Immunolocalization Of Protein Research Articles

Immunolocalization of intermediate filaments in the kidney of the dromedary camel (Camelus dromedarius)

Intermediate filaments belong to a large family of proteins which contribute to the formation of the cytoskeleton. The immunolocalization of cytoskeletal proteins has been used extensively in the diagnosis of various renal pathologies. The present study described the immunolocalization of the cytoskeletal proteins vimentin, desmin, smooth muscle actin, and cytokeratin 19 in the normal kidney of the dromedary camel. Kidney samples from eight adult camels were processed for histology and immunohistochemistry. The kidney was enclosed in a renal capsule composed of vimentin immunoreactive fibroblasts and smooth muscle actin immunoreactive smooth muscle cells. The smooth muscle cells in the renal capsule did not exhibit desmin immunoreactivity. Podocytes forming the visceral layer of the glomerular capsule were immunoreactive for vimentin. Immunoreactivity for vimentin and smooth muscle actin in the parietal layer of the glomerular capsule varied, with both reactive and non-reactive cells observed. Intraglomerular mesangial cells were immunoreactive for smooth muscle actin and desmin, but non-reactive to vimentin. The endothelial lining of blood vessels was vimentin immunoreactive, while smooth muscle actin and desmin were demonstrated in the smooth muscle cells of the vessels. The thin limbs of the loops of Henle in cortical nephrons displayed vimentin immunoreactivity. The proximal and distal convoluted tubules, as well as the collecting ducts were negative to vimentin, smooth muscle actin, desmin and cytokeratin 19 immunostaining. In conclusion, the present study has revealed that similarities and differences exist in the immunolocalization of cytoskeletal proteins in the camel when compared to other mammals. The presence of smooth muscle actin in the parietal cells of the glomerular capsule suggests a contractile function of these cells. The results of the study indicate that vimentin and smooth muscle actin can be used as markers for the identification of podocytes and intraglomerular mesangial cells, respectively, in the camel kidney.

Robust, versatile DNA FISH probes for chromosome-specific repeats in Caenorhabditis elegans and Pristionchus pacificus.

Repetitive DNA sequences are useful targets for chromosomal fluorescence in situ hybridization. We analyzed recent genome assemblies of Caenorhabditis elegans and Pristionchus pacificus to identify tandem repeats with a unique genomic localization. Based on these findings, we designed and validated sets of oligonucleotide probes for each species targeting at least 1 locus per chromosome. These probes yielded reliable fluorescent signals in different tissues and can easily be combined with the immunolocalization of cellular proteins. Synthesis and labeling of these probes are highly cost-effective and require no hands-on labor. The methods presented here can be easily applied in other model and nonmodel organisms with a sequenced genome.

Immunolocalization of adenomatous polyposis coli protein (apc) in the regenerating lizard tail suggests involvement in tissue differentiation and regulation of growth

Lizard tail regeneration is likely regulated by the balanced activity of oncogenes and tumor suppressors that control cell proliferation avoiding tumorigenic degeneration. One of the main tumor suppressor genes present in the regenerating tail is the "adenomatous polyposis coli (apc)" but the localization of its coded protein (apc) is not known. This protein may be involved in regulation of apical-basal tail regeneration in lizards. The present immunohistochemical study shows that apc is localized in apical wound epidermis and regenerating ependyme, two tissues that proliferate and also express onco-genes. Apc is not present in blastema cells but localizes in differentiating cells of regenerating scales, muscles and less intensely in the non-apical ependymal epithelium and cartilage. This suggests that apc is involved in the induction of their differentiation. The apc immunolabeling is mainly nuclear in the basal epidermal layer of the apical wound epidermis where it may be involved in modulating keratinocytes proliferation, like in the forming scales. In regenerating muscle and cartilage apc is mainly cytoplasmic while sparse labeled nuclei are seen in proliferative areas of these tissues. In the regenerating spinal cord, the nuclear and cytoplasmic apc labeling is present in ependymal cells of the distal-most ependymal ampulla but the labeling fades in more proximal regions and mainly remains in the cytoplasm facing the central canal and in sparse nuclei. It is suggested that the pattern of immunolabeling for apc indicates that this tumor suppressor may contribute to tissue differentiation within the regenerating tail.

Immunolocalization of Vasa, PIWI, and TDRKH proteins in male germ cells during spermatogenesis of the teleost fish Poecilia reticulata

Vasa, PIWI and TDRKH are conserved components of germ granules that in metazoans are involved in germline specification and differentiation, as documented by mutational experiments in some model animals. So far, investigations on PIWI during spermatogenesis of fish has been limited to a few species, and no information is available for TDRKH, another protein involved in the piRNA pathway. In this study, the immunolocalization of these three germline determinants was analyzed in male gonads of the teleost fish Poecilia reticulata to document their localization pattern in the different stages of germ cell differentiation.To analyze their distribution pattern during the different stages of spermatogenesis we performed immunohistochemistry (IHC) and immunofluorescence (IF) assays using primary polyclonal antibodies after testing their specificity with Western Blot. Moreover, sections of testis stained with haematoxylin and eosin clarified the structural organization of P. reticulata testis, while the use of the confocal microscope and the nuclear staining clarified the different stages of germ cell differentiation during spermatogenesis.The results showed that Vasa, PIWI and TDRKH were specifically immunolocalized in the germ cells of P. reticulata, with no specific signal detected in Sertoli cells and in other somatic cells of the gonad. These markers were detected in all stages of differentiation from early spermatogonia to advanced spermatids. Vasa staining was the strongest in spermatogonia, and then decreases throughout differentiation. Instead, both PIWI and TDRKH staining increases during differentiation, and their distribution pattern, similar to what observed in the mouse, suggests their concerted participation in the piRNA pathway also in this fish.

The molecular identification, odor binding characterization, and immunolocalization of odorant-binding proteins in Liriomyza trifolii

The Liriomyza trifolii is a highly invasive polyphagia pest. Understanding the physiological functions of odorant binding proteins (OBPs) in the chemical communication of L. trifolii can lead to effective pest management strategies. Seven full-length OBPs were identified by transcriptome screening of L. trifolii adults. Bioinformatics analyses classified the seven OBPs into two subfamilies (six classic OBPs, one minus-C OBP). The analysis of their expression in different development stages revealed that LtriOBP5 was highly expressed in the larval stage, LtriOBP4 in the pupa stage, and LtriOBP1, 2, 3, 6, 7 in the adult stage; the expression levels were higher in male adults than in females. The analysis of different tissues showed high expression of LtriOBP1, 3, 6, 7 in the antennae, which were selected for in vitro purification. To explore the ligand compounds of OBPs, fluorescence competitive binding experiments were performed. Immunofluorescence localization revealed that LtriOBP1, 3, 6, 7 showed strong binding abilities to plant volatiles and were located in the antennae, implying that LtriOBP1, 3, 6, 7 may play key roles in olfaction, such as host location. LtriOBP6 and LtriOBP7 had strong binding abilities to specific herbivore-induced plant volatiles, suggesting LtriOBP6 and LtriOBP7 may also play critical roles in chemoreception. This study provides preliminary exploration of the olfactory perception mechanism of L. trifolii, which can be used as a basis to design insect behavior regulators and develop highly effective insecticides using mixture of ligands and known pesticides.

Evidence for conifer sucrose transporters' functioning in the light-dependent adjustment of sugar allocation.

Sucrose is the central unit of carbon and energy in plants. Active intercellular transport of sucrose is mediated by sucrose transporters (SUTs), genes for which have been found in the genomes of all land plants. However, they have only been assigned functions in angiosperm species. Here, we cloned two types of SUTs from two gymnosperms, the conifers Cedrus deodara (Roxb. G. Don) and Pinus massoniana Lambert, and analyzed their sucrose transport activities. Uptake of the fluorescent sucrose-analog esculin into tobacco epidermis cells expressing the conifer SUT confirmed their transport ability. To determine their function in planta, we investigated their mRNA abundance in relation to photosynthesis and sugar levels in leaves, inner bark, wood and roots. Combined with measurements of protein abundance and immunolocalization of C. deodara SUTs, our results suggest a role for CdSUT1G and CdSUT2 in supporting phloem transport under varying environmental conditions. The implications of these findings regarding conifer physiology and SUT evolution are discussed.

Zn2+-Dependent Nuclease Is Involved in Nuclear Degradation during the Programmed Cell Death of Secretory Cavity Formation in Citrus grandis 'Tomentosa' Fruits.

Zn2+- and Ca2+-dependent nucleases exhibit activity toward dsDNA in the four classes of cation-dependent nucleases in plants. Programmed cell death (PCD) is involved in the degradation of cells during schizolysigenous secretory cavity formation in Citrus fruits. Recently, the Ca2+-dependent DNase CgCAN was proven to play a key role in nuclear DNA degradation during the PCD of secretory cavity formation in Citrus grandis ‘Tomentosa’ fruits. However, whether Zn2+-dependent nuclease plays a role in the PCD of secretory cells remains poorly understood. Here, we identified a Zn2+-dependent nuclease gene, CgENDO1, from Citrus grandis ‘Tomentosa’, the function of which was studied using Zn2+ ions cytochemical localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The full-length cDNA of CgENDO1 contains an open reading frame of 906 bp that encodes a protein 301 amino acids in length with a S1/P1-like functional domain. CgENDO1 degrades linear double-stranded DNA at acidic and neutral pH. CgENDO1 is mainly expressed in the late stage of nuclear degradation of secretory cells. Further spatiotemporal expression patterns of CgENDO1 showed that CgENDO1 is initially located on the endoplasmic reticulum and then moves into intracellular vesicles and nuclei. During the late stage of nuclear degradation, it was concentrated in the area of nuclear degradation involved in nuclear DNA degradation. Our results suggest that the Zn2+-dependent nuclease CgENDO1 plays a direct role in the late degradation stage of the nuclear DNA in the PCD of secretory cavity cells of Citrus grandis ‘Tomentosa’ fruits.

Germline-Restricted Chromosome (GRC) in Female and Male Meiosis of the Great Tit (Parus major, Linnaeus, 1758).

All songbirds studied so far have a germline-restricted chromosome (GRC), which is present in the germ cells and absent in the somatic cells. It shows a wide variation in size, morphology, and genetic content between the songbird species. In this paper, we analyzed GRC behavior in female and male meiosis of the great tit, using immunolocalization of meiotic proteins and FISH with GRC-derived DNA probes. We found that, despite dozens of million years of independent evolution, the great tit GRC displays a striking similarity with the GRCs of two species of martins and two species of estrildid finches examined earlier. It was usually present in two copies in females forming recombining bivalent and in one copy in males forming a condensed heterochromatic body with dotted-like axial elements of the synaptonemal complex. We observed mosaicism for the GRC copy number in the female and male great tit. This indicates that one of the GRC copies might be passively lost during premeiotic germ cell divisions. After the meiotic prophase, the GRC was ejected from most male germ cells. The reverse and interspecies FISH with GRC-specific microdissected DNA probes indicates that GRCs of the great tit, pale martin, and zebra finch differ substantially in their genetic content despite similarities in the meiotic behavior.

Femoral µCT Analysis, Mechanical Testing and Immunolocalization of Bone Proteins in β-Hydroxy β-Methylbutyrate (HMB) Supplemented Spiny Mouse in a Model of Pregnancy and Lactation-Associated Osteoporosis.

A metabolite of leucine, ß-hydroxy-ß-methylbutyrate (HMB), used as a dietary supplement effects muscle tissue gain and bone tissue quality. Since there are no studies on the effects of HMB during pregnancy yet, the aim of the current study was to determine the effects of HMB supplementation during pregnancy on osteoporotic bone quality postpartum and post-lactation using spiny mice (Acomys cahirinus) as the animal models. The six-month-old dams were divided into four groups: pregnant and lactating controls, and pregnant and lactating HMB-treated (during the second trimester of pregnancy) females. The intensity of the immunoreaction of osteocalcin (OC), osteoprotegerin (OPG), bone morphogenetic protein 2 (BMP-2), tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 and 13 (MMP-8 and MMP-13) and proteins involved in bone turnover, was measured in femoral trabecular and compact bone, as well as in the hyaline and epiphyseal cartilage of the femora. The analysis of the trabecular bone microarchitecture showed that the administration of HMB to pregnant females, by influencing the proteins responsible for bone cell activity and collagen remodeling, can provide protection from bone loss. Based on the results of the current study it can be assumed that HMB administration to pregnant females has a more positive impact on trabecular than compact bone.

Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?

Spheroid culture might stabilize the ligamentocyte phenotype. Therefore, the phenotype of lapine cruciate ligamentocyte (L-CLs) minispheroids prepared either by hanging drop (HD) method or by using a novel spheroid plate (SP) and the option of methyl cellulose (MC) for tuning spheroid formation was tested. A total of 250 and 1000 L-CLs per spheroid were seeded as HDs or on an SP before performing cell viability assay, morphometry, gene expression (qRT-PCR) and protein immunolocalization after 7 (HD/SP) and 14 (SP) days. Stable and viable spheroids of both sizes could be produced with both methods, but more rapidly with SP. MC accelerated the formation of round spheroids (HD). Their circular areas decreased significantly during culturing. After 7 days, the diameters of HD-derived spheroids were significantly larger compared to those harvested from the SP, with a tendency of lower circularity suggesting an ellipsoid shape. Gene expression of decorin increased significantly after 7 days (HD, similar trend in SP), tenascin C tended to increase after 7 (HD/SP) and 14 days (SP), whereas collagen type 1 decreased (HD/SP) compared to the monolayer control. The cruciate ligament extracellular matrix components could be localized in all mini-spheroids, confirming their conserved expression profile and their suitability for ligament tissue engineering.

Immunolocalization of protease-activated receptors in endothelial cells of splenic sinuses.

The immunolocalization of protease-activated receptors (PARs) and related proteins in splenic sinus endothelial cells was examined using immunofluorescence and electron microscopy. Immunofluorescence microscopy showed that PAR1 colocalized with PAR2, PAR3, and PAR4. PAR4 colocalized with PAR3 and P2Y12. Myosin heavy chain IIA localized to the outer shape and at the base of cells, but did not colocalize with α-catenin. The localization of di-phosphorylated myosin regulatory light chains (ppMLC) was partially detected on the outer circumference and conspicuously at the base of cells. Macrophage migration inhibitory factor (MIF) also localized in cells. Immunogold electron microscopy revealed the localization of PAR1 on the caveolar membrane, plasma membrane, and junctional membrane of cells. PAR2 and PAR3 localized to the plasma membrane of cells. PAR4 localized to the plasma membrane, depressions in the plasma membrane, and cytoplasmic vesicles. PpMLC was detected in stress fibers, but rarely near the adherens junctions of neighboring cells. MIF localized in vesicles on the apical and basal sides of the Golgi apparatus. Electron microscopy of endothelial cells with saponin extraction showed the depression of many coated pits formed by clathrin from the plasma membrane. Stress fibers developed at the base of cells; however, few actin filaments were observed near adherens junctions. These results indicate that PARs play important roles in splenic sinus endothelial cells, such as in endothelial barrier protection and the maintenance of firm adhesion to ring fibers.

Photobiomodulation therapy does not depend on the differentiation of dental pulp cells to enhance functional activity associated with angiogenesis and mineralization.

The purpose of this study is to analyze the influence of InGaAlP diode laser (660nm) with or without an odontogenic medium (OM) in the functional activity of OD-21 cells. Undifferentiated OD-21 pulp cells were cultivated with or without OM and divided into four groups (n = 5): nonirradiated control (C -), nonirradiated + OM (C +), irradiated (L -), and irradiated + OM (L +). Laser application was performed in two sessions of a 24-h interval with an irradiance of 11.3 mW/cm2, energy density of 1J/cm2, and total cumulative energy/well of 4.6J. Cell proliferation, VEGF-164 expression, mineralization, and expression of Alp, Runx2, and Dmp1 genes, as well as immunolocalization of RUNX2 and MEPE proteins, were evaluated. Data were analyzed by statistical tests (α = 0.05). All studied groups showed a similar increase in cell proliferation with or without OM. After 7 and 10days, a significatively higher concentration of VEGF-164 in L - group when compared to C - group was observed. A significant increase in mineralized nodules in the L + was noted when compared to C + in the same conditions. Photobiomodulation upregulated significantly Runx2 and Dmp1 expression after 10days in L - and after 7days in L + , with downregulation of Dmp1 after 10days in L + group. Immunolocalization of RUNX2 and MEPE was expressive after 7days of culture in the cytoplasm adjacent to the nucleus with a decrease after 10days, regardless of the presence of OM. Photobiomodulation enhances metabolism associated with angiogenesis, gene expression, and mineralization regardless of the odontogenic medium in OD-21 cells.

GENE EXPRESSION LEVEL, IMMUNOLOCALIZATION, AND FUNCTION OF FATTY ACID-BINDING PROTEIN FROM SCHISTOSOMA JAPONICUM.

The Schistosoma japonicum fatty acid-binding protein (FABP) is used in the cell membrane to absorb and transport fatty acids, which cannot be resynthesized by the organism and combined with hydrophobic ligands. Among the 5 stages of the worm life cycle examined, FABP messenger ribonucleic acid (mRNA) expression was highest in male adult worms, followed by the liver-stage schistosome, and was the lowest in the lung-stage schistosome. The fabp gene-coding region was cloned and expressed to obtain recombinant S. japonicum FABP (rSjFABP) with a molecular weight of approximately 18 kDa. Mice were then immunized against rSjFABP to prepare anti-FABP serum. Using immunohistochemical techniques, FABP protein was found to localize to the eggshell, parenchyma, and digestive tract. Double-stranded RNA-mediated knockdown of FABP mRNA by RNA interference decreased the number of transcripts by >70%. Moreover, the egg production rate decreased, whereas the abnormal egg ratio was significantly increased in the FABP-silenced group compared with the negative control group (P < 0.05). These results demonstrate that FABP localizes in adults and in various stages. FABP contributes to the egg-laying capacity of adults, which may be related to the reproductive function of S. japonicum.

Immunolocalization of some HOX proteins in immature and mature feline testes.

Homeobox (HOX) proteins are known for their critical role in body shape formation and tissue differentiation of developing vertebrate embryos. Recent research has shown that HOX proteins have many physiological roles such as cell proliferation, cell cycle, apoptosis and cell differentiation in adults, as well as the development of the vertebrate nerve and reproductive system. This study was conducted to determine the possible physiological functions and expression intensities of HOXA10, HOXA11, HOXB6 and HOXC6 proteins in the male reproductive system (testes, epididymis and deferens ducts), which are important for the continuity of some specific cat breeds in different age ranges. In the study, a total of 18 testicular tissues were used, divided into two groups: less than 6months (immature) and more than 1year (mature). Tissue samples were then subjected to immunohistochemical staining with protein-specific antibodies examined in the study. In the findings obtained in the research; it was observed that HOXA10, HOXA11, HOXB6 and HOXC6 produced different intensities of immunolocalization in the epididymis and ductus deferens layers in the immature and mature testicular cells. In addition, it was found that HOXA10 immunoreaction was also seen in some vascular endothelial cells. As a result, it was concluded that the HOX proteins could contribute to the physiological functions of testes, epididymis and ductus deferens and affect male fertility.

Association between mucosal barrier disruption by Pseudomonas aeruginosa exoproteins and asthma in patients with chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a common chronic respiratory condition, frequently associated with asthma and affecting the majority of cystic fibrosis (CF) patients. Pseudomonas aeruginosa infections and biofilms have been implicated in recalcitrant CRS. One of the mechanisms of action for bacteria in CRS and CF is mucosal barrier disruption by secreted products that contribute to the inflammation. However, the role of biofilm and planktonic forms of P. aeruginosa in this process is not known. The aim is to determine the effect of P. aeruginosa exoproteins isolated from CF and non-CF CRS patients on the mucosal barrier. Exoproteins from 40 P. aeruginosa isolates were collected in planktonic and biofilm forms and applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs). Mucosal barrier integrity was evaluated by transepithelial electrical resistance (TEER), passage of FITC-dextrans and immunofluorescence of tight junction proteins. Cytotoxicity assays were performed to measure cell viability, and IL-6 ELISA was carried out to evaluate pro-inflammatory effects. Planktonic exoproteins from 20/40 (50%) clinical isolates had a significant detrimental effect on the barrier and significantly increased IL-6 production. Barrier disruption was characterized by a reduced TEER, increased permeability of FITC-dextrans and discontinuous immunolocalization of tight junction proteins and was significantly more prevalent in isolates harvested from patients with comorbid asthma (P<.05). Exoproteins from planktonic P. aeruginosa clinical isolates from asthmatic CRS patients have detrimental effects on the mucosal barrier and induce IL-6 production potentially contributing to the mucosal inflammation in CRS patients.

FC 001ACIDOSIS AND ALKALI THERAPY ARE ASSOCIATED WITH TRANSCRIPTIONAL CHANGES AND ALTERED ABUNDANCE OF GENES INVOLVED IN CELL METABOLISM AND BICARBONATE TRANSPORT IN KIDNEY TRANSPLANT RECIPIENTS

Abstract Background and Aims Metabolic acidosis is a common event in kidney transplant recipients and has been associated to a higher risk of graft loss and mortality. In patients with CKD and acidosis, alkali therapy ameliorating acidosis appears to protect kidney function. However, it is still poorly understood how acidosis causes the detrimental effects to kidney graft function and how alkali therapy would interact with these mechanisms. Here we aim to identify transcriptomic alterations in kidney transplant recipients without metabolic acidosis in comparison to patients with metabolic acidosis with and without alkali therapy. Moreover, we examined immunolocalization of key proteins involved in acid-base base regulation in biopsies from these patients. Method We obtained 22 biopsies of patients 4-6 years after kidney transplantation. Among these patients, nine were not acidotic (serum [HCO3-] ≥ 22 mM), nine had acidosis ([HCO3-] &amp;lt; 22 mM), and four had acidosis and received sodium bicarbonate (alkali therapy) fully correcting acidosis. Age, immunosuppressive drugs, time after transplantation, and eGFR were not statistically different between groups. RNA was extracted from biopsies and RNAseq was performed. Immunohistochemistry was performed for key proteins involved in the renal regulation of acid-base balance. Additionally, a control group of 6 non-transplanted healthy kidneys was included in the histology analysis. Results RNAseq analysis revealed 40 genes differentially expressed between acidosis and no acidosis groups. While most of the genes tended to be recovered by alkali therapy, only three fully recovered with bicarbonate supplementation (p-value &amp;lt; 0.05 and log2(fold change) above 0.5). These genes were KCNJ15 (Kir4.2), SHMT1, and ACADSB. Renal localization of the genes was determined using single-cell RNA sequencing data (Ransick et al., Developmental Cell, 2019, doi.org/10.1016/j.devcel.2019.10.005). Most of the genes were expressed in the proximal tubule and were organized in the model shown in Figure 1A. Several of these genes participate in cell metabolism, such as beta-oxidation, and iron, folate, and methionine metabolism. Moreover, the K+-channel Kir4.2 regulates the activity of the electrogenic sodium bicarbonate cotransporter 1 (NBCe1, SLC4A4) and ammoniagenesis in renal proximal tubules. Immunofluorescence analysis showed that NBCe1 expression in proximal tubules was strongly reduced in patients who developed acidosis and was partially recovered in patients who received alkali therapy (Figure 1B). In type B intercalated cells, a similar pattern was observed for Pendrin (SLC26A4). No alteration in the expression of GDH (GLUD1), AE1 (SLC4A1), AQP2, CA2, RhCG (SLC42A3), and B1 subunit of the H+ATPase (ATP6V1B1) was observed in kidneys of treated or untreated patients with acidosis. Conclusion Kidney transplant recipients suffering from metabolic acidosis show distinct expression pattern of genes involved in cell metabolism and acid-base transport.

Expression patterns of iron regulatory proteins after intense light exposure in a cone-dominated retina.

Iron is implicated in ocular diseases such as in age-related macular degeneration. Light is also considered as a pathological factor in this disease. Earlier, two studies reported the influence of constant light environment on the pattern of expressions of iron-handling proteins. Here, we aimed to see the influence of light in 12-h light-12-h dark (12L:12D) cycles on the expression of iron-handling proteins in chick retina. Chicks were exposed to 400 lx (control) and 5000 lx (experimental) light at 12L:12D cycles and sacrificed at variable timepoints. Retinal ferrous ion (Fe2+) level, ultrastructural changes, lipid peroxidation level, immunolocalization and expression patterns of iron-handling proteins were analysed after light exposure. Both total Fe2+ level (p = 0.0004) and lipid peroxidation (p = 0.002) significantly increased at 12-, 48- and 168-h timepoint (for Fe2+) and 48- and 168-h timepoint (for lipid peroxidation), and there were degenerative retinal changes after 168 h of light exposure. Intense light exposure led to an increase in the levels of transferrin and transferrin receptor-1 (at 168-h) and ferroportin-1, whereas the levels of ferritins, hephaestin, (at 24-, 48- and 168-h timepoint) and ceruloplasmin (at 168-h timepoint) were decreased. These changes in iron-handling proteins after light exposure are likely due to a disturbance in the iron storage pool evident from decreased ferritin levels, which would result in increased intracellular Fe2+ levels. To counteract this, Fe2+ is released into the extracellular space, an observation supported by increased expression of ferroportin-1. Ceruloplasmin was able to convert Fe2+ into Fe3+ until 48 h of light exposure, but its decreased expression with time (at 168-h timepoint) resulted in increased extracellular Fe2+ that might have caused oxidative stress and retinal cell damage.

Reproductive Isolation Between Taxonomically Controversial Forms of the Gray Voles (Microtus, Rodentia; Arvicolinae): Cytological Mechanisms and Taxonomical Implications.

The formation of hybrid sterility is an important stage of speciation. The voles of the genus Microtus, which is the most speciose genus of rodents, provide a good model for studying the cytological mechanisms of hybrid sterility. The voles of the “mystacinus” group of the subgenus Microtus (2n = 54) comprising several recently diverged forms with unclear taxonomic status are especially interesting. To resolve the taxonomic status of Microtus mystacinus and Microtus kermanensis, we crossed both with Microtus rossiaemeridionalis, and M. kermanensis alone with Microtus arvalis “obscurus” and M. transcaspicus and examined the reproductive performance of their F1 hybrids. All interspecies male hybrids were sterile. Female M. kermanensis × M. arvalis and M. kermanensis × M. transcaspicus hybrids were sterile as well. Therefore, M. mystacinus, M. kermanensis, and M. rossiaemeridionalis could be considered valid species. To gain an insight into the cytological mechanisms of male hybrid sterility, we carried out a histological analysis of spermatogenesis and a cytological analysis of chromosome synapsis, recombination, and epigenetic chromatin modifications in the germ cells of the hybrids using immunolocalization of key meiotic proteins. The hybrids showed wide variation in the onset of spermatogenesis arrest stage, from mature (although abnormal) spermatozoa to spermatogonia only. Chromosome asynapsis was apparently the main cause of meiotic arrest. The degree of asynapsis varied widely across cells, individuals, and the crosses—from partial asynapsis of several small bivalents to complete asynapsis of all chromosomes. The asynapsis was accompanied by a delayed repair of DNA double-strand breaks marked by RAD51 antibodies and silencing of unpaired chromatin marked by γH2A.X antibodies. Overall, the severity of disturbances in spermatogenesis in general and in chromosome synapsis in particular increased in the hybrids with an increase in the phylogenetic distance between their parental species.

CDH12 as a Candidate Gene for Kidney Injury in Posterior Urethral Valve Cases: A Genome-wide Association Study Among Patients with Obstructive Uropathies

BackgroundPosterior urethral valves (PUVs) and ureteropelvic junction obstruction (UPJO) are congenital obstructive uropathies that may impair kidney development. ObjectiveTo identify genetic variants associated with kidney injury in patients with obstructive uropathy. Design, setting, and participantsWe included 487 patients born in 1981 or later who underwent pyeloplasty or valve resection before 18 yr of age in the discovery phase, 102 PUV patients in a first replication phase, and 102 in a second replication phase. Outcome measurements and statistical analysisSigns of kidney injury were defined as dialysis, nephrectomy, kidney transplantation, estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m2, high blood pressure, antihypertensive medication use, proteinuria, and/or one kidney functioning at <45%. We used χ2 tests to calculate p values and odds ratios for >600 000 single-nucleotide polymorphisms (SNPs) in the discovery sample comparing patients with and without signs of kidney injury within 5 yr after surgery. We performed stratified analyses for PUV and UPJO and Kaplan-Meier and Cox regression analyses in the discovery and two replication samples for the associated SNPs, and RNA and protein expression analyses for the associated gene in fetal tissues. Results and limitationsDespite the small and nonhomogeneous sample, we observed suggestive associations for six SNPs in three loci, of which rs6874819 in the CDH12 gene was the most clear (p = 7.5 × 10–7). This SNP also seemed to be associated with time to kidney injury in the PUV discovery and replication samples. RNA expression analyses showed clear CDH12 expression in fetal kidneys, which was confirmed by protein immunolocalization. ConclusionsThis study identified CDH12 as a candidate gene for kidney injury in PUV. Patient summaryWe found that variants of the CDH12 gene increase the risk of kidney injury in patients with extra flaps of tissue in the urethra (posterior urethral valves). This is the first report on this gene in this context. Our study provides interesting new information about the pathways involved and important leads for further research for this condition.

Immunolocalization of epidermal differentiation complex proteins reveals distinct molecular compositions of cells that control structure and mechanical properties of avian skin appendages

The epidermal differentiation complex (EDC) is a cluster of genes that encode structural proteins of skin derivatives with variable mechanical performances, from the scales of reptiles and birds to the hard claws and beaks, and to the flexible but resistant corneous material of feathers. Corneous proteins with or without extended beta-regions are produced from avian genomes, and include the largely prevalent corneous beta proteins (CβPs, formerly indicated as beta-keratins), and minor contribution from histidine-rich proteins, trichohyalin-like proteins (scaffoldin), loricrin, and other proteins rich in cysteine or other types of amino acids. The light-microscopic and ultrastructural immunolocalization of major and minor EDC-proteins in avian skin (feather CβPs, EDKM, EDWM, EDMTFH, EDDM, and scaffoldin) suggests that each specific appendage consists of a particular mix of these proteins in addition to the main proteins containing a peculiar beta-region of 34 amino acids, indicated as feather/scale/claw/beak CβPs (fCβPs, sCβPs, cCβPs, bCβPs). This indicates that numerous proteins of the EDC are added to the variable meshwork of intermediate filament keratins to produce avian epidermis with different mechanical and functional properties. Although the specific roles for these proteins are not known they likely make an important contribution to the final material properties of the different skin appendages of birds. The highest number of sauropsid CβPs is found in birds, suggesting a relation to the evolution of feathers, and additional epidermal differentiation proteins have contributed to the evolutionary adaptations of avian skin.