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The discovery that extracellular vesicles (EVs) can transfer functional extracellular RNAs (exRNAs) between cells opened new avenues into the study of EVs in health and disease. Growing interest in EV RNAs and other forms of exRNA has given rise to research programmes including but not limited to the Extracellular RNA Communication Consortium (ERCC) of the US National Institutes of Health. In 2017, the International Society for Extracellular Vesicles (ISEV) administered a survey focusing on EVs and exRNA to canvass-related views and perceived needs of the EV research community. Here, we report the results of this survey. Overall, respondents emphasized opportunities for technical developments, unraveling of molecular mechanisms and standardization of methodologies to increase understanding of the important roles of exRNAs in the broader context of EV science. In conclusion, although exRNA biology is a relatively recent emphasis in the EV field, it has driven considerable interest and resource commitment. The ISEV community looks forward to continuing developments in the science of exRNA and EVs, but without excluding other important molecular constituents of EVs.

Extracellular vesicles, including exosomes, are released by all cells, including those of the nervous system. Capable of delivering lipid, protein and nucleic acids to both nearby and distal cells, exosomes have been hypothesized to play a role in progression of many diseases of the nervous system. To date, most analyses on the role of these vesicles in the healthy and diseased state have relied on studying vesicles from in vitro sources, such as conditioned cell culture media, or body fluids. Here we have taken a critical approach to the enrichment and characterization of exosomes from human frontal cortex. This method maintains the integrity of the vesicles and their cargo, and comprehensive proteomic and genomic characterization confirms the legitimacy of the resulting extracellular vesicles as endosome-derived exosomes. This method will enable neuroscientists to acquire more detailed information about exosomes in the brain and explore the role(s) this form of intercellular communication and unique source of lipid, protein and RNA has in healthy brain function and pathogenic conditions. Furthermore, this method may have important utility in the isolation of exosomes from other tissues.

Allogeneic mesenchymal stromal cells (MSCs) have been used clinically for decades, without cross‐matching, on the assumption that they are immune‐privileged. In the equine model, we demonstrate innate and adaptive immune responses after repeated intra‐articular injection with major histocompatibility complex (MHC) mismatched allogeneic MSCs, but not MHC matched allogeneic or autologous MSCs. We document increased peri‐articular edema and synovial effusion, increased synovial cytokine and chemokine concentrations, and development of donor‐specific antibodies in mismatched recipients compared with recipients receiving matched allogeneic or autologous MSCs. Importantly, in matched allogeneic and autologous recipients, but not mismatched allogeneic recipients, there was increased stromal derived factor‐1 along with increased MSC concentrations in synovial fluid. Until immune recognition of MSCs can be avoided, repeated clinical use of MSCs should be limited to autologous or cross‐matched allogeneic MSCs. When non–cross‐matched allogeneic MSCs are used in single MSC dose applications, presensitization against donor MHC should be assessed., Autologous and matched mesenchymal stromal cell (MSC) intra‐articular transplantation resulted in increased stromal derived factor‐1 and endogenous progenitors, whereas mismatched MSCs resulted in local inflammation, not different to lipopolysaccharide (LPS) injection, and antibody dependent cytotoxicity of MSCs.

Cell therapies for neonatal morbidities are progressing to early phase clinical trials. However, protocols for intravenous (IV) delivery of cell therapies to infants have not been evaluated. It has been assumed the cell dose prescribed is the dose delivered. Early in our clinical trial of human amnion epithelial cells (hAECs), we observed cells settling in the syringe and IV tubing used to deliver the suspension. The effect on dose delivery was unknown. We aimed to quantify this observation and determine an optimal protocol for IV delivery of hAECs to extremely preterm infants. A standard pediatric infusion protocol was modeled in the laboratory. A syringe pump delivered the hAEC suspension over 60 minutes via a pediatric blood transfusion set (200‐μm filter and 2.2 mL IV line). The infusion protocol was varied by agitation methods, IV‐line volumes (0.2‐2.2 mL), albumin concentrations (2% vs 4%), and syringe orientations (horizontal vs vertical) to assess whether these variables influenced the dose delivered. The influence of flow rate (3‐15 mL/h) was assessed after other variables were optimized. The standard infusion protocol delivered 17.6% ± 9% of the intended hAEC dose. Increasing albumin concentration to 4%, positioning the syringe and IV line vertically, and decreasing IV‐line volume to 0.6 mL delivered 99.7% ± 13% of the intended hAEC dose. Flow rate did not affect dose delivery. Cell therapy infusion protocols must be considered. We describe the refinement of a cell infusion protocol that delivers intended cell doses and could form the basis of future neonatal cell delivery protocols., Cell therapy is neonatal medicine's new frontier. While the challenges of translation have been much discussed, we have overlooked a simple yet fundamental hurdle; a protocol that delivers the intended cell dose intravenously to infants. Our existing protocol delivered less than 20% of the intended dose of human amnion epithelial cells. Here, we demonstrate simple measures can optimize cell dose delivery.

Epiblast stem cells (EpiSCs) derived from postimplantation epiblast are pluripotent stem cells, epigenetically distinct from embryonic stem cells (ESCs), which are widely used in reprogramming studies. Recent achieved haploid cell lines in mammalian species open a new era for high‐throughput genetic screening, due to their homozygous phenotypes. Here, we report the generation of mouse haploid EpiSCs (haEpiSCs) from postimplantation chimeric embryos at embryonic day 6.5 (E6.5). These cells maintain one set of chromosomes, express EpiSC‐specific genes, and have potentials to differentiate into three germ layers. We also develop a massive mutagenesis protocol with haEpiSCs, and subsequently perform reprogramming selection using this genome‐wide mutation library. Multiple modules related to various pathways are implicated. The validation experiments prove that knockout of Hst3st3b1 (one of the candidates) can promote reprogramming of EpiSCs to the ground state efficiently. Our results open the feasibility of utilizing haEpiSCs to elucidate fundamental biological processes including cell fate alternations., Mouse haploid epiblast stem cells (haEpiSCs) can be derived from E6.5 postimplantation chimeric epiblast, which are pluripotent with a single genome. Genome‐scale gene trapping in haEpiSCs provides a platform to uncover essential genes regulating cellular reprograming. Knocking out one of the candidate genes, Hs3st3b1, greatly improves the efficiency of reprogramming from EpiSCs to embryonic stem cells.

Islet transplantation is an emerging treatment for type 1 diabetes which offers the prospect of physiological control of blood glucose and reductions in acute hypoglycaemic episodes. However, current protocols are limited by a rapid decline in islet functional viability during the isolation process, culture period, and post‐transplantation. Much of this can be attributed to the deleterious effects of hypoxic and cytokine stressors on β cells. One experimental strategy to improve the functional viability of islets is coculture or cotransplantation with mesenchymal stromal cells (MSCs). Numerous studies have shown that MSCs have the capacity to improve islet survival and insulin secretory function, and the mechanisms of these effects are becoming increasingly well understood. In this review, we will focus on recent studies demonstrating the capacity for MSCs to protect islets from hypoxia‐ and cytokine‐induced stress. Islets exposed to acute hypoxia (1%‐2% O2) or to inflammatory cytokines (including IFN‐γ, TNF‐α, and IL‐B) in vitro undergo apoptosis and a rapid decline in glucose‐stimulated insulin secretion. Coculture of islets with MSCs, or with MSC‐conditioned medium, protects from these deleterious effects, primarily with secreted factors. These protective effects are distinct from the immunomodulatory and structural support MSCs provide when cotransplanted with islets. Recent studies suggest that MSCs may support secretory function by the physical transfer of functional mitochondria, particularly to metabolically compromised β cells. Understanding how MSCs respond to stressed islets will facilitate the development of MSC secretome based, cell‐free approaches to supporting islet graft function during transplantation by protecting or repairing β cells., The functional viability of pancreatic islets rapidly declines during isolation, culture, and post‐transplantation. Transplantation stressors can be modeled in vitro by exposing islets to acute hypoxia or inflammatory cytokines. This review covers research demonstrating that coculture of islets with mesenchymal stromal cells (MSCs), or with MSC‐conditioned medium, protects from these deleterious effects, primarily by MSC‐secreted factors, and the clinical implications of this.

Acute myocardial infarction (MI) resulting from coronary ischemia is a major cause of disability and death worldwide. Transplantation of human embryonic stem cell (hESC)‐derived cardiovascular progenitor cells (hCVPCs) promotes the healing of infarcted hearts by secreted factors. However, the hCVPC‐secreted proteins contributing to cardiac repair remain largely unidentified. In this study, we investigated protective effects of neurotrophin (NT)‐3 secreted from hCVPCs in hearts against myocardial ischemia/reperfusion (I/R) injury and explored the underlying mechanisms to determine the potential of using hCVPC products as a new therapeutic strategy. The implantation of hCVPCs into infarcted myocardium at the beginning of reperfusion following 1 hour of ischemia improved cardiac function and scar formation of mouse hearts. These beneficial effects were concomitant with reduced cardiomyocyte death and increased angiogenesis. Moreover, hCVPCs secreted a rich abundance of NT‐3. The cardioreparative effect of hCVPCs in the I/R hearts was mimicked by human recombinant NT‐3 (hNT‐3) but canceled by NT‐3 neutralizing antibody (NT‐3‐Ab). Furthermore, endogenous NT‐3 was detected in mouse adult cardiomyocytes and its level was enhanced in I/R hearts. Adenovirus‐mediated NT‐3 knockdown exacerbated myocardial I/R injury. Mechanistically, hNT‐3 and endogenous NT‐3 inhibited I/R‐induced cardiomyocyte apoptosis through activating the extracellular signal‐regulated kinase (ERK) and reducing the Bim level, resulting in the cardioreparative effects of infarcted hearts together with their effects in the improvement of angiogenesis. These results demonstrate for the first time that NT‐3 is a cardioprotective factor secreted by hCVPCs and exists in adult cardiomyocytes that reduces I/R‐induced cardiomyocyte apoptosis via the ERK‐Bim signaling pathway and promotes angiogenesis. As a cell product, NT‐3 may represent as a noncell approach for the treatment of myocardial I/R injury., Neurotrophin (NT)‐3 contributes to the cardioreparative effects of human embryonic stem cell (hESC)‐derived cardiovascular progenitor cells (hCVPCs) characterized by reduction of ischemia/reperfusion (I/R)‐induced cardiomyocyte apoptosis in adult mice via the extracellular signal‐regulated kinase (ERK)‐Bim signaling pathway and promotion of angiogenesis, resulting in the cardiac function improvement and fibrosis limitation in the infarcted hearts.

Human antigen R (HuR) is a widespread RNA-binding protein involved in homeostatic regulation and pathological processes in many diseases. Atherosclerosis is the leading cause of cardiovascular disease and acute cardiovascular events. However, the role of HuR in atherosclerosis remains unknown. In this study, mice with smooth muscle-specific HuR knockout (HuRSMKO) were generated to investigate the role of HuR in atherosclerosis. HuR expression was reduced in atherosclerotic plaques. As compared with controls, HuRSMKO mice showed increased plaque burden in the atherosclerotic model. Mechanically, HuR could bind to the mRNAs of adenosine 5′-monophosphate-activated protein kinase (AMPK) α1 and AMPKα2, thus increasing their stability and translation. HuR deficiency reduced p-AMPK and LC3II levels and increased p62 level, thereby resulting in defective autophagy. Finally, pharmacological AMPK activation induced autophagy and suppressed atherosclerosis in HuRSMKO mice. Our findings suggest that smooth muscle HuR has a protective effect against atherosclerosis by increasing AMPK-mediated autophagy.

Galectin 3-binding protein (LGALS3BP, also known as 90K) is a multifunctional glycoprotein involved in immunity and cancer. However, its precise role in colon inflammation and tumorigenesis remains unclear. Here, we showed that Lgals3bp−/− mice were highly susceptible to colitis and colon tumorigenesis, accompanied by the induction of inflammatory responses. In acute colitis, NF-κB was highly activated in the colon of Lgals3bp−/− mice, leading to the excessive production of pro-inflammatory cytokines, such as IL-6, TNFα, and IL-1β. Mechanistically, Lgals3bp suppressed NF-κB through the downregulation of TAK1 in colon epithelial cells. There was no significant difference in the pro-inflammatory cytokine levels between wild-type and Lgals3bp−/− mice in a chronic inflammatory state, during colon tumorigenesis. Instead, Lgals3bp−/− mice showed elevated levels of GM-CSF, compared to those in WT mice. We also found that GM-CSF promoted the accumulation of myeloid-derived suppressor cells and ultimately increased colon tumorigenesis in Lgals3bp−/− mice. Taken together, Lgals3bp plays a critical role in the suppression of colitis and colon tumorigenesis through the downregulation of the TAK1-NF-κB-cytokine axis. These findings suggest that LGALS3BP is a novel immunotherapeutic target for colon inflammation and tumorigenesis.

Transcription factor EB (TFEB), a well-known master regulator of autophagy and lysosomal biogenesis, is a member of the microphthalmia family of transcription factors (MiT family). Over the years, TFEB has been shown to have diverse roles in various physiological processes such as clearance for intracellular pathogenic factors and having developmental functions such as dendritic maturation, as well as osteoclast, and endoderm differentiation. However, in the present study, we propose a novel mechanism for TFEB governing pluripotency of mouse ESCs (mESCs) by regulating the pluripotency transcriptional network (PTN) in these cells. We observed high levels of TFEB mRNA and protein levels in undifferentiated mESCs. Interestingly, we found a reduction of Nanog and Sox2 levels in TFEB knockout (KO) mESCs while pluripotency was maintained as there was an upregulation of TFE3, a potent stem cell maintenance factor. In consistent, double knockout of TFEB/TFE3 (TFEB/3 DKO) reduced mESC pluripotency, as indicated by the loss of ESC morphology, reduction of ESC markers, and the emergence of differentiation markers. We further discovered that Nanog was a TFEB target gene in undifferentiated mESCs. TFEB also promoted sex-determining region Y-box2 (Sox2) transcription by forming a heterodimer with Sox2 in mESCs. Notably, Sox2, Oct4, and Nanog were also binding to the TFEB promoter and thus generating a feed-forward loop in relation to TFEB. Although high levels of nuclear TFEB are expected to enhance autophagy–lysosomal activity, undifferentiated mESC remarkably displayed low basal autophagy–lysosomal activity. Overexpression or knockout of TFEB did not affect the expression of TFEB lysosomal–autophagy target genes and TFEB also had a lesser binding affinity to its own lysosomal promoter-target genes in mESCs compared to differentiated cells. Collectively, these findings define a newly incorporative, moonlighting function for TFEB in regulating PTN, independent of its autophagy–lysosomal biogenesis roles.

Autologous bone marrow mononuclear cell (BMMNC) transplantation has been widely studied in recent years. The fresh cell cocktail in BMMNCs, without going through the in vitro culture process, helps to establish a stable microenvironment for osteogenesis, and each cell type may play a unique role in bone regeneration. Our study compared the efficacy of concentrated fresh BMMNCs and cultured bone marrow-derived mesenchymal stem cells (BMSCs) in Beagle dogs for the first time. Fifteen-millimeter segmental bone defects were created in the animals' tibia bones. In BMMNCs group, the defects were repaired with concentrated fresh BMMNCs combined with β-TCP (n = 5); in cultured BMSC group, with in vitro cultured and osteo-induced BMSCs combined with β-TCP (n = 5); in scaffold-only group, with a β-TCP graft alone (n = 5); and in blank group, nothing was grafted (n = 3). The healing process was monitored by X-rays and single photon emission computed tomography. The animals were sacrificed 12 months after surgery and their tibias were harvested and analyzed by microcomputed tomography and hard tissue histology. Moreover, the microstructure, chemical components, and microbiomechanical properties of the regenerated bone tissue were explored by multiphoton microscopy, Raman spectroscopy and nanoindentation. The results showed that BMMNCs group promoted much more bone regeneration than cultured BMSC group. The grafts in BMMNCs group were better mineralized, and they had collagen arrangement and microbiomechanical properties similar to the contralateral native tibia bone. These results indicate that concentrated fresh bone marrow mononuclear cells may be superior to in vitro expanded stem cells in segmental bone defect repair.

Osteosarcoma is the most common primary malignant tumor of bone derived from osteoblasts, which is a noteworthy threat to the health of children and adolescents. In this study, we found that MCM8 has significantly higher expression level in osteosarcoma tissues in comparison with normal tissues, which was also correlated with more advanced tumor grade and pathological stage. In agreement with the role of MCM proteins as indicators of cell proliferation, knockdown/overexpression of MCM8 inhibited/promoted osteosarcoma cell proliferation in vitro and tumor growth in vivo. Also, MCM8 knockdown/overexpression was also significantly associated with the promotion/inhibition of cell apoptosis and suppression/promotion of cell migration. More importantly, mechanistic study identified CTGF as a potential downstream target of MCM8, silencing of which could enhance the regulatory effects of MCM8 knockdown and alleviate the effects of MCM8 overexpression on osteosarcoma development. In summary, MCM8/CTGF axis was revealed as critical participant in the development and progression of osteosarcoma and MCM8 may be a promising therapeutic target for osteosarcoma treatment.

The posttranslational modifications (PTMs) of microtubules have been reported to play an important role in cancer aggressiveness, including apoptosis resistance. In this study, we aimed to investigate the biological role of microtubule PTMs in the regulation of paclitaxel responsiveness. The acetylated tubulin (Ace-tub) level was strongly associated with paclitaxel sensitivity, as observed in patient-derived primary lung cancer cells and xenografted immunodeficient mice. We showed that paclitaxel-resistant H460 lung cancer cells, generated by a stepwise increase in paclitaxel, exhibited markedly increased tubulin acetylation and consequently acquired paclitaxel resistance. Upregulation of tubulin acetylation by overexpression of α-tubulin acetyltransferase 1 wild-type (αTAT1wt), an enzyme required for acetylation, or by treatment with trichostatin A (TSA), a histone deacetylase 6 (HDAC6) inhibitor, significantly attenuated paclitaxel-induced apoptosis. Investigation of the underlying mechanism revealed that the levels of antiapoptotic Mcl-1 appeared to increase in αTAT1wt-overexpressing and TSA-treated cells compared to control cells, whereas the levels of other antiapoptotic regulatory proteins were unchanged. On the other hand, decreased tubulin acetylation by αTAT1 RNA interference downregulated Mcl-1 expression in patient-derived primary lung cancer and paclitaxel-resistant lung cancer cells. A microtubule sedimentation assay demonstrated that Mcl-1 binds to microtubules preferentially at Ace-type, which prolongs the Mcl-1 half-life (T1/2). Furthermore, immunoprecipitation analysis revealed that polyubiquitination of Mcl-1 was extensively decreased in response to TSA treatment. These data indicate that tubulin acetylation enhances the resistance to paclitaxel-induced cell death by stabilizing Mcl-1 and protecting it from ubiquitin–proteasome-mediated degradation.

Progenitor cells from adipose tissue are able to induce bone repair; however, inconsistent or unreliable efficacy has been reported across preclinical and clinical studies. Soluble inhibitory factors, such as the secreted Wnt signaling antagonists Dickkopf‐1 (DKK1), are expressed to variable degrees in human adipose‐derived stem cells (ASCs), and may represent a targetable “molecular brake” on ASC mediated bone repair. Here, anti‐DKK1 neutralizing antibodies were observed to increase the osteogenic differentiation of human ASCs in vitro, accompanied by increased canonical Wnt signaling. Human ASCs were next engrafted into a femoral segmental bone defect in NOD‐Scid mice, with animals subsequently treated with systemic anti‐DKK1 or isotype control during the repair process. Human ASCs alone induced significant but modest bone repair. However, systemic anti‐DKK1 induced an increase in human ASC engraftment and survival, an increase in vascular ingrowth, and ultimately improved bone repair outcomes. In summary, anti‐DKK1 can be used as a method to augment cell‐mediated bone regeneration, and could be particularly valuable in the contexts of impaired bone healing such as osteoporotic bone repair., Systemic anti‐DKK1 treatment promotes engrafted stem cell survival, induces vascular ingrowth, and catalyzes bone matrix deposition among human adipose stem cell (ASC) treated femoral segmental bone defects. BM, bone marrow; CB, cortical bone; S, scaffold.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy that is primarily detected at the metastatic stage. Most HGSOC originates from the fallopian tube epithelium (FTE) and metastasizes to the ovary before invading the peritoneum; therefore, it is crucial to study disease initiation and progression using FTE-derived models. We previously demonstrated that loss of PTEN from the FTE leads to ovarian cancer. In the present study, loss of PTEN in FTE led to the enrichment of cancer stem cell markers such as LGR5, WNT4, ALDH1, CD44. Interestingly, loss of the transcription factor PAX2, which is a common and early alteration in HGSOC, played a pivotal role in the expression of cancer stem-like cells (CSC) markers and cell function. In addition, loss of PTEN led to the generation of two distinct subpopulations of cells with different CSC marker expression, tumorigenicity, and chemoresistance profiles. Taken together, these data suggest that loss of PTEN induces reprogramming of the FTE cells into a more stem-like phenotype due to loss of PAX2 and provides a model to study early events during the FTE-driven ovarian cancer tumor formation.

The origin and intermediate host for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is yet to be determined. Coronaviruses found to be closely related to SARS-CoV-2 include RaTG13 derived from bat and two clusters (PCoV-GD and PCoV-GX) of coronaviruses identified in pangolin. Here, we studied the infectivity and antigenicity patterns of SARS-CoV-2 and the three related coronaviruses. Compared with the other three viruses, RaTG13 showed almost no infectivity to a variety of cell lines. The two pangolin coronaviruses and SARS-CoV-2 showed similar infectious activity. However, in SARS-CoV-2-susceptible cell lines, the pangolin coronaviruses presented even higher infectivity. The striking difference between the SARS-CoV-2 and pangolin coronaviruses is that the latter can infect porcine cells, which could be partially attributed to an amino acid difference at the position of 498 of the spike protein. The infection by SARS-CoV-2 was mainly mediated by Furin and TMPRSS2, while PCoV-GD and PCoV-GX mainly depend on Cathepsin L. Extensive cross-neutralization was found between SARS-CoV-2 and PCoV-GD. However, almost no cross-neutralization was observed between PCoV-GX and SARS-CoV-2 or PCoV-GD. More attention should be paid to pangolin coronaviruses and to investigate the possibility of these coronaviruses spreading across species to become zoonoses among pigs or humans.

BackgroundOsteosarcoma is an aggressive malignant tumor which has attracted worldwide attention. MEX3A may be associated with tumors while has not yet seen its coverage on osteosarcoma. Herein, this study was to investigate the correlation between MEX3A and the progression of osteosarcoma.MethodsFirstly, we determined that expression of MEX3A was significantly higher in osteosarcoma tissues than that in marginal bone by immunohistochemical staining. Additionally, MEX3A expression was downregulated by the RNAi‐mediated knockdown. The functions of MEX3A knockdown on proliferation, apoptosis, cell cycle, migration was assessed by MTT assay, flow cytometry, wound-healing assay and Transwell assay, respectively. Knockdown of MEX3A resulted in suppressing cell proliferation, increasing cell apoptosis, inducing the G2 phase cell cycle arrest, and attenuating cellular migration. Furthermore, mouse xenograft model confirmed inhibitory effects of MEX3A knockdown on osteosarcoma formation.ResultsThe preliminary exploration on the molecular mechanism of MEX3A in osteosarcoma cells showed that the induction of apoptosis needs the participation of a series of apoptosis- associated factors, such as upregulation of Caspase 3, Caspase 8 and HSP60, downregulation of HSP27 and XIAP.ConclusionsIn summary, these findings predicated that therapy directed at decreasing MEX3A expression is a potential osteosarcoma treatment.

Colorectal cancer (CRC) is the third most common cause of cancer globally and the fourth attributable cause of mortality and morbidity due to cancer. An emerging factor contributing to CRC is the gut microbiota and the cellular changes associated with it. Further insights on this may help in the prevention, diagnosis and new therapeutic approaches to colorectal cancer. In most cases of CRC, genetic factors appear to contribute less to its aetiology than environmental and epigenetic factors; therefore, it may be important to investigate these environmental factors, their effects, and the mechanisms that may contribute to this cancer. The gut microbiota has recently been highlighted as a potential risk factor that may affect the structural components of the tumor microenvironment, as well as free radical and enzymatic metabolites directly, or indirectly. Many studies have reported changes in the gut microbiota of patients with colorectal cancer. What is controversial is whether the cancer is the cause or consequence of the change in the microbiota. There is strong evidence supporting both possibilities. The presence of Fusobacterium nucleatum in human colorectal specimens has been demonstrated by RNA-sequencing. F. nucleatum has been shown to express high levels of virulence factors such as FadA, Fap2 and MORN2 proteins. Our review of the published data suggest that F. nucleatum may be a prognostic biomarker of CRC risk, and hence raises the potential of antibiotic treatment of F. nucleatum for the prevention of CRC.

A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (hUGT1A1) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-hUGT1A1, and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs., Graphical Abstract, At postnatal day 28, AAV-hUGT1A1 establishes persistent correction of hyperbilirubinemia in Ugt1a1 deficient rats while earlier treatment results in (partial) loss of efficacy overtime. Only in naive animals does immune suppression by rapamycin effectively bloc NAb induction, allowing effective re-treatment. In primed animals, this induction was reduced but not completely prevented.

Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling., Graphical Abstract, Yahata et al. refined a previous G13513A-mpTALEN to more efficiently manipulate mtDNA heteroplasmy in MELAS-iPSCs. Mutation-rich MELAS-iPSCs showed unusual differentiation properties, which were recovered by decreasing the heteroplasmy levels with new G13513A-mpTALEN. They further demonstrated that drug-inducible, MYOD-transfected MELAS-iPSCs could efficiently differentiate into MyHC-positive myocytes, with or without mutant mtDNA.

Acute myeloid leukemia (AML) is a hematological malignancy with high incidence and recurrence rates. Gene expression profiling has revealed that transcriptional overexpression of glioma‐associated oncogene 1 (GLI1), a vital gene in the Hedgehog (Hh) signaling pathway, occurs in poor-prognosis AML, and high levels of phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) and AKT3 predict shorter overall survival in AML patients. In this study, we discovered that GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity in AML cells while knocking down GLI1 has the opposite effect. Moreover, GLI1 promoted cell cycle progression and led to elevated protein levels of cyclins and cyclin-dependent kinases (CDKs) in AML cells. By luciferase assays and co-immunoprecipitation, we demonstrated that the PI3K/AKT pathway is directly activated by GLI1. GLI1 overexpression significantly accelerates tumor growth and upregulated p-AKT, CDK4, and cyclinD3 in vivo. Notably, the GLI1 inhibitor GANT61 and the CDK4/6 inhibitor PD 0332991 had synergistic effects in promoting Ara-c sensitivity in AML cell lines and patient samples. Collectively, our data demonstrate that GLI1 reduces drug sensitivity by regulating cell cycle through the PI3K/AKT/GSK3/CDK pathway, providing a new perspective for involving GLI1 and CDK4/6 inhibitors in relapsed/refractory (RR) patient treatment.

Systemic or localized application of glucocorticoids (GCs) can lead to iatrogenic ocular hypertension, which is a leading cause of secondary open-angle glaucoma and visual impairment. Previous work has shown that dexamethasone increases zonula occludens-1 (ZO-1) protein expression in trabecular meshwork (TM) cells, and that an antisense oligonucleotide inhibitor of ZO-1 can abolish the dexamethasone-induced increase in trans-endothelial flow resistance in cultured Schlemm’s canal (SC) endothelial and TM cells. We have previously shown that intracameral inoculation of small interfering RNA (siRNA) targeting SC endothelial cell tight junction components, ZO-1 and tricellulin, increases aqueous humor outflow facility ex vivo in normotensive mice by reversibly opening SC endothelial paracellular pores. In this study, we show that targeted siRNA downregulation of these SC endothelial tight junctions reduces intraocular pressure (IOP) in vivo, with a concomitant increase in conventional outflow facility in a well-characterized chronic steroid-induced mouse model of ocular hypertension, thus representing a potential focused clinical application for this therapy in a sight-threatening scenario., Graphical Abstract, Cassidy and colleagues show that intracameral inoculation of siRNA targeting Schlemm’s canal endothelial cell tight junction proteins, ZO-1 and tricellulin, can reduce intraocular pressure and increase outflow facility in a murine model of steroid-induced ocular hypertension.

Chronic graft-versus-host disease (cGVHD) is the main cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mesenchymal stem cells (MSCs) in bone marrow (BM) remain unclear in the pathophysiology of cGVHD. In this study, we analyzed BM-MSCs from 66 patients after allo-HSCT, including 33 with active cGVHD and 33 without cGVHD. BM-MSCs showed similar morphology, frequency, phenotype, and proliferation in patients with or without cGVHD. MSCs from the active cGVHD group showed a decreased apoptosis rate (P P P P r = 0.534, r = 0.476, r = −0.796, and r = −0.747, respectively in RUNX2, COL1A1, PPAR-γ, and FABP4). The expression of Wnt/β-catenin pathway ligand Wnt3a was increased in cGVHD-MSCs. The dysfunction of cGVHD-MSCs could be reversed by Dickkopf related protein 1(DKK1) to inhibit the binding of Wnt3a. In summary, the differentiation of BM-MSCs was abnormal in active cGVHD, and its underlying mechanism is the upregulated of Wnt3a through Wnt/β-catenin signaling pathway of MSCs.

Exosome-derived microRNAs (miRNAs) are potential diagnostic biomarkers. However, little is known about their effectiveness as diagnostic biomarkers of fulminant myocarditis (FM). This study aimed to explore serum exosomal miRNAs as potential biomarkers for FM diagnosis. Peripheral blood samples were collected from 99 patients with FM, 32 patients with nonfulminant myocarditis (NFM), and 105 healthy controls (HCs). The miRNA expression profiles of serum exosomes were determined using next-generation sequencing, and differentially expressed miRNAs were further analyzed by quantitative reverse transcriptase polymerase chain reaction. A logistic regression model was constructed using a training cohort (n = 120) and then validated using an independent cohort (n = 106). The area under the receiver operating characteristic curve was used to evaluate diagnostic accuracy. In FM patients, hsa-miR-30a, hsa-miR-192, hsa-miR-146a, hsa-miR-155, and hsa-miR-320a were validated as significantly and differentially expressed candidates that could serve as potential markers for diagnosing FM. In addition, the miRNA panel (hsa-miR-155 and hsa-miR-320a) from the multivariate logistic regression model demonstrated high accuracy in the diagnosis of FM and was able to distinguish FM from HCs and NFM. Moreover, the diagnostic value of the miRNA panel was greater than that of CRP and cTn alone or together. The miRNA panel provided the excellent diagnostic capability for FM., Graphical Abstract, This study investigated the exosome-derived miRNA profiles in serum from patients with FM and found that the miRNA panel for FM diagnosis exhibited high accuracy in distinguishing FM from HCs and NFM. Our results support the hypothesis that circulating exosomal miRNA profiles can serve as noninvasive biomarkers of FM.

Critical limb ischemia is a condition in which tissue necrosis occurs due to arterial occlusion, resulting in limb amputation in severe cases. Both endothelial cells (ECs) and vascular smooth muscle cells (SMCs) are needed for the regeneration of peripheral arteries in ischemic tissues. However, it is difficult to isolate and cultivate primary EC and SMC from patients for therapeutic angiogenesis. Induced pluripotent stem cells (iPSCs) are regarded as useful stem cells due to their pluripotent differentiation potential. In this study, we explored the therapeutic efficacy of human iPSC‐derived EC and iPSC‐derived SMC in peripheral artery disease model. After the induction of mesodermal differentiation of iPSC, CD34+ progenitor cells were isolated by magnetic‐activated cell sorting. Cultivation of the CD34+ progenitor cells in endothelial culture medium induced the expression of endothelial markers and phenotypes. Moreover, the CD34+ cells could be differentiated into SMC by cultivation in SMC culture medium. In a murine hindlimb ischemia model, cotransplantation of EC with SMC improved blood perfusion and increased the limb salvage rate in ischemic limbs compared to transplantation of either EC or SMC alone. Moreover, cotransplantation of EC and SMC stimulated angiogenesis and led to the formation of capillaries and arteries/arterioles in vivo. Conditioned medium derived from SMC stimulated the migration, proliferation, and tubulation of EC in vitro, and these effects were recapitulated by exosomes isolated from the SMC‐conditioned medium. Together, these results suggest that iPSC‐derived SMC enhance the therapeutic efficacy of iPSC‐derived EC in peripheral artery disease via an exosome‐mediated paracrine mechanism., Co‐transplantation of the induced pluripotent stem cell (iPSC)‐derived endothelial cells and smooth muscle cells enhanced the therapeutic efficacies of endothelial cells and smooth muscle cells in a murine hindlimb ischemia model. Smooth muscle cells promoted angiogenesis and therapy of peripheral artery disease through exosome‐mediated paracrin mechanism.

Relevance Pien Tze Huang (PZH) is a well-known Traditional Chinese Medicine (TCM), characterized by a multitude of pharmacological effects, such as hepatoprotection and inhibition of inflammation and cell proliferative conditions. Many of these effects have been validated at the cellular, molecular and physiological levels but, to date, most of these findings have not been comprehensively disclosed. Objectives This review aims to provide a critical summary of recent studies focusing on PZH and its multiple pharmacological effects. As a result, we further discuss some novel perspectives related to PZH’s mechanisms of action and a holistic view of its therapeutic activities. Methods A systematic review was performed focusing on PZH studies originated from original scientific resources. The scientific literature retrieved for this work was obtained from International repositories including NCBI/PubMed, Web of Science, Science Direct and China National Knowledge Infrastructure (CNKI) databases. Results The major active componentes and their potential functions, including hepatoprotective and neuroprotective effects, as well as anti-cancer and anti-inflammatory activities, were summarized and categorized accordingly. As indicated, most of the pharmacological effects were validated in vitro and in vivo. The identification of complex bioactive components in PZH may provide the basis for further therapeutic initiatives. Conclusion Here we have collectively discussed the recent evidences covering most, if not all, pharmacological effects driven by PZH. This review provides novel perspectives on understanding the modes of action and the holistic view of TCM. The rational development of future clinical trials will certainly provide evidence-based medical evidences that will also confirm the therapeutic advantages of PZH, based on the current information available.

Human induced pluripotent stem cell-derived intestinal epithelial cells (hiPSC-IECs) are expected to be utilized in regenerative medicine. To perform a safe transplantation without the risk of tumor formation, residual undifferentiated hiPSCs must be removed from hiPSC-IECs. In this study, we examined whether vinblastine (a multiple drug resistance 1 [MDR1] substrate) could remove residual undifferentiated hiPSCs in hiPSC-IECs and attempted to generate hiPSC-IECs applicable to transplantation medicine. We found that the expression levels of pluripotent markers were largely decreased and those of intestinal markers were increased by vinblastine treatment. The treatment of undifferentiated hiPSCs with vinblastine significantly decreased their viability. These results suggested that undifferentiated hiPSCs can be eliminated from hiPSC-IECs by vinblastine treatment. We hypothesized that MDR1-negative cells (such as undifferentiated hiPSCs) die upon vinblastine treatment because they are unable to excrete vinblastine. As expected, the cell viability of MDR1-knockout hiPSC-IECs was significantly decreased by vinblastine treatment. Furthermore, teratomas were formed by subcutaneous transplantation of hiPSC-IECs mixed with undifferentiated hiPSCs into mice, but they were not observed when the transplanted cells were pre-treated with vinblastine. Vinblastine-treated hiPSC-IECs would be an effective cell source for safe regenerative medicine., Graphical Abstract, This study showed that vinblastine (a multiple drug resistance 1 substrate) can remove residual undifferentiated human induced pluripotent stem cells (hiPSCs) in hiPSC-derived intestinal epithelial cells (hiPSC-IECs) and enhances the functions of hiPSC-IECs. Vinblastine-treated hiPSC-IECs would be an effective cell source for safe regenerative medicine.

Background Cholangiocarcinoma (CCA) is the second most common primary liver malignancy worldwide. Several microRNAs (miRNAs) have been implicated as potential tumor suppressors in CCA. This study aims to explore the potential effects of miR-1182 and let-7a on CCA development. Methods Bioinformatics analysis was conducted to screen differentially expressed genes in CCA, Western blot analysis detected NUAK1 protein expression and RT-qPCR detected miR-1182, let-7a and NUAK1 expression in CCA tissues and cell lines. Dual luciferase reporter gene assay and RIP were applied to validate the relationship between miR-1182 and NUAK1 as well as between let-7a and NUAK1. Functional experiment was conducted to investigate the role of miR-1182, let-7a and NUAK1 in cell migration, proliferation and autophagy. Then, the CCA cells that received various treatments were implanted to mice to establish animal model, followed by tumor observation and HE staining to evaluate lung metastasis. Results CCA tissues and cells were observed to have a high expression of NUAK1 and poor expression of miR-1182 and let-7a. NUAK1 was indicated as a target gene of miR-1182 and let-7a. Importantly, upregulation of either miR-1182 or let-7a induced autophagy, and inhibited cell progression and in vivo tumor growth and lung metastasis; moreover, combined treatment of miR-1182 and let-7a overexpression presented with enhanced inhibitory effect on NUAK1 expression and CCA progression, but such synergistic effect could be reversed by overexpression of NUAK1. Conclusion Taken together, the findings suggest the presence of a synergistic antitumor effect of miR-1182 and let-7a on the development of CCA via the down-regulation of NUAK1, providing novel insight into the targeted therapy against CCA.

Approximately 25% of all cases of ovarian cancer (OVCA) cases are associated with inherited risk. However, accurate risk assessment is limited by the presence of variants of unknown significance (VUS). Previously, we performed whole-exome sequencing on 48 OVCA patients with familial predisposition, yet negative for pathogenic BRCA1/2 mutations. In our cohort, we uncovered thirteen truncating mutations in genes associated with apoptosis (~35% of our patient cohort). The TP53I3 p.S252X premature stop gain was identified in two unrelated patients. TP53I3 is transcriptionally activated by p53 and believed to play a role in DNA damage response and reactive oxygen species-induced apoptosis. In addition, nonsense variants in apoptosis-related genes TP53AIP1, BCLAF1, and PIK3C2G were identified in our cohort; highlighting the potential relevance of genes involved in apoptotic processes to hereditary cancer. In the current study, we employed functional assays and demonstrated that cells expressing TP53I3 p.S252X displayed decreased homologous recombination repair efficiency and increased sensitivity to chemotherapeutic drugs bleomycin, mitomycin c, and etoposide. In addition, in the presence of oxidative stress from hydrogen peroxide or etoposide we observed a reduction in the formation of reactive oxygen species, an important precursor to apoptosis with this variant. Our findings suggest that the combination of in silico and wet laboratory approaches can better evaluate VUSs, establish novel germline predisposition genetic loci, and improve individual cancer risk estimates.

Lithium is neuroprotective in preclinical stroke models. In addition to that, poststroke neuroregeneration is stimulated upon transplantation of mesenchymal stem cells (MSCs). Preconditioning of MSCs with lithium further enhances the neuroregenerative potential of MSCs, which act by secreting extracellular vesicles (EVs). The present work analyzed whether MSC preconditioning with lithium modifies EV secretion patterns, enhancing the therapeutic potential of such derived EVs (Li‐EVs) in comparison with EVs enriched from native MSCs. Indeed, Li‐EVs significantly enhanced the resistance of cultured astrocytes, microglia, and neurons against hypoxic injury when compared with controls and to native EV‐treated cells. Using a stroke mouse model, intravenous delivery of Li‐EVs increased neurological recovery and neuroregeneration for as long as 3 months in comparison with controls and EV‐treated mice, albeit the latter also showed significantly better behavioral test performance compared with controls. Preconditioning of MSCs with lithium also changed the secretion patterns for such EVs, modifying the contents of various miRNAs within these vesicles. As such, Li‐EVs displayed significantly increased levels of miR‐1906, which has been shown to be a new regulator of toll‐like receptor 4 (TLR4) signaling. Li‐EVs reduced posthypoxic and postischemic TLR4 abundance, resulting in an inhibition of the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) signaling pathway, decreased proteasomal activity, and declined both inducible NO synthase and cyclooxygenase‐2 expression, all of which culminated in reduced levels of poststroke cerebral inflammation. Conclusively, the present study demonstrates, for the first time, an enhanced therapeutic potential of Li‐EVs compared with native EVs, interfering with a novel signaling pathway that yields both acute neuroprotection and enhanced neurological recovery., Lithium‐treated extracellular vesicles (Li‐EVs) induce neuroprotection, neuroregeneration, and neurological recovery under in vitro and in vivo stroke conditions. Increased concentrations of intravesicular miR‐1906 due to lithium treatment reduce toll‐like receptor 4 expression patterns. The latter results in reduction of NF‐κB activation and reduced proteasomal activity followed by decreased levels of pro‐inflammatory mediators within the ischemic brain. Likewise, Li‐EVs reverse the poststroke peripheral immunosuppression.

Background Gastric intestinal metaplasia (GIM) is a significant risk factor for gastric cancer. Risk of gastric cancer/dysplasia between complete intestinal metaplasia (CIM) and incomplete intestinal metaplasia (IIM) was controversial. Our study aimed to pool relative risk (RR) of cancer/dysplasia of IIM compared with CIM in GIM patients. Methods PubMed, EMBASE, Cochrane Library and Web of Science were searched for studies concerning cancer/dysplasia in GIM patients. Random-effects or fixed-effects model was utilized for pooling RR. Sensitivity and publication bias analyses were conducted. Stability of results would be evaluated in case of publication bias. Results 12 studies were included. Compared with CIM, pooled RR of cancer/dysplasia in IIM patients was 4.48 (95% CI 2.50–8.03), and the RR was 4.96 (95% CI 2.72–9.04) for cancer, and 4.82 (95% CI 1.45–16.0) for dysplasia. The pooled RR for cancer/dysplasia in type III IM was 6.27 (95% CI 1.89–20.77) compared with type II + I IM, while it was 5.55 (95% CI 2.07–14.92) compared with type II IM. Pooled RR between type II IM and type I IM was 1.62 (95% CI 1.16–2.27). Subgroup analyses showed that IIM was associated with a higher risk of gastric cancer/dysplasia in Western population (pooled RR = 4.65 95% CI 2.30–9.42), but not in East Asian population (pooled RR = 4.01 95% CI 0.82–19.61). Conclusions IIM was related to a higher risk of cancer/dysplasia compared with CIM. Risk of developing cancer/dysplasia from type I, II, and III intestinal metaplasia increased gradually.

The exact mechanism of myocardial hypertrophy has not been completely elucidated. NOD-like receptor protein 3 (NLRP3) and the pyroptotic cascade play a critical role in cardiac hypertrophy and inflammation. The myokine irisin can inhibit NLRP3 activation, although its exact mechanism of action is unknown. In this study, we induced cardiac hypertrophy in a mouse model via aortic constriction (TAC) to further explore the pathological role of NLRP3 inflammasome-mediated pyroptosis and the potential therapeutic effects of irisin. Cardiac hypertrophy significantly increased the percentage of apoptotic cells and upregulated IL-1β, cleaved caspase-1, and GSDMD-N that lie downstream of the NLRP3 inflammasome. Subsequently, irisin was co-administered to the TAC mice or angiotensin II (Ang-II)-treated cardiomyocytes to observe whether it could attenuate pyroptosis and cardiac hypertrophy. We established a direct association between pyroptosis and cardiac hypertrophy and found that pharmacological or genetic inhibition of NLRP3 attenuated cardiac hypertrophy. Furthermore, ectopic overexpression of NLRP3 abrogated the cardioprotective effects of irisin. To summarize, pyroptosis is a pathological factor in cardiac hypertrophy, and irisin is a promising therapeutic agent that inhibits NLRP3-mediated pyroptosis of cardiomyocytes.

Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary fluid with a high mortality rate, but the underlying mechanism is not yet fully understood, causing absent specific therapeutic drugs to treat with ARDS. In recent years, more and more studies have applied proteomics to ARDS. Non-targeted studies of proteomics in ARDS are just beginning and have the potential to identify novel drug targets and key pathways in this disease. This paper will provide a brief review of the recent advances in the application of non-targeted proteomics to ARDS.

BackgroundTUBA1C is a microtubule component that is involved in a variety of cancers. Our main objective was to investigate TUBA1C expression, its prognostic value, its potential biological functions, and its impact on the immune system of patients with lung adenocarcinoma (LUAD).MethodsThe Cancer Genome Atlas (TCGA), Gene Expression Profiling Interactive Analysis (GEPIA) and Immunohistochemistry Analysis were used to analyze TUBA1C expression, its clinicopathology, overall survival (OS), and disease-free survival (DFS) in LUAD patients. We also determined the correlation between TUBA1C and tumor-infiltrating immune cells (TIICs) by using CIBERSORT and GEPIA databases. To determine the expression of TUBA1C in LUAD, we analyzed a collection of immune infiltration levels and cumulative survival of LUAD tissues in TIMER database. By using UALCAN, STRING, and GeneMANIA databases, we investigated the protein-coding genes related to TUBA1C and its co-expression genes in LUAD tissues. Gene set enrichment analysis (GSEA) was performed by using the TCGA dataset.ResultsThe mRNA and the protein expression of TUBA1C were found to be up-regulated in LUAD tissues. The univariate analysis indicated that an increased expression of TUBA1C was significantly correlated to the following parameters: age, stage, and lymph node metastasis. An over-expression of TUBA1C was associated with a poor prognosis of LUAD. In TIMER and CIBERSORT databases, we found that TUBA1C is correlated with 13 types of TIICs: activated B cell, activated CD4 T cell, central memory CD4 T cell, effector memory CD8 T cell, eosinophils, immature B cell, gamma-delta T cell, immature dendritic cell, mast cell, memory B cell, natural killer T cell, regulatory T cell, and type 2T helper cell. By performing GSEA, we found that TUBA1C is closely correlated to cell cycle, p53 signaling pathway, glycolysis, and gluconeogenesis.ConclusionsOur findings indicate that TUBA1C is associated with TIICs in tumor microenvironment. Therefore, it serves as a novel prognostic biomarker and a target for future treatment methods of LUAD.

Background Emerging evidence continues to highlight the significant role of microRNAs (miRNAs) in the regulation of cancer growth and metastasis. Herein, the current study aimed to elucidate the role of exosomal miR-183 in prostate cancer development. Methods Initially, public microarray-based gene expression profiling of prostate cancer was employed to identify differentially expressed miRNAs. The putative target gene TPM1 of miR-183 was subsequently predicted, followed by the application of a luciferase reporter assay and examination of the expression patterns in prostate cancer patients and cell lines. The effects of miR-183 and TPM1 on processes such as cell proliferation, invasion and migration were evaluated using in vitro gain- and loss-of-function experiments. The effect of PC3 cells-derived exosomal miR-183 was validated in LNCaP cells. In vivo experiments were also performed to examine the effect of miR-183 on prostate tumor growth. Results High expression of miR-183 accompanied with low expression of TPM1 was detected in prostate cancer. Our data indicated that miR-183 could target and downregulate TPM1, with the overexpression of miR-183 and exosomal miR-183 found to promote cell proliferation, migration, and invasion in prostate cancer. Furthermore, the tumor-promoting effect of exosome-mediated delivery of miR-183 was subsequently confirmed in a tumor xenograft model. Conclusions Taken together, the key findings of our study demonstrate that prostate cancer cell-derived exosomal miR-183 enhance prostate cancer cell proliferation, invasion and migration via the downregulation of TPM1, highlighting a promising therapeutic target against prostate cancer.

Efficient delivery of nucleic acids for the engineering of primary T cells is central to the study of the basic biology of these key immune effector cells and has clinical implications. To date, lentiviral vectors delivering guide RNAs for CRISPR-Cas9 editing are not optimal for use in primary cells. Herein, we describe the T cell optimized for packaging (TOP) vector for delivering guide RNAs and transgenes into primary T cells. The TOP vector produces high-titer virus compared to a routinely used guide RNA vector, resulting in a ~10-fold increase in transduction in T cells. Moreover, a TOP vector expressing a chimeric antigen receptor and a guide RNA targeting the T cell receptor showed an ~5- to 9-fold increased transduction efficiency with ~2- to 3-fold higher expression compared to the commonly used epHIV7 vector and was simultaneously able to mediate efficient knockout of the endogenous T cell receptor in >71% of transduced cells upon Cas9 electroporation. The increased packaging of the TOP vector genome into viral particles appears to contribute to its higher transduction efficiency. The TOP vector represents an optimal tool for tandem delivery of transgenes and guide RNAs to primary T cells for use in functional screens and immunotherapy applications., Graphical Abstract, The T cell optimized for packaging (TOP) vector is a lentiviral construct for efficient dual delivery of guide RNAs and transgenes into primary T cells. The TOP vector provides a useful tool for CRISPR screening and potential immunotherapy applications such as delivering chimeric antigen receptor while depleting endogenous T cell receptor expression.

Therapeutic payload delivery to the central nervous system (CNS) remains a major challenge in gene therapy. Recent studies using function-driven evolution of adeno-associated virus (AAV) vectors have successfully identified engineered capsids with improved blood-brain barrier (BBB) penetration and CNS tropism in mouse. However, these strategies require transgenic animals and thus are limited to rodents. To address this issue, we developed a directed evolution approach based on recovery of capsid library RNA transcribed from CNS-restricted promoters. This RNA-driven screen platform, termed TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA), was tested in the mouse with AAV9 peptide display libraries and showed rapid emergence of dominant sequences. Ten individual variants were characterized and showed up to 400-fold higher brain transduction over AAV9 following systemic administration. Our results demonstrate that the TRACER platform allows rapid selection of AAV capsids with robust BBB penetration and CNS tropism in non-transgenic animals., Graphical Abstract, In this article, Nonnenmacher and colleagues establish TRACER, a cell-specific RNA-driven directed evolution platform for functional biopanning of adeno-associated virus (AAV) capsid libraries in animal tissue. Neuron- and glial-specific selection of an AAV9 library in mouse brain identified several AAV variants with unprecedented brain and spinal cord transduction.

Effective detection of microbiological contaminations present in medicinal cellular products is a crucial step to ensure patients’ safety. In recent decades, several rapid microbiological methods have been developed and validated, but variabilities linked to the use of different resources have led to discordant validation of methods and performance results. Considering this, while developing an in-house BacT/Alert-based method, we evaluated all of the materials used in its validation. Of particular importance, we noticed that the syringe gauge used to inject the samples into the bottles was crucial to obtain robust results. We chose to conduct a comparative test between the BacT/Alert system and the compendial method described in the European Pharmacopoeia, using five dilutions of nine reference microorganism strains and 21G or 27G needles. Our results confirmed that the BacT/Alert system is a valid and faster alternative method to assess sterility of clinical cell therapy products, and that the use of 27G needles increases its sensitivity to detect reference anaerobe microorganisms., Graphical Abstract, The BacT/Alert system is used in GMP control activities to test the sterility of ATMPs. The use of 27G needles decreases the TTD of most of the tested microorganisms, when compared to the European Pharmacopeia method, and allows less air intrusion and improves the growth and sensitivity of the method for anaerobic microorganisms.

Diabetic nephropathy (DN) is the primary microvascular complication of diabetes mellitus and may result in end-stage renal disease. The overproduction of various inflammatory factors is involved in the pathogenesis of DN. Protein tyrosine phosphatase 1B (PTP1B) modulates the expression of a series of cytokines and nuclear factor kappa B (NF-κB) activity. cAMP response element-binding protein (CREB) and lysine methyltransferase 5A (KMT5A) have been reported to participate in the maintenance of a healthy endothelium. In the present study, we hypothesise that CREB associates with KMT5A to modulate PTP1B expression, thus contributing to high glucose-mediated glomerular endothelial inflammation. Our analyses revealed that plasma inflammatory factor levels, glomerular endothelial p65 phosphorylation and PTP1B expression were increased in DN patients and rats. In vitro, high glucose increased endothelial inflammatory factor levels and p65 phosphorylation by augmenting PTP1B expression in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose decreased CREB and KMT5A expression. CREB overexpression and KMT5A overexpression both inhibited high glucose-induced PTP1B expression, p65 phosphorylation and endothelial inflammatory factor levels. si-CREB- and sh-KMT5A-induced p65 phosphorylation and endothelial inflammatory factor levels were reversed by si-PTP1B. Furthermore, CREB was associated with KMT5A. Mechanistic research indicated that CREB and histone H4 lysine 20 methylation (H4K20me1, a downstream target of KMT5A) occupy the PTP1B promoter region. sh-KMT5A augmented PTP1B promoter activity and activated the positive effect of si-CREB on PTP1B promoter activity. Our in vivo study demonstrated that CREB and KMT5A were downregulated in glomerular endothelial cells of DN patients and rats. In conclusion, CREB associates with KMT5A to promote PTP1B expression in vascular endothelial cells, thus contributing to hyperglycemia-induced inflammatory factor levels in DN patients and rats.

Background Hepatocellular carcinoma (HCC) is the leading cause of tumor-related death worldwide due to high morbidity and mortality, yet lacking effective biomarkers and therapies. Circular RNAs (circRNAs) are a group of non-coding RNAs that regulate gene expression through interacting with miRNAs, implicating in the tumorigenesis and progression. A novel circRNA, circTP63, was reported to be an oncogene in HCC. However, its role in HCC remains unclear. Methods qRT-PCR was used to assess the mRNA levels of CircTP63 in 90 pairs of tumor and adjacent normal tissues from HCC patients, one human normal hepatic epithelial cell line and HCC cell lines. CCK-8, colony formation, transwell, and flow cytometry assays were performed to detect the cellular function of circTP63/miR-155-5p/ZBTB18 in HCC cells. HCC xenograft mice models were established to assess the in vivo effect of circTP63. Bioinformatic analysis, RNA pull-down and luciferase assays were used to determine the interaction among circTP63/miR-155-5p/ZBTB18. Results circTP63 was significantly upregulated in HCC tissues and cell lines. High circTP63 expression is closely associated with the tumor stages, lymph node metastasis, and poor prognosis of HCC patients. Functionally, knockdown of circTP63 inhibited cell proliferation, migration, invasion, and promoted cell apoptosis of HCC. Meanwhile, overexpression of circTP63 enhanced HCC progression. Mechanically, circTP63 was a sponge of miR-155-5p to facilitate the ZBTB18 expression, and the ZBTB18 expression in HCC tissues was negatively associated with the survival rate of HCC patients. Furthermore, rescued assays revealed that the reduced tumor-promoting effect on HCC cells induced by knockdown of circTP63 can be reversed by miR-155-5p inhibitor or ZBTB18 overexpression. Conclusion Our data highlight a critical circTP63-miR-155-5p-ZBTB18 regulatory network involved in the HCC progression, gaining mechanistic insights into the function of circRNAs in HCC progression, and providing effective biomarkers and therapeutic targets for HCC treatment.

TEFM (transcription elongation factor of mitochondria) has been identified as a novel nuclear-encoded transcription elongation factor in the transcription of mitochondrial genome. Our bioinformatics analysis of TCGA data revealed an aberrant over-expression of TEFM in hepatocellular carcinoma (HCC). We analyzed its biological effects and clinical significance in this malignancy. TEFM expression was analyzed by quantitative real-time PCR, western blot, and immunohistochemistry analysis in HCC tissues and cell lines. The effects of TEFM on HCC cell growth and metastasis were determined by cell proliferation, colony formation, flow cytometric cell cycle and apoptosis, migration, and invasion assays. TEFM expression was significantly increased in HCC tissues mainly caused by down-regulation of miR-194-5p. Its increased expression is correlated with poor prognosis of HCC patients. TEFM promoted HCC growth and metastasis both in vitro and in vivo by promoting G1–S cell transition, epithelial-to-mesenchymal transition (EMT), and suppressing cell apoptosis. Mechanistically, TEFM exerts its tumor growth and metastasis promoting effects at least partly through increasing ROS production and subsequently by activation of ERK signaling. Our study suggests that TEFM functions as a vital oncogene in promoting growth and metastasis in HCC and may contribute to the targeted therapy of HCC.

Bovine lactoferricin (LFcinB) has antimicrobial and immunomodulatory properties; however, the effects on diabetic wound healing remain poorly understood. The wound healing potential of LFcinB was investigated with in vitro, ex vivo, and in vivo models. Cell migration and proliferation were tested on keratinocytes and on porcine ears. A type 1 diabetic mouse model was also used to evaluate wound healing kinetics, bacterial diversity patterns, and the effect of LFcinB on oxidative stress, macrophage phenotype, angiogenesis, and collagen deposition. LFcinB increased keratinocyte migration in vitro (p < 0.05) and ex vivo (p < 0.001) and improved wound healing in diabetic mice (p < 0.05), though not in normoglycemic control mice. In diabetic mouse wounds, LFcinB treatment led to the eradication of Bacillus pumilus, a decrease in Staphylococcus aureus, and an increase in the Staphylococcus xylosus prevalence. LFcinB increased angiogenesis in diabetic mice (p < 0.01), but this was decreased in control mice (p < 0.05). LFcinB improved collagen deposition in both diabetic and control mice (p < 0.05). Both oxidative stress and the M1-to-M2 macrophage ratios were decreased in LFcinB-treated wounds of diabetic animals (p < 0.001 and p < 0.05, respectively) compared with saline, suggesting a downregulation of inflammation in diabetic wounds. In conclusion, LFcinB treatment demonstrated noticeable positive effects on diabetic wound healing., Graphical Abstract, Topical delivery of bovine lactoferricin (LFcinB) has antimicrobial and immunomodulatory actions and improves wound closure and changes the bacterial diversity of diabetic wounds. Hallmarks of diabetic wound healing impairments like inflammation, oxidative stress, disrupted angiogenesis, and collagen deposition improved by LFcinB indicate its therapeutic potential.

Triple-negative breast cancer (TNBC) patients exhibit variable responses to chemotherapy, suggesting an underlying molecular heterogeneity. In the current study, we analyzed publicly available transcriptome data from 360 TNBC and 88 normal breast tissues, which revealed activation of nucleosome and cell cycle as the hallmarks of TNBC. Mechanistic network analysis identified activation of FOXM1 and ERBB2, and suppression of TP53 and NURP1 networks in TNBC. Employing Iterative Clustering and Guide-gene Selection (ICGS), Uniform Manifold Approximation and Projection (UMAP), and dimensionality reduction analyses, we classified TNBC into seven molecular subtypes, each exhibiting a unique molecular signature, including immune infiltration (CD19, CD8, and macrophages) and mesenchymal signature, which correlated with variable disease outcomes in a larger cohort (1,070) of BC. Mechanistically, depletion of TTK, TPX2, UBE2C, CDCA7, MELK, NFE2L3, DDX39A, and LRP8 led to substantial inhibition of colony formation of TNBC models, which was further enhanced in the presence of paclitaxel. Our data provide novel insights into the molecular heterogeneity of TNBC and identified TTK, TPX2, UBE2C, and LRP8 as main drivers of TNBC tumorigenesis., Graphical Abstract, TNBC patients exhibit variable responses to therapy, suggesting an underlying molecular heterogeneity. Herein, we classified TNBC into seven molecular subtypes with distinct molecular signatures. Our data highlight pivotal roles for TTK, TPX2, and LRP8 in TNBC biology and their sensitivity to paclitaxel, suggesting their potential utilization as therapeutic targets.

To optimize the regenerative proficiency of stem cells, a cardiopoietic protein‐based cocktail consisting of multiple growth factors has been developed and advanced into clinical trials for treatment of ischemic heart failure. Streamlining the inductors of cardiopoiesis would address the resource intensive nature of the current stem cell enhancement protocol. To this end, the microencapsulated‐modified‐mRNA (M3RNA) technique was here applied to introduce early cardiogenic genes into human adipose‐derived mesenchymal stem cells (AMSCs). A single mesodermal transcription factor, Brachyury, was sufficient to trigger high expression of cardiopoietic markers, Nkx2.5 and Mef2c. Engineered cardiopoietic stem cells (eCP) featured a transcriptome profile distinct from pre‐engineered AMSCs. In vitro, eCP demonstrated protective antioxidant capacity with enhanced superoxide dismutase expression and activity; a vasculogenic secretome driving angiogenic tube formation; and macrophage polarizing immunomodulatory properties. In vivo, in a murine model of myocardial infarction, intramyocardial delivery of eCP (600 000 cells per heart) improved cardiac performance and protected against decompensated heart failure. Thus, heart repair competent stem cells, armed with antioxidant, vasculogenic, and immunomodulatory traits, are here engineered through a protein‐independent single gene manipulation, expanding the available regenerative toolkit., Cardiopoietic stem cells are originally derived using a cocktail of cardiogenic growth factors. This study unveils the feasibility of achieving cardiopoiesis by a single gene (Brachyury) based induction of the human stem cell source. Engineered cardiopoietic stem cell progeny features pro‐reparative traits and rescues heart failure postinfarction, introducing a scalable platform for regenerative therapy.

X-linked agammaglobulinemia (XLA) is an immune disorder caused by mutations in Bruton’s tyrosine kinase (BTK). BTK is expressed in B and myeloid cells, and its deficiency results in a lack of mature B cells and protective antibodies. We previously reported a lentivirus (LV) BTK replacement therapy that restored B cell development and function in Btk and Tec double knockout mice (a phenocopy of human XLA). In this study, with the goal of optimizing both the level and lineage specificity of BTK expression, we generated LV incorporating the proximal human BTK promoter. Hematopoietic stem cells from Btk−/−Tec−/− mice transduced with this vector rescued lineage-specific expression and restored B cell function in Btk−/−Tec−/− recipients. Next, we tested addition of candidate enhancers and/or ubiquitous chromatin opening elements (UCOEs), as well as codon optimization to improve BTK expression. An Eμ enhancer improved B cell rescue, but increased immunoglobulin G (IgG) autoantibodies. Addition of the UCOE avoided autoantibody generation while improving B cell development and function and reducing vector silencing. An optimized vector containing a truncated UCOE upstream of the BTK promoter and codon-optimized BTK cDNA resulted in stable, lineage-regulated BTK expression that mirrored endogenous BTK, making it a strong candidate for XLA therapy., Graphical Abstract, Mutations in BTK lead to XLA characterized by absent B cells and antibody responses. LVs containing the BTK promoter and other candidate elements were tested in a murine XLA model. Use of a 0.7-kb UCOE and codon optimization restored B cell development, antibody responses, and lineage-appropriate expression without evidence of toxicity.

Background Surface plasmon resonance is a label-free biophysical technique that is widely used in investigating biomolecular interactions, including protein-protein, protein-DNA, and protein-small molecule binding. Surface plasmon resonance is a very powerful tool in different stages of small molecule drug development and antibody characterization. Both academic institutions and pharmaceutical industry extensively utilize this method for screening and validation studies involving direct molecular interactions. In most applications of the surface plasmon resonance technology, one of the studied molecules is immobilized on a microchip, while the second molecule is delivered through a microfluidic system over the immobilized molecules. Changes in total mass on the chip surface is recorded in real time as an indicator of the molecular interactions. Main body Quality and accuracy of the surface plasmon resonance data depend on experimental variables, including buffer composition, type of sensor chip, coupling chemistry of molecules on the sensor surface, and surface regeneration conditions. These technical details are generally included in materials and methods sections of published manuscripts and are not easily accessible using the common internet browser search engines or PubMed. Herein, we introduce a surface plasmon resonance database, www.sprdatabase.info that contains technical details extracted from 5140 publications with surface plasmon resonance data. We also provide an analysis of experimental conditions preferred by different laboratories. These experimental variables can be searched within the database and help future users of this technology to design better experiments. Conclusion Amine coupling and CM5 chips were the most common methods used for immobilizing proteins in surface plasmon resonance experiments. However, number of different chips, capture methods and buffer conditions were used by multiple investigators. We predict that the database will significantly help the scientific community using this technology and hope that users will provide feedback to improve and expand the database indefinitely. Publicly available information in the database can save a great amount of time and resources by assisting initial optimization and troubleshooting of surface plasmon resonance experiments.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete silencing of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Previously, we showed that CRISPR inhibition could successfully target and repress DUX4 in FSHD myocytes. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. Thus, we re-engineered our platform to allow in vivo delivery of more potent epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive skeletal muscle-specific expression of dCas9 from Staphylococcus aureus fused to HP1α, HP1γ, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain. Targeting each regulator to the DUX4 promoter/exon 1 increased chromatin repression at the locus, specifically suppressing DUX4 and its target genes in FSHD myocytes and in a mouse model of the disease. Importantly, minimizing the regulatory cassette and using the smaller Cas9 ortholog allowed our therapeutic cassettes to be effectively packaged into adeno-associated virus (AAV) vectors for in vivo delivery. By engineering a muscle-specific epigenetic CRISPR platform compatible with AAV vectors for gene therapy, we have laid the groundwork for clinical use of dCas9-based chromatin effectors in skeletal muscle disorders., Graphical Abstract, FSHD is caused by incomplete epigenetic silencing, leading to pathogenic misexpression of DUX4 in skeletal muscles. We designed an FSHD-optimized cassette to express dCas9 fused to each of four epigenetic repressors. This epigenetic CRISPR inhibition effectively and specifically represses DUX4-fl and its targets in primary FSHD myocytes and in vivo.

Triple-negative breast cancer (TNBC) patients with upregulated Wnt/β-catenin signaling often have poor clinical prognoses. During pathological examinations of breast cancer sections stained for β-catenin, we made the serendipitous observation that relative to non-TNBC, specimens from TNBC patients have a greater abundance of nucleoli. There was a remarkable direct relationship between nuclear β-catenin and greater numbers of nucleoli in TNBC tissues. These surprising observations spurred our investigations to decipher the differential functional relevance of the nucleolus in TNBC versus non-TNBC cells. Comparative nucleolar proteomics revealed that the majority of the nucleolar proteins in TNBC cells were potential targets of β-catenin signaling. Next, we undertook an analysis of the nucleolar proteome in TNBC cells in response to β-catenin inhibition. This effort revealed that a vital component of pre-rRNA processing, LAS1 like ribosome biogenesis factor (LAS1L) was significantly decreased in the nucleoli of β-catenin inhibited TNBC cells. Here we demonstrate that LAS1L protein expression is significantly elevated in TNBC patients, and it functionally is important for mammary tumor growth in xenograft models and enables invasive attributes. Our observations highlight a novel function for β-catenin in orchestrating nucleolar activity in TNBCs.

Background Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA. Results We established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5′ exonuclease is induced from a chromosomally-integrated gene in the same cell. Conclusions The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.

During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (β2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the β2AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level β2AR signaling dominated over β1AR . Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of β2AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact., Graphical Abstract, During normal- and patho-physiological situations, the behavior of beta2-adrenoreceptor (β2AR) is determined by its polymorphic variants. A human-based isogenic model system represents a promising tool for systematic analysis of mechanisms of β2AR variant-mediated cellular response under normal and stressed conditions. This allows important subtleties of polymorphisms in β2AR to be unraveled.