Insights into microstructure and expression of markers of proliferation, apoptosis and T cells in the spleen of cattle egret (Bubulcus ibis).

The spleen is the largest secondary lymphoid organ with significant roles in pathogen clearance. It is involved in several avian diseases. The cattle egret is a wild insectivorous bird of agricultural and socioeconomic importance. Data related to microstructural features of cattle egret spleen are lacking. The present study investigated the gross anatomical, histological and immunohistochemical characteristics of the cattle egret spleen. Proliferation (PCNA and PHH3), apoptosis (cleaved caspase 3, C.CASP3) and T-cell (CD3 and CD8) markers were assessed. Grossly, the spleen appeared brownish red, oval-shaped and located at the oesophago-proventricular junction. Histologically, the spleen was surrounded by a thin capsule sending a number of trabeculae which contained branches of the splenic vessels. The white pulp consisted of the periarteriolar lymphoid sheath and periellipsoidal lymphatic sheath (PELS). The red pulp was formed of sinusoids and cords. The penicillar capillaries, which represent the terminal segments of the splenic arterial tree were highly branched, wrapped by prominent ellipsoids and directly connected to the splenic sinusoids, suggesting a closed type of circulation. Immunohistochemically, proliferating cell nuclear antigen (PCNA)-expressing cells were distributed with high counts throughout the splenic parenchyma, being highest within the splenic cords and PELS. Both PHH3- and C.CASP3-expressing cells revealed a similar pattern to that of PCNA, although with fewer counts. Large numbers of T cells were observed throughout the splenic parenchyma, mainly within the cords, as revealed by CD3 and CD8 immunoreaction. The present study provides a clear insight into the precise structure of the spleen in cattle egrets and thus improves our understanding about birds' immunity.

Age-related morphological and ultrastructural changes in the palate and pharyngeal masticatory apparatus of grass carp (Ctenopharyngodon idella) juveniles.

Grass carp (Ctenopharyngodon idella or C. idella) is a Cyprinid fish frequently utilized for aquaculture, medical, and research purposes. In C. idella, the palate is followed by a well-developed pharyngeal masticatory apparatus. The latter consists of an upper chewing pad superimposing a paired set of teeth. The present study investigated morphological, morphometric, histochemical, and surface ultrastructural changes involving these structures in C. idella juveniles at three different timepoints namely 35 mm total length (TL; 59 days posthatching (dph); fry-fingerling transition), 70 mm TL (90 dph; fingerling), and 210 mm TL ( 365 dph: yearling). The palatal epithelium revealed a constant number of taste buds. However, the height and width of these buds revealed an age-dependent increase. The number of palatal acidic goblet cells increased gradually with age. Enhanced keratinization of pad epithelium, and increased teeth dimensions were age-associated characteristics. Ultrastructurally, the palatal surface of C. idella was slightly papillated at 35 mm TL after which it formed brick-like structural units that tended to cluster into longitudinally paralleled rows toward the palate-pad junction. Goblet cell openings appeared oval at 35 mm TL and became club-shaped by 210 mm TL at which the epithelium appeared compact and heavily coated in mucus. Indentations of pad surface and signs of dental wear and tear were evident ultrastructurally at 70 mm TL and onwards. The current study reports for the first time age-related developmental features of the palate and pharyngeal masticatory apparatus of grass carp. Results of the present work will help to understand aging-associated factors involving the studied fish and other related aquatic species.

Bladder cancer: therapeutic challenges and role of 3D cell culture systems in the screening of novel cancer therapeutics.

Bladder cancer (BC) is the sixth most common worldwide urologic malignancy associated with elevated morbidity and mortality rates if not well treated. The muscle-invasive form of BC develops in about 25% of patients. Moreover, according to estimates, 50% of patients with invasive BC experience fatal metastatic relapses. Currently, resistance to drug-based therapy is the major tumble to BC treatment. The three-dimensional (3D) cell cultures are clearly more relevant not only as a novel evolving gadget in drug screening but also as a bearable therapeutic for different diseases. In this review, various subtypes of BC and mechanisms of drug resistance to the commonly used anticancer therapies are discussed. We also summarize the key lineaments of the latest cell-based assays utilizing 3D cell culture systems and their impact on understanding the pathophysiology of BC. Such knowledge could ultimately help to address the most efficient BC treatment.

Histological and ultrastructural characterization of the dorso-ventral skin of the juvenile and the adult starry puffer fish (Arothron stellatus, Anonymous 1798).

BACKGROUND: The starry puffer fish (Arothron stellatus, Anonymous, 1798) is a poisonous tetradontidae fish inhabiting the Red sea. The skin constitutes an important defense against any external effects. The study aims to characterize the dorso-ventral skin of the juvenile and the adult starry puffer fish using light and scanning electron microscopies. Twenty specimens of juvenile and adult fresh fishes were used. RESULTS: The scanning electron microarchitecture of the skin of the juvenile and adult fish showed delicate irregular-shaped protrusions, and well-defined bricks-like elevations on the dorsal side and interrupted folds as well as irregular-shaped protrusions on the ventral side. In adult fish, the patterned microridges of the superficial and deep epithelial cells (keratinocytes) were larger and well-defined in the dorsal skin than in the ventral side, the contrary was seen in the juvenile fish. The microridges were arranged in a fingerprint or honeycomb patterns. The openings of the mucous cells were more numerous in the dorsal skin in both age stages but more noticeable in adult. Furthermore, the sensory cells were more dominant in the juveniles than the adults. The odontic spines were only seen in adult. Histologically, few taste buds were observed in the epidermis of the dorsal skin surface of the adult fish. Both mucous and club cells were embedded in the epidermis of the juvenile and adult fish with different shapes and sizes. Melanophores were observed at the dorsal skin of both juvenile and adult fishes while fewer numbers were noticed at the ventral surfaces. Several dermal bony plates with different shapes and sizes were demonstrated in the skin of both adult and juvenile fishes. CONCLUSION: The structural variations of skin of the juvenile and adult fishes may reflect the various environmental difficulties that they confront.

Calpain signaling: from biology to therapeutic opportunities in neurodegenerative disorders.

Neurodegenerative disorders represent a major and growing healthcare challenge globally. Among the numerous molecular pathways implicated in their pathogenesis, calpain signaling has emerged as a crucial player in neuronal dysfunction and cell death. Calpain is a family of calcium-dependent cysteine proteases that is involved in many biological processes, such as signal transduction, cytoskeleton remodeling, and protein turnover. Dysregulation of calpain activation and activity has been associated with several neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. Understanding the intricate structure of calpains is crucial for unraveling their roles in cellular physiology and their implications in pathology. In addition, the identification of diverse abnormalities in both humans and other animal models with deficiencies in calpain highlights the significant progress made in understanding calpain biology. In this comprehensive review, we delve into the recent roles attributed to calpains and provide an overview of the mechanisms that govern their activity during the progression of neurodegenerative diseases. The possibility of utilizing calpain inhibition as a potential therapeutic approach for treating neuronal dysfunctions in neurodegenerative disorders would be an area of interest in future calpain research.

Outer and inner mitochondrial membrane proteins TOMM40 and TIMM50 are intensively concentrated and localized at Purkinje and pyramidal neurons in the New Zealand white rabbit brain.

Mitochondria are involved in a variety of developmental processes and neurodegenerative diseases. The translocase complexes of the outer and inner mitochondrial membranes (TOM and TIM) are protein complexes involved in transporting protein precursors across mitochondrial membranes. Although rabbits are important animal models for neurodegenerative diseases, the expression of TOM and TIM complexes has yet to be examined in the rabbit brain. In the present study, we quantitatively evaluated the protein expression of the translocase of outer mitochondrial membrane 40 (TOMM40) and inner mitochondrial membrane 50 (TIMM50) complexes, two of the TOM/TIM complexes, in the cerebral, cerebellar, and hippocampal cortices of the New Zealand white rabbit brain, using immunohistochemistry. Sections from brain specimens were initially stained for cytochrome c oxidase (COX), a well-known mitochondrial marker, which was found to be homogeneously expressed in the cerebrum, but localized to the Purkinje and pyramidal neurons of the cerebellum and hippocampus, respectively. TOMM40 and TIMM50 proteins consistently revealed a similar expression pattern, although at different ratios. In the cerebrum, TOMM40 and TIMM50 immunoreactions were homogeneously distributed within the cytoplasm of various neurons. Meanwhile, Purkinje cells in the cerebellum and pyramidal neurons in the hippocampus displayed higher intensities in their cytoplasm. The specific cellular localization of TOMM40 and TIMM50 proteins in various regions of the rabbit brain suggests a distinct function of each protein in these regions. Further analysis will be required to evaluate the molecular functions of these proteins.

Anatomical, histochemical, and immunohistochemical observations on the gastrointestinal tract of Gallinula chloropus (Aves: Rallidae).

BACKGROUND: Gallinula chloropus (Linnaeus, 1758) is a wild aquatic omnivorous bird characterized by a marked resistance to harsh environmental conditions and a worldwide distribution. In this study, anatomical, morphometrical, histochemical, and immunohistochemical techniques were employed to study the structure of the gastrointestinal tract of Gallinula chloropus. RESULTS: The esophagus appeared tubular with no distinct crop. Both superficial (SPG) and deep (DPG) proventricular glands were present. The DPG filled about two-thirds of the total wall thickness. Histochemically, the mucosubstances revealed mixed alcian blue-PAS positive reactions. They were mainly localized in the acini of the esophageal glands and SPG, gastric surface epithelium, duct system of DPG, and intestinal goblet cells. The highest number of goblet cells per every 1 mm2 of the intestinal mucosa was seen within the ileum and rectum, 2555 ± 468 and 2607 ± 653 respectively. Notably, glucagon immunoreactive (IR) cells were abundant in the mucosa of the small and large intestines and the proventriculus, while somatostatin IR cells were concentrated within the acini of the DPG. IR cells for the mitosis marker phospho-histone H3 (PHH3) were highest within the entire intestinal crypts and mucosa-associated lymphoid tissues (MALT). In contrast, cells IR for the apoptosis marker C.CASP3 were remarkable in epithelial cells at the tips of intestinal villi and in MALT, reflecting the dynamic nature of the latter mentioned structures. CONCLUSIONS: The findings of the present study advance our knowledge of the gross and microscopic anatomy of the gastrointestinal tract in wild birds and could help to enhance the productivity of Aves via improving gut health.

Evaluating the distribution of T-lymphocytes and S-phase proliferating cells across the nasal mucosa of dromedary camel (Camelus dromedarius).

The lining mucosa of the nasal cavity performs important roles for the host adaptation to the external environment. Camels are unique in their adaptation to the lifestyle of nomadic deserts. The present study aimed to evaluate the distribution pattern of T lymphocytes and S-phase proliferating cells within the nasal mucosa of camel using antibodies against CD3 and PCNA, respectively. The mucosa of the rostral, middle, and caudal parts of the nasal cavity was collected and processed for immunohistochemical staining. CD3-immunoreactive (-IR) cells were observed within the epithelium and lamina propria of all examined parts. However, the numbers of these cells were significantly higher in the rostral part of the nasal mucosa compared to its middle and caudal parts (P < 0.05). Such expression of CD3-IR cells within the rostral nasal mucosa was most pronounced within its lamina propria which also revealed aggregations of lymphoid cells. The increased frequency of CD3 expressing cells at the rostral part of the nasal mucosa suggests a potential role of the nasal vestibule in limiting the infection via constant clearance of encountered pathogens. PCNA-IR cells were mainly found within the basal layers of the nasal epithelium at the rostral part of the nasal cavity, though they showed a significant decrease in their frequencies on moving caudad. The results of the present work will form a basis for assessment of various nasal pathologies affecting camels particularly those associated with increased rates of T lymphocytes infiltration and/or cell proliferation.

Structure of the Eurasian moorhen spleen: A comprehensive study using gross anatomy, light, and transmission electron microscopy.

The spleen is the largest secondary lymphoid organ with major roles in the removal of blood-borne antigens. The Eurasian moorhen is a wild aquatic bird that revealed the adaptation to harsh environmental conditions. Information regarding the structural features of moorhen's spleen is lacking. The present study aimed to describe the composition of moorhen's spleen using anatomical dissection, histology, histochemistry, immunohistochemistry, and transmission electron microscopy. The spleen appeared as a brownish red sickle-shaped organ close to the proventriculus and gizzard. The splenic capsule was very thin, and the trabeculae were poorly developed. The white pulp formed of the periarteriolar lymphoid sheath, lymphoid follicles, and periellipsoidal lymphatic sheath. The red pulp composed of sinusoids and cords and contained various types of blood cells. Blood vessels were observed within the splenic capsule and inside the parenchyma. Notably, penicillar capillaries (PCs) appeared branched and surrounded by well-developed ellipsoids. Direct connections were observed between PCs and splenic sinusoids suggesting a closed type of circulation. Ultrastructurally, intercellular spaces and vascular channels were evident in the wall of PCs. Ellipsoid-associated cells, lymphocytes, monocytes, and heterophils were observed within splenic ellipsoids. Ellipsoids were thus suggested as a crucial component of moorhen's spleen. Numerous MafB-immunoreactive (IR) macrophages were seen within the red pulp. Splenic cords contained the highest number of PHH3-IR cells, while CCASP3-IR cells were exclusive to follicles of the white pulp. In conclusion, the structure of moorhen's spleen revealed species-specific features. The findings of the present study could help to improve the immunity of domestic birds.

c-MAF deletion in adult C57BL/6J mice induces cataract formation and abnormal differentiation of lens fiber cells.

The transcription factor c-MAF is a member of the large MAF family, members of which possess transactivation and bZIP domains. c-MAF plays an important role in lens formation, T-lymphocyte differentiation, hypertrophic chondrocyte differentiation, and kidney development in mouse embryos. However, because homozygous deletion of c-Maf in C57BL/6J mice causes embryonic lethality, the functions of c-MAF in adult mice remain largely uninvestigated. To address this issue, we generated c-Maf floxed (c-Maffl/fl) C57BL/6J mice and established tamoxifen-inducible c-Maf knockout mice (c-Maffl/fl; CAG-Cre-ERTM mice, c-MafΔTAM). After tamoxifen injection, adult c-MafΔTAM mice showed successful deletion of c-Maf protein and developed severe cataracts; cataracts are also seen in human patients who have mutations in the c-MAF DNA binding domain. Furthermore, adult c-MafΔTAM mice exhibited abnormal lens structure and impaired differentiation of lens fiber cells. In summary, we established c-Maffl/fl and c-MafΔTAM C57BL/6J mice, which can be useful animal models for the investigation of c-MAF function in various developmental stages and can also be used as a disease model for cataracts.

Uncovering the role of MAFB in glucagon production and secretion in pancreatic α-cells using a new α-cell-specific Mafb conditional knockout mouse model.

Cre/loxP is a site-specific recombination system extensively used to enable the conditional deletion or activation of target genes in a spatial- and/or temporal-specific manner. A number of pancreatic-specific Cre driver mouse lines have been broadly established for studying the development, function and pathology of pancreatic cells. However, only a few models are currently available for glucagon-producing α-cells. Disagreement exists over the role of the MAFB transcription factor in glucagon expression during postnatal life, which might be due to the lack of α-cell-specific Cre driver mice. In the present study, we established a novel Gcg-Cre knock-in mouse line with the Cre transgene expressed under the control of the preproglucagon (Gcg) promoter without disrupting the endogenous Gcg gene expression. Then, we applied this newly developed Gcg-Cre mouse line to generate a new α-cell-specific Mafb conditional knockout mouse model (MafbΔGcg). Not only α-cell number but also glucagon production were significantly decreased in MafbΔGcg mice compared to control littermates, suggesting an indispensable role of MAFB in both α-cell development and function. Taken together, our newly developed Gcg-Cre mouse line, which was successfully utilized to uncover the role of MAFB in α-cells, is a useful tool for genetic manipulation in pancreatic α-cells, providing a new platform for future studies in this field.

Human Islet Response to Selected Type 1 Diabetes-Associated Bacteria: A Transcriptome-Based Study.

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from destruction of pancreatic ß-cells. T1D subjects were recently shown to harbor distinct intestinal microbiome profiles. Based on these findings, the role of gut bacteria in T1D is being intensively investigated. The mechanism connecting intestinal microbial homeostasis with the development of T1D is unknown. Specific gut bacteria such as Bacteroides dorei (BD) and Ruminococcus gnavus (RG) show markedly increased abundance prior to the development of autoimmunity. One hypothesis is that these bacteria might traverse the damaged gut barrier, and their constituents elicit a response from human islets that causes metabolic abnormalities and inflammation. We have tested this hypothesis by exposing human islets to BD and RG in vitro, after which RNA-Seq analysis was performed. The bacteria altered expression of many islet genes. The commonly upregulated genes by these bacteria were cytokines, chemokines and enzymes, suggesting a significant effect of gut bacteria on islet antimicrobial and biosynthetic pathways. Additionally, each bacteria displayed a unique set of differentially expressed genes (DEGs). Ingenuity pathway analysis of DEGs revealed that top activated pathways and diseases included TREM1 signaling and inflammatory response, illustrating the ability of bacteria to induce islet inflammation. The increased levels of selected factors were confirmed using immunoblotting and ELISA methods. Our data demonstrate that islets produce a complex anti-bacterial response. The response includes both symbiotic and pathogenic aspects. Both oxidative damage and leukocyte recruitment factors were prominent, which could induce beta cell damage and subsequent autoimmunity.

Current understanding of the role of gut dysbiosis in type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disorder that results from destruction of the insulin-producing pancreatic ß-cells. The disease mainly affects juveniles. Changes in the composition of the gut microbiota (dysbiosis) and changes in the properties of the gut barrier have been documented in T1D subjects. Because these factors affect immune system functions, they are likely to play a role in disease pathogenesis. However, their exact role is currently not fully understood and is under intensive investigation. In this article we discuss recent advancements depicting the role of intestinal dysbiosis on immunity and autoimmunity in T1D. We also discuss therapies aimed at maintaining a healthy gut barrier as prevention strategies for T1D.

Normal magnetic resonance anatomy of the hind foot of Egyptian buffalo (Bubalus bubalis): A correlative low-field T1- and T2-weighted MRI and sectional anatomy atlas.

Buffaloes represent a major source of milk production, especially in developing countries including Egypt. The buffalo foot is frequently involved in a large proportion of lameness cases. The relatively small size and complexity of its structures often render the radiographic evaluation of the foot challenging. Magnetic resonance imaging (MRI) is a noninvasive imaging technique that is regarded as both safe and accurate for assessment of the foot disorders in both man and animals. The purpose of the current investigation was to describe the MRI anatomy of buffalo foot using cadaveric hind feet. The feet were subjected to consecutive MRI scanning using a 0.3 Tesla scanner. Both T1-weighted (T1-W) and T2-weighted (T2-W) spin-echo pulse sequences were applied in dorsal, sagittal and transverse planes. The heterogeneity of signal intensities noted amongst foot components allowed for clear differentiation of bones, tendons, ligaments, adipose tissue and synovial fluid. The T1-W images provided an excellent overview of the foot. They were valuable for visualizing the bones and the alignment of tendons and ligaments. The T2-W images were particularly useful for the evaluation of synovial structures such as tendon sheaths and joint cavities. A communication between the two plantar sacs of the metatarsophalangeal (fetlock) joints was evident in T2-W images. MRI findings were further confirmed using relevant gross anatomical sections. The present study establishes a detailed MRI anatomic reference of buffalo foot that could help veterinary researchers, clinicians and surgeons for increasing the accuracy of interpretation of foot MRI scans of both healthy and diseased animals.

MAFB is dispensable for the fetal testis morphogenesis and the maintenance of spermatogenesis in adult mice.

The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using Mafb-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not. MAFB was localized in Leydig and Sertoli cells at embryonic day (E) 18.5 but in Leydig cells, Sertoli cells, and pachytene spermatocytes in adults. Mafb-deficient testes at E18.5 showed fully formed seminiferous tubules with no abnormal structure or differences in testicular somatic cell numbers compared with those of control wild-type mice. Additionally, the expression levels of genes related to development and function of testicular cells were unchanged between genotypes. In adults, the expression of MAFB in Sertoli cells was shown to be stage specific and induced by RA. By generating Mafbfl/fl CAG-CreER™ (Mafb-cKO) mice, in which Cre recombinase was activated upon tamoxifen treatment, we found that the neonatal cKO mice died shortly upon Mafb deletion, but adult cKO mice were alive upon deletion. Adult cKO mice were fertile, and spermatogenesis maintenance was normal, as indicated by histological analysis, hormone levels, and germ cell stage-specific markers. Moreover, there were no differences in the proportion of seminiferous stages between cKO mice and controls. However, RNA-Seq analysis of cKO Sertoli cells revealed that the down-regulated genes were related to immune function and phagocytosis activity but not spermatogenesis. In conclusion, we found that MAFB is dispensable for fetal testis morphogenesis and spermatogenesis maintenance in adult mice, despite the significant gene expression in different cell types, but MAFB might be critical for phagocytosis activity of Sertoli cells.

ß-Cell-Specific Mafk Overexpression Impairs Pancreatic Endocrine Cell Development.

The MAF family transcription factors are homologs of v-Maf, the oncogenic component of the avian retrovirus AS42. They are subdivided into 2 groups, small and large MAF proteins, according to their structure, function, and molecular size. MAFK is a member of the small MAF family and acts as a dominant negative form of large MAFs. In previous research we generated transgenic mice that overexpress MAFK in order to suppress the function of large MAF proteins in pancreatic ß-cells. These mice developed hyperglycemia in adulthood due to impairment of glucose-stimulated insulin secretion. The aim of the current study is to examine the effects of ß-cell-specific Mafk overexpression in endocrine cell development. The developing islets of Mafk-transgenic embryos appeared to be disorganized with an inversion of total numbers of insulin+ and glucagon+ cells due to reduced ß-cell proliferation. Gene expression analysis by quantitative RT-PCR revealed decreased levels of ß-cell-related genes whose expressions are known to be controlled by large MAF proteins. Additionally, these changes were accompanied with a significant increase in key ß-cell transcription factors likely due to compensatory mechanisms that might have been activated in response to the ß-cell loss. Finally, microarray comparison of gene expression profiles between wild-type and transgenic pancreata revealed alteration of some uncharacterized genes including Pcbd1, Fam132a, Cryba2, and Npy, which might play important roles during pancreatic endocrine development. Taken together, these results suggest that Mafk overexpression impairs endocrine development through a regulation of numerous ß-cell-related genes. The microarray analysis provided a unique data set of differentially expressed genes that might contribute to a better understanding of the molecular basis that governs the development and function of endocrine pancreas.

Role of large MAF transcription factors in the mouse endocrine pancreas.

The members of the MAF family of transcription factors are homologs of v-Maf -the oncogenic component of the avian retrovirus AS42. The MAF family is subdivided into 2 groups, small and large MAFs. To elucidate the role of the large MAF transcription factors in the endocrine pancreas, we analyzed large MAF gene knockout mice. It has been shown that Mafa(-/-) mice develop phenotypes including abnormal islet structure soon after birth. This study revealed that Ins1 and Ins2 transcripts and the protein contents were significantly reduced in Mafa(-/-) mice at embryonic day 18.5. In addition, Mafa(-/-);Mafb(-/-) mice contained less than 10% of the insulin transcript and protein of those of wild-type mice, suggesting that Mafa and Mafb cooperate to maintain insulin levels at the embryonic stage. On the other hand, the number of insulin-positive cells in Mafa(-/-) mice was comparable to that of wild-type mice, and even under a Mafb-deficient background the number of insulin-positive cells was not decreased, suggesting that Mafb plays a dominant role in embryonic ß-cell development. We also found that at 20 weeks of age Mafa(-/-);Mafb(+/-) mice showed a higher fasting blood glucose level than single Mafa(-/-) mice. In summary, our results indicate that Mafa is necessary for the maintenance of normal insulin levels even in embryos and that Mafb is important for the maintenance of fasting blood glucose levels in the Mafa-deficient background in adults.