The Therapeutic Potential of Zinc-Alpha2-Glycoprotein (AZGP1) in Fibrotic Kidney Disease

Chronic kidney disease (CKD) is characterized by a long-term loss of kidney function and, in most cases, by progressive fibrosis. Zinc-alpha2-glycoprotein (AZGP1) is a secreted protein, which is expressed in many different tissues and has been associated with a variety of functions. In a previous study, we have shown in cell culture and in AZGP1 deficient mice that AZGP1 has protective anti-fibrotic effects. In the present study, we tested the therapeutic potential of an experimental increase in AZGP1 using two different strategies. (1) C57Bl/6J mice were treated systemically with recombinant AZGP1, and (2) a transgenic mouse strain was generated to overexpress AZGP1 conditionally in proximal tubular cells. Mice underwent unilateral uretic obstruction as a pro-fibrotic kidney stress model, and kidneys were examined after 14 days. Recombinant AZGP1 treatment was accompanied by better preservation of tubular integrity, reduced collagen deposition, and lower expression of injury and fibrosis markers. Weaker but similar tendencies were observed in transgenic AZGP1 overexpressing mice. Higher AZGP1 levels led to a significant reduction in stress-induced accumulation of tubular lipid droplets, which was paralleled by improved expression of key players in lipid metabolism and fatty acid oxidation. Together these data show beneficial effects of elevated AZGP1 levels in fibrotic kidney disease and highlight a novel link to tubular cell lipid metabolism, which might open up new opportunities for CKD treatment.

The pro-fibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression

Increased senescence and expression of pro-fibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and pro-fibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and pro-fibrotic genes were measured in primary fibroblasts from the lungs of old (24 months) and young (3 months) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53 and b-galactosidase) and increased expression of pro-fibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1 and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and pro-fibrotic genes. Young cells were induced to express the senescence and pro-fibrotic phenotype by sulfasalazine, an Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, an Slc7a11 inducer, decreased senescence without affecting pro-fibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased pro-fibrotic markers. In short, old lung fibroblasts manifest a pro-fibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

Macrophage polarisation in caesarean scar diverticulum

AimsTo determine immunohistochemical features and correlations between M1/M2 polarisation status with disease severity of post-caesarean scar diverticulum (CSD).MethodsHistological and immunohistological stainings were performed and inflammatory (CD16, CD163 and tumour necrosis factor-α (TNF-α)), fibrosis (α-smooth muscle actin (α-SMA)) and angiogenic (CD31) markers were examined in uterine tissues collected from patients with uterine scar diverticula (CSD) (n=37) and caesarean section (CS) (n=3).ResultsCSD tissues have higher expression of α-SMA, TNF-α, CD16 and CD31 and lower expression of CD163 than CS tissue (p<0.05). Compared with adjacent tissues, thick-walled blood vessels, glands and fibrotic sites have higher expression of α-SMA, TNF-α and CD16. Statistical correlation was observed between the expression of CD16 and TNF-α (R=0.693, p<0.001), α-SMA (R=0.404, p<0.05) and CD31 (R=0.253, p<0.05) in CSD tissues, especially with the ratio of CD16/CD163 (R=0.590, p<0.01). A more significant difference was observed between the expression of CD16/CD163 and α-SMA (R=0.556, p<0.001), TNF-α (R=0.633, p<0.0001) and CD31 (R=0.336, p<0.05).ConclusionsIn this study, TNF-α, α-SMA, CD16 and CD31 proteins were overexpressed in all CSD cases, and CD16/CD163 was positively correlated with tissue inflammation, fibrosis and neovascularisation. Abnormal mononuclear macrophage infiltration may be involved in the origin and progression of CSD.

The Use of κ-Carrageenan in Egg Yolk Free Extender Improves the Efficiency of Canine Semen Cryopreservation

κ-Carrageenan is a plant polysaccharide derived from red seaweeds reported to possess potential medicinal and antioxidants activities. The present study aimed to identify the cryoprotective effects of κ-carrageenan on the quality of frozen-thawed canine semen. Twenty-eight ejaculates were collected and diluted in a Tris egg-yolk-free extender supplemented with various concentrations of κ-carrageenan (0.0%, 0.1%, 0.2%, 0.3%, and 0.5%). The addition of κ-carrageenan to the extender at a 0.2% concentration induced a significant increase in the total motility (TM) and the rapid progressive motility (RPM) of canine sperm. Among the experimental groups, the highest percentage of sperms with intact acrosomes was found in the 0.5% κ-carrageenan group (p < 0.05). Apoptosis levels were significantly lower in the 0.1% and 0.2% κ-carrageenan treatment. Moreover, sperm in the κ-carrageenan supplemented group showed a significantly higher expression of antiapoptotic (Bcl-2) and lower expression of NADPH oxidase (NOX5), spermine synthase (SMS), and spermine oxidase (SMOX) genes than those in the control group. In conclusion, the addition of κ-carrageenan to the freezing extender improved the overall efficiency of frozen-thawed dog spermatozoa.

Association between peripheral lncRNA expression and coping style in schizophrenia patients

In order to explore the relationship between peripheral lncRNA expression and coping style in schizophrenia patients, this study screened the peripheral blood mononuclear cells in 5 patients and 5 controls, and 10 differentially expressed lncRNAs were selected and validated in 96 patients and 50 controls by qPCR. Compared to control group, three lncRNAs (NONHSAT089447, NONHSAT021545, NONHSAT041499) were up-regulated in schizophrenia group. Among them, NONHSAT089447, NONHSAT021545 were negatively associated with positive coping style, and other four lncRNAs (ENST00000394742, NONHSAT089447, NONHSAT021545, NONHSAT041499) were positively related to negative coping style. Positive coping style scores in higher-expression of PR4 and PR6 subgroup were significantly lower than those in lower-expression subgroup. While on the other hand, negative coping style scores in higher-expression of NONHSAT089447, NONHSAT021545, NONHSAT041499 subgroup were significantly higher than those in lower-expression subgroup. In conclusion, three lncRNAs (NONHSAT089447, NONHSAT021545, NONHSAT041499) were over-expressed in schizophrenia patients, probably playing a role in the epigenetic process of choosing coping style.

Effect of Topical PTH 1-34 Functionalized to Biogran® in the Process of Alveolar Repair in Rats Submitted to Orchiectomy

(1) Background: There are many therapies for osteoporosis control and bone maintenance; anabolic drugs such as teriparatide and bone grafts help in the repair process and stimulate bone formation. Thus, the aim of the present study was to evaluate the behavior of repaired bone in the presence of PTH (teriparatide) associated with Biogran® (biomaterial) through a sonochemical procedure after extraction in rats. (2) Methods: The insertion of Biogran® with PTH in the alveolus was performed 30 days after incisor extraction. Euthanasia occurred after 60 days. (3) Results: The use of local treatment of PTH loaded with Biogran® in healthy rats promoted good results for micro-CT, with an increase in percentage and bone volume, number and trabecular separation and less total porosity. Greater immunostaining for Wnt, β-Catenin and osteocalcin proteins and lower expression for Thrombospondin-Related Adhesive Protein (TRAP), which shows an increase in the number of osteoblasts and inhibition of osteoclast action. However, the treated orchiectomized groups did not obtain such expressive results. (4) Conclusion: The use of Biogran® with PTH improved alveolar repair in rats. However, new researches with more efficient doses must be studied to collaborate effectively with the formation of a quality bone after the orchiectomy.

MyD88 Mediates Colitis- and RANKL-Induced Microfold Cell Differentiation

Intestinal microfold (M) cells are critical for sampling antigens in the gut and initiating the intestinal mucosal immune response. In this study, we found that the oral administration of dextran sulfate sodium (DSS) and Salmonella infection induced colitis. In the process, the expression levels of M cell differentiation-related genes were synchronized with the kinetics of pro-inflammatory cytokines. Compared to wild-type (WT) mice, MyD88−/− mice exhibited significantly lower expression levels of M cell differentiation-related genes. However, DSS induced colitis in MyD88−/− mice but failed to promote the transcription of M cell differentiation related genes. Furthermore, the receptor activator of the Nuclear Factor-κB ligand (RANKL) upregulated the transcription of M cell differentiation related genes in murine intestinal organoids prepared from both WT and MyD88−/− mice. Meanwhile, fewer changes in M cell differentiation related genes were found in MyD88−/− mice as compared to WT mice. Hence, we concluded that myeloid differentiation factor 88 (MyD88) is an essential molecule for colitis- and RANKL-related differentiation of M cells.

Expression of Proton-Sensitive GPR31, GPR151, TASK1 and TASK3 in Common Skin Tumors

TWIK-related acid-sensitive potassium channels TASK1 and TASK3, as well as the G-protein-coupled receptors GPR31 and GPR151, are proton-sensitive membrane proteins. They can be activated or inhibited by low extracellular pH (pHe), which is a hallmark of the tumor microenvironment in solid tumors. However, the role of these channels in the development of skin tumors is still unclear. In this study, we investigated the expression profiles of TASK1, TASK3, GPR31 and GPR151 in squamous cell carcinomas (SCCs), basal cell carcinomas (BCCs), nevus cell nevi (NCN), and malignant melanomas (MMs). We performed immunohistochemistry using paraffin-embedded tissue samples from patients and found that most skin tumors express TASK1/3 and GPR31/151. The results show that BCCs are often negative for GPR31/151 as well as for TASK1/3, while nearly all SCCs express these markers. MMs and NCN show similar expression patterns. However, some tumors show a decreasing TASK1/3 expression in deeper dermal tumor tissue, while GPCRs were expressed more evenly. The lower frequency of GPR31/151 and TSAK1/3 expression in BCCs when compared to SCCs is a novel histological feature distinguishing these two entities. Moreover, BCCs also show lower expression of GPR31/151 and TASK1/3 as compared to NCN and MMs.

CRABP2 involvement in a mechanism of Golgi stress and tumor dry matter in non-small cell lung cancer cells via ER dependent Hippo pathway

Objective: The paper aimed to explore the mechanism of cellular retinoic acid binding protein 2 (CRABP2) involvement in Golgi stress and tumor dryness in non-small cell lung cancer (NSCLC) cells through the estrogen receptor (ER) dependent Hippo pathway. Methods: Human NSCLC cell line A549 was purchased from ATCC andcultured in RPMI-1640 with 10% FBS. Attractene reagent was used for plasmid transfection. ER (sh) RNA was designed using RNAi Designer. Seventy-six hours after infection, stable cells were obtained after treated with puromycin for 3 weeks. ER silencing cells (with inhibited ER expression) were compared to the control cells (normal cultured NSCLC cell line A549, CRABP2 normal expression). CRABP2 and ER expression levels were detected by RT-PCR. MTT assay was used to detect cell proliferation, and the cell localization of ER and Golgi was observed by confocal microscopy. The invasion and metastasis of cells were analyzed by Boden chamber invasion and migration assays. Western blotting assays was used for detecting the protein expression of E-cadherin, vimentin, ZO-1 protein and epithelial-mesenchymal transition (EMT) related factors. Results: The lower expression level of mRNA was detected in the ER-silencing group compared to the control group (P<0.05). We also found a higher proliferation level of cells, the number of invading and metastatic cells, the expression of vimentin, p-Lats1T1079, Lats1 and p-YAPS127 mRNA in the control group compared to the ER silencing group (P<0.05). And the expression level of protein kinase RNA-like endoplasmic reticulum kinase (PERK), phosphorylate eukaryotic initiation factor 2 (p-eIF2 alpha), activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP) in the control group was higher than that in the ER silencing group (P<0.05). Adversely, a lower expression level of E-cadherin and ZO-1 protein was found in the control group compared to the ER silencing group (P<0.05). Conclusion: The expression of CRABP2 in NSCLC cells was regulated by ER, and cell proliferation and invasion were regulated by the Hippo pathway. At the same time, it was found that decreased expression of CRABP2 enhanced endoplasmic reticulum/Golgi stress response.

The Biases in Flavonoids and Anthocyanin Biosynthesis of a Red Flesh Table Grape Revealed by Metabolome and Transcriptome Co-analysis

Red flesh is a welcomed fruit trait, yet the regulation of red flesh formation in grape is not well understood. ‘Mio Red’ is a seedless table grape variety with light red flesh and blue-purple skin, the flesh color developed in the late stage of berry ripening, remarkably later than the skin coloring at veraison. The flesh and skin flavonoids metabolome and the transcriptome were analyzed. A total of 173 flavonoids including 17 anthocyanins were identified, 68 were found significantly different (Fold change ≥ 2 or ≤ 0. 5, VIP ≥ 1). Quercetin 3-O-glucoside, epicatechin-epiafzelechin, apigenin 6,8-C-diglucoside and hesperetin 5-O-glucoside were of higher content in the flesh, while the rest flavonoids were of higher content in the skin. The main anthocyanin in the flesh was pelargonidin derivatives in contrast to peonidin derivatives in the skin. Transcriptome comparison recruited 3970 differentially expressed genes (DEGs, log2Fold change > = 1, FDR < 0.05, FPKM ≥ 1), among them 57 were structural genes of flavonoid metabolism pathway. Two anthocyanin synthase (ANS) DEGs were annotated, ANS1 (Vitvi11g00565) and ANS2 (Vitvi02g00435) led the expression in the flesh and skin respectively. In the flesh, anthocyanin biosynthesis structural gene UFGT, positive regulators MYBA1/2/3, and anthocyanin transporters GST14 and MATE5 were of significantly lower expression, while negative regulators MYBC2-L1 and MYB3 were of higher transcription. The results of this study provide new information in the coloring mechanism of red flesh grape and assisting breeding of future table grapes having higher content of phytonutrient providing the health benefit as red wines.