Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

OrganoidChip facilitates hydrogel-free immobilization for fast and blur-free imaging of organoids.

Organoids are three-dimensional structures of self-assembled cell aggregates that mimic anatomical features of in vivo organs and can serve as in vitro miniaturized organ models for drug testing. The most efficient way of studying drug toxicity and efficacy requires high-resolution imaging of a large number of organoids acquired in the least amount of time. Currently missing are suitable platforms capable of fast-paced high-content imaging of organoids. To address this knowledge gap, we present the OrganoidChip, a microfluidic imaging platform that incorporates a unique design to immobilize organoids for endpoint, fast imaging. The chip contains six parallel trapping areas, each having a staging and immobilization chamber, that receives organoids transferred from their native culture plates and anchors them, respectively. We first demonstrate that the OrganoidChip can efficiently immobilize intestinal and cardiac organoids without compromising their viability and functionality. Next, we show the capability of our device in assessing the dose-dependent responses of organoids' viability and spontaneous contraction properties to Doxorubicin treatment and obtaining results that are similar to off-chip experiments. Importantly, the chip enables organoid imaging at speeds that are an order of magnitude faster than conventional imaging platforms and prevents the acquisition of blurry images caused by organoid drifting, swimming, and fast stage movements. Taken together, the OrganoidChip is a promising microfluidic platform that can serve as a building block for a multiwell plate format that can provide high-throughput and high-resolution imaging of organoids in the future.

Changes of Enterocyte Morphology and Enterocyte: Goblet Cell Ratios in Dogs with Protein-Losing and Non-Protein-Losing Chronic Enteropathies.

This study aimed to assess the morphometry of enterocytes as well as the goblet cell-to-enterocyte ratio in different intestinal segments of dogs with chronic enteropathies (CE). Histopathological intestinal samples from 97 dogs were included in the study (19 healthy juveniles, 21 healthy adults, 24 dogs with protein-losing enteropathy (PLE), and 33 CE dogs without PLE). Healthy adult small intestinal enterocytes showed progressively reduced epithelial cell height in the aboral direction, while juvenile dogs showed progressively increased epithelial cell height in the aboral direction. CE dogs had increased epithelial cell height in the duodenum, while PLE dogs had decreased epithelial cell heights compared to healthy adult dogs. Both the CE and PLE dogs showed decreased enterocyte width in the duodenal segment, and the ileal and colonic enterocytes of CE dogs were narrower than those of healthy adult dogs. CE dogs had a lower goblet cell-to-enterocyte ratio in the colon segment compared to healthy dogs. This study provides valuable morphometric information on enterocytes during canine chronic enteropathies, highlighting significant morphological enterocyte alterations, particularly in the small intestine, as well as a reduced goblet cell-to-enterocyte ratio in the colon of CE cases compared to healthy adult dogs.

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability. Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells. The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol describes the tasks directly preceding an experiment, including in vitro validation of experimental results. Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research.

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they represent relevant large animal models with the translational potential to human medicine. Organoids are 3-dimensional (3D), self-assembled structures derived from stem cells that mimic the microanatomy and physiology of their organ of origin. These translational in vitro models can be used for drug permeability and discovery applications, toxicology assessment, and to provide a mechanistic understanding of the pathophysiology of multifactorial chronic diseases. Furthermore, canine organoids can enhance the lives of companion dogs, providing input in various areas of veterinary research and facilitating personalized treatment applications in veterinary medicine. A small group of donors can create a biobank of organoid samples, reducing the need for continuous tissue harvesting, as organoid cell lines can be sub-cultured indefinitely. Herein, three protocols that focus on the culture of intestinal and hepatic canine organoids derived from adult stem cells are presented. The Canine Organoid Isolation Protocol outlines methods to process tissue and embedding of the cell isolate in a supportive matrix (solubilized extracellular membrane matrix). The Canine Organoid Maintenance Protocol describes organoid growth and maintenance, including cleaning and passaging along with appropriate timing for expansion. The Organoid Harvesting and Biobanking Protocol describes ways to extract, freeze, and preserve organoids for further analysis.

Multiomic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals novel markers of almost all major cell types.

MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal (SI) epithelium. However, it is not known which microRNAs are expressed in each of the cell types of the SI epithelium. To bridge this important knowledge gap, we performed comprehensive microRNA profiling in all major cell types of the mouse SI epithelium. We used flow cytometry and fluorescence-activated cell sorting with multiple reporter mouse models to isolate intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, tuft cells, and secretory progenitors. We then subjected these cell populations to small RNA-sequencing. The resulting atlas revealed highly enriched microRNA markers for almost every major cell type (https://sethupathy-lab.shinyapps.io/SI_miRNA/). Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin-run-on sequencing to determine which of these LEMs are likely cotranscribed with their host genes. We then performed single-cell RNA-sequencing to define the cell type specificity of the host genes and embedded LEMs. We observed that the two most enriched microRNAs in secretory progenitors are miR-1224 and miR-672, the latter of which we found is deleted in hominin species. Finally, using several in vivo models, we established that miR-152 is a Paneth cell-specific microRNA.NEW & NOTEWORTHY In this study, first, microRNA atlas (and searchable web server) across all major small intestinal epithelial cell types is presented. We have demonstrated microRNAs that uniquely mark several lineages, including enteroendocrine and tuft. Identification of a key marker of mouse secretory progenitor cells, miR-672, which we show is deleted in humans. We have used several in vivo models to establish miR-152 as a specific marker of Paneth cells, which are highly understudied in terms of microRNAs.

Tightly linked Rps12 and Rps13 genes provide broad-spectrum Phytophthora resistance in soybean.

The Phytophtora root and stem rot is a serious disease in soybean. It is caused by the oomycete pathogen Phytophthora sojae. Growing Phytophthora resistant cultivars is the major method of controlling this disease. Resistance is race- or gene-specific; a single gene confers immunity against only a subset of the P. sojae isolates. Unfortunately, rapid evolution of new Phytophthora sojae virulent pathotypes limits the effectiveness of an Rps ("resistance to Phytophthora sojae") gene to 8-15 years. The current study was designed to investigate the effectiveness of Rps12 against a set of P. sojae isolates using recombinant inbred lines (RILs) that contain recombination break points in the Rps12 region. Our study revealed a unique Rps gene linked to the Rps12 locus. We named this novel gene as Rps13 that confers resistance against P. sojae isolate V13, which is virulent to recombinants that contains Rps12 but lack Rps13. The genetic distance between the two Rps genes is 4 cM. Our study revealed that two tightly linked functional Rps genes with distinct race-specificity provide broad-spectrum resistance in soybean. We report here the molecular markers for incorporating the broad-spectrum Phytophthora resistance conferred by the two Rps genes in commercial soybean cultivars.

Emerging Roles of Urine-Derived Components for the Management of Bladder Cancer: One Man's Trash Is Another Man's Treasure.

Urinary bladder cancer (UBC) is the most common malignancy of the urinary tract in humans, with an estimated global prevalence of 1.1 million cases over 5 years. Because of its high rates of recurrence and resistance to chemotherapy, UBC is one of the most expensive cancers to treat, resulting in significant health care costs. The development of innovative molecular and cellular tools is necessary to refine patient stratification and help predict response to treatment. Urine is an underused resource of biological components shed from bladder tumors, such as exfoliated cells and extracellular vesicles, that could serve as molecular fingerprints and provide valuable biological insights into tumor phenotype and mechanisms of resistance to chemotherapy. Additionally, characterization of urine-derived extracellular vesicles and cells could be used as reliable biomarkers for prediction of response to neoadjuvant therapy.

Overexpression of a plasma membrane protein generated broad-spectrum immunity in soybean.

Plants fight-off pathogens and pests by manifesting an array of defence responses using their innate immunity mechanisms. Here we report the identification of a novel soybean gene encoding a plasma membrane protein, transcription of which is suppressed following infection with the fungal pathogen, Fusarium virguliforme. Overexpression of the protein led to enhanced resistance against not only against F. virguliforme, but also against spider mites (Tetranychus urticae, Koch), soybean aphids (Aphis glycines, Matsumura) and soybean cyst nematode (Heterodera glycines). We, therefore, name this protein as Glycine max disease resistance 1 (GmDR1; Glyma.10g094800). The homologues of GmDR1 have been detected only in legumes, cocoa, jute and cotton. The deduced GmDR1 protein contains 73 amino acids. GmDR1 is predicted to contain an ecto- and two transmembrane domains. Transient expression of the green fluorescent protein fused GmDR1 protein in soybean leaves showed that it is a plasma membrane protein. We investigated if chitin, a pathogen-associated molecular pattern (PAMP), common to all pathogen and pests considered in this study, can significantly enhance defence pathways among the GmDR1-overexpressed transgenic soybean lines. Chitin induces marker genes of the salicylic- and jasmonic acid-mediated defence pathways, but suppresses the defence pathway regulated by ethylene. Chitin induced SA- and JA-regulated defence pathways may be one of the mechanisms involved in generating broad-spectrum resistance among the GmDR1-overexpressed transgenic soybean lines against two serious pathogens and two pests including spider mites, against which no known resistance genes have been identified in soybean and among the most other crop species.

Harnessing the Biology of Canine Intestinal Organoids to Heighten Understanding of Inflammatory Bowel Disease Pathogenesis and Accelerate Drug Discovery: A One Health Approach.

In a recent issue of the Lancet, the prevalence of Inflammatory Bowel Disease (IBD) was estimated at 7 million worldwide. Overall, the burden of IBD is rising globally, with direct and indirect healthcare costs ranging between $14.6 and $31.6 billion in the U.S. alone in 2014. There is currently no cure for IBD, and up to 40% of patients do not respond to medical therapy. Although the exact determinants of the disease pathophysiology remain unknown, the prevailing hypothesis involves complex interplay among host genetics, the intestinal microenvironment (primarily bacteria and dietary constituents), and the mucosal immune system. Importantly, multiple chronic diseases leading to high morbidity and mortality in modern western societies, including type II diabetes, IBD and colorectal cancer, have epidemiologically been linked to the consumption of high-calorie, low-fiber, high monosaccharide, and high-fat diets (HFD). More specifically, data from our laboratory and others have shown that repeated consumption of HFD triggers dysbiotic changes of the gut microbiome concomitant with a state of chronic intestinal inflammation and increased intestinal permeability. However, progress in our understanding of the effect of dietary interventions on IBD pathogenesis has been hampered by a lack of relevant animal models. Additionally, current in vitro cell culture systems are unable to emulate the in vivo interplay between the gut microbiome and the intestinal epithelium in a realistic and translatable way. There remains, therefore, a critical need to develop translatable in vitro and in vivo models that faithfully recapitulate human gut-specific physiological functions to facilitate detailed mechanistic studies on the impact of dietary interventions on gut homeostasis. While the study of murine models has been pivotal in advancing genetic and cellular discoveries, these animal systems often lack key clinical signs and temporal pathological changes representative of IBD. Specifically, some limitations of the mouse model are associated with the use of genetic knockouts to induce immune deficiency and disease. This is vastly different from the natural course of IBD developing in immunologically competent hosts, as is the case in humans and dogs. Noteworthily, abundant literature suggests that canine and human IBD share common clinical and molecular features, such that preclinical studies in dogs with naturally occurring IBD present an opportunity to further our understanding on disease pathogenesis and streamline the development of new therapeutic strategies. Using a stepwise approach, in vitro mechanistic studies investigating the contribution of dietary interventions to chronic intestinal inflammation and "gut leakiness" could be performed in intestinal organoids and organoid derived monolayers. The biologic potential of organoids stems from the method's ability to harness hard-wired cellular programming such that the complexity of the disease background can be reflected more accurately. Likewise, the effect of therapeutic drug candidates could be evaluated in organoids prior to longitudinal studies in dog and human patients with IBD. In this review, we will discuss the value (and limitations) of intestinal organoids derived from a spontaneous animal disease model of IBD (i.e., the dog), and how it can heighten understanding of the interplay between dietary interventions, the gut microbiota and intestinal inflammation. We will also review how intestinal organoids could be used to streamline the preclinical development of therapeutic drug candidates for IBD patients and their best four-legged friends.

Identification of target genes for RNAi-mediated control of the Twospotted Spider Mite.

RNA interference (RNAi) is being developed for the management of pests that destroy crops. The twospotted Spider Mite (TSSM), Tetranychus urticae is a worldwide pest due to its unique physiological and behavioral characteristics including extraordinary ability to detoxify a wide range of pesticides and feed on many host plants. In this study, we conducted experiments to identify target genes that could be used for the development of RNAi-based methods to control TSSM. Leaf disc feeding assays revealed that knockdown in the expression genes coding for proteins involved in the biosynthesis and action of juvenile hormone (JH) and action of ecdysteroids [Methoprene-tolerant (Met), retinoid X receptor ß, farnesoic acid O-methyltransferase, and CREB-binding protein] caused 35-56% mortality. Transgenic tobacco plants expressing hairpin dsRNA targeting Met gene were generated and tested. About 48% mortality was observed in TSSM raised on transgenic tobacco plants expressing dsMet. These studies not only broaden our knowledge on understanding hormone action in TSSM but also identified target genes that could be used in RNAi-mediated control of TSSM.

A Novel Phytophthora sojae Resistance Rps12 Gene Mapped to a Genomic Region That Contains Several Rps Genes.

Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance.

Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells.

The gene Par-4 (Prostate Apoptosis Response 4) was originally identified in prostate cancer cells undergoing apoptosis and its product Par-4 showed cancer specific pro-apoptotic activity. Particularly, the SAC domain of Par-4 (SAC-Par-4) selectively kills cancer cells leaving normal cells unaffected. The therapeutic significance of bioactive SAC-Par-4 is enormous in cancer biology; however, its large scale production is still a matter of concern. Here we report the production of SAC-Par-4-GFP fusion protein coupled to translational enhancer sequence (5' AMV) and apoplast signal peptide (aTP) in transgenic Nicotiana tabacum cv. Samsun NN plants under the control of a unique recombinant promoter M24. Transgene integration was confirmed by genomic DNA PCR, Southern and Northern blotting, Real-time PCR, and Nuclear run-on assays. Results of Western blot analysis and ELISA confirmed expression of recombinant SAC-Par-4-GFP protein and it was as high as 0.15% of total soluble protein. In addition, we found that targeting of plant recombinant SAC-Par-4-GFP to the apoplast and endoplasmic reticulum (ER) was essential for the stability of plant recombinant protein in comparison to the bacterial derived SAC-Par-4. Deglycosylation analysis demonstrated that ER-targeted SAC-Par-4-GFP-SEKDEL undergoes O-linked glycosylation unlike apoplast-targeted SAC-Par-4-GFP. Furthermore, various in vitro studies like mammalian cells proliferation assay (MTT), apoptosis induction assays, and NF-κB suppression suggested the cytotoxic and apoptotic properties of plant-derived SAC-Par-4-GFP against multiple prostate cancer cell lines. Additionally, pre-treatment of MAT-LyLu prostate cancer cells with purified SAC-Par-4-GFP significantly delayed the onset of tumor in a syngeneic rat prostate cancer model. Taken altogether, we proclaim that plant made SAC-Par-4 may become a useful alternate therapy for effectively alleviating cancer in the new era.

Overexpression of the synthetic chimeric native-T-phylloplanin-GFP genes optimized for monocot and dicot plants renders enhanced resistance to blue mold disease in tobacco (N. tabacum L.).

To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP) was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco.

Expression of an apoplast-directed, T-phylloplanin-GFP fusion gene confers resistance against Peronospora tabacina disease in a susceptible tobacco.

KEY MESSAGE: Phylloplanins are plant-derived, antifungal glycoproteins produced by leaf trichomes. Expression of phylloplanin-GFP fusion gene to the apoplast of a blue mold susceptible tobacco resulted in increased resistance to this pathogen. ABSTRACT: Tobaccos and certain other plants secrete phylloplanin glycoproteins to aerial surfaces where they appear to provide first-point-of-contact resistance against fungi/fungi-like pathogens. These proteins can be collected by water washing of aerial plant surfaces, and as shown for tobacco and a sunflower phylloplanins, spraying concentrated washes onto, e.g., turf grass aerial surfaces can provide resistance against various fungi/fungi-like pathogens, in the laboratory. These results suggest that natural-product, phylloplanins may be useful as broad-selectivity fungicides. An obvious question now is can a tobacco phylloplanin gene be introduced into a disease-susceptible plant to confer endogenous resistance. Here we demonstrate that introduction of a tobacco phylloplanin gene--as a fusion with the GFP gene--targeted to the apoplasm can increase resistance to blue mold disease in a susceptible host tobacco.

Manipulating cellulose biosynthesis by expression of mutant Arabidopsis proM24::CESA3(ixr1-2) gene in transgenic tobacco.

Manipulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create modified cellulose for anthropogenic use. Evidence exists that cellulose microfibril structure and its recalcitrance to enzymatic digestion can ameliorated via mis-sense mutation in the primary cell wall-specific gene AtCELLULOSE SYNTHASE (CESA)3. This mis-sense mutation has been identified based on conferring drug resistance to the cellulose inhibitory herbicide isoxaben. To examine whether it would be possible to introduce mutant CESA alleles via a transgenic approach, we overexpressed a modified version of CESA3, AtCESA3(ixr1-2) derived from Arabidopsis thaliana L. Heynh into a different plant family, the Solanceae dicotyledon tobacco (Nicotiana tabacum L. variety Samsun NN). Specifically, a chimeric gene construct of CESA3(ixr1-2) , codon optimized for tobacco, was placed between the heterologous M24 promoter and the rbcSE9 gene terminator. The results demonstrated that the tobacco plants expressing M24-CESA3(ixr1-2) displayed isoxaben resistance, consistent with functionality of the mutated AtCESA3(ixr1-2) in tobacco. Secondly, during enzymatic saccharification, transgenic leaf- and stem-derived cellulose is 54%-66% and 40%-51% more efficient, respectively, compared to the wild type, illustrating translational potential of modified CESA loci. Moreover, the introduction of M24-AtCESA3(ixr1-2) caused aberrant spatial distribution of lignified secondary cell wall tissue and a reduction in the zone occupied by parenchyma cells.

Compromised Rat Testicular Antioxidant Defence System by Hypothyroidism before Puberty.

Altered thyroid function during early stages of development is known to affect adversely testicular growth, physiology, and antioxidant defence status at adulthood. The objective of the present study is to investigate the modulation of antioxidant defence status in neonatal persistent hypothyroid rats before their sexual maturation and also to identify the specific testicular cell populations vulnerable to degeneration during neonatal hypothyroidism in immature rats. Hypothyroidism was induced in neonates by feeding the lactating mother with 0.05% 6-n-propyl-2-thiouracil (PTU) through the drinking water. From the day of parturition till weaning (25 day postpartum), the pups received PTU through mother's milk (or) drinking water and then directly from drinking water containing PTU for the remaining period of experimentation. On the 31st day postpartum, the animals were sacrificed for the study. An altered antioxidant defence system marked by elevated SOD, CAT, and GR activities, with decreased GPx and GST activities were observed along with increased protein carbonylation, disturbed redox status in hypothyroid immature rat testis. This compromised testicular antioxidant status might have contributed to poor growth and development by affecting the spermatogenesis and steroidogenesis in rats before puberty as indicated by reduced germ cell number, complete absence of round spermatids, decreased seminiferous tubule diameter, and decreased testosterone level.

Keratinase production and biodegradation of whole chicken feather keratin by a newly isolated bacterium under submerged fermentation.

A new feather-degrading bacterium PKD 5 was isolated from feather dumping soil and identified as Bacillus weihenstephanensis based on morphological and physiochemical characteristics as well as 16S rRNA gene analysis. Extracellular keratinase was produced during submerged aerobic cultivation in a medium containing chicken feather as sole carbon and energy source and supplemented with salt solutions (NaCl 5.0, MgSO4 1.0, K2HPO4 1.0, (NH4)2SO4, 2.0 g/l). The optimal conditions for keratinase production include initial pH of 7.0, inoculum size of 2% (v/v), and cultivation at 40 °C. The maximum keratinase production and keratinolytic activity of PKD 5 was achieved after 7 days of fermentation under shaking condition (120 rpm). The enzyme has found application in developing cost-effective feather by-products for feeds and fertilizers. The manuscript first time describes B. weihenstephanensis PKD 5-mediated keratinase production under submerged fermentation and whole chicken feather biodegradation.

Development of useful recombinant promoter and its expression analysis in different plant cells using confocal laser scanning microscopy.

BACKGROUND: Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. METHODOLOGY/PRINCIPAL FINDINGS: We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, -271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. CONCLUSION AND SIGNIFICANCE: We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells.

Thiol redox status critically influences mitochondrial response to thyroid hormone-induced hepatic oxidative injury: A temporal analysis.

Liver is a major target organ for thyroid hormone. The objective of the present study was to investigate temporal regulation of mitochondrial glutathione and protein-bound thiol redox status in hyperthyroid liver. Mitochondria were isolated from control and hyperthyroid rat liver tissues at different time intervals, i.e., 24, 72, and 120 h following treatment, and sub-fractionated into sub-mitochondrial particles (SMPs) and matrix fractions. Increased prooxidant levels were indicative of oxidative stress in hyperthyroid mitochondria. Sensitivity to membrane lipid peroxidation (LPx) was maximal after 24 h, which subsided with time. Oxidative damage to proteins was evident as high carbonylation after 72 h; thiol residue damage was an early phenomenon. Reduced and oxidized glutathione (GSH and GSSG) pools of mitochondria were progressively depleted, thereby, impairing matrix antioxidant capacity. However, adaptations to withstand oxidative challenge were elicited in both SMPs and matrix fractions over the long term. It is concluded that maintenance of appropriate intra-mitochondrial glutathione and protein-bound thiol redox status could be instrumental in attenuating thyroid hormone-induced oxidative stress.