective study that involves pregnant women who died from SCD complications in the UK between 1952-2012. Data were collected from the UK Confidential Enquiries into Maternal Death and its causes between 1952–2012. Prior to 1985, exact cause of death in this cohort was not recorded. Results: 33 deaths reported between 1964 and 1984. 17 deaths were reported due to sickle cell disease between 1985 and 2012. Five women in this group died of sickle cell crisis, one woman had liver sequestration crisis, two women died of venous thromboembolism, two had myocardial fibrosis and three died of sepsis. Remaining women died of amniotic fluid embolism, SUDEP, myocardial ischemia and intracranial haemorrhage. Conclusion: The leading causes of death in sickle cell sick pregnant women are sickle cell crises, sepsis, venous thrombosis and thromboembolism. Prenatal care for women with SCD should be managed by a multidisciplinary team that includes an obstetrician, nutritionist, primary care physician, and haematologist. In every sick Sickle Cell woman Sickle Cell crises should be on the top of the list of differential diagnosis. Aggressive treatment of complications with low threshold to commence broad-spectrum antibiotics and LMWH contribute to better outcomes.

Keywords: incidence, maternal mortality, sickle cell disease (SCD), uk

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Authors: Rafael Mineiro, André Furtado, Isabel Gonçalves, Cecília Santos, Telma Quintela

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The choroid plexus (CP) epithelial cells form the blood-cerebrospinal fluid barrier. This barrier is highly important for brain protection by physically separating the blood from the cerebrospinal fluid, controlling the trafficking of molecules, including therapeutic drugs, from blood to the brain. The control is achieved by tight junctions between epithelial cells, membrane receptors and transport proteins from the solute carrier and ATP-binding cassette superfamily on the choroid plexus epithelial cells membrane. Previous research of our group showed a functional molecular clock in the CP. The key findings included a rhythmic expression of Bmal1, Per2, and Cry2 in female rat CP. and a rhythmic expression of Cry2 and Per2 in male rat CP. Furthermore, in cultured rat CP epithelial cells we already showed that 17β-estradiol upregulates the expression of Bmal1 and Per1, where the Per1 and Per2 upregulation was abrogated in the presence of the estrogen receptors antagonist ICI. These findings, together with the fact that the CP produces robust rhythms, prompt us to understand the impact of sex hormones and circadian rhythms in CP drug transporters expression, which is a step towards the development and optimization of therapeutic strategies for efficiently delivering drugs to the brain. For that, we analyzed the circadian rhythmicity of the Abcb1, Abcc2, Abcc4 Abcg2, and Oat3 drug transporters at the CP of male and female rats. This analysis was performed by accessing the gene expression of the mentioned transporters at 4 time points by RT-qPCR and the presence of rhythms was evaluated by the CircWave software. Our findings showed a rhythmic expression of Abcc1 in the CP of male rats, of Abcg2 in female rats, and of Abcc4 and Oat3 in both male and female rats with an almost antiphasic pattern between male and female rats for Abcc4. In conclusion, these findings translated to a functional point of view may account for daily variations in brain permeability for several therapeutic drugs, making our findings important data for the future establishment and development of therapeutic strategies according to daytime.

Keywords: choroid plexus, circadian rhythm, membrane transporters, sex hormones

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Authors: Roshni Raha, Karthikeyan S.

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The world's largest livestock population resides in India. Existing strategies must be modified to increase the production of livestock and their by-products in order to meet the demands of the growing human population. Even though India leads the world in both milk production and the number of cows, average production is not very healthy and productive. This may be due to the animals' poor nutrition caused by a chronic under-availability of high-quality fodder and feed. This article explores Azolla pinnata to be a promising source to produce high-quality unconventional feed and fodder for effective livestock production and good quality breeding in India. This article is an exploratory study using a literature survey and experimentation analysis. In the realm of agri-biotechnology, azolla sp gained attention for helping farmers achieve sustainability, having minimal land requirements, and serving as a feed element that doesn't compete with human food sources. It has high methionine content, which is a good source of protein. It can be easily digested as the lignin content is low. It has high antioxidants and vitamins like beta carotene, vitamin A, and vitamin B12. Using this concept, the paper aims to investigate and develop a model of using azolla plants as a novel, high-potential feed source to combat the problems of low production and poor quality of animals in India. A representative sample of animal feed is collected where azolla is added. The sample is ground into a fine powder using mortar. PITC (phenylisothiocyanate) is added to derivatize the amino acids. The sample is analyzed using HPLC (High-Performance Liquid Chromatography) to measure the amino acids and monitor the protein content of the sample feed. The amino acid measurements from HPLC are converted to milligrams per gram of protein using the method of amino acid profiling via a set of calculations. The amino acid profile data is then obtained to validate the proximate results of nutrient enhancement of the composition of azolla in the sample. Based on the proximate composition of azolla meal, the enhancement results shown were higher compared to the standard values of normal fodder supplements indicating the feed to be much richer and denser in nutrient supply. Thus azolla fed sample proved to be a promising source for animal fodder. This would in turn lead to higher production and a good breed of animals that would help to meet the economic demands of the growing Indian population. Azolla plants have no side effects and can be considered as safe and effective to be immersed in the animal feed. One area of future research could begin with the upstream scaling strategy of azolla plants in India. This could involve introducing several bioreactor types for its commercial production. Since azolla sp has been proved in this paper as a promising source for high quality animal feed and fodder, large scale production of azolla plants will help to make the process much quicker, more efficient and easily accessible. Labor expenses will also be reduced by employing bioreactors for large-scale manufacturing.

Keywords: azolla, fodder, nutrient, protein

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Authors: Ahmed Bahieldin, Ahmed Atef, Jamal S. M. Sabir, Nour O. Gadalla, Sherif Edris, Ahmed M. Alzohairy, Nezar A. Radhwan, Mohammed N. Baeshen, Ahmed M. Ramadan, Hala F. Eissa, Sabah M. Hassan, Nabih A. Baeshen, Osama Abuzinadah, Magdy A. Al-Kordy, Fotouh M. El-Domyati, Robert K. Jansen

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Wild salt-tolerant barley (Hordeum spontaneum) is the ancestor of cultivated barley (Hordeum vulgare or H. vulgare). Although the cultivated barley genome is well studied, little is known about genome structure and function of its wild ancestor. In the present study, RNA-Seq analysis was performed on young leaves of wild barley treated with salt (500 mM NaCl) at four different time intervals. Transcriptome sequencing yielded 103 to 115 million reads for all replicates of each treatment, corresponding to over 10 billion nucleotides per sample. Of the total reads, between 74.8 and 80.3% could be mapped and 77.4 to 81.7% of the transcripts were found in the H. vulgare unigene database (unigene-mapped). The unmapped wild barley reads for all treatments and replicates were assembled de novo and the resulting contigs were used as a new reference genome. This resultedin94.3 to 95.3%oftheunmapped reads mapping to the new reference. The number of differentially expressed transcripts was 9277, 3861 of which were uni gene-mapped. The annotated unigene- and de novo-mapped transcripts (5100) were utilized to generate expression clusters across time of salt stress treatment. Two-dimensional hierarchical clustering classified differential expression profiles into nine expression clusters, four of which were selected for further analysis. Differentially expressed transcripts were assigned to the main functional categories. The most important groups were ‘response to external stimulus’ and ‘electron-carrier activity’. Highly expressed transcripts are involved in several biological processes, including electron transport and exchanger mechanisms, flavonoid biosynthesis, reactive oxygen species (ROS) scavenging, ethylene production, signaling network and protein refolding. The comparisons demonstrated that mRNA-Seq is an efficient method for the analysis of differentially expressed genes and biological processes under salt stress.

Keywords: electron transport, flavonoid biosynthesis, reactive oxygen species, rnaseq

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Authors: Daniela E. Marin, Cornelia Braicu, Gina C. Pistol, Roxana Cojocneanu-Petric, Ioana Berindan Neagoe, Mihail A. Gras, Ionelia Taranu

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Aristolochic acid (AA) is a carcinogenic, mutagenic, and nephrotoxic compound commonly found in the Aristolochiaceae family of plants. AA is frequently associated with urothelial carcinoma of the upper urinary tract in human and animals and is considered as being responsible for Balkan Endemic Nephropathy. The pig provides a good animal model because the porcine urological system is very similar to that of humans, both in aspects of physiology and anatomy. MicroRNA (miRNA) are small non-coding RNAs that have an impact on a wide range of biological processes by regulating gene expression at post-transcriptional level. The objective of this study was to analyze the miRNA profiling in the kidneys of AA intoxicated swine. For this purpose, ten TOPIGS-40 crossbred weaned piglets, 4-week-old, male and females with an initial average body weight of 9.83 ± 0.5 kg were studied for 28 days. They were given ad libitum access to water and feed and randomly allotted to one of the following groups: control group (C) or aristolochic acid group (AA). They were fed a maize-soybean-meal-based diet contaminated or not with 0.25mgAA/kg. To profile miRNA in the kidneys of pigs, microarrays and bioinformatics approaches were applied to analyze the miRNA in the kidney of control and AA intoxicated pigs. After normalization, our results have shown that a total of 5 known miRNAs and 4 novel miRNAs had different profiling in the kidney of intoxicated animals versus control ones. Expression of miR-32-5p, miR-497-5p, miR-423-3p, miR-218-5p, miR-128-3p were up-regulated by 0.25mgAA/kg feed, while the expression of miR-9793-5p, miR-9835-3p, miR-9840-3p, miR-4334-5p was down-regulated. The microRNA profiling in kidney of intoxicated animals was associated with modified expression of target genes as: RICTOR, LASP1, SFRP2, DKK2, BMI1, RAF1, IGF1R, MAP2K1, WEE1, HDGF, BCL2, EIF4E etc, involved in cell division cycle, apoptosis, cell differentiation and cell migration, cell signaling, cancer etc. In conclusion, this study provides new data concerning the microRNA profiling in kidney after aristolochic acid intoxications with important implications for human and animal health.

Keywords: aristolochic acid, kidney, microRNA, swine

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Authors: Madhusudana S. N., Reeta Mani, Ashwini S. Satishchandra P., Netravati, Udhani V., Fiaz A., Karande S.

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Rabies is an acute encephalitis which is considered 100% fatal despite occasional reports of survivors. However, in recent times more cases of human rabies survivors are being reported. In the last 5 years, there are six laboratories confirmed human rabies survivors in India alone. All cases were children below 15 years and all contracted the disease by dog bites. All of them also had received the full or partial course of rabies vaccination and 4 out of 6 had also received rabies immunoglobulin. All cases were treated in intensive care units in hospitals at Bangalore, Mumbai, Chandigarh, Lucknow and Goa. We report here the results of immunological and virological studies conducted at our laboratory on these patients. The clinical samples that were obtained from these patients were Serum, CSF, nuchal skin biopsy and saliva. Serum and CSF samples were subjected to standard RFFIT for estimation of rabies neutralizing antibodies. Skin biopsy, CSF and saliva were processed by TaqMan real-time PCR for detection of viral RNA. CSF, saliva and skin homogenates were also processed for virus isolation by inoculation of suckling mice. The PBMCs isolated from fresh blood was subjected to ELISPOT assay to determine the type of immune response (Th1/Th2). Both CSF and serum were also investigated for selected cytokines by Luminex assay. The level of antibodies to virus G protein and N protein were determined by ELISA. All survivors had very high titers of RVNA in serum and CSF 100 fold higher than non-survivors and vaccine controls. A five-fold rise in titer could be demonstrated in 4 out of 6 patients. All survivors had a significant increase in antibodies to G protein in both CSF and serum when compared to non-survivors. There was a profound and robust Th1 response in all survivors indicating that interferon gamma could play an important factor in virus clearance. We could isolate viral RNA in only one patient four years after he had developed symptoms. The partial N gene sequencing revealed 99% homology to species I strain prevalent in India. Levels of selected cytokines in CSF and serum did not reveal any difference between survivors and non-survivors. To conclude, survival from rabies is mediated by virus-specific immune responses of the host and clearance of rabies virus from CNS may involve the participation of both Th2 and Th1 immune responses.

Keywords: rabies, rabies treatment, rabies survivors, immune reponse in rabies encephalitis

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Authors: Sweta Pandey, Samridhi Dhyani, Susmita Chaudhuri

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Klebsiella pneumoniae bacteria is a global threat to human health due to an increase in multi-drug resistance among strains. The hypervirulent strains of Klebsiella pneumoniae is a major trouble due to their association with life-threatening infections in a healthy population. One of the major virulence factors of hyper virulent strains of Klebsiella pneumoniae is the T6SS (Type six secretary system) which is majorly involved in microbial antagonism and causes interaction with the host eukaryotic cells during infections. T6SS mediates some of the crucial factors for establishing infection by the bacteria, such as cell adherence, invasion, and subsequent in vivo colonisation. The antibacterial activity and the cell invasion property of the T6SS system is a major requirement for the establishment of K. pneumoniae infections within the gut. The T6SS can be an appropriate target for developing therapeutics. The T6SS consists of an inner tube comprising hexamers of Hcp (Haemolysin -regulated protein) protein, and at the top of this tube sits VgrG (Valine glycine repeat protein G); the tip of the machinery consists of PAAR domain containing proteins which act as a delivery system for bacterial effectors. For this study, immune response to recombinant VgrG protein was generated to establish this protein as a potential immunogen for the development of therapeutic leads. The immunogenicity of the selected protein was determined by predicting the B cell epitopes by the BCEP analysis tool. The gene sequence for multiple domains of VgrG protein (phage_base_V, T6SS_Vgr, DUF2345) was selected and cloned in pMAL vector in E. coli. The construct was subcloned and expressed as a fusion protein of 203 residue protein with mannose binding protein tag (MBP) to enhance solubility and purification of this protein. The purified recombinant VgrG fusion protein was used for mice immunisation. The antiserum showed reactivity with the recombinant VgrG in ELISA and western blot. The immunised mice were challenged with K. pneumoniae bacteria and showed bacterial clearance in immunised mice. The recombinant VgrG protein can further be used for studying downstream signalling of VgrG protein in mice during infection and for therapeutic MAb development to eradicate K. pneumoniae infections.

Keywords: immune response, Klebsiella pneumoniae, multi-drug resistance, recombinant protein expression, T6SS, VgrG

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Authors: Camelia Vizireanu, Daniela Istrati, Alina Georgiana Profir, Rodica Mihaela Dinica

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Globally, there is a great interest in promoting the consumption of fruit and vegetables to improve health. Due to the content of essential compounds such as antioxidants, important amounts of fruits and vegetables should be included in the daily diet. Juices are good sources of vitamins and can also help increase overall fruit and vegetable consumption. Starting from this trend (introduction into the daily diet of vegetables and fruits) as well as the desire to diversify the range of functional products for both adults and children, a fermented juice was made using probiotic microorganisms based on root vegetables, with potential beneficial effects in the diet of children, vegetarians and people with lactose intolerance. The three vegetables selected for this study, red beet, carrot, and celery bring a significant contribution to functional compounds such as carotenoids, flavonoids, betalain, vitamin B and C, minerals and fiber. By fermentation, the functional value of the vegetable juice increases due to the improved stability of these compounds. The combination of probiotic microorganisms and vegetable fibers resulted in a nutrient-rich synbiotic product. The stability of the nutritional and sensory qualities of the obtained synbiotic product has been tested throughout its shelf life. The evaluation of the physico-chemical changes of the synbiotic drink during storage confirmed that: (i) vegetable juice enriched with honey and vegetable pulp is an important source of nutritional compounds, especially carbohydrates and fiber; (ii) microwave treatment used to inhibit pathogenic microflora did not significantly affect nutritional compounds in vegetable juice, vitamin C concentration remained at baseline and beta-carotene concentration increased due to increased bioavailability; (iii) fermentation has improved the nutritional quality of vegetable juice by increasing the content of B vitamins, polyphenols and flavonoids and has a good antioxidant capacity throughout the shelf life; (iv) the FTIR and Raman spectra have highlighted the results obtained using physicochemical methods. Based on the analysis of IR absorption frequencies, the most striking bands belong to the frequencies 3330 cm⁻¹, 1636 cm⁻¹ and 1050 cm⁻¹, specific for groups of compounds such as polyphenols, carbohydrates, fatty acids, and proteins. Statistical data processing revealed a good correlation between the content of flavonoids, betalain, β-carotene, ascorbic acid and polyphenols, the fermented juice having a stable antioxidant activity. Also, principal components analysis showed that there was a negative correlation between the evolution of the concentration of B vitamins and antioxidant activity. Acknowledgment: This study has been founded by the Francophone University Agency, Project Réseau régional dans le domaine de la santé, la nutrition et la sécurité alimentaire (SaIN), No. at Dunarea de Jos University of Galati 21899/ 06.09.2017 and by the Sectorial Operational Programme Human Resources Development of the Romanian Ministry of Education, Research, Youth and Sports trough the Financial Agreement POSDRU/159/1.5/S/132397 ExcelDOC.

Keywords: bioactive compounds, fermentation, synbiotic drink from vegetables, stability during storage

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Authors: Osman Adiguzel

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Shape-memory alloys are a new class of functional materials with a peculiar property known as shape memory effect. These alloys return to a previously defined shape on heating after deformation in low temperature product phase region and take place in a class of functional materials due to this property. The origin of this phenomenon lies in the fact that the material changes its internal crystalline structure with changing temperature. Shape memory effect is based on martensitic transitions, which govern the remarkable changes in internal crystalline structure of materials. Martensitic transformation, which is a solid state phase transformation, occurs in thermal manner in material on cooling from high temperature parent phase region. This transformation is governed by changes in the crystalline structure of the material. Shape memory alloys cycle between original and deformed shapes in bulk level on heating and cooling, and can be used as a thermal actuator or temperature-sensitive elements due to this property. Martensitic transformations usually occur with the cooperative movement of atoms by means of lattice invariant shears. The ordered parent phase structures turn into twinned structures with this movement in crystallographic manner in thermal induced case. The twinned martensites turn into the twinned or oriented martensite by stressing the material at low temperature martensitic phase condition. The detwinned martensite turns into the parent phase structure on first heating, first cycle, and parent phase structures turn into the twinned and detwinned structures respectively in irreversible and reversible memory cases. On the other hand, shape memory materials are very important and useful in many interdisciplinary fields such as medicine, pharmacy, bioengineering, metallurgy and many engineering fields. The choice of material as well as actuator and sensor to combine it with the host structure is very essential to develop main materials and structures. Copper based alloys exhibit this property in metastable beta-phase region, which has bcc-based structures at high temperature parent phase field, and these structures martensitically turn into layered complex structures with lattice twinning following two ordered reactions on cooling. Martensitic transition occurs as self-accommodated martensite with inhomogeneous shears, lattice invariant shears which occur in two opposite directions, <110 > -type directions on the {110}-type plane of austenite matrix which is basal plane of martensite. This kind of shear can be called as {110}<110> -type mode and gives rise to the formation of layered structures, like 3R, 9R or 18R depending on the stacking sequences on the close-packed planes of the ordered lattice. In the present contribution, x-ray diffraction and transmission electron microscopy (TEM) studies were carried out on two copper based alloys which have the chemical compositions in weight; Cu-26.1%Zn 4%Al and Cu-11%Al-6%Mn. X-ray diffraction profiles and electron diffraction patterns reveal that both alloys exhibit super lattice reflections inherited from parent phase due to the displacive character of martensitic transformation. X-ray diffractograms taken in a long time interval show that locations and intensities of diffraction peaks change with the aging time at room temperature. In particular, some of the successive peak pairs providing a special relation between Miller indices come close each other.

Keywords: Shape memory effect, martensite, twinning, detwinning, self-accommodation, layered structures

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Authors: Omaima A. Sharaf, Tarek A. Moussa, Said M. Badr El-Din, H. Moawad

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Background: Polycyclic aromatic hydrocarbons (PAHs) pose great environmental and human health concerns for their widespread occurrence, persistence, and carcinogenic properties. PAHs releases due to anthropogenic activities to the wider environment have led to higher concentrations of these contaminants than would be expected from natural processes alone. This may result in a wide range of environmental problems that can accumulate in agricultural ecosystems, which threatened to become a negative impact on sustainable agricultural development. Thus, this study aimed to evaluate the physico-chemical, and microbial properties of the compost leachate (CL) to assess its role as nutrient and microbial source (biostimulation/bioaugmentation) for developing a cost-effective bioremediation technology for PAHs contaminated sites. Material and Methods: PAHs-degrading bacteria were isolated from CL that was collected from a composting site located in central Scotland, UK. Isolation was carried out by enrichment using phenanthrene (PHR), pyrene (PYR) and benzo(a)pyrene (BaP) as the sole source of carbon and energy. The isolates were characterized using a variety of phenotypic and molecular properties. Six different isolates were identified based on the difference in morphological and biochemical tests. The efficiency of these isolates in PAHs utilization was assessed. Further analysis was performed to define taxonomical status and phylogenic relation between the most potent PAHs-utilizing bacterial strains and other standard strains, using molecular approach by partial 16S rDNA gene sequence analysis. Results indicated that the 16S rDNA sequence analysis confirmed the results of biochemical identification, as both of biochemical and molecular identification of the isolates assigned them to Bacillus licheniformis, Pseudomonas aeruginosa, Alcaligenes faecalis, Serratia marcescens, Enterobacter cloacae and Providenicia which were identified as the prominent PAHs-utilizers isolated from CL. Conclusion: This study indicates that the CL samples contain a diverse population of PAHs-degrading bacteria and the use of CL may have a potential for bioremediation of PAHs contaminated sites.

Keywords: polycyclic aromatic hydrocarbons, physico-chemical analyses, compost leachate, microbial and biochemical analyses, phylogenic relations, 16S rDNA sequence analysis

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Authors: Giorgio Bertolazzi, Panayiotis Benos, Michele Tumminello, Claudia Coronnello

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MicroRNAs are small non-coding RNAs that post-transcriptionally regulate the expression levels of messenger RNAs. MicroRNA regulation activity depends on the recognition of binding sites located on mRNA molecules. ComiR (Combinatorial miRNA targeting) is a user friendly web tool realized to predict the targets of a set of microRNAs, starting from their expression profile. ComiR incorporates miRNA expression in a thermodynamic binding model, and it associates each gene with the probability of being a target of a set of miRNAs. ComiR algorithms were trained with the information regarding binding sites in the 3’UTR region, by using a reliable dataset containing the targets of endogenously expressed microRNA in D. melanogaster S2 cells. This dataset was obtained by comparing the results from two different experimental approaches, i.e., inhibition, and immunoprecipitation of the AGO1 protein; this protein is a component of the microRNA induced silencing complex. In this work, we tested whether including coding region binding sites in the ComiR algorithm improves the performance of the tool in predicting microRNA targets. We focused the analysis on the D. melanogaster species and updated the ComiR underlying database with the currently available releases of mRNA and microRNA sequences. As a result, we find that the ComiR algorithm trained with the information related to the coding regions is more efficient in predicting the microRNA targets, with respect to the algorithm trained with 3’utr information. On the other hand, we show that 3’utr based predictions can be seen as complementary to the coding region based predictions, which suggests that both predictions, from 3'UTR and coding regions, should be considered in a comprehensive analysis. Furthermore, we observed that the lists of targets obtained by analyzing data from one experimental approach only, that is, inhibition or immunoprecipitation of AGO1, are not reliable enough to test the performance of our microRNA target prediction algorithm. Further analysis will be conducted to investigate the effectiveness of the tool with data from other species, provided that validated datasets, as obtained from the comparison of RISC proteins inhibition and immunoprecipitation experiments, will be available for the same samples. Finally, we propose to upgrade the existing ComiR web-tool by including the coding region based trained model, available together with the 3’UTR based one.

Keywords: AGO1, coding region, Drosophila melanogaster, microRNA target prediction

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Authors: Ezzati Givi M., Hoveizi E., Nezhad Marani N.

Abstract:

Platinum-based anticancer therapeutics are the most widely used drugs in clinical chemotherapy but have major limitations and various side effects in clinical applications. Gonadotoxicity and sterility is one of the most common complications for cancer survivors, which seem to be drug-specific and dose-related. Therefore, many efforts have been dedicated to discovering a new structure of platinum-based anticancer agents with improved therapeutic index, fewer side effects. In this regard, new Pt(II)-phosphane complexes containing heterocyclic thionate ligands (PCTL) have been synthesized, which show more potent antitumor activities in comparison to cisplatin. Cisplatin, the best leading metal-based antitumor drug in the field, induces testicular toxicity on Leydig and Sertoli cells leading to serious side effects such as azoospermia and infertility. Therefore in the present study, we aimed to investigate the cytotoxicity effect of PCTL on mice TM4 Sertoli cells with particular emphasis on the role of autophagy in comparison to cisplatin. In this study, an MTT assay was performed to evaluate the IC50 of PCTL and to analyze the TM3 Leydig cell's viability. Cells morphology was evaluated via invert microscope and Changing in morphology for nuclei swelling or autophagic vacuoles formation were assessed by DAPI and MDC staining. Testosterone production in the culture medium was measured using an ELISA kit. Finally, the expression of Autophagy-related genes, Atg5, Beclin1 and p62, were analyzed by qPCR. Based on the obtained results by MTT, the IC50 value of PCTL was 50 μM in TM3 cells and cytotoxic effects was in a dose- and time-dependent manner. Cells morphological changes investigated by inverted microscopy, DAPI, and MDC staining which showed the cytotoxic concentrations of PCTL was significantly higher than cisplatin in the treated TM3 Leydig cells. The results of PCR showed a lack of expression of the p62, Atg5 and Beclin1 gene in TM3 cells treated with PCTL in comparison to cisplatin and control groups. It should be noted that the effects of 25 μM PCTL concentration on TM3 cells have been associated with increased testosterone production and secretion, which requires further study to explain the possible causes and involved molecular mechanisms. The results of the study showed that the PCTL had less-lethal effects on TM3 cells in comparison to cisplatin and probably did not induce autophagy in TM3 cells.

Keywords: platinum-based anticancer agents, cisplatin, Leydig TM3 cells, autophagy

Procedia PDF Downloads 127

Authors: Mohammadjavad Sotoudeheian

Abstract:

COVID-19, which began in December 2019, uses the angiotensin-converting enzyme 2 (ACE2) receptor to enter and spread through the cells. ACE2 mRNA is present in almost every organ, including nasopharynx, lung, as well as the brain. Ports of entry of SARS-CoV-2 into the central nervous system (CNS) may include arterial circulation, while viremia is remarkable. However, it is imperious to develop neurological symptoms evaluation CSF analysis in patients with COVID-19, but theoretically, ACE2 receptors are expressed in cerebellar cells and may be a target for SARS-CoV-2 infection in the brain. Recent evidence agrees that SARS-CoV-2 can impact the brain through direct and indirect injury. Two biomarkers for CNS injury, glial fibrillary acidic protein (GFAP) and neurofilament light chain (NFL) detected in the plasma of patients with COVID-19. NFL, an axonal protein expressed in neurons, is related to axonal neurodegeneration, and GFAP is over-expressed in CNS inflammation. GFAP cytoplasmic accumulation causes Schwan cells to misfunction, so affects myelin generation, reduces neuroskeletal support over NfLs during CNS inflammation, and leads to axonal degeneration. Interleukin-6 (IL-6), which extensively over-express due to interleukin storm during COVID-19 inflammation, regulates gene expression, as well as GFAP through STAT molecular pathway. IL-6 also impresses the phosphoinositide 3-kinase (PI3K)/STAT/smads pathway. The PI3K/ protein kinase B (Akt) pathway is the main modulator upstream of the mammalian target of rapamycin (mTOR), and alterations in this pathway are common in neurodegenerative diseases. Most neurodegenerative diseases show a disruption of autophagic function and display an abnormal increase in protein aggregation that promotes cellular death. Therefore, induction of autophagy has been recommended as a rational approach to help neurons clear abnormal protein aggregates and survive. The mTOR is a major regulator of the autophagic process and is regulated by cellular stressors. The mTORC1 pathway and mTORC2, as complementary and important elements in mTORC1 signaling, have become relevant in the regulation of the autophagic process and cellular survival through the extracellular signal-regulated kinase (ERK) pathway.

Keywords: mTORC1, COVID-19, PI3K, autophagy, neurodegeneration

Procedia PDF Downloads 85