Identification of a novel m6A-related lncRNAs signature and immunotherapeutic drug sensitivity in pancreatic adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) ranks as the fourth leading cause for cancer-related deaths worldwide. N6-methyladenosine (m6A) and long non-coding RNAs (lncRNAs) are closely related with poor prognosis and immunotherapeutic effect in PDAC. The aim of this study is to construct and validate a m6A-related lncRNAs signature and assess immunotherapeutic drug sensitivity in PDAC. METHODS: RNA-seq data for 178 cases of PDAC patients and 167 cases of normal pancreatic tissue were obtained from TCGA and GTEx databases, respectively. A set of 21 m6A-related genes were downloaded based on the previous report. Co-expression network was conducted to identify m6A-related lncRNAs in PDAC. Cox analyses and least absolute shrinkage and selection operator (Lasso) regression model were used to construct a risk prognosis model. The relationship between signature genes and immune function was explored by single-sample GSEA (ssGSEA). The tumor immune dysfunction and exclusion (TIDE) score and tumor mutation burden (TMB) were utilized to evaluate the response to immunotherapy. Furthermore, the expression levels of 4 m6A-related lncRNAs on PDAC cell lines were measured by the quantitative real-time PCR (qPCR). The drug sensitivity between the high- and low-risk groups was validated using PDAC cell lines by Cell-Counting Kit 8 (CCK8). RESULTS: The risk prognosis model was successfully constructed based on 4 m6A-related lncRNAs, and PDAC patients were divided into the high- and low-risk groups. The overall survival (OS) of the high-risk groups was more unfavorable compared with the low-risk groups. Receiver operating characteristic (ROC) curves demonstrated that the risk prognosis model reasonably predicted the 2-, 3- and 5-year OS of PDAC patients. qPCR analysis confirmed the decreased expression levels of 4 m6A-related lncRNAs in PDAC cells compared to the normal pancreatic cells. Furthermore, CCK8 assay revealed that Phenformin exhibited higher sensitivity in the high-risk groups, while Pyrimethamine exhibited higher sensitivity in the low-risk groups. CONCLUSION: The prognosis of patients with PDAC were well predicted in the risk prognosis model based on m6A-related lncRNAs, and selected immunotherapy drugs have potential values for the treatment of pancreatic cancer.

Extracellular vesicles for ischemia/reperfusion injury-induced acute kidney injury: a systematic review and meta-analysis of data from animal models.

BACKGROUND: Acute kidney injury (AKI) induced by ischemia/reperfusion injury significantly contribute to the burden of end-stage renal disease. Extracellular vesicles (EVs), especially for stem/progenitor cell-derived EVs (stem/progenitor cell-EVs), have emerged as a promising therapy for ischemia/reperfusion injury-induced AKI. However, their regulatory effects remain poorly understood, and their therapeutic efficiency in clinical trials is controversial. Here, we performed this systematic review and meta-analysis to assess the stem/progenitor cell-EV efficacy in treating ischemia/reperfusion injury-induced AKI in preclinical rodent models. METHODS: A literature search was performed in PubMed, Embase, Scopus, and Web of Science to identify controlled studies about the therapeutic efficiency of stem/progenitor cell-EVs on ischemia/reperfusion injury-induced AKI rodent models. The level of SCr, an indicator of renal function, was regarded as the primary outcome. Meta-regression analysis was used to reveal the influential factors of EV therapy. Sensitivity analysis, cumulative meta-analysis, and assessment of publication bias were also performed in our systematic review and meta-analysis. A standardized mean difference (SMD) was used as the common effect size between stem/progenitor cell-EV-treated and control groups, with values of 0.2, 0.5, 0.8, and 1.0 defined as small, medium, large, and very large effect sizes, respectively. RESULTS: A total of 30 studies with 985 ischemia/reperfusion injury-induced AKI rodent models were included. The pooled results showed that EV injection could lead to a remarkable sCr reduction compared with the control group (SMD, - 3.47; 95%CI, - 4.15 to - 2.80; P < 0.001). Meanwhile, the EV treatment group had lower levels of BUN (SMD, - 3.60; 95%CI, - 4.25 to - 2.94; P < 0.001), indexes for tubular and endothelial injury, renal fibrosis (fibrosis score and α-SMA), renal inflammation (TNF-α, IL-1ß, iNOS, and CD68 + macrophages), but higher levels of indexes for tubular proliferation, angiogenesis-related VEGF, and reactive oxygen species. However, our meta-regression analysis did not identify significant associations between sCr level and cell origins of EVs, injection doses, delivery routes, and therapy and outcome measurement time (all P values > 0.05). Significant publication bias was observed (Egger's test, P < 0.001). CONCLUSION: Stem/progenitor cell-EVs are effective in improving renal function in rodent ischemia/reperfusion injury-induced AKI model. These vesicles may help (i) reduce cell apoptosis and stimulate cell proliferation, (ii) ameliorate inflammatory injury and renal fibrosis, (iii) promote angiogenesis, and (iv) inhibit oxidative stress. However, the current systematic review and meta-analysis did not identify significant influential factors associated with treatment effects. More preclinical studies and thoughtfully designed animal studies are needed in the future.

miR-6869-5p Transported by Plasma Extracellular Vesicles Mediates Renal Tubule Injury and Renin-Angiotensin System Activation in Obesity.

Obesity increases the risk of other diseases, including kidney disease. Local renal tubular renin-angiotensin system (RAS) activation may play a role in obesity-associated kidney disease. Extracellular vehicles (EVs) transmit necessary information in obesity and cause remote organ damage, but the mechanism is unclear. The aim of the study was to investigate whether the plasma EVs cargo miR-6869-5p causes RAS activation and renal tubular damage. We isolated plasma EVs from obese and lean subjects and analyzed differentially-expressed miRNAs using RNA-seq. Then, EVs were co-cultured with human proximal renal tubular epithelial cells (PTECs) in vitro. Immunohistochemical pathology was used to assess the degree of RAS activation and tubule injury in vivo. The tubule damage-associated protein and RAS activation components were detected by Western blot. Obesity led to renal tubule injury and RAS activation in humans and mice. Obese-EVs induce RAS activation and renal tubular injury in PTECs. Importantly, miR-6869-5p-treated PTECs caused RAS activation and renal tubular injury, similar to Obese-EVs. Inhibiting miR-6869-5p decreased RAS activation and renal tubular damage. Our findings indicate that plasma Obese-EVs induce renal tubule injury and RAS activation via miR-6869-5p transport. Thus, miR-6869-5p in plasma Obese-EVs could be a therapeutic target for local RAS activation in obesity-associated kidney disease.

Critical roles of cytokine storm and secondary bacterial infection in acute kidney injury development in COVID-19: A multi-center retrospective cohort study.

Acute kidney injury (AKI) may develop in patients with coronavirus disease 2019 (COVID-19) and is associated with in-hospital death. We investigated the incidence of AKI in 223 hospitalized COVID-19 patients and analyzed the influence factors of AKI. The incidence of cytokine storm syndrome and its correlation with other clinicopathologic variables were also investigated. We retrospectively enrolled adult patients with virologically confirmed COVID-19 who were hospitalized at three hospitals in Wuhan and Guizhou, China between February 13, 2020, and April 8, 2020. We included 124 patients with moderate COVID-19 and 99 with severe COVID-19. AKI was present in 35 (15.7%) patients. The incidence of AKI was 30.3% for severe COVID-19 and 4.0% for moderate COVID-19 (p < 0.001). Furthermore, cytokine storm was found in 30 (13.5%) patients and only found in the severe group. Kidney injury at admission (odds ratio [OR]: 3.132, 95% confidence interval [CI]: 1.150-8.527; p = 0.025), cytokine storm (OR: 4.234, 95% CI: 1.361-13.171; p = 0.013), and acute respiratory distress syndrome (ARDS) (OR: 7.684, 95% CI: 2.622-22.523; p < 0.001) were influence factors of AKI. Seventeen (48.6%) patients who received invasive mechanical ventilation developed AKI, of whom 64.7% (11/17) died. Up to 86.7% of AKI patients with cytokine storms may develop a secondary bacterial infection. The leukocyte counts were significantly higher in AKI patients with cytokine storm than in those without (13.0 × 109/L, interquartile range [IQR] 11.3 vs. 8.3 × 109/L, IQR 7.5, p = 0.005). Approximately 1/6 patients with COVID-19 eventually develop AKI. Kidney injury at admission, cytokine storm and ARDS are influence factors of AKI. Cytokine storm and secondary bacterial infections may be responsible for AKI development in COVID-19 patients.

Potential role of extracellular vesicles in the pathophysiology of glomerular diseases.

Extracellular vesicles (EVs) are membrane-bound vesicles released by most cells and are found in diverse biological fluids. The release of EVs provides a new mechanism for intercellular communication, allowing cells to transfer their functional cargoes to target cells. Glomerular diseases account for a large proportion of end-stage renal disease (ESRD) worldwide. In recent years, an increasing number of research groups have focused their effort on identifying the functional role of EVs in renal diseases. However, the involvement of EVs in the pathophysiology of glomerular diseases has not been comprehensively described and discussed. In this review, we first briefly introduce the characteristics of EVs. Then, we describe the involvement of EVs in the mechanisms underlying glomerular diseases, including immunological and fibrotic processes. We also discuss what functions EVs derived from different kidney cells have in glomerular diseases and how EVs exert their effects through different signaling pathways. Furthermore, we summarize recent advances in the knowledge of EV involvement in the pathogenesis of various glomerular diseases. Finally, we propose future research directions for identifying better management strategies for glomerular diseases.

Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells.

Myelin-associated glycoprotein (MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB) was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase (ROCK) phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

Attenuation of TRPV1 by AMG-517 after nerve injury promotes peripheral axonal regeneration in rats.

Aims The main objective was to investigate the effects of the transient receptor potential cation channel subfamily V member 1 (TRPV1) on nerve regeneration following sciatic transection injury by functional blockage of TRPV1 using AMG-517, a specific blocker of TRPV1. Methods AMG-517 was injected into the area surrounding ipsilateral lumbar dorsal root ganglia 30 min after unilateral sciatic nerve transection. The number of sciatic axons and the expression of growth-associated protein-43 (GAP-43) and glial fibrillary acidic protein was examined using semithin sections, Western blot, and immunofluorescence analyses. Results Blockage of TRPV1 with AMG-517 markedly promoted axonal regeneration, especially at two weeks after sciatic injury; the number of axons was similar to the uninjured control group. After sciatic nerve transection, expression of glial fibrillary acidic protein was decreased and GAP-43 was increased at the proximal stump. However, the expression of both glial fibrillary acidic protein and GAP-43 increased significantly in AMG-517-treated groups. Conclusions TRPV1 may be an important therapeutic target to promote peripheral nerve regeneration after injury.

The Effect of Botulinum Neurotoxin Serotype a Heavy Chain on the Growth Related Proteins and Neurite Outgrowth after Spinal Cord Injury in Rats.

(1) Background: The botulinum toxin A (BoNT-A) heavy chain (HC) can stimulate the growth of primary motor neurites. (2) Methods: A recombinant BoNT/A HC was injected locally plus interval intrathecal catheter of BoNT/A HC to rats with ipsilateral semi-dissociated lumbar spinal cord injuries (SCIs). First, 2D gel with a silver nitrate stain was applied to detect the general pattern of protein expression. Growth associated protein 43 (GAP-43) and superior cervical ganglion 10 (SCG10) were chosen to represent the altered proteins, based on their molecular weight and pI, and were used to further detect their expression. Meanwhile, the neuronal processes were measured. The measurements of thermal hyperalgesia and grasp power at the ipsilateral hindlimb were used to evaluate spinal sensory and motor function, respectively. (3) Results: The local injection of BoNT/A HC followed by its intrathecal catheter intervally altered the spinal protein expression pattern after an SCI; protein expression was similar to normal levels or displayed a remarkable increase. The changes in the expression and distribution of phosphorylated growth associated protein 43(p-GAP 43) and superior cervical ganglion 10 (SCG 10) indicated that the administration of BoNT/A HC to the SCI significantly amplified the expression of p-GAP43 and SCG10 (p < 0.05). Meanwhile, the positive immunofluorescent staining for both p-GAP43 and SCG10 was mainly present near the rostral aspect of the injury, both in the cytoplasm and the neuronal processes. Moreover, the outgrowth of neurites was stimulated by the BoNT/A HC treatment; this was evident from the increase in neurite length, number of branches and the percentage of cells with neuronal processes. The results from the spinal function tests suggested that the BoNT/A HC did not affect sensation, but had a large role in improving the ipsilateral hindlimb grasp power (p < 0.05). (4) Conclusions: The local injection with the intermittent intrathecal administration of BoNT/A heavy chain to rats with SCI increased the local expression of GAP-43 and SCG 10, which might be affiliated with the regeneration of neuronal processes surrounding the injury, and might also be favorable to the relief of spinal motor dysfunction.

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury.

The transient receptor potential cation channel subfamily V member 1 (TRPV1) provides the sensation of pain (nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517 (300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.

Distribution of paired immunoglobulin-like receptor B in the nervous system related to regeneration difficulties after unilateral lumbar spinal cord injury.

Paired immunoglobulin-like receptor B (PirB) is a functional receptor of myelin-associated inhibitors for axonal regeneration and synaptic plasticity in the central nervous system, and thus suppresses nerve regeneration. The regulatory effect of PirB on injured nerves has received a lot of attention. To better understand nerve regeneration inability after spinal cord injury, this study aimed to investigate the distribution of PirB (via immunofluorescence) in the central nervous system and peripheral nervous system 10 days after injury. Immunoreactivity for PirB increased in the dorsal root ganglia, sciatic nerves, and spinal cord segments. In the dorsal root ganglia and sciatic nerves, PirB was mainly distributed along neuronal and axonal membranes. PirB was found to exhibit a diffuse, intricate distribution in the dorsal and ventral regions. Immunoreactivity for PirB was enhanced in some cortical neurons located in the bilateral precentral gyri. Overall, the findings suggest a pattern of PirB immunoreactivity in the nervous system after unilateral spinal transection injury, and also indicate that PirB may suppress repair after injury.

Respiratory syncytial virus (RSV) infects neuronal cells and processes that innervate the lung by a process involving RSV G protein.

Respiratory syncytial virus (RSV) is a primary cause of morbidity and life-threatening lower respiratory tract disease in infants and young children. Children with acute RSV bronchiolitis often develop respiratory sequelae, but the disease mechanisms are poorly understood. Mounting evidence suggests that RSV may mediate persistent infection. Using immunohistochemistry to identify RSV and RSV-infected cell types, we show that RSV infects primary neurons and neuronal processes that innervate the lungs through a process that involves RSV G protein and the G protein CX3C motif. These findings suggest a mechanism for disease chronicity and have important implications for RSV disease intervention strategies.

Glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system of mice.

Induction of apoptosis by rabies virus (RV) has been reported to be associated with the expression of the glycoprotein (G), but inversely correlated with pathogenicity. To further delineate the association between the expression of the G and the induction of apoptosis, recombinant RVs with replacement of only the G gene were used to infect mice by the intracerebral route. Recombinant viruses expressing the G from attenuated viruses expressed higher level of the G and induced more apoptosis in mice than recombinant RV expressing the G from wild-type (wt) or pathogenic RV, demonstrating that it is the G gene that determines the level of G expression and, consequently, the induction of apoptosis. Likewise, recombinant viruses expressing the G from wt or pathogenic RV are more pathogenic in mice than those expressing G from attenuated RV, confirming the inverse correlation between RV pathogenicity and the induction of apoptosis. To investigate the mechanism by which induction of apoptosis attenuates viral pathogenicity, mice were infected with wt or attenuated RV by the intramuscular route. It was found that low doses of attenuated RV induced apoptosis in the spinal cord and failed to spread to the brain or produce neurological disease. On the other hand, apoptosis was not observed in the spinal cord of mice infected with the same doses of wt RV and the virus spread to various parts of the brain and induced fatal neurologic disease. These results suggest that glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system (CNS) of mice.

Attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system.

Rabies virus (RV) induces encephalomyelitis in humans and animals. However, the pathogenic mechanism of rabies is not fully understood. To investigate the host responses to RV infection, we examined and compared the pathology, particularly the inflammatory responses, and the gene expression profiles in the brains of mice infected with wild-type (wt) virus silver-haired bat RV (SHBRV) or laboratory-adapted virus B2C, using a mouse genomic array (Affymetrix). Extensive inflammatory responses were observed in animals infected with the attenuated RV, but little or no inflammatory responses were found in mice infected with wt RV. Furthermore, attenuated RV induced the expression of the genes involved in the innate immune and antiviral responses, especially those related to the alpha/beta interferon (IFN-alpha/beta) signaling pathways and inflammatory chemokines. For the IFN-alpha/beta signaling pathways, many of the interferon regulatory genes, such as the signal transduction activation transducers and interferon regulatory factors, as well as the effector genes, for example, 2'-5'-oligoadenylate synthetase and myxovirus proteins, are highly induced in mice infected with attenuated RV. However, many of these genes were not up-regulated in mice infected with wt SHBRV. The data obtained by microarray analysis were confirmed by real-time PCR. Together, these data suggest that attenuated RV activates, while pathogenic RV evades, the host innate immune and antiviral responses.

Degeneration of neuronal processes after infection with pathogenic, but not attenuated, rabies viruses.

The structural alterations of neuronal processes in mice were investigated after the mice were infected with rabies virus (RV). Silver staining of infected brain sections showed severe destruction and disorganization of neuronal processes in mice infected with pathogenic RV but not with attenuated RV. However, neuronal bodies showed very little pathological changes. Electron microscopy revealed the disappearance of intracellular organelles, as well as the disappearance of synaptic structures and vesicles. Infection of primary neurons with pathogenic, but not attenuated, RV resulted in the destruction of neuronal processes and disappearance of microtubule-associated protein 2 and neurofilament immunoreactivity, which suggests that pathogenic RV causes degeneration of neuronal processes possibly by interrupting cytoskeletal integrity.

CGRP peptide and regenerating sensory axons.

CGRP peptide, a widely expressed constituent of sensory neurons, plays important roles in nerve function and repair when axons are severed. CGRP synthesis declines, yet peptide nonetheless accumulates in severed axon endbulbs. In this work we explore an apparent selective and ongoing expression of CGRP peptide in regenerative sensory axon sprouts. Following sural nerve crush in rats out to 14 days, regenerating and branching sensory axons had intense and selective expression of CGRP, not associated with endbulbs. Parent L4 and L5 perikarya and axons in the sural nerve proximal to crush, however, did not exhibit such heightened CGRP presence. Instead, back labeling of regenerating axons with fluorogold or diamidino yellow labeled perikarya with reduced CGRP expression. Similarly, ATF-3, a robust marker of axotomized neurons, was associated with reduced, rather than elevated expression of alphaCGRP mRNA. Unexpectedly, however, we identified an enlarged secondary population of intact uninjured neurons, frequently smaller and projecting to the dorsal horn with new and heightened intense CGRP expression but not ATF-3- or tracer-labeled. Distal regenerating sensory axons selectively express CGRP peptide despite reduced perikaryal content, a phenomenon not explained by simple accumulation. Having an injured neighbor neuron, however, may also paradoxically alter how CGRP is expressed in intact neurons.

Mu opioid receptors and analgesia at the site of a peripheral nerve injury.

Opioid ligands may exert antinociception through receptors expressed on peripheral afferent axons. Whether local opioid receptors might attenuate neuropathic pain is uncertain. In this work, we examined the function and expression of local mu opioid receptors (MORs) associated with the chronic constriction injury (CCI) model of sciatic neuropathic pain in rats. Low-dose morphine or its carrier were percutaneously superfused over the CCI site with the injector blinded to the identity of the injectate. Morphine, but not its carrier, and not equimolar systemic doses of morphine reversed thermal hyperalgesia in a dose-related, naloxone-sensitive fashion. Moreover, analgesia was conferred at both 48 hours and 14 days after CCI, times associated with very different stages of nerve repair. Equimolar local DAGO ([D-Ala2, N-Me-Phe4, Gly5-(ol)] enkephalin), a selective MOR ligand, provided similar analgesia. Local morphine also attenuated mechanical allodynia. MOR protein was expressed in axonal endbulbs of Cajal just proximal to the injury site, in aberrantly regenerating small axons in the epineurial sheath around the CCI site and in residual small axons distal to the CCI lesion. Sensory neurons ipsilateral to CCI had an increase in the proportion of neurons expressing MOR. We suggest that local MOR expressed in axons may be exploited to modulate some forms of neuropathic pain.