Bone Marrow Mesenchymal Stem Cells (BMSCs) Enhance Endometrial Stromal Cell Migration and Epithelial-Mesenchymal Transition in Adenomyosis Through Upregulation of Neuropilin 1

Hormone support (estrogen and progesterone) is a key factor in decidualization and embryo implantation. Elevated levels of estrogen lead to luteal phase defects through Neuropilin 1, a membranecytoskeleton junction protein. This study aimed to explore the effect of BMSCs on endometrial stromal cells (ESCs) in adenomyosis. ESCs obtained from patients with adenomyosis were cocultured with BMSCs in the absence of presence of Neuropilin 1 inhibitor followed by analysis of expression of decidualization-related genes by RT-qPCR and western blot, cell viability by MTT assay, cell invasion and migration by Transwell assay, oxidative stress factors by ROS kit. Treatment with Neuropilin 1 inhibitor significantly decreased ESC proliferation and invasion, blocked epithelialmesenchymal transition (EMT) process, and restrained decidualization with a downregulation of decidualization-related genes. Furthermore, inhibition of Neuropilin 1 exerted effects through estrogen regulation. However, co-culture with BMSCs restored ESC activity by promoting Neuropilin expression and enhanced intrauterine ESC decidualization. In conclusion, Neuropilin 1 inhibitor restrains decidualization through estrogen regulation which can be abrogated by estrogen receptor antagonists. BMSCs restore the damaged ESC decidualization through increasing Neuropilin 1 expression, which provides new insights into the adverse effect of Neuropilin 1 on human ESCs, suggesting that BMSC is a potential therapeutic drug candidate for adenomyosis.

miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability

Background The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer’s disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. Methods Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. Results Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ß-catenin signaling, a key pathway required for the BBB maintenance. Conclusion For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier’s function.

Clinical outcomes of surgical management for rare types of progressive familial intrahepatic cholestasis: a case series

Background Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of genetic autosomal recessive diseases that cause severe cholestasis, which progresses to cirrhosis and liver failure, in infancy or early childhood. We herein report the clinical outcomes of surgical management in patients with four types of PFIC. Case presentation Six patients diagnosed with PFIC who underwent surgical treatment between 1998 and 2020 at our institution were retrospectively assessed. Living-donor liver transplantation (LDLT) was performed in 5 patients with PFIC. The median age at LDLT was 4.8 (range: 1.9–11.4) years. One patient each with familial intrahepatic cholestasis 1 (FIC1) deficiency and bile salt export pump (BSEP) deficiency died after LDLT, and the four remaining patients, one each with deficiency of FIC1, BSEP, multidrug resistance protein 3 (MDR3), and tight junction protein 2 (TJP2), survived. One FIC1 deficiency recipient underwent LDLT secondary to deterioration of liver function, following infectious enteritis. Although he underwent LDLT accompanied by total external biliary diversion, the patient died because of PFIC-related complications. The other patient with FIC1 deficiency had intractable pruritus and underwent partial internal biliary diversion (PIBD) at 9.8 years of age, pruritus largely resolved after PIBD. One BSEP deficiency recipient, who had severe graft damage, experienced recurrence of cholestasis due to the development of antibodies against BSEP after LDLT, and eventually died due to graft failure. The other patient with BSEP deficiency recovered well after LDLT and there was no evidence of posttransplant recurrence of cholestasis. In contrast, recipients with MDR3 or TJP2 deficiency showed good courses and outcomes after LDLT. Conclusions Although LDLT was considered an effective treatment for PFIC, the clinical courses and outcomes after LDLT were still inadequate in patients with FIC1 and BSEP deficiency. LDLT accompanied by total biliary diversion may not be as effective for patients with FIC1 deficiency.

Connexin 43 confers chemoresistance through activating PI3K

Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1–a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide–inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit β (PIK3CB, also called PI3Kβ or p110β), suggesting that Cx43 activates PIK3CB/p110β independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110β, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110β-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110β together is an effective therapeutic approach for overcoming chemoresistance.

Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation

Spindle orientation is often achieved by a complex of Pins/LGN, Mud/NuMa, Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, laser ablation, and depletion of an adherens junction protein disrupted Pins planar polarity and spindle orientation. Furthermore, snail depletion, which abrogates ventral furrow forces, disrupted Pins polarization and spindle orientation, suggesting that morphogenetic movements and resulting forces transmitted through the tissue can polarize Pins and orient division. Thus, morphogenetic forces associated with mesoderm invagination result in planar polarized Pins to mediate division orientation at a distant region of the embryo during morphogenesis. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

Alterations in intestinal microbiota composition coincide with impaired intestinal morphology and dysfunctional ileal immune response in growing-finishing pigs under constant chronic heat stress

Background Previous studies had shown that short-term acute heat stress (HS) affected the host’s metabolism and intestinal microbiota independent of feed intake (FI) reduction, and long-term calorie restriction caused intestinal morphological injuries and gut microbial alterations. However, research on the effects of constant chronic HS on intestinal microbial composition and the roles of FI reduction played in is limited. This study aimed to investigate the effects of 7-day constant chronic HS on the composition of intestinal microbes in growing-finishing pigs, and its relationship with pigs’ performance, intestinal morphology, and ileal immune response. Twenty-four growing-finishing pigs (Duroc × Large White × Landrace, 30 ± 1 kg body weight) were randomly assigned to three treatments (n = 8), 1) thermal neutral (TN) conditions (25 ± 1 °C) with ad libitum FI, 2) HS conditions (35 ± 1 °C) with ad libitum FI, 3) pair-fed (PF) with HS under TN conditions to discriminate the confounding effects of dissimilar FI, and the FI was the previous day’s average FI of HS. The small intestinal segments (duodenum, jejunum, and ileum) and feces were collected on d 8. Results Results indicated that HS drastically declined (P < 0.05) average daily gain (ADG) and average daily feed intake (ADFI) (about 61%) in comparison with TN, and caused hyperpyrexia, meanwhile PF caused hypothermia. Morphological observation by light and electron microscopes showed that both HS and PF treatment decreased (P < 0.05) the villus and microvillus height compared with TN. Additionally, HS increased (P < 0.05) protein expression of heat shock protein 70 in the duodenum, jejunum, and ileum. Furthermore, the expression of tight junction protein zonula occluden-1 (ZO-1) in the duodenum and ileum, and Occludin in the ileum were enhanced (P < 0.05) compared with TN and PF. Moreover, HS significantly enhanced (P < 0.05) the mRNA relative expression of inflammatory cytokines (TLR-2, TLR-4, and tumor necrosis factor-α (TNF-α), IL-6, IL-8, PG1–5, β-defensin 2 (pBD-2)), mucins (mucin-1 and mucin-2) and P65 protein level in the ileal mucosa tissue. Intestinal microbiota analysis by 16S rRNA sequencing showed lower (P < 0.10) α diversity in both HS and PF, and a separated cluster of β diversity among groups. Compared with TN, HS but not PF mainly reduced (FDR < 0.05) Bacteroidetes (phylum), Bacteroidia (class) and elevated the proportions of Proteobacteria (phylum, FDR < 0.05), Bacillales (order, FDR < 0.05), Planococcaceae (family, FDR < 0.05), Kurthia (genus, FDR < 0.05), Streptococcaceae (family, FDR < 0.10) and Streptococcus (genus, FDR < 0.10). Notably, Lactobacillales (order) was decreased (FDR < 0.05) by PF alone. Furthermore, the Spearman correlation analysis indicated that the microbes prevalent in HS were positively (P < 0.05) associated with intestinal morphological injuries indicators and ileal immune response parameters, and the microbes reduced in HS were negatively (P < 0.05) with the performance data. Conclusions Intestinal morphological injuries and ileal immune response caused by constant chronic HS independent of FI showed close connections with alterations in intestinal microbiota in growing-finishing pigs.

Differential Mechanisms of Action and Efficacy of Vitamin E Components in Antioxidant Cytoprotection of Human Retinal Pigment Epithelium

The purpose of this study was to determine if different vitamin E components exhibit similar efficacy and mechanism of action in protecting Retinal pigment epithelium (RPE) cells from oxidative damage. We hypothesized that α-tocopherol (αT) is unique among vitamin E components in its cytoprotective mechanism of action against oxidative stress in RPE cells and that it requires protein synthesis for optimal antioxidant effect. We used cell viability assays, fluorescent chemical labeling of DNA and actin and immuno-labeling of the antioxidant proteins Nrf2 and Sod2 and of the tight junction protein, ZO-1, and confocal microscopy to determine the effects of αT and γT against oxidative stress in immortalized human RPE cells (hTERT-RPE). Using the four main vitamin E components, αT, γT, δ-tocopherol (δT) and α-tocotrienol (αTr), we ascertained that they exhibit similar, but not identical, antioxidant activity as αT when used at equimolar concentrations. In addition, we determined that the exposure time of RPE cells to α-tocopherol is critical for its ability to protect against oxidative damage. Lastly, we determined that αT, but not γT, partially requires the synthesis of new proteins within a 24-h period and prior to exposure to tBHP for optimal cytoprotection. We conclude that, unlike γT and δT, αT appears to be unique in its requirement for transport and/or signaling for it to be an effective antioxidant. As a result, more focus should be paid to which vitamin E components are used for antioxidant interventions.

Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

COX5B-Mediated Bioenergetic Alterations Modulate Cell Growth and Anticancer Drug Susceptibility by Orchestrating Claudin-2 Expression in Colorectal Cancers

Oxidative phosphorylation (OXPHOS) consists of four enzyme complexes and ATP synthase, and is crucial for maintaining physiological tissue and cell growth by supporting the main bioenergy pool. Cytochrome c oxidase (COX) has been implicated as a primary regulatory site of OXPHOS. Recently, COX subunit 5B (COX5B) emerged as a potential biomarker associated with unfavorable prognosis by modulating cell behaviors in specific cancer types. However, its molecular mechanism remains unclear, particularly in colorectal cancers (CRCs). To understand the role of COX5B in CRCs, the expression and postoperative outcome associations using independent in-house patient cohorts were evaluated. A higher COX5B tumor/nontumor expression ratio was associated with unfavorable clinical outcomes (p = 0.001 and 0.011 for overall and disease-free survival, respectively. In cell-based experiments, the silencing of COX5B repressed cell growth and enhanced the susceptibility of CRCs cells to anticancer drugs. Finally, downstream effectors identified by RNA sequencing followed by RT-qPCR and functional compensation experiments revealed that the tight junction protein Claudin-2 (CLDN2) acts downstream of COX5B-mediated bioenergetic alterations in controlling cell growth and the sensitivity to anticancer drugs in CRCs cells. In conclusion, it was found that COX5B promoted cell growth and attenuated anticancer drugs susceptibility in CRCs cells by orchestrating CLDN2 expression, which may contribute to unfavorable postoperative outcomes of patients with CRCs.