Regulatory B cells protect against chronic hypoxia-induced pulmonary hypertension by modulating the Tfh/Tfr immune balance.

Hypoxia-induced pulmonary hypertension (HPH) is a progressive and lethal disease characterized by the uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) and obstructive vascular remodelling. Previous research demonstrated that Breg cells were involved in the pathogenesis of pulmonary hypertension. This work aimed to evaluate the regulatory function of Breg cells in HPH. HPH mice model were established and induced by exposing to chronic hypoxia for 21 days. Mice with HPH were treated with anti-CD22 or adoptive transferred of Breg cells. The coculture systems of Breg cells with CD4+ T cells and Breg cells with PASMCs in vitro were constructed. Lung pathology was evaluated by HE staining and immunofluorescence staining. The frequencies of Breg cells, Tfh cells and Tfr cells were analysed by flow cytometry. Serum IL-21 and IL-10 levels were determined by ELISA. Protein levels of Blimp-1, Bcl-6 and CTLA-4 were determined by western blot and RT-PCR. Proliferation rate of PASMCs was measured by EdU. Compared to the control group, mean PAP, RV/(LV + S) ratio, WA% and WT% were significantly increased in the model group. Anti-CD22 exacerbated abnormal hemodynamics, pulmonary vascular remodelling and right ventricle hypertrophy in HPH, which ameliorated by adoptive transfer of Breg cells into HPH mice. The proportion of Breg cells on day 7 induced by chronic hypoxia was significantly higher than control group, which significantly decreased on day 14 and day 21. The percentage of Tfh cells was significantly increased, while percentage of Tfr cells was significantly decreased in HPH than those of control group. Anti-CD22 treatment increased the percentage of Tfh cells and decreased the percentage of Tfr cells in HPH mice. However, Breg cells restrained the Tfh cells differentiation and expanded Tfr cells differentiation in vivo and in vitro. Additionally, Breg cells inhibited the proliferation of PASMCs under hypoxic condition in vitro. Collectively, these findings suggested that Breg cells may be a new therapeutic target for modulating the Tfh/Tfr immune balance in HPH.

HOX transcript antisense RNA is elevated in gastric carcinogenesis and regulated by the NF-κB pathway.

The expression pattern of HOX transcript antisense RNA (HOTAIR) in the progression of gastric cancer and the regulation of its expression are still unclear. In the current study, HOTAIR expressions in gastric tissues collected from patients with superficial gastritis, atrophic gastritis, atypical hyperplasia, and gastric cancer as well as normal controls was quantitatively examined. The results showed that the expression of HOTAIR was higher in gastric cancer than in normal tissues, but reached the highest level in atrophic gastritis, suggesting that HOTAIR may be involved in the molecular process of nonresolving inflammation. Then tumor necrosis factor-α-induced protein-8 like-2 (TIPE2), a known gene associated with nonresolving inflammation, was overexpressed and the results showed that the promotion in TIPE2 expression triggered HOTAIR reduction, this result was further verified by microarray analysis and TIPE2 knockout mice. Subsequently, the data obtained from HOTAIR knockdown experiment showed that it significantly enhanced colony forming capability and inhibited p27 expression in AGS cells. Furthermore, deletion constructs and luciferase-based activity assays indicated that the -475 to -443bp region of HOTAIR promoter contained a crucial regulatory element. Transcription factor prediction with software TRANSFAC revealed that nuclear factor-κB signaling protein p65 had a binding site in this region and might have roles in HOTAIR expression. The binding of phosphor-p65 to HOTAIR promoter was verified by chromatin immunoprecipitation, and succeeding experiment results demonstrated that p65 reduction by p65 small interfering RNA and TIPE2 overexpression also decreased HOTAIR expression. Conclusively, our results suggest that HOTAIR was associated with nonresolving inflammation, and its expression is regulated by p65.

Hypercapnia downregulates hypoxia-induced lysyl oxidase expression in pulmonary artery smooth muscle cells via inhibiting transforming growth factor ß1 signalling.

Hypoxic pulmonary arterial hypertension is characterized by elevated pulmonary vascular resistance and remodelling. Transforming growth factor-ß1 (TGF-ß1 ) is the master regulator in cellular response to hypoxia which can directly target lysyl oxidase (LOX). This study aimed to determine whether hypercapnia attenuates hypoxic pulmonary hypertension via regulating TGF-ß1 and LOX signalling. We found that exposure to hypercapnia ameliorated the increase in mean pulmonary artery pressure (mPAP) and ratio of right ventricle to left ventricle plus septum (RV/(LV + S)) induced by hypoxia but had no effect on mPAP and RV/(LV + S) in normoxia-exposed control. In addition, exposure to hypoxia upregulated the mRNA and protein levels of LOX and TGF-ß1 in rat PASMCs both in vivo and in vitro, but these effects were abrogated by concurrent exposure to hypercapnia. The downregulation of LOX in rat PASMCs induced by hypercapnia was reversed by the administration with TGF-ß1 , while TGF-ß1 knockdown repressed the upregulation of LOX in hypoxia-exposed rat PASMCs. In conclusion, hypoxia upregulates LOX and TGF-ß1 expression in PASMCs and contributes to pulmonary hypertension. Hypercapnia downregulates hypoxia-induced LOX expression and alleviates hypoxia-associated pulmonary hypertension via inhibiting TGF-ß1 signalling. SIGNIFICANCE OF THE STUDY: Hypoxia-induced upregulation of TGF-ß1 , PDGF, and HIF-1α plays a pivotal role in PAH, but molecular mechanism of how hypoxia regulates LOX expression is not clear. In the present study, we showed that mRNA and protein expression levels of LOX were substantially increased when TGF-ß1 was induced by hypoxia, and the effects were reversed by TGF-ß1 knockdown. Our study indicates that TGF-ß1 is implicated in the regulation of LOX.

Tumor necrosis factor-α-induced protein-8 like-2 (TIPE2) upregulates p27 to decrease gastic cancer cell proliferation.

The pathogenesis of gastric cancer is not completely understood. Tumor necrosis factor-α-induced protein-8 like-2 (TIPE2) has recently been identified as a novel negative regulator gene of the immune system, and studies in mice and humans have suggested its inhibitory action in both inflammation and cancer. In this study, we examined the expression levels of TIPE2 in human gastric cancer tissues and also samples of paraneoplastic control tissue, and found that TIPE2 expression was reduced in gastric cancer. To investigate the role of TIPE2 in gastric cell carcinogenesis, a TIPE2 plasmid was introduced into gastric cell lines and TIPE2 function was examined. Colony-forming assays showed that restoration of TIPE2 expression in gastric cells significantly suppressed cell proliferation. Analysis by flow cytometry showed that the number of cells in the S phase of the cell cycle was reduced concomitant with TIPE2 expression, and cell apoptosis was maintained at a low level. Microarray and western blot analyses revealed that TIPE2 selectively up-regulated N-ras and p27 expression. The role of p27 in mediating TIPE2-associated cell growth inhibition was verified by a p27 siRNA interference assay. In this study, we proved that TIPE2 is an inhibitor of gastric cancer cell growth, and suggest that TIPE2 might promote a p27-associated signaling cascade that leads to restored control of the cell cycle and cell division. Our results provide a new molecular mechanism by which TIPE2 may regulate proliferation of gastric cells.

Size-fitting effect for hybridization of DNA/mercaptohexanol mixed monolayers on gold.

In this article we proposed a simple hexagonal model for exploring the hybridization of thiol-modified probe DNA self-assembled monolayers (SAMs) on gold with target DNA molecules in solution. The size-fitting coefficient d(c)/d(t) from the model was used to discuss the principle for DNA optimal hybridization, where d(c) was the channel diameter among three adjacent probe DNA molecules on gold and dt was the gyration diameter of the target DNA molecules in solution. Experimentally we investigated the hybridization effect (hybridization efficiency H(E) and hybridization density H(D)) of thiol-modified probe DNA (DNA base amount m = 15, 25 or 35)/mercaptohexanol (MCH) mixed SAMs on gold in 1 M electrolyte solution by chronocoulometry (CC) and electrochemical impedance spectroscopy (EIS). The surface coverage Γ(m) of the probe DNA on gold was adjusted by changing the mixed concentration ratio of probe DNA with MCH (C(DNA)/C(MCH)) in the assembly solution. Results indicated that with the increase of C(DNA)/C(MCH), H(E) decreased gradually; H(D) first increased and then decreased, which arrived at the biggest at C(DNA)/C(MCH) = 1 for all the probe DNA/MCH mixed SAMs (m = 15, 25, 35). The optimal Γ(m) for achieving the biggest H(D) in DNA hybridization decreased with the increase of m from 15 to 35. The experimental conclusions obtained by CC and EIS measurements verified each other. Combining the simple model with our experimental results, we ascertained that d(c)/d(t) decreased with the increase of m, which showed a good linear relationship. These conclusions provided an important reference and guidance for controllably constructing DNA sensors with optimal performance.

Comparative Genomic Analysis of Two-Component Signal Transduction Systems in Probiotic Lactobacillus casei.

Lactobacillus casei has traditionally been recognized as a probiotic, thus needing to survive the industrial production processes and transit through the gastrointestinal tract before providing benefit to human health. The two-component signal transduction system (TCS) plays important roles in sensing and reacting to environmental changes, which consists of a histidine kinase (HK) and a response regulator (RR). In this study we identified HKs and RRs of six sequenced L. casei strains. Ortholog analysis revealed 15 TCS clusters (HK-RR pairs), one orphan HKs and three orphan RRs, of which 12 TCS clusters were common to all six strains, three were absent in one strain. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. Some TCS clusters are involved with the response under the stress of the bile salts, acid, or oxidative, which contribute to survive the difficult journey through the human gastrointestinal tract. Computational predictions of 15 TCSs were verified by PCR experiments. This genomic level study of TCSs should provide valuable insights into the conservation and divergence of TCS proteins in the L. casei strains.

Eco-friendly Nanoparticles Synthesized from Salvia sclarea Ethanol Extract Protect against STZ-induced Diabetic Nephropathy in Rats via Antioxidant, Anti-inflammatory, and Apoptosis Mechanisms.

Recent global scientific attention has been directed towards eco-friendly synthesis and versatile applications of silver nanoparticles (AgNPs) due to their effectiveness against specific cells and tissues. This study aimed to develop a green synthesis method for AgNPs using ethanolic extract from Salvia sclarea aerial parts, and to assess their protective efficacy against streptozotocin (STZ)-induced diabetic nephropathy in rats. Additionally, antioxidant, anti-inflammatory, and apoptosis studies were conducted to understand their mode of action. Characterization via ultraviolet-visible (UV-Vis) spectroscopy, infrared (IR) spectroscopy, and X-ray diffraction (XRD) confirmed the formation of ethanol extract of Salvia sclarea silver nanoparticles (EESS AgNPs), with a distinctive absorption peak at 400 nm. Scanning electron microscopy (SEM) analysis revealed predominantly spherical and quasi-spherical shapes of the synthesized nanoparticles. The treatment procedure spanned for a period of 12 weeks in diabetic rats and were evaluated for inflammatory markers (tumor necrosis factor-α, antioxidant markers (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) and apoptosis markers (Bcl-2, Bax, cleaved-caspase-3). Results demonstrated that treatment with EESS AgNPs significantly reduced blood glucose levels compared to the diabetic group. Additionally, EESS AgNPs treatment led to a significant decrease in levels of pro-inflammatory cytokines TNF-α, IL-1ß, and PKC-ꞵ in renal cells. Furthermore, EESS AgNPs effectively modulated antioxidant enzyme concentrations, including GSH, SOD, GPx, and CAT, bringing them to acceptable levels. Administration of EESS AgNPs also resulted in a significant decrease in protein levels of Bax and activated caspase-3, while increasing expression of the anti-apoptotic protein Bcl-2 in renal cells of STZ-induced diabetic rats. In conclusion, EESS AgNPs demonstrate potent anti-hyperglycemic effects, potentially mitigating diabetic nephropathy by suppressing hyperglycemiainduced oxidative stress, apoptosis, and inflammation in renal cells of diabetic rats.

Effect of Perioperative Blood Transfusion on Prognosis after Radical Resection in Patients with Non-Metastatic Renal Cell Carcinoma: A Retrospective Analysis.

OBJECTIVE: Advancements in medical science have improved non-metastatic renal cell carcinoma (NM-RCC) treatment strategies, but long-term survival is influenced by various factors, including perioperative blood transfusion. This study aims to analyse prognostic factors in patients with NM-RCC after radical nephrectomy. METHODS: From January 2018 to December 2021, a total of 132 patients with NM-RCC after radical nephrectomy were studied. According to 2-year follow-up data, the patients were categorised into case (with poor outcomes, including pneumothorax, renal issues, recurrence or death) and control groups. Data on demographics, clinical characteristics and perioperative blood transfusion were collected, and key prognostic factors were identified through logistic regression. RESULTS: A total of 32 patients with poor prognosis were included in the case group, accounting for 24.24% (32/132), and 100 patients without poor prognosis were included in the control group, accounting for 75.76% (100/132). Tumour stage, tumour size and perioperative blood transfusion were all risk factors for the prognosis of patients, and odds ratio (OR) >1. The above indicators had high predictive value for the prognosis of patients after surgery. CONCLUSIONS: The prognostic factors of patients with NM-RCC after radical nephrectomy include tumour stage, tumour size and perioperative blood transfusion, and each factor had predictive value.

Effects of Rehabilitation Therapy at Different Intervention Times on Daily Living Activity and Motor Function in Patients with Traumatic Spinal Cord Injury.

Objectives: We investigated the impact of traumatic spinal cord injury (TSCI) on daily living activities and motor function of TSCI patients. Methods: A total of 88 TSCI patients were randomly divided into Group A (N=44) and Group B (N=44). Group A received rehabilitation treatment 7 days after the stabilization of vital signs, and Group B received rehabilitation treatment 30 days after hospitalization. Results: The compliance rate of Group A (93.18%) was higher than that of Group B (72.73%) (χ 2 =6.510, p<.05); The scores of American Spinal Injury Association (ASIA) and Activities of Daily Living (ADL) in Group A were higher than those in Group B. The self-rating score of anxiety and depression was lower than that of Group B (p<.05). Conclusion: For the rehabilitation treatment of TSCI patients, it is better to choose the intervention after the vital signs are stable to improve patients' ability for daily living activities and motor function.

Evironmental pollutant perfluorodecanoic acid upregulates cIAP2 to suppress gastric cell senescence.

The role of perfluorodecanoic acid (PFDA) in gastric carcinogenesis and its mechanism remains unknown. Our previous research revealed that PFDA regulated the growth of human gastric cells. However, its core molecules and basic mechanisms are still not clear. In the present study, cDNA microarrays were used to determine mRNA changes in AGS cells after treatment with PFDA. DAVID analysis of the genes with >2­fold increased expression in microarray data revealed five genes which were involved in cancer pathways. The most upregulated gene was cIAP2, whose upregulation in AGS was confirmed by western blot analysis and quantitative PCR (qPCR) analyses. In order to investigate the role of cIAP2 in cell proliferation, cIAP2 siRNA was employed to regulate cIAP2 expression following PFDA treatment. The results revealed that the growth rate of cIAP2­knockdown cells was reduced by about 50% compared to the control. Given that our previous flow cytometric assays revealed no significant change (3.7 vs. 6.4%) in the percentage of apoptotic cells when PFDA was added to the medium and cIAP2 expression was upregulated, we next applied flow cytometry to assess whether cIAP2 would lead to cell cycle variations. The research data revealed that the proportion of cells in the G1, S and G2 phases was not significantly altered with the decrease of cIAP2 expression. Finally, the role of cIAP2 in AGS cell senescence was investigated, and the results indicated that cell senescence was significantly increased in the cIAP2 siRNA group in comparison to the control siRNA group. Since p53 has been identified as a tumor suppressor and its molecular alterations are common in different human tumors, we investigated the relationship of p53 with cIAP2. The experimental results demonstrated that cIAP2 regulated the expression of p53 and thus was likely to be a potential mechanism for PFDA­induced growth promotion. Overall, the results revealed that PFDA may suppress cellular senescence induced by p53 through the regulation of cIAP2 protein expression.

Perfluorodecanoic acid stimulates NLRP3 inflammasome assembly in gastric cells.

Perfluorodecanoic acid (PFDA), a perfluorinated carboxylic acid, presents in the environment and accumulates in human blood and organs, but its association with tumor promotion are not clear. Given that inflammation plays a significant role in the development of gastric malignancies, we evaluated the effects of PFDA on activation of the inflammasome and inflammation regulation in the gastric cell line AGS. When added to cell cultures, PFDA significantly stimulated IL-1ß and IL18 secretion and their mRNA levels compared with control cells. By RT-PCR and western-blot we found that up-regulation of NLRP3 were associated with promotion of IL-1ß and IL-18 production. Then expression variation of cIAP1/2, c-Rel and p52 were analyzed, the results demonstrated raised mRNA expression in all the tested genes concomitant with enhanced inflammasome activity after exposure to PFDA. Assays with cIAP2 siRNA and NFκB reporter provided additional evidence that these genes were involved in PFDA-induced inflammasome assembly. Furthermore, increased secretion of IL-1ß and IL-18 were detected in stomach of PFDA-treated mice, disorganized alignment of epithelial cells and inflammatory cell infiltration were also observed in the stomach tissues upon PFDA treatment. This study reports for the first time that PFDA regulates inflammasome assembly in human cells and mice tissues.

Perfluorodecanoic acid (PFDA) promotes gastric cell proliferation via sPLA2-IIA.

The association of perfluorodecanoicacid (PFDA) with tumor promotion and associated effects is not clear. Given that PDFA is mostly consumed with food and drinking water, we evaluated the effects of PFDA on a gastric cell line. When added to cell cultures, PFDA significantly increased growth rate and colony forming ability compared with control treatment. We found that suppression of cell senescence, but not apoptosis or autophagy was associated with PFDA-induced promotion of cell amount. To determine the molecular mechanism that was involved, DNA microarray assays was used to analyze changes in gene expression in response to PFDA treatment. Data analysis demonstrated that the vascular endothelial growth factor signaling pathway had the lowest p-value, with sPLA2-IIA (pla2g2a) exhibits the most altered expression pattern within the pathway. Moreover, sPLA2-IIA and its transcription factor TCF4, known as a direct target and a binding partner of Wnt/ß-catenin signaling in gastric cells respectively, were the third and second most varied genes globally. Cells transfected with expression plasmids pENTER-tcf4 and pENTER-pla2g2a show reduced cell proliferation by more than 60% and 30% respectively. Knockdown with sPLA2-IIA siRNA provided additional evidence that sPLA2-IIA was a mediator of PFDA-induced cell senescence suppression. The results suggest for the first time that PFDA induced suppression of cell senescence through inhibition of sPLA2-IIA protein expression and might increased the proliferative capacity of an existing tumor.

TIPE2, a negative regulator of TLR signaling, regulates p27 through IRF4-induced signaling.

Targeted inhibition of specific toll-like receptor (TLR) pathways may provide an effective strategy to prevent the development of selected gastric malignancies. Tumor necrosis factor (TNF)-α-induced protein 8-like-2 (TIPE2) was identified as a novel negative regulator of TLR signaling. Our previous study identified TIPE2 as an inhibitor of gastric cancer cell growth; it promotes p27 expression, which leads to restored control of the cell cycle and cell division. However, the molecular mechanism by which TIPE2 regulates p27 remains unclear. In the present study, we examined the expression patterns of TIPE2 in serial clinical gastritis tissues as well as gastric cancer, and found a negative correlation between TIPE2 expression and progression of gastritis to gastric cancer. This negative correlation verified the role of TIPE2 in preventing the occurrence and development of gastric cancer, suggesting that TIPE2 may be a potential biomarker for gastric cancer progression. To determine the mechanism employed by TIPE2 in gastric cell carcinogenesis, a TIPE2-expressing plasmid was introduced into gastric cell lines, and microarray and western blot analysis revealed that TIPE2 selectively upregulates the expression of interferon regulatory factor 4 (IRF4). Variations in IRF4 expression were additionally verified in knockout mice. Next, the effect of IRF4 on p27 expression was tested by an IRF4 siRNA interference assay. Finally, we explored the signaling pathways used by TIPE2 to regulate IRF4. An experiment using pathway inhibitors and a nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) luciferase reporter assay showed that NF-κB plays a crucial role in regulating IRF4 expression. Our data provide evidence that TIPE2, a potential biomarker for gastric cancer progression, stimulates an IRF4-associated signaling cascade that promotes p27 expression and controls cell growth. To the best of our knowledge, this is the first study to demonstrate that IRF4 acts as an inhibitor of epithelial cell proliferation and mediates the expression of TIPE2, a negative regulator of TLR signaling, to control cell growth.

The effect of prenatal morphine exposure on memory consolidation in the chick.

The central nervous system exhibits remarkable plasticity in early life and can be altered significantly by prenatal morphine exposure. Previous studies show that prenatal morphine exposure may alter the capacity for learning and memory in post-partum chicks. The one-trial passive avoidance learning paradigm with 1-day-old chicks is an excellent model to study several mechanisms of memory formation, including STM, ITM, and LTM. The following represents our investigation of the effect of prenatal morphine exposure on learning and memory deficits in the chick. In these experiments, morphine was injected into the airspace of eggs (20 mg/kg) and the one-trial passive avoidance learning paradigm was used to test the effect of prenatal morphine exposure on memory consolidation. The data suggest that chicks injected with morphine daily from E12 to E16 had significantly impaired long-term memory at 120 min after training (p<0.001) but not intermediate-term memory at 30 min after training.

Telemetric recordings of single neuron activity and visual scenes in monkeys walking in an open field.

This paper describes a portable recording system and methods for obtaining chronic recordings of single units and tracking rhesus monkey behavior in an open field. The integrated system consists of four major components: (1) microelectrode assembly; (2) head-stage; (3) recording station; and (4) data storage station, the first three of which are carried by the monkey and weigh 800 g. Our system provides synchronized video and electrophysiological signals, which are transmitted by a wireless system to a distance of 50 m. Its major advantages are that neuronal recordings are made in freely moving monkeys, and well-separated action potentials with amplitude five times higher than the background noise are usually recorded and readily kept for many hours. Using this system, we were able to study "place cells" in non-human primate brains. The described methods provide a new way to examine correlations between single neuron activity and primate behaviors, and can also be used to study the cellular basis of social behaviors in non-human primates.

[Methods for single unit recording in behavioring morphine craving rat].

In this paper, one method was introduced, which was a combination of the cue-related morphine addiction model and a technique for obtaining chronic extracellular recordings of single unit in freely moving rats. With the combination and improvement of this technique, we have successfully applied this new method to study the neuronal activity of the hippocampus CA1 region in morphine withdrawal rats. In all, we found some more accurate and objective cellular characteristics of hippocampal neurons, and considered these characteristics as one of electrophysiological indexes of morphine addiction rats.

Vascular endothelial growth factor plays a critical role in the formation of the pre-metastatic niche via prostaglandin E2.

Factors secreted by primary tumors can alter the microenvironment at distant organ sites, generating pre-metastatic niches for subsequent metastatic cancer cell colonization. Breast cancer cells have a propensity to home preferentially to the lung, but the underlying molecular mechanisms whereby primary breast carcinoma-derived factors affect the pre-metastatic lung environment before the arrival of the tumor cells are poorly understood. In this study, 4T1 mammary carcinoma cells were subcutaneously injected into the mammary glands of mice, resulting in the induction of inflammation, increased total vessel density and recruitment of bone marrow-derived cells (BMDCs) in pre-metastatic lungs. Subsequent examination revealed that the sites of inflammatory cell clusters in the lungs were tumor metastasis sites. Moreover, vascular endothelial growth factor (VEGF) induced prostaglandin E2 (PGE2) production in mouse pulmonary microvascular endothelial cells (MPVECs) and enhanced the adhesion of 4T1 cells. Treatment with the cyclooxygenase-2 inhibitor celecoxib significantly reduced 4T1 cell adhesion to MPVECs, and also reduced cancer metastasis and the inflammatory response. These results suggest that VEGF may be an underlying carcinoma-derived factor responsible for formation of the pre-metastatic niche in the lung of 4T1 cell-bearing mice. This study, therefore, demonstrated that primary tumors can alter the lung microenvironment during the pre-metastatic phase by triggering an inflammatory response and PGE2 production. Primary tumor-derived VEGF might thus be a crucial factor responsible for the formation of the pre-metastatic niche by inducing PGE2 production.