The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models.

Fetal skin achieves scarless wound repair. Dermal fibroblasts play a central role in extracellular matrix deposition and scarring outcomes. Both fetal and gingival wound repair share minimal scarring outcomes. We tested the hypothesis that compared to adult skin fibroblasts, human fetal skin fibroblast diversity is unique and partly overlaps with gingival skin fibroblasts. Human fetal skin (FS, n = 3), gingiva (HGG, n = 13), and mature skin (MS, n = 13) were compared at single-cell resolution. Dermal fibroblasts, the most abundant cluster, were examined to establish a connectome with other skin cells. Annexin1-FPR1 signaling pathway was dominant in both FS as well as HGG fibroblasts and related myeloid cells while scanty in MS fibroblasts. Myeloid-specific FPR1-ORF delivered in murine wound edge using tissue nanotransfection (TNT) technology significantly enhanced the quality of healing. Pseudotime analyses identified the co-existence of an HGG fibroblast subset with FPR1high myeloid cells of fetal origin indicating common underlying biological processes.

Outbreaks of the immunosuppressive infectious bursal disease (IBD) are frequently reported worldwide, despite the vaccination regimes. A 2009 Californian IBD outbreak caused by rA and rB isolates was described as very virulent (vv) IBD virus (IBDV); however, molecular factors beyond this virulence were not fully uncovered. Therefore, segments of both isolates were amplified, successfully cloned, whole genome sequenced by Next Generation Sequencing, genotyped, and the leading virulence factors were entirely investigated in terms of phylogenetic and amino acid analysis and protein modeling for positive selection orientation and interaction analysis. rA and rB isolates displayed the highest amino acid identity (97.84-100%) with Genotype 3 strains. Interestingly, rA and rB contained all virulence hallmarks of hypervariable (HVR), including 222A, 242I, 249Q, 256I, 284A, 286T, 294I, 299S, and 318G, as well as the serine-rich heptapeptide sequence. Moreover, we pinpointed the A3B2 genotype of rA and rB, predominant in non-reassortants, and we highlighted the absence of recombination events. Furthermore, gene-wise phylogenetic analysis showed the entire genes of rA and rB clustered with the vvIBDVs and emphasized their share in IBDV virulence. VP5 showed a virulence marker, MLSL (amino acid sequence). VP2 encountered three significant novel mutations apart from the HVR, including G163E in rA and Y173C and V178A in rB, all residing within interacting motifs. VP4 contained 168Y, 173N, 203S, and 239D characteristic for the vv phenotype. A235V mutation was detected at the dsRNA binding domain of VP3. In VP1, the TDN triplet and the mutation (V4I) were detected, characteristic of hypervirulence occurring at the N-terminus responsible for protein priming. Although selection analysis revealed seven sites, codon 222 was the only statistically significant selection site. The VP2 modeling of rA and rB highlighted great structure fitness, with 96.14% Ramachandran favored positioning including the 222A, i.e., not influencing the structure stability. The 222A was found to be non-interface surface residue, associated with no interaction with the attachment-mediated ligand motif. Our findings provide pivotal insights into the evolution and underlying virulence factors and will assist in the development of control strategies via sequence-based continuous monitoring for the early detection of novel vv strains.

Zebrafish is an elegant vertebrate employed to model the pathological etiologies of human maladies such as cardiac diseases. Persistent physiological stresses can induce abnormalities in heart functions such as cardiac hypertrophy (CH), which can lead to morbidity and mortality. In the present study, using zebrafish as a study model, efficacy of the traditional Indian Ayurveda medicine "Yogendra Ras" (YDR) was validated in ameliorating drug-induced cardiac hypertrophy. YDR was prepared using traditionally described methods and composed of nano- and micron-sized metal particles. Elemental composition analysis of YDR showed the presence of mainly Au, Sn, and Hg. Cardiac hypertrophy was induced in the zebrafish following a pretreatment with erythromycin (ERY), and the onset and reconciliation of disease by YDR were determined using a treadmill electrocardiogram, heart anatomy analysis, C-reactive protein release, and platelet aggregation time-analysis. YDR treatment of CH-induced zebrafish showed comparable results with the Standard-of-care drug, verapamil, tested in parallel. Under in-vitro conditions, treatment of isoproterenol (ISP)-stimulated murine cardiomyocytes (H9C2) with YDR resulted in the suppression of drug-stimulated biomarkers of oxidative stress: COX-2, NOX-2, NOX-4, ANF, troponin-I, -T, and cardiolipin. Taken together, zebrafish showed a strong disposition as a model for studying the efficacy of Ayurvedic medicines towards drug-induced cardiopathies. YDR provided strong evidence for its capability in modulating drug-induced CH through the restoration of redox homeostasis and exhibited potential as a viable complementary therapy.

The microRNAs expression has emerged as a potential biomarker for the diagnosis and prognosis of prostate cancer. This study investigated the expression of miRNA-182 and miRNA-187 in prostate cancer patients and established a correlation between miRNA expression and staging of prostate cancer.

Avian reovirus (ARV) is an emerging pathogen which causes significant economic challenges to the chicken and turkey industry in the USA and globally, yet the molecular characterization of most ARV strains is restricted to a single particular gene, the sigma C gene. The genome of arthrogenic reovirus field isolates (R18-37308 and R18-38167), isolated from broiler chickens in North Carolina (NC), USA in 2018, was sequenced using long-read next-generation sequencing (NGS). The isolates were genotyped based on the amino acid sequence of sigma C (σC) followed by phylogenetic and amino acid analyses of the other 11 genomically encoded proteins for whole genomic constellation and genetic variation detection. The genomic length of the NC field strains was 23,494 bp, with 10 dsRNA segments ranging from 3959 bp (L1) to 1192 bp (S4), and the 5' and 3' untranslated regions (UTRs) of all the segments were found to be conserved. R18-37308 and R18-38167 were found to belong to genotype (G) VI based on the σC analysis and showed nucleotide and amino acid sequence identity ranging from 84.91-98.47% and 83.43-98.46%, respectively, with G VI strains. Phylogenetic analyses of individual genes of the NC strains did not define a single common ancestor among the available completely sequenced ARV strains. Nevertheless, most sequences supported the Chinese strain LY383 as a probable ancestor of these isolates. Moreover, amino acid analysis revealed multiple amino acid substitution events along the entirety of the genes, some of which were unique to each strain, which suggests significant divergence owing to the accumulation of point mutations. All genes from R18-37308 and R18-38167 were found to be clustered within genotypic clusters that included only ARVs of chicken origin, which negates the possibility of genetic pooling or host variation. Collectively, this study revealed sequence divergence between the NC field strains and reference ARV strains, including the currently used vaccine strains could help updating the vaccination regime through the inclusion of these highly divergent circulating indigenous field isolates.

MicroRNAs (miRNAs) are small, conserved RNAs known to regulate several biological processes by influencing gene expression in eukaryotes. The implication of miRNAs as another player of regulatory layers during heart development and diseases has recently been explored. However, there is no study which elucidates the profiling of miRNAs during development of heart till date. Very limited miRNAs have been reported to date in cardiac context. In addition, integration of large scale experimental data with computational and comparative approaches remains an unsolved challenge.The present study was designed to identify the microRNAs implicated in heart development using next generation sequencing, bioinformatics and experimental approaches. We sequenced six small RNA libraries prepared from different developmental stages of the heart using chicken as a model system to produce millions of short sequence reads. We detected 353 known and 703 novel miRNAs involved in heart development. Out of total 1056 microRNAs identified, 32.7% of total dataset of known microRNAs displayed differential expression whereas seven well studied microRNAs namely let-7, miR-140, miR-181, miR-30, miR-205, miR-103 and miR-22 were found to be conserved throughout the heart development. The 3'UTR sequences of genes were screened from Gallus gallus genome for potential microRNA targets. The target mRNAs were appeared to be enriched with genes related to cell cycle, apoptosis, signaling pathways, extracellular remodeling, metabolism, chromatin remodeling and transcriptional regulators. Our study presents the first comprehensive overview of microRNA profiling during heart development and prediction of possible cardiac specific targets and has a big potential in future to develop microRNA based therapeutics against cardiac pathologies where fetal gene re-expression is witnessed in adult heart.

An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.

Biliary atresia (BA) is a neonatal obstructive cholangiopathy that progresses to end-stage liver disease, often requiring transplantation. The murine model of BA, employing rhesus rotavirus (RRV), parallels human disease and has been used to elucidate mechanistic aspects of a virus induced biliary cholangiopathy. We previously reported that the RRV VP4 gene plays an integral role in activating the immune system and induction of BA. Using rotavirus binding and blocking assays, this study elucidated how RRV VP4 protein governs cholangiocyte susceptibility to infection both in vitro and in vivo in the murine model of BA. We identified the amino acid sequence on VP4 and its cholangiocyte binding protein, finding that the sequence is specific to those rotavirus strains that cause obstructive cholangiopathy. Pretreatment of murine and human cholangiocytes with this VP4-derived peptide (TRTRVSRLY) significantly reduced the ability of RRV to bind and infect cells. However, the peptide did not block cholangiocyte binding of TUCH and Ro1845, strains that do not induce murine BA. The SRL sequence within TRTRVSRLY is required for cholangiocyte binding and viral replication. The cholangiocyte membrane protein bound by SRL was found to be Hsc70. Inhibition of Hsc70 by small interfering RNAs reduced RRV's ability to infect cholangiocytes. This virus-cholangiocyte interaction is also seen in vivo in the murine model of BA, where inoculation of mice with TRTRVSRLY peptide significantly reduced symptoms and mortality in RRV-injected mice.

Biliary atresia (BA) is a neonatal disease that results in obliteration of the biliary tree. The murine model of BA, which mirrors the human disease, is based upon infection of newborn mice with rhesus rotavirus (RRV), leading to an obstructive cholangiopathy. The purpose of this study was to characterize the temporal relationship between viral infection and the induction of this model. BALB/c mice were infected with RRV on day of life (DOL) 0, 3, 5, and 7. Groups were characterized as early-infection (infection by DOL 3) or late-infection (infection after DOL 5). Early RRV infection induced symptoms in 95% of pups with a mortality rate of 80%. In contrast, late infection caused symptoms in only 50% of mice, and 100% of pups survived. The clinical findings correlated with histological analysis of extrahepatic biliary trees, cytokine expression, and viral titers. Primary murine cholangiocytes isolated, cultured, and infected with RRV yielded higher titers of infectious virus in those harvested from DOL 2 versus DOL 9 mice. Less interferon alpha and beta was produced in DOL 2 versus DOL 9 RRV infected primary cholangiocytes. Injection of BALB/c interferon alpha/beta receptor knockout (IFN-αβR(-/-)) pups at DOL 7 showed increased symptoms (79%) and mortality (46%) when compared to late infected wild type mice. In conclusion, the degree of injury sustained by relatively immature cholangiocytes due to more robust RRV replication correlated with more severe clinical manifestations of cholangiopathy and higher mortality. Interferon alpha production by cholangiocytes appears to play a regulatory role. These findings confirm a temporal dependence of RRV infection in murine BA and begin to define a pathophysiologic role of the maturing cholangiocyte.

Biliary atresia (BA) is a devastating neonatal cholangiopathy that progresses to fibrosis and end-stage liver disease by 2 years of age. Portoenterostomy may reestablish biliary drainage, but, despite drainage, virtually all afflicted patients develop fibrosis and progress to end-stage liver disease requiring liver transplantation for survival.

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.

MicroRNA (miRNA) biogenesis requires AtDRB1 (double-stranded RNA binding protein)/HYL1 (Hyponastic Leaves1) protein for processing and maturation of miRNA precursors. The AtDRB1/HYL1 protein associates with AtDCL1 (Dicer-Like1) and accurately processes primary-miRNAs (pri-mRNAs) first to precursor-miRNAs (pre-miRNAs) and finally to mature miRNAs. The dephosphorylation of AtDRB1/HYL1 protein is very important for the precise processing of miRNA precursors. The monocot model crop plant Oryza sativa encodes four orthologues of AtDRB1/HYL1 protein, the only one encoded by Arabidopsis thaliana. The present study focuses on the functionality of the O. sativa DRBs as the orthologues of AtDRB1/HYL1 by using RNA binding assays and in planta protein-protein interaction analysis. Further, mitogen-activated protein kinase MPK3 is established as the kinase phosphorylating DRB1 protein in both the model plants, O. sativa and Arabidopsis. MicroRNA microarray analysis in atmpk3 and atmpk6 mutants indicate the importance of AtMPK3 in maintaining the level of miRNAs in the plant.

Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers.

MicroRNAs (miRNAs) are the naturally expressed small, 18~25 nts long non-coding single stranded RNAs, which inhibit the translation by interacting with the 3' untranslated region (UTR) of specific mRNA targets or by repression of posttranscriptional modification of mRNAs. MiRNAs are found to regulate the differentiation, development, function and stress responsive growth of cardiac cells. Their role and association with several disease progressions is of interest in recent years. Our interest is to study their role in cardiac hypertrophy (characterized by increased cell size, protein synthesis and reactivation of gene pathways). Therefore, we analyzed their features using a dataset (# ≈1400 #) of potential intronic and 3'UTR targeted miRNAs from known cardiac marker genes. We report 10 uncharacterized miRNAs regulating cardiac marker genes during cardiac hypertrophy and other cardiac diseases.

The Rhesus rotavirus (RRV) induced murine model of biliary atresia (BA) is a useful tool in studying the pathogenesis of this neonatal biliary obstructive disease. In this model, the mitogen associated protein kinase pathway is involved in RRV infection of biliary epithelial cells (cholangiocytes). We hypothesized that extracellular signal-related kinase (ERK) phosphorylation is integral to calcium influx, allowing for viral replication within the cholangiocyte. Utilizing ERK and calcium inhibitors in immortalized cholangiocytes and BALB/c pups, we determined that ERK inhibition resulted in reduced viral yield and subsequent decreased symptomatology in mice. In vitro, the RRV VP6 protein induced ERK phosphorylation, leading to cellular calcium influx. Pre-treatment with an ERK inhibitor or Verapamil resulted in lower viral yields. We conclude that the pathogenesis of RRV-induced murine BA is dependent on the VP6 protein causing ERK phosphorylation and triggering calcium influx allowing replication in cholangiocytes.

Biliary atresia (BA) is a neonatal inflammatory cholangiopathy that requires surgical intervention by Kasai portoenterostomy to restore biliary drainage. Even with successful portoenterostomy, most patients diagnosed with BA progress to end-stage liver disease, necessitating a liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of neonatal mice induces an inflammatory obstructive cholangiopathy that parallels human BA. The model is triggered by RRV viral protein (VP)4 binding to cholangiocyte cell-surface proteins. High mobility group box 1 (HMGB1) protein is a danger-associated molecular pattern that when released extracellularly moderates innate and adaptive immune response. In this study, we investigated how mutations in three RRV VP4-binding sites, RRVVP4-K187R (sialic acid-binding site), RRVVP4-D308A (integrin α2β1-binding site), and RRVVP4-R446G (heat shock cognate 70 [Hsc70]-binding site), affects infection, HMGB1 release, and the murine model of BA. Newborn pups injected with RRVVP4-K187R and RRVVP4-D308A developed an obstruction within the extrahepatic bile duct similar to wild-type RRV, while those infected with RRVVP4-R446G remained patent. Infection with RRVVP4-R446G induced a lower level of HMGB1 release from cholangiocytes and in the serum of infected pups. RRV infection of HeLa cells lacking Hsc70 resulted in no HMGB1 release, while transfection with wild-type Hsc70 into HeLa Hsc70-deficient cells reestablished HMGB1 release, indicating a mechanistic role for Hsc70 in its release. Conclusion: Binding to Hsc70 contributes to HMGB1 release; therefore, Hsc70 potentially serves as a therapeutic target for BA.