Native myocardium has limited regenerative potential post injury. Advances in lineage reprogramming have provided promising cellular sources for regenerative medicine in addition to research applications. Recently we have shown that adult mouse fibroblasts can be reprogrammed to expandable, multipotent, induced cardiac progenitor cells (iCPCs) by employing forced expression of five cardiac factors along with activation of canonical Wnt and JAK/STAT signaling. Here we aim to further characterize iCPCs by highlighting their safety, ease of attainability, and functionality within a three-dimensional cardiac extracellular matrix scaffold. Specifically, iCPCs did not form teratomas in contrast to embryonic stem cells when injected into immunodeficient mice. iCPC reprogramming was achieved in wild type mouse fibroblasts without requiring a cardiac-specific reporter, solely utilizing morphological changes to identify, clonally isolate, and expand iCPCs, thus increasing the versatility of this technology. iCPCs also show the ability to repopulate decellularized native heart scaffolds and differentiated into organized structures containing cardiomyocytes, smooth muscle, and endothelial cells. Optical mapping of recellularized scaffolds shows field-stimulated calcium transients that propagate across islands of reconstituted tissue and bipolar local stimulation demonstrates cell-cell coupling within scaffolds. Overall, iCPCs provide a readily attainable, scalable, safe, and functional cell source for a variety of application including drug discovery, disease modeling, and regenerative therapy.

The comparative analysis of innate immunity across different insect taxa has revealed unanticipated evolutionary plasticity, providing intriguing examples of immunity-related effector gene expansion and loss. Phasmatodea, the stick and leaf insects, is an order of hemimetabolous insects that can provide insight into ancestral innate immunity genes lost by later insect clades. We injected the stick insect Peruphasma schultei with a mixture of microbial elicitors to activate a strong immune response, followed by RNA-Seq analysis to screen for induced immunity-related effector genes. This revealed a highly diverse spectrum of antimicrobial peptides (AMPs) belonging to the attacin, coleoptericin, defensin, thaumatin, and tachystatin families. In addition, we identified a large group of short, cysteine-rich putative AMPs, some of which were strongly elicited. The immunity-related effector gene repertoire also included c-type and i-type lysozymes and several pattern-recognition proteins, such as proteins that recognize Gram-negative bacteria and peptidoglycans. Finally, we identified 45 hemolymph lipopolysaccharide-binding protein sequences, an unusually large number for insects. Taken together, our results indicate that at least some phasmids synthesize a broad spectrum of diverse AMPs that deserve further in-depth analysis.

Multiple sclerosis (MS) is an inflammatory disease in central nervous system (CNS) with limited therapeutic drugs. In the present study, we explored the anti-inflammatory/neuroprotective properties of Acteoside (AC), an active compound from medicinal herb Radix Rehmanniae (RR), and neuroprotective effects of AC on MS pathology by using an experimental autoimmune encephalomyelitis (EAE) model. We tested the hypothesis that AC could alleviate EAE pathogenesis through inhibiting inflammation and ONOO--mediated mitophagy activation in vivo and in vitro. The results showed that AC treatment effectively ameliorated neurological deficit score and postponed disease onset in the EAE mice. AC treatment inhibited inflammation/demyelination, alleviated peripheral activation and CNS infiltration of encephalitogenic CD4+ T cells and CD11b+ activated microglia/macrophages in the spinal cord of EAE mice. Meanwhile, AC treatment reduced ONOO- production, down-regulated the expression of iNOS and NADPH oxidases, and inhibited neuronal apoptotic cell death and mitochondrial damage in the spinal cords of the EAE mice. Furthermore, AC treatment decreased the ratio of LC3-II to LC3-I in mitochondrial fraction, and inhibited the translocation of Drp1 to the mitochondria. In vitro studies further proved that AC possessed strong ONOO- scavenging capability and protected the neuronal cells from nitrative cytotoxicity via suppressing ONOO--mediated excessive mitophagy. Taken together, Acteoside could be a potential therapeutic agent for multiple sclerosis treatment. The suppression of ONOO--induced excessive mitophagy activation could be one of the critical mechanisms contributing to its anti-inflammatory and anti-demyelinating properties.

3,4-Dimethylmethcathinone (3,4-DMMC) is a new psychoactive substance whose recreational use and trade have recently increased. Given the absence of information on the toxicokinetics of 3,4-DMMC, the present work aimed at validating a GC-MS methodology for the drug quantification in biological matrices, and further characterizing its biodistribution in Wistar rats. The method was validated based on the evaluation of the drug stability, limit of detection and quantification, linearity, selectivity, precision, accuracy and recovery. To characterize biodistribution, Wistar rats were administered with 20 or 40 mg/Kg of 3,4-DMMC i.p.. After 1 h or 24 h, rats were anaesthetized, euthanized and blood, brain, liver, heart, kidneys, lungs, spleen, urine (only at 24 h), and a portion of gut, muscle and adipose tissue were collected for analysis. After 1 h, 3,4-DMMC was present in all analysed matrices, and the presence of two metabolites was further detected in all of them. The drug accumulation was higher in kidneys, lungs, spleen and brain. After 24 h, 3,4-DMMC was only present in urine, along with five metabolites. All metabolites were tentatively identified. Through elucidation of the most appropriate analytical matrices and the metabolites that may have the largest detection windows, these data are expected to assist in future clinical and forensic investigations.

The presence of GBA1 gene mutations increases risk for Parkinson's disease (PD), but the pathogenic mechanisms of GBA1 associated PD remain unknown. Given that impaired α-synuclein turnover is a hallmark of PD pathogenesis and cathepsin D is a key enzyme involved in α-synuclein degradation in neuronal cells, we have examined the relationship of glucocerebrosidase (GCase), cathepsin D and monomeric α-synuclein in human neural crest stem cell derived dopaminergic neurons. We found that normal activity of GCase is necessary for cathepsin D to perform its function of monomeric α-synuclein removal from neurons. GBA1 mutations lead to a lower level of cathepsin D protein and activity, and higher level of monomeric α-synuclein in neurons. When GBA1 mutant neurons were treated with GCase replacement or chaperone therapy; cathepsin D protein levels and activity were restored, and monomeric α-synuclein decreased. When cathepsin D was inhibited, GCase replacement failed to reduce monomeric α-synuclein levels in GBA1 mutant neurons. These data indicate that GBA1 gene mutations increase monomeric α-synuclein levels via an effect on lysosomal cathepsin D in neurons.

Histological analysis is a fundamental and principal method used in biological research and even for disease diagnosis. The result shows the status of cells and tissues in organs and enables us to infer the condition of the whole body. The tissue staining method known as hematoxylin and eosin staining (HE) is one of the most general methods of investigating the status of cells and tissues. Hematoxylin stains the nucleus violet and eosin stains cytosol pink. HE staining shows the unique morphologies of tissues and cells. However, after being stained with HE, tissues are very difficult to use in another histological analysis because hematoxylin is hard to remove from the sections due to its stain stability. Therefore, serial sections of the tissue are used to obtain more information through another staining, including immunohistochemistry. The adjacent tissue section is not the same as the HE-stained section, however, so the results from the adjacent sections can cause confusion or ambiguity. The present study showed that our decolorization solution can decolor the hematoxylin or iron hematoxylin stain from stained structures, including the nucleus, and the decolored section could be stained again in another staining, including immunohistochemistry. This decolorization method is very valuable, in that it can determine the accurate distribution of substances and features in cells and tissues, and thus it can improve the robustness of the resulting data.

Prediction of gene function and gene regulatory networks is one of the most active topics in bioinformatics. The accumulation of publicly available gene expression data for hundreds of plant species, together with advances in bioinformatical methods and affordable computing, sets ingenuity as one of the major bottlenecks in understanding gene function and regulation. Here, we show how a credit card-sized computer retailing for <50 USD can be used to rapidly predict gene function and infer regulatory networks from RNA sequencing data. To achieve this, we constructed a bioinformatical pipeline that downloads and allows quality-control of RNA sequencing data; and generates a gene co-expression network that can reveal enzymes and transcription factors participating and controlling a given biosynthetic pathway. We exemplify this by first identifying genes and transcription factors involved in the biosynthesis of secondary cell wall in the plant Artemisia annua, the main natural source of the anti-malarial drug artemisinin. Networks were then used to dissect the artemisinin biosynthesis pathway, which suggest potential transcription factors regulating artemisinin biosynthesis. We provide the source code of our pipeline (https://github.com/mutwil/LSTrAP-Lite) and envision that the ubiquity of affordable computing, availability of biological data and increased bioinformatical training of biologists will transform the field of bioinformatics. This article is part of a Special Issue entitled: Transcriptional Profiles and Regulatory Gene Networks edited by Dr. Dr. Federico Manuel Giorgi and Dr. Shaun Mahony.

Micronuclei (MNs) are extranuclear DNA-containing bodies and determining MN frequencies is a measure of genomic instability. An age-related increase in MN frequencies in lymphocytes has been quantified, but this effect has not yet been measured in nasal and buccal cells.

Lactobacillus sakei S1 strongly inhibits the expression of interleukin (IL)-6 and IL-1β in lipopolysaccharide-induced peritoneal macrophages by a mechanism for which lactic acid bacteria from kimchi that inhibit tumor necrosis factor-alpha (TNF-κ) were isolated. Therefore, we further evaluated the protective effect of this strain on the colitis mouse model induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). TNBS significantly elevated myeloperoxidase (MPO) expression, macroscopic scores, and colon shortening. Oral L. sakei S1 administration resulted in reduction of TNBS-induced loss in body weight, colon shortening, MPO activity, expression of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB). L. sakei S1 inhibited the expression of inflammatory cytokines IL-1β, IL-6 and TNF-κ, induced by TNBS, but enhanced IL-10 expression. L. sakei S1 showed resistance to artificial digestive juices and adherence to intestinal epithelial Caco-2 cells. Thus, L. sakei S1 may inhibit the NF-κB pathway and be used in functional food to treat colitis.

Glycerol dehydrogenase (GlyDH) catalyzes the oxidation of glycerol to dihydroxyacetone (DHA), which is the first step in the glycerol metabolism pathway. GlyDH has attracted great interest for its potential industrial applications, since DHA is a precursor for the synthesis of many commercially valuable chemicals and various drugs. In this study, GlyDH from Klebsiella pneumoniae (KpGlyDH) was overexpressed in E. coli and purified to homogeneity for biochemical and molecular characterization. KpGlyDH exhibits an exclusive preference for NAD+ over NADP+. The enzymatic activity of KpGlyDH is maximal at pH 8.6 and pH 10.0. Of the three common polyol substrates, KpGlyDH showed the highest kcat/Km value for glycerol, which is three times higher than for racemic 2,3-butanediol and 32 times higher than for ethylene glycol. The kcat value for glycerol oxidation is notably high at 87.1 ± 11.3 sec-1. KpGlyDH was shown to exist in an equilibrium between two different oligomeric states, octamer and hexadecamer, by size-exclusion chromatography analysis. KpGlyDH is structurally thermostable, with a Tm of 83.4°C, in thermal denaturation experiment using circular dichroism spectroscopy. The biochemical and biophysical characteristics of KpGlyDH revealed in this study should provide the basis for future research on its glycerol metabolism and possible use in industrial applications.

Bipolar disorder (BD) is a highly heritable psychiatric disorder characterized by episodes of manic and depressed mood states and associated with cortical brain abnormalities. Although the course of BD is often progressive, longitudinal brain imaging studies are scarce. It remains unknown whether brain abnormalities are static traits of BD or result from pathological changes over time. Moreover, the genetic effect on implicated brain regions remains unknown.

Long non-coding RNAs (lncRNAs) are crucial regulators of tumorigenesis and progression in human cancer, including hepatocellular carcinoma (HCC). However, the role of most lncRNAs that are dysregulated in HCC remains to be elucidated. Here, we investigated the role of OSER1-AS1 in the progression of HCC. The results of database and qRT-PCR analysis demonstrated that OSER1-AS1 was highly expressed in HCC tissues and the high expression of OSER1-AS1 was closely associated with larger tumor size, advanced tumor stages, lower disease free survival and overall survival of HCC patients. OSER1-AS1 knockdown significantly inhibited the proliferation, invasion and migration of HCC cells, and induced the apoptosis. In addition, the dual luciferase reporter assay directly demonstrated that OSER1-AS1 functioned as a molecular sponge for miR-372-3p to promote Rab23 expression. Moreover, the results of immunohistochemistry and western blot analysis showed that Rab23 was highly expressed in HCC tissues, and the high expression of Rab23 was closely associated with the poor overall survival of HCC patients. Immunofluorescence assay also found the subcellular localization of Rab23 in HCC cells. Rab23 was obviously downregulated in cells that were transfected with miR-372-3p mimics. MiR-372-3p mimics significantly inhibited the proliferation and invasion of HCC cells). Rab23 restoration partially reversed miR-372-3p-induced tumor suppressive effects on HCC cells. In conclusion, we found that OSER1-AS1 acted as a ceRNA to sponge miR-372-3p, thereby positively regulating the Rab23 expression and ultimately acting as a tumor suppressor gene in HCC progression.

Liver sinusoidal endothelial cells play a key role maintaining the hepatic homeostasis, the disruption of which is associated with such end-stage liver diseases as hepatocellular carcinoma and cirrhosis. In the present study we investigated the role of brahma-related gene 1 (BRG1), a chromatin remodeling protein, in regulating endothelial transcription and the implication in liver fibrosis. We report that endothelial-specific deletion of BRG1 in mice attenuated liver fibrosis induced by injection with thioacetamide (TAA). Coincidently, alleviation of liver fibrosis as a result of endothelial BRG1 deletion was accompanied by an up-regulation of eNOS activity and NO bioavailability. In cultured endothelial cells, exposure to lipopolysaccharide (LPS) suppressed eNOS activity whereas BRG1 depletion with small interfering RNA restored eNOS-dependent NO production. Further analysis revealed that BRG1 was recruited to the caveolin-1 (CAV1) promoter by Sp1 and activated transcription of CAV1, which in turn inhibited eNOS activity. Mechanistically, BRG1 interacted with the H3K4 trimethyltransferase MLL1 to modulate H3K4 trimethylation surrounding the CAV1 promoter thereby contributing to LPS-induced CAV1 activation. In conclusion, our data unveil a novel role for BRG1 in the regulation of endothelial function and liver fibrosis.

Burn patients are at high risk of infection, and as sepsis contributes significantly to morbidity and mortality, early diagnosis is essential. Procalcitonin (PCT) is a biomarker released in response to inflammation and specifically bacterial infection. This study aimed to determine the value of PCT as a diagnostic biomarker of sepsis in burns patients in Johannesburg, South Africa.

Large bodies of studies have shown that the CRISPR/Cas9-based library screening is a very powerful tool for the identification of gene functions. However, most of these studies have focused on protein-coding genes, and, furthermore, very few studies have used gene reporters for screening. In the present study, we generated DNA methyltransferase 3B (DNMT3B) reporter and screened a CRISPR/Cas9 synergistic activation mediator (SAM) library against a focused group of lncRNAs. With this screening approach, we identified Rhabdomyosarcoma 2-Associated Transcript (RMST) as a positive regulator for DNMT3B. This was confirmed by activation of the endogenous RMST by SAM or ectopic expression of RMST. Moreover, RMST knockout (KO) suppresses DNMT3, while rescue with RMST in the KO cells restores the DNMT3 level. Finally, RMST KO suppresses global DNA methylation, leading to the upregulation of methylation-regulated genes. Mechanistically, RMST promotes the interaction between the RNA-binding protein HuR and DNMT3B 3' UTR, increasing the DNMT3B stability. Together, these results not only provide the feasibility of a reporter system for CRISPR library screening but also demonstrate the previously uncharacterized factor RMST as an important player in the modulation of DNA methylation.

Follicular helper T (Tfh) cells provide essential help for humoral immune response. Transcriptional factor Bcl6 is the master regulator for Tfh generation and is induced very early after T cell activation in a CD28-dependent manner, but how CD28 signal promotes Bcl6 early expression remains unknown. Here we found that CD28 signal quickly induces expression of the H3K36me2 methytransferase Nsd2, which is required for Bcl6 expression as early as the first cell division after T cell activation. Nsd2 deficiency in T cells leads to decreased Bcl6 expression, impaired Tfh generation, compromised germinal center response, and delayed virus clearance. Ectopic Bcl6 expression rescues the Tfh defect of Nsd2 KO cells. ICOS signal is dispensable for early Nsd2 induction but required for sustained Nsd2 expression, which is critical for Tfh maintenance. Overexpression of Nsd2 increases Bcl6 expression and enhances Tfh generation; 4-mo-old mice even develop spontaneous Tfh. Overall, our study reveals Nsd2 as a critical epigenetic regulator for Tfh differentiation.

Doublecortin-like kinase 1 (DCLK1) is a cancer stem cell marker for the colorectal cancer (CRC). It plays critical roles in the oncogenesis, progression, and metastasis of CRC. DCLK1 can be an intriguing therapeutic target for CRC treatment. However, the molecular mechanism of how DCLK1 functions is unclear currently. In our research, we aim to apply RNA-Sequencing (RNA-Seq) technology, a high throughput massively Next-generation sequencing approach, to monitor transcriptome changes due to DCLK1 overexpression in the CRC cells. In order to achieve our goal, RNA from quadruplicate samples from two clones of isogenic DCLK1 stable overexpression cells and the parental wild-type HCT116 cells was sent for RNA-Seq on the Illumina NextSeq500 platform. Differentially expressed (DE) genes were evaluated by t-test (P < 0.05 and fold-change ± 1.5 or greater) using two methods: (1) FWER; and (2) Benjamani and Hochberg FDR (false discovery rate) which corrects for multiple comparisons. Gene networks and functional analysis were evaluated using Ingenuity Pathways Analysis (IPA). We identified 1463 DE genes common for both DCLK1 overexpression clone A and clone B cells. IPA results indicated that 72 canonical pathways were significantly modified by DCLK1 overexpression (P < 0.05), among which nine out of the top ten pathways are involved in the cell cycle regulation, indicating that DCLK1 might play its tumorigenesis role via activation of pathways facilitating cell proliferation, repression of pathways inhibiting cells proliferation, and function against pathways facilitating cell apoptosis. Cell cycle analysis results confirmed the IPA findings, which demonstrated that DCLK1 overexpression cells had much less G0/G1 cells but much more S and G2/M cells (P < 0.05). In conclusion, DCLK1 overexpression significantly modified transcriptome profile of CRC cancer cells. Control of the cell cycle regulation might be one of the critical mechanism for DCLK1 function. Our findings provide more direct evidence for the development of DCLK1 as a therapeutic target for CRC treatment, and will be of great benefit for the discovery of novel therapeutic target within the DCLK1 molecular network for the treatment of colorectal cancer patients.

Inappropriate localization of proteins can interfere with normal cellular function and drive tumor development. To understand how this contributes to the development of acute myeloid leukemia (AML), we compared the nuclear proteome and transcriptome of AML blasts with normal human CD34+ cells. Analysis of the proteome identified networks and processes that significantly affected transcription regulation including misexpression of 11 transcription factors with seven proteins not previously implicated in AML. Transcriptome analysis identified changes in 40 transcription factors but none of these were predictive of changes at the protein level. The highest differentially expressed protein in AML nuclei compared with normal CD34+ nuclei (not previously implicated in AML) was S100A4. In an extended cohort, we found that over-expression of nuclear S100A4 was highly prevalent in AML (83%; 20/24 AML patients). Knock down of S100A4 in AML cell lines strongly impacted their survival whilst normal hemopoietic stem progenitor cells were unaffected. These data are the first analysis of the nuclear proteome in AML and have identified changes in transcription factor expression or regulation of transcription that would not have been seen at the mRNA level. These data also suggest that S100A4 is essential for AML survival and could be a therapeutic target in AML.

Ion channels can regulate the plasma membrane potential (Vm ) and cell migration as a result of altered ion flux. However, the mechanism by which Vm regulates motility remains unclear. Here, we show that the Nav 1.5 sodium channel carries persistent inward Na+ current which depolarizes the resting Vm at the timescale of minutes. This Nav 1.5-dependent Vm depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically encoded FRET biosensor of Rac1 activation shows that depolarization-induced Rac1 activation results in acquisition of a motile phenotype. By identifying Nav 1.5-mediated Vm depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase-dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.

The SMYD2 protein lysine methyltransferase methylates various histone and non-histone proteins and is overexpressed in several cancers. Using peptide arrays, we investigated the substrate specificity of the enzyme, revealing a recognition of leucine (or weaker phenylalanine) at the -1 peptide site and disfavor of acidic residues at the +1 to +3 sites. Using this motif, novel SMYD2 peptide substrates were identified, leading to the discovery of 32 novel peptide substrates with a validated target site. Among them, 19 were previously reported to be methylated at the target lysine in human cells, strongly suggesting that SMYD2 is the protein lysine methyltransferase responsible for this activity. Methylation of some of the novel peptide substrates was tested at the protein level, leading to the identification of 14 novel protein substrates of SMYD2, six of which were more strongly methylated than p53, the best SMYD2 substrate described so far. The novel SMYD2 substrate proteins are involved in diverse biological processes such as chromatin regulation, transcription, and intracellular signaling. The results of our study provide a fundament for future investigations into the role of this important enzyme in normal development and cancer.