Immunofluorescent Localization Research Articles

Embryonic growth retardation and altered expression of IGF-II is reciprocal induced by phytocompounds during early gestation in mice

Methanolic crude extract of Scoparia dulcis (CESD) was orally administered to female mice during the early gestation (day 4–day 8) at a dose of 500 mg/kg/day. It induces embryo resorption and morphological changes of fetal maternal tissue. Histomorphology was studied by routine hematoxylin eosin stain. In situ immunofluorescence localization of IGF-II using Texas red showed an ordered expression of the growth factor in the maternal decidual cells, trophoblast cells and the embryo. Western blot analysis showed a gradual increase of IGF-II from D4 to D8 of control females. In contrast, the CESD-treated females showed resorption of embryo on D8 with disorganized in situ expression and lowered IGF-II in fetal maternal tissue. The phytocompounds present in the CESD could modulate either the ER or IGF-II receptors causing reduced IGF-II expression in the target tissues which lead to the failure of embryonic growth during periimplantation.

MiPDCD6 effector suppresses host PAMP‐triggered immunity to facilitate Meloidogyne incognita parasitism in tomato

AbstractPlant‐parasitic nematodes secrete several effectors that suppress host PAMP‐triggered immunity for successful parasitism. Here, we have characterized a novel effector from the root‐knot nematode Meloidogyne incognita, named MiPDCD6. The MiPDCD6 gene is expressed in the subventral oesophageal glands and is upregulated in the mid‐stages of M. incognita parasitism in plants. Tobacco rattle virus‐induced gene silencing (VIGS) targeting MiPDCD6 attenuated M. incognita parasitism. Compared with the control, in tomato plants with MiPDCD6 VIGS, the numbers of root knots were significantly reduced by 46.3%, giant cell development was delayed by 7 days (as shown by the improved paraffin section technique), and the expression levels of both PR‐1 and PR‐5 tomato genes were significantly higher, as shown by reverse transcription‐PCR. Although MiPDCD6 lacks a classic signal peptide for secretion at its N‐terminus, results of immunofluorescence localization showed that MiPDCD6 could be secreted by M. incognita, transported and function in giant cells of tomato. Transient expression of MiPDCD6 in Nicotiana benthamiana suppressed cell death triggered by BAX and callose deposition induced by PIAvh142, inhibited reactive oxygen species accumulation induced by flg22, and reduced expression of PR1 and PR5, which are marker genes of the salicylic acid (SA)‐mediated immune signalling pathway. These results indicate that M. incognita deploys MiPDCD6 to suppress SA‐mediated plant immunity to promote its parasitism.

Identification and expression analysis of a new small ubiquitin-like modifier from Taenia pisiformis

The small ubiquitin-like modifier (SUMO) plays important roles, with the SUMOylation pathway as one of its core components. In the present work, a single SUMO gene was initially identified from Taenia pisiformis and designated as TpSUMO. Bioinformatic analysis showed that the TpSUMO gene contained a 309 bp open reading frame (ORF), encoding 102 amino acids, and had a predicted molecular weight of ∼12 kDa. The amino acid sequence of TpSUMO was deduced and it shared 44.00% identity with human SUMO2 (HsSUMO2) and exhibited more than 97.78% identity with SUMOs from Taenia and Echinococcus. TpSUMO possessed a putative non-consensus site (FK11MG) within its N-terminus and a typical di-glycine (GG) motif at the C-terminus. Basic local alignment search tool (BLAST) analysis showed that only a single SUMO-related ortholog was present in each set of known genome data for fourteen tapeworm species. The precursor His-TpSUMO-FL, mature His-TpSUMO-GG and mutant His-TpSUMO-GGK11R proteins (∼18 kDa) were expressed in Escherichia coli Rosseta (DE3), and rabbit polyclonal anti-TpSUMO was generated with a high titer of 1.28 × 105. In vitro SUMOylation assay results showed that TpSUMO multimer formation in the His-TpSUMO-GG reaction could be catalyzed by the human SAE1/SAE2 and UBC9 conjugation system, but K11R mutation disrupted TpSUMO chain synthesis. Quantitative real-time PCR (qRT-PCR) further revealed that TpSUMO was ubiquitously expressed in different stages of T. pisiformis and in higher levels during an early development phase (day 14) of adult worms. Immunofluorescence localization showed that TpSUMO was detected in the bladder wall of cysticerci, in the testis in immature segment, and within eggs in the gravid proglottids. These findings indicated that TpSUMO is a new member of the SUMO protein family and may play a vital role in regulation of functions within proteins involved in worm growth and development.

Beneficial Effect of Proline Supplementation on Goat Spermatozoa Quality during Cryopreservation.

Simple SummarySperm suffers damage from reactive oxygen species stress (ROS) during cryopreservation. Supplementation of antioxidants is suggested to reduce sperm cryodamage induced by ROS. Proline has been regarded as a powerful antioxidant. However, there is no information on the role proline plays during goat sperm cryopreservation. Thus, this study aims to investigate the effects of proline during goat sperm cryopreservation procedures. In the present study, supplementation of 2 mM proline significantly enhanced post-thaw goat sperm quality by regulating sperm redox homeostasis. Proline supplementation protects goat sperm against ROS stress through PRODH-mediated metabolism.Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.

Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells.

Activation of the integrin phagocytic receptors CR3 (αMβ2, CD11b/CD18) and CR4 (αXβ2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (β2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMβ2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMβ2 and αXβ2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.

Phosphorylation of the Autographa californica multiple nucleopolyhedrovirus polyhedron envelope protein plays an important role in the formation of the occlusion body envelope.

During the life cycle of a baculovirus, a crystallized protein matrix, formed by polyhedrin (POLH), is produced. The protein matrix is surrounded by a multilayered protein/carbohydrate envelope, and matrix and envelope together form a mature occlusion body (OB). The polyhedron envelope plays an important role in resistance against adverse external environments. The polyhedron envelope protein (PEP) is the main protein that forms the polyhedron envelope, but the mechanism of formation of the polyhedron envelope is unclear. Here, through immunofluorescence localization observations, we found that PEP interacted with both POLH and P10 during formation of the polyhedron envelope in the late stages of infection, and PEP was also required for P10 incorporation on the surface of OBs. In this process, the phosphorylation of PEP played an important role. PEP was determined to be a phosphorylated protein using the Phos-tag technique, and PK1 was determined to be the phosphokinase of PEP by co-immunoprecipitation and in vitro phosphorylation. Immunofluorescence localization revealed that PEP was continuously phosphorylated by PK1 after PEP entered the nucleus until PEP was correctly packaged on the OB surface. Multi-point mutations of PEP conservative potential phosphorylation sites showed that the simultaneous mutation of S85, T86 and Y92 caused changes in the location of PEP and P10 in the late stages of infection, and resulted in an OB surface that lacked the polyhedron envelope. These data suggested that the phosphorylation of PEP at particular sites, i.e. S85, T86 and Y92, plays an important role in the formation of the polyhedron envelope.

A prospective pilot study of narrowband UV-B treatment of lichen planopilaris

A prospective pilot study of narrowband UV-B treatment of lichen planopilaris

New Insights Into the Local Auxin Biosynthesis and Its Effects on the Rapid Growth of Moso Bamboo (Phyllostachys edulis).

Auxin plays a crucial regulatory role in higher plants, but systematic studies on the location of auxin local biosynthesis are rare in bamboo and other graminaceous plants. We studied moso bamboo (Phyllostachys edulis), which can grow up to 1 m/day and serves as a reference species for bamboo and other fast-growing species. We selected young tissues such as root tips, shoot tips, young culm sheaths, sheath blades, and internode divisions for local auxin biosynthesis site analysis. IAA immunofluorescence localization revealed that auxin was similarly distributed in different stages of 50-cm and 300-cm bamboo shoots. Shoot tips had the highest auxin content, and it may be the main site of auxin biosynthesis in the early stage of rapid growth. A total of 22 key genes in the YUCCA family for auxin biosynthesis were identified by genome-wide identification, and these had obvious tissue-specific and spatio-temporal expression patterns. In situ hybridization analysis revealed that the localization of YUCCA genes was highly consistent with the distribution of auxin. Six major auxin synthesis genes, PheYUC3-1, PheYUC6-1, PheYUC6-3, PheYUC9-1, PheYUC9-2, and PheYUC7-3, were obtained that may have regulatory roles in auxin accumulation during moso bamboo growth. Culm sheaths were found to serve as the main local sites of auxin biosynthesis and the auxin required for internode elongation may be achieved mainly by auxin transport.

Andrographolide‐Mediated Protection of Barrier Integrity and Permeability is Mediated Through NLRP3 Inflammasome Inhibition in Blood‐Brain Barrier Endothelial Cells

The blood‐brain barrier (BBB) that consists of the inter‐endothelial tight junctions, acts as the major blood‐brain interface and regulator of brain microvascular hyperpermeability. In the brain, the loss of BBB integrity and hyperpermeability leads to cerebral edema formation and intracranial pressure elevation in traumatic brain injury and ischemic strokes. Recent studies demonstrated that reactive oxygen species are potential signals for the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation that leads to interleukin‐1β (IL‐1β) secretion. IL‐1β plays a major role in promoting BBB hyperpermeability in various brain pathologies, including TBI. Furthermore, recent studies from our lab have demonstrated a tri‐phasic role for hydrogen peroxide (H2O2) in BBB endothelial cells where it promotes, angiogenesis, hyperpermeability and apoptosis in a tri‐phasic and concentration dependent manner. Hydrogen peroxide plays a major role physiologically as the second messenger and pathologically as an inducer of oxidative stress and associated cellular signaling. We hypothesized that in BBB endothelial cells, H2O2‐induced barrier dysfunction and hyperpermeability are NLRP3 inflammasome‐dependent. The main objective of our study was to evaluate the role of NLRP3 inflammasome signaling in H2O2‐mediated barrier dysfunction and hyperpermeability in human brain microvascular endothelial cells and to test whether andrographolide, a labdane diterpenoid possessing potent anti‐inflammatory properties will attenuate such effects. Th effect of H2O2 treatment on tight junction integrity/permeability was studied using zonula occludens‐1 (ZO‐1) immunofluorescence localization, ZO‐1 immunoblot analysis, Transwell monolayer permeability assay using FITC‐dextran (10‐kDa) as a fluorescent marker and cell viability/apoptosis assay. The effect of NLRP3 inhibitor (MCC950) and andrographolide on H2O2‐induced hyperpermeability was studied using Transwell permeability assay. Our results show that, CRISPR/Cas‐9‐mediated knockdown of ZO‐1 resulted in monolayer hyperpermeability demonstrating the significance of ZO‐1 in regulating barrier functions. CRISPR‐based activation of NLRP3 induced monolayer hyperpermeability whereas CRISPR/Cas‐9‐based NLRP3 knockdown had no significant effect on permeability. H2O2 (10µM) induced monolayer hyperpermeability and the effect was decreased by MCC950 and andrographolide treatment. Hydrogen peroxide induced cathepsin B (an activator of the NLRP3 pathway) activity significantly, and the effect was decreased by andrographolide treatment. H2O2‐mediated barrier dysfunction and hyperpermeability were not due to changes in cell viability/apoptosis. These results suggest that NLRP3 is a mediator of H2O2‐induced barrier dysfunction and hyperpermeability in human BBB endothelial cells and andrographolide is an effective inhibitor of this pathway.

Inhibitory effect of artesunate on bone destruction in rheumatoid arthritis: an exploration based on AhR/ARNT/NQO1 signaling pathway

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 μmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 μmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.

Inhibition of SGK1 ameliorates ventriculomegaly in a genetic rat model via regulation of TRPV4 in the choroid plexus

Hydrocephalus is a clinical manifestation of cerebrospinal fluid (CSF) accumulation, leading to ventriculomegaly and increased intracranial pressure. Diuretics and other pharmacotherapies have failed to treat hydrocephalus in the human population; therefore, treatment options remain exclusively surgical. The need to develop pharmacotherapies for the treatment of hydrocephalus remains an unmet clinical need and paramount to this need is the study of brain fluid regulation. Previous studies in our laboratory identified the transient receptor potential vanilloid 4 (TRPV4) channel as a potential regulatory hub protein in the choroid plexus responsible, in part, for the regulation of CSF production. Antagonists of the TRPV4 channel were shown to ameliorate hydrocephalus in a genetic rat model. The current studies highlight a novel drug target: serum‐ and glucocorticoid‐induced kinase 1 (SGK1) in the choroid plexus. A specific small molecular weight, membrane permeant inhibitor of SGK1, SI113, has shown promising results in cancer and metabolic research in recent years. In other tissues, SGK1 has been shown to phosphorylate and activate TRPV4 at the membrane, indicating SGK1 as an important upstream regulatory kinase. SI113 blocked TRPV4‐mediated transepithelial ion flux and barrier conductance increases in a cell culture model of human choroid plexus epithelia (HIBCPP line), and ameliorated ventriculomegaly in the genetic rat model. In hydrocephalic animals, there is an increased apical membrane signal in the choroid plexus epithelial cells via immunofluorescent localization of p‐SGK1 and TRPV4, but not of pan‐SGK1. This is not accompanied by a significant increase in either protein or transcript abundance as measured by western blotting or qPCR, respectively. The change in localization is, however, attenuated by treatment with SI113. Animals treated with SI113 show no change in amounts of TRPV4 or total SGK1, but do show significantly reduced levels of p‐SGK1. Thus, phosphorylation of SGK1 appears to be crucial for the activation of TRPV4 which is, in turn, a channel protein that is activated during the development of hydrocephalus. Like Trpv4‐/‐mice, Sgk1‐/‐mice do not show overt phenotypes, indicating pharmacological antagonism of the kinase would have minimal off‐target effects. Overall, these data provide a strong preclinical basis for the use of SGK1 inhibitors in the treatment of hydrocephalus.

Osteopontin-induced vascular hyperpermeability through tight junction disruption in diabetic retina

Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.

ADRV 12L: A Ranaviral Putative Rad2 Family Protein Involved in DNA Recombination and Repair.

The Andrias davidianus ranavirus (ADRV) is a member of the family Iridoviridae and belongs to the nucleocytoplasmic large DNA viruses. Based on genomic analysis, an ADRV-encoding protein, ADRV 12L, and its homologs from other iridoviruses were predicted as Rad2 family proteins based on the conserved amino acids, domains, and secondary structures. Expression analysis showed that the transcription of ADRV 12L started at 4 h post infection, and its expression was not inhibited by a DNA-replication inhibitor. Meanwhile, immunofluorescence localization showed that ADRV 12L mainly localized in viral factories and colocalized with the viral nascent DNA, which hinted at a possible role in DNA replication. Furthermore, a mutant ADRV lacking 12L (ADRV-Δ12L) was constructed. In both luciferase assays based on homologous recombination (HR) and double-strand break repair (DSBR) that followed, ADRV-Δ12L induced less luciferase activity than the wild-type ADRV, indicating that HR and DSBR were impaired in ADRV-Δ12L infected cells. In addition, infection with ADRV-Δ12L resulted in smaller plaque sizes and lower viral titers than that with wild-type ADRV, indicating an important role for 12L in efficient virus infection. Therefore, the results suggest that Rad2 homologs encoded by iridovirus have important roles in HR- and DSBR-process of the viral DNA and, thus, affect virus replication and the production of progeny virions.

Multiple cell types in the oviduct express the prolactin receptor.

Little is known about the physiological role of prolactin in the oviduct. Examining mRNA for all four isoforms of the prolactin receptor (PRLR) in mice by functional oviduct segment and stage of the estrous cycle, we found short form 3 (SF3) to be the most highly expressed, far exceeding the long form (LF) in highly ciliated areas such as the infundibulum, whereas in areas of low ciliation, the SF3 to LF ratio was ~1. SF2 expression was low throughout the oviduct, and SF1 was undetectable. Only in the infundibulum did PRLR ratios change with the estrous cycle. Immunofluorescent localization of SF3 and LF showed an epithelial (both mucosal and mesothelial) distribution aligned with the mRNA results. Despite the high SF3/LF ratio in densely ciliated regions, these regions responded to an acute elevation of prolactin (30 min, intraperitoneal), with LF‐tyrosine phosphorylated STAT5 seen within cilia. Collectively, these results show ciliated cells are responsive to prolactin and suggest that prolactin regulates estrous cyclic changes in ciliated cell function in the infundibulum. Changes in gene expression in the infundibulum after prolonged prolactin treatment (7‐day) showed prolactin‐induced downregulation of genes necessary for cilium development/function, a result supporting localization of PRLRs on ciliated cells, and one further suggesting hyperprolactinemia would negatively impact ciliated cell function and therefore fertility. Flow cytometry, single‐cell RNAseq, and analysis of LF‐td‐Tomato transgenic mice supported expression of PRLRs in at least a proportion of epithelial cells while also hinting at additional roles for prolactin in smooth muscle and other stromal cells.

Molecular characterization of glyceraldehyde-3-phosphate dehydrogenase from Eimeria tenella.

Chicken coccidiosis is an extremely common and lethally epidemic disease caused by Eimeria spp. The control measures of coccidiosis depend mainly on drugs. However, the ensuing drug resistance problem has brought considerable economic loss to the poultry industry. In our previous study, comparative transcriptome analyses of a drug-sensitive (DS) strain and two drug-resistant strains (diclazuril-resistant (DZR) and maduramicin-resistant (MRR) strains) of Eimeria tenella were carried out by transcriptome sequencing. The expression of glyceraldehyde-3-phosphate dehydrogenase of E. tenella (EtGAPDH) was upregulated in the two resistant strains. In this study, we cloned and characterized EtGAPDH. Indirect immunofluorescence localization was used to observe the distribution of EtGAPDH in E. tenella. The results showed that the protein was distributed mainly on the surface of sporozoites and merozoites, and in the cytoplasm of merozoites. qPCR was performed to detect the transcription level of EtGAPDH in the different developmental stages of the E. tenella DS strain. The transcription level of EtGAPDH was significantly higher in second-generation merozoites than in the other three stages. The transcription level of EtGAPDH in the different drug-resistant strains and DS strain of E. tenella was also analyzed by qPCR. The results showed that the transcription level was significantly higher in the two drug-resistant strains (MRR and DZR) than in the DS strain. As the concentration of diclazuril and maduramicin increased, the transcription levels also increased. Western blot results showed that EtGAPDH protein was upregulated in the DZR and MRR strains. Enzyme activity showed that the enzyme activity of EtGAPDH was higher in the two resistant strains than in the DS strain. These results showed that EtGAPDH possess several roles that separate and distinct from its glycolytic function and maybe involved in the development of E. tenella resistance to anticoccidial drugs.

Molecular characterization, immunofluorescent localization, and expression levels of two bicarbonate anion transporters in the whitish mantle of the giant clam, Tridacna squamosa, and the implications for light-enhanced shell formation

Giant clams conduct light-enhanced shell formation, which requires the increased transport of Ca2+ and inorganic carbon (Ci) from the hemolymph through the shell-facing epithelium of the whitish inner mantle to the extrapallial fluid where CaCO3 deposition occurs. The major form of Ci in the hemolymph is HCO3−, but the mechanisms of HCO3− transport through the basolateral and apical membranes of the shell-facing epithelial cells remain unknown. This study aimed to clone from the inner mantle of Tridacna squamosa the complete coding cDNA sequences of electrogenic Na+-HCO3−cotransporter 1 homolog (NBCe1-like-b) and electrogenic Na+-HCO3−cotransporter 2 homolog (NBCe2-like). NBCe1-like-b comprised 3360 bp, encoding a 125.7 kDa protein with 1119 amino acids. NBCe1-like-b was slightly different from NBCe1-like-a of the ctenidium reported elsewhere, as it had a serine residue (Ser1025), which might undergo phosphorylation leading to the transport of Na+: HCO3− at a ratio of 1: 2 into the cell. NBCe1-like-b was localized at the basolateral membrane of the shell-facing epithelial cells, and its gene and protein expression levels increased significantly in the inner mantle during illumination, indicating a role in the light-enhanced uptake of HCO3− from the hemolymph. The sequence of NBCe2-like obtained from the inner mantle was identical to that reported previously for the outer mantle. In the inner mantle, NBCe2-like had an apical localization in the shell-facing epithelial cells, and its protein abundance was upregulated during illumination. Hence, NBCe2-like might take part in the light-enhanced transport of HCO3− through the apical membrane of these cells into the extrapallial fluid.

A Novel Idiopathic Atrial Calcification: Pathologic Manifestations and Potential Mechanism.

BackgroundCardiac calcification is a type of ectopic pathologic calcification of unknown etiology and mechanisms. Once diagnosed, the location, extent and morphology of the calcified lesions, as well as their functional significance in the heart, are usually the focus of case reports. Calcification is mostly distributed in myocardium, but rarely reported in atrium. In addition, because of limited sampling and complex pathological mechanisms, the etiology underlying the formation of these calcified lesions also remains unclear.MethodsTwo cardiac calcifications were found in a patient, died of trauma-induced subarachnoid hemorrhage after slightly drinking, during a standard autopsy. The location and morphological characteristics of the calcified lesions were determined by computed tomography (CT) and CT-based 3D reconstruction. The specific histopathological characteristics of the lesions were determined by multi-staining. The concentration of free calcium and inorganic pyrophosphate (PPi) in plasma reflected the change of calcium metabolism. The expression and membranal localization of the ATP Binding Cassette Subfamily C Member 6 (ABCC6) in hepatocytes were detected by immunofluorescence. The variants of the ABCC6 were detected by Sanger sequencing and potential pathogenic variants were further identified by in silico analysis.ResultsThe present study describes a patient with idiopathic calcification with two pear-shaped and irregularly hollow lesions symmetrically distributed in the patient's atrium. Massive accumulation of calcium salts was identified by multi-staining. For this patient, the plasma concentration of free calcium was higher than the control, indicating that calcium metabolism was disturbed. Furthermore, the plasma PPi of the patient was lower than the normal. By using immunofluorescence, the expression and membranal localization of ABCC6 was decreased and impaired in hepatocytes, respectively. Combined with Sanger sequencing and in silico analysis, 7 variants were identified.ConclusionsThis study described a novel patient with symmetrically distributed idiopathic atrial calcifications. Furthermore, all the results indicated that these pathologic calcifications may be secondary to reduced plasma PPi content due to ABCC6 dysfunction in hepatocytes. Moreover, these findings provided novel clues to the pathogenesis, clinical diagnosis and treatment of idiopathic atrial calcification in future.

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.

Rare missense mutations and compound heterozygous mutations in ABCA7 contribute to Alzheimer's disease in Belgian patients

Initially, ABCA7 was associated with Alzheimer's disease (AD) in large genome-wide association studies. Targeted resequencing of ABCA7 suggested a role for rare premature termination codon (PTC) mutations. We observed loss of ABCA7 in PTC carriers, although with high variability due to differences in transcript rescue. Additionally, rare missense mutations are present in ABCA7 with unknown effect on the protein. We aimed to investigate the contribution of rare (MAF≤1%) mutations or compound heterozygous mutations in ABCA7 in Belgian AD patient and control cohorts, as well as the effect of rare missense mutations on the subcellular localization of the protein. Targeted resequencing or whole exome sequencing of ABCA7 exons and splice sites in 1375 Belgian AD patients and 976 controls. We validated all rare, non-synonymous coding and splice mutations. Allele-specific PCR, haplotype sharing analysis and long-read sequencing to determine cis/trans configuration of compound heterozygous mutations. In vitro mutagenesis to introduce missense mutations of interest in an entry vector. Gateway cloning to generate wild type and mutant ABCA7 pCR3 expression vectors with a C-terminal EmGFP-tag. Transfection of HeLa and HEK293 cells for immunofluorescence microscopy to localize wild type versus mutant ABCA7. We identified rare mutations in 12.7% (175/1375) of the patients and in 7.8% (76/976) of the controls. We observed co-segregation of p.G1820S with AD in a family with apparent autosomal dominant inheritance. We observed compound heterozygous mutations in 10 patients (0.73%) and five controls (0.51%). We confirmed trans configuration in four of the patients and cis configuration in four other patients. In the remaining two patients, configuration remains unknown. In the five controls, we confirmed trans configuration in one and cis configuration in the other four. We selected eight missense mutations present in Belgian AD patients (Figure) to introduce in an entry vector. Mutant and wild type entry clones were subcloned in the expression vector to transfect and generate immunofluorescence localization data. We observed an enrichment in patients of rare single mutations and compound heterozygous mutations. In addition, trans compound heterozygous mutations might modify penetrance and pathogenicity. Functional studies of missense mutations are necessary to determine their potential pathogenic effect.

Immune Response in Myocardial Injury: In Situ Hybridization and Immunohistochemistry Techniques for SARS-CoV-2 Detection in COVID-19 Autopsies.

Coronavirus disease-19 (COVID-19) is caused by the newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the lung remains the primary target site of COVID-19 injury, damage to myocardium, and other organs also contribute to the morbidity and mortality of this disease. There is also increasing demand to visualize viral components within tissue specimens. Here we discuss the cardiac autopsy findings of 12 intensive care unit (ICU) naïve and PCR-positive COVID-19 cases using a combination of histological, Immunohistochemical/immunofluorescent and molecular techniques. We performed SARS-CoV-2 qRT-PCR on fresh tissue from all cases; RNA-ISH and IHC for SARS-CoV-2 were performed on selected cases using FFPE tissue from heart. Eight of these patients also had positive post-mortem serology for SARS-CoV-2. Histopathologic changes in the coronary vessels and inflammation of the myocardium as well as in the endocardium were documented which support the reports of a cardiac component to the viral infection. As in the pulmonary reports, widespread platelet and fibrin thrombi were also identified in the cardiac tissue. In keeping with vaccine-induced activation of virus-specific CD4+ and CD8+ T cells, and release of cytokines such as interferon-gamma (IFNγ), we observed similar immune cellular distribution and cytokines in these patients. Immunohistochemical and immunofluorescent localisation for the viral Spike (S-protein) protein and the nucleocapsid protein (NP) were performed; presence of these aggregates may possibly contribute to cardiac ischemia and even remodelling.