Possible Influence of Ethnicity on CT Perfusion Parameter Thresholds in Acute Ischemic Stroke.

Introduction Tissue at risk, as estimated by CT perfusion utilizing Tmax+6, correlates with final infarct volume (FIV) in acute ischemic stroke (AIS) without reperfusion. Tmax thresholds are derived from Western ethnic populations but not from ethnic Asian populations. We aimed to investigate the influence of ethnicity on Tmax thresholds. Methods From a clinical-imaging registry of Australian and Indonesian stroke patients, we selected a participant subgroup with the following inclusion criteria: AIS under 24 hours and absence of reperfusion therapy. Clinical data included demographics, time metrics, stroke severity, premorbid, and 3-month Modified Rankin Score. Baseline CTP and MRI <72 hours were performed. Volumes of Tmax utilizing different thresholds and final infarct volumes (FIV) were calculated. Spearman correlation was used to evaluate relationship involving ordinal variables and calculate the optimal Tmax threshold against FIV in both populations. Results Two hundred patients were included in the study sample 100 in Jakarta and 100 in Geelong. The median National Institutes Health Stroke Scale (IQR) were 6(3-11) and 3(1-5), respectively. The median Tmax+6(IQR) was 0 (0-46.5) in Jakarta group and 0(0-7.5) in Geelong group. The median FIV(IQR) was 0 (0-30.5) and 0 (0-5.5). Tmax +8s in Jakarta population against FIV showed Spearman's coefficient =0.72, representing the optimal Tmax threshold. Tmax+6s showed Spearman's coefficient =0.51 against FIV in the Geelong population. Conclusions Tmax thresholds approximating FIV were possibly different in the Asian when compared with the non-Asian populations. Future studies are required to extend and confirm the validity of our findings.

SINE RNA of the imprinted miRNA clusters mediates constitutive type III interferon expression and antiviral protection in hemochorial placentas.

Hemochorial placentas have evolved defense mechanisms to prevent the vertical transmission of viruses to the immunologically underdeveloped fetus. Unlike somatic cells that require pathogen-associated molecular patterns to stimulate interferon production, placental trophoblasts constitutively produce type III interferons (IFNL) through an unknown mechanism. We demonstrate that transcripts of short interspersed nuclear elements (SINEs) embedded in miRNA clusters within the placenta trigger a viral mimicry response that induces IFNL and confers antiviral protection. Alu SINEs within primate-specific chromosome 19 (C19MC) and B1 SINEs within rodent-specific microRNA cluster on chromosome 2 (C2MC) produce dsRNAs that activate RIG-I-like receptors (RLRs) and downstream IFNL production. Homozygous C2MC knockout mouse trophoblast stem (mTS) cells and placentas lose intrinsic IFN expression and antiviral protection, whereas B1 RNA overexpression restores C2MCΔ/Δ mTS cell viral resistance. Our results uncover a convergently evolved mechanism whereby SINE RNAs drive antiviral resistance in hemochorial placentas, placing SINEs as integral players in innate immunity.

Hepatic stellate cells maintain liver homeostasis through paracrine neurotrophin-3 signaling that induces hepatocyte proliferation.

Organ size is maintained by the controlled proliferation of distinct cell populations. In the mouse liver, hepatocytes in the midlobular zone that are positive for cyclin D1 (CCND1) repopulate the parenchyma at a constant rate to preserve liver mass. Here, we investigated how hepatocyte proliferation is supported by hepatic stellate cells (HSCs), pericytes that are in close proximity to hepatocytes. We used T cells to ablate nearly all HSCs in the murine liver, enabling the unbiased characterization of HSC functions. In the normal liver, complete loss of HSCs persisted for up to 10 weeks and caused a gradual reduction in liver mass and in the number of CCND1+ hepatocytes. We identified neurotrophin-3 (Ntf-3) as an HSC-produced factor that induced the proliferation of midlobular hepatocytes through the activation of tropomyosin receptor kinase B (TrkB). Treating HSC-depleted mice with Ntf-3 restored CCND1+ hepatocytes in the midlobular region and increased liver mass. These findings establish that HSCs form the mitogenic niche for midlobular hepatocytes and identify Ntf-3 as a hepatocyte growth factor.

Serum MicroRNA Transcriptomics and Acute Rejection or Recurrent Hepatitis C Virus in Human Liver Allograft Recipients: A Pilot Study.

BACKGROUND: Acute rejection (AR) and recurrent hepatitis C virus (R-HCV) are significant complications in liver allograft recipients. Noninvasive diagnosis of intragraft pathologies may improve their management. METHODS: We performed small RNA sequencing and microRNA (miRNA) microarray profiling of RNA from sera matched to liver allograft biopsies from patients with nonimmune, nonviral (NINV) native liver disease. Absolute levels of informative miRNAs in 91 sera matched to 91 liver allograft biopsies were quantified using customized real-time quantitative PCR (RT-qPCR) assays: 30 biopsy-matched sera from 26 unique NINV patients and 61 biopsy-matched sera from 41 unique R-HCV patients. The association between biopsy diagnosis and miRNA abundance was analyzed by logistic regression and calculating the area under the receiver operating characteristic curve. RESULTS: Nine miRNAs-miR-22, miR-34a, miR-122, miR-148a, miR-192, miR-193b, miR-194, miR-210, and miR-885-5p-were identified by both sRNA-seq and TLDA to be associated with NINV-AR. Logistic regression analysis of absolute levels of miRNAs and goodness-of-fit of predictors identified a linear combination of miR-34a + miR-210 (P < 0.0001) as the best statistical model and miR-122 + miR-210 (P < 0.0001) as the best model that included miR-122. A different linear combination of miR-34a + miR-210 (P < 0.0001) was the best model for discriminating NINV-AR from R-HCV with intragraft inflammation, and miR-34a + miR-122 (P < 0.0001) was the best model for discriminating NINV-AR from R-HCV with intragraft fibrosis. CONCLUSIONS: Circulating levels of miRNAs, quantified using customized RT-qPCR assays, may offer a rapid and noninvasive means of diagnosing AR in human liver allografts and for discriminating AR from intragraft inflammation or fibrosis due to R-HCV.

Plasma microRNA Interindividual Variability in Healthy Individuals, Pregnant Women, and an Individual with a Stably Altered Neuroendocrine Phenotype.

BACKGROUND: Extracellular RNAs (exRNAs) in biofluids are amenable to quantitative analysis and proposed as noninvasive biomarkers for monitoring organ function. Cell-lineage-specific microRNAs (miRNAs) are present in plasma as soluble ribonucleoproteins or enclosed in exRNA carriers and transported through the vasculature. However, more extensive studies of healthy individuals are needed to gain insights into the variability of plasma miRNA abundance and composition. METHODS: The exRNA composition of platelet-depleted plasma collected twice from 236 healthy individuals was characterized by small RNA sequencing. Plasma of pregnant women featuring dramatically increased placental miRNAs and samples from subject P12 with noticeably increased epithelial- and neuroendocrine-origin miRNAs were included for comparison. The miRNA content of 10 000g and 100 000g pellet fractions of plasma generated by ultracentrifugation was also determined. Data analysis methods included Pearson correlation, differential gene expression, and unsupervised clustering. RESULTS: The abundance changes for more variable miRNAs in plasma of normal individuals correlated between coexpressed cell-lineage-specific miRNAs of the liver, neuroendocrine organs, epithelial cells, and muscle. ExRNA of pellet fractions contained <2% of total plasma miRNA with modest enrichment of lineage-specific and variable miRNAs compared to supernatant. The abundance fold changes of miRNAs observed in pregnancy and P12 compared to normal exceeded interquartile variability of healthy individuals. The neuroendocrine miRNA signature of P12 persisted for more than 4 years and was absent in other individuals. CONCLUSIONS: This study defines the framework and effect size for screening of extensive plasma collections for miRNA phenotypes and biomarker discovery.

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo.

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss-of-function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

Chromosome 19 microRNA cluster enhances cell reprogramming by inhibiting epithelial-to-mesenchymal transition.

During implantation, cytotrophoblasts undergo epithelial-to-mesenchymal transition (EMT) as they differentiate into invasive extravillous trophoblasts (EVTs). The primate-specific microRNA cluster on chromosome 19 (C19MC) is exclusively expressed in the placenta, embryonic stem cells and certain cancers however, its role in EMT gene regulation is unknown. In situ hybridization for miR-517a/c, a C19MC cistron microRNA, in first trimester human placentas displayed strong expression in villous trophoblasts and a gradual decrease from proximal to distal cell columns as cytotrophoblasts differentiate into invasive EVTs. To investigate the role of C19MC in the regulation of EMT genes, we employed the CRISPR/dCas9 Synergistic Activation Mediator (SAM) system, which induced robust transcriptional activation of the entire C19MC cistron and resulted in suppression of EMT associated genes. Exposure of human iPSCs to hypoxia or differentiation of iPSCs into either cytotrophoblast-stem-like cells or EVT-like cells under hypoxia reduced C19MC expression and increased EMT genes. Furthermore, transcriptional activation of the C19MC cistron induced the expression of OCT4 and FGF4 and accelerated cellular reprogramming. This study establishes the CRISPR/dCas9 SAM as a powerful tool that enables activation of the entire C19MC cistron and uncovers its novel role in suppressing EMT genes critical for maintaining the epithelial cytotrophoblasts stem cell phenotype.

Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis.

Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. We performed conventional small-RNA-sequencing (sRNA-seq) and sRNA-seq with T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor EDTA, ACD, and heparin plasma to study the effect on ex-mRNA capture. Compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs reads up to 50-fold. The exRNA pool was dominated by hematopoietic cells and platelets, with additional contribution from the liver. About 60% of the 15- to 42-nt reads originated from the coding sequences, in a pattern reminiscent of ribosome-profiling. Blood sample type had a considerable influence on the exRNA profile. On average approximately 350 to 1,100 distinct ex-mRNA transcripts were detected depending on plasma type. In serum, additional transcripts from neutrophils and hematopoietic cells increased this number to near 2,300. EDTA and ACD plasma showed a destabilizing effect on ex mRNA and non-coding RNA ribonucleoprotein complexes compared to other plasma types. In a proof-of-concept study, we investigated differences between the exRNA profiles of patients with acute coronary syndrome (ACS) and healthy controls. The improved tissue resolution of ex mRNAs after PNK-treatment enabled us to detect a neutrophil-signature in ACS that escaped detection by ex miRNA analysis.

Single-Cell RNA Profiling of Glomerular Cells Shows Dynamic Changes in Experimental Diabetic Kidney Disease.

BACKGROUND: Recent single-cell RNA sequencing (scRNA-seq) analyses have offered much insight into cell-specific gene expression profiles in normal kidneys. However, in diseased kidneys, understanding of changes in specific cells, particularly glomerular cells, remains limited. METHODS: To elucidate the glomerular cell-specific gene expression changes in diabetic kidney disease, we performed scRNA-seq analysis of isolated glomerular cells from streptozotocin-induced diabetic endothelial nitric oxide synthase (eNOS)-deficient (eNOS-/-) mice and control eNOS-/- mice. RESULTS: We identified five distinct cell populations, including glomerular endothelial cells, mesangial cells, podocytes, immune cells, and tubular cells. Using scRNA-seq analysis, we confirmed the expression of glomerular cell-specific markers and also identified several new potential markers of glomerular cells. The number of immune cells was significantly higher in diabetic glomeruli compared with control glomeruli, and further cluster analysis showed that these immune cells were predominantly macrophages. Analysis of differential gene expression in endothelial and mesangial cells of diabetic and control mice showed dynamic changes in the pattern of expressed genes, many of which are known to be involved in diabetic kidney disease. Moreover, gene expression analysis showed variable responses of individual cells to diabetic injury. CONCLUSIONS: Our findings demonstrate the ability of scRNA-seq analysis in isolated glomerular cells from diabetic and control mice to reveal dynamic changes in gene expression in diabetic kidneys, with variable responses of individual cells. Such changes, which might not be apparent in bulk transcriptomic analysis of glomerular cells, may help identify important pathophysiologic factors contributing to the progression of diabetic kidney disease.

Deciphering human ribonucleoprotein regulatory networks.

RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i.e. specific combinations of RBPs that bind to specific sets of RNAs and targeted regions. These regulatory modules represented functionally related proteins and exhibited distinct differences in RNA metabolism, expression variance, as well as subcellular localization. This integrative investigation of experimental RBP-RNA interaction evidence and RBP regulatory function in a human cell line will be a valuable resource for understanding the complexity of post-transcriptional regulation.

Autophagy is a gatekeeper of hepatic differentiation and carcinogenesis by controlling the degradation of Yap.

Activation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.

Longstanding unilateral dislocation of the temporomandibular joint in a 6-year-old girl.

Unilateral longstanding dislocation of the temporomandibular joint (TMJ) is rare in children. These patients may demonstrate some function in opening and closing of the mouth due to the formation of a pseudo-joint in the dislocated position. In this report we describe a case of longstanding unilateral dislocation of the TMJ in a 6-year-old girl, highlighting the diagnostic difficulty and management of the condition.

Dynamic risk stratification for predicting the recurrence in differentiated thyroid cancer.

AIM: To analyze the predictive value of the dynamic risk stratification (DRS) system for assessing the risk of recurrent/persistent disease in our large group of differentiated thyroid carcinoma (DTC) patients. PATIENTS AND METHODS: We retrospectively included 2184 consecutive patients who received radioiodine ablation therapy following a total or near total thyroidectomy in our department between 1998 and 2014. The American Thyroid Association (ATA) classification was used for initial risk classification. At the second year of follow-up period after radioiodine ablation therapy, DRS was performed also. The ATA and DRS risk classification results were compared with clinical outcome. RESULTS: According to DRS, more than half of the ATA high-risk patients (73.2%) moved to the DRS low-risk category and the 6.4% of ATA low-risk patients comprised the DRS high-risk category. In comparison of variables within the ATA and the DRS risk groups with clinical outcome, combined use of the ATA and the DRS systems was statistically significant to predict the recurrent/persistent disease (P<0.005). CONCLUSION: The present study revealed that the DRS system is a necessary stratification system in addition to the initial risk evaluation. The DRS can discriminate those patients who does not require closer follow-up in the long-term period.

Single cell RNA sequencing to dissect the molecular heterogeneity in lupus nephritis.

Lupus nephritis is a leading cause of mortality among systemic lupus erythematosus (SLE) patients, and its heterogeneous nature poses a significant challenge to the development of effective diagnostics and treatments. Single cell RNA sequencing (scRNA-seq) offers a potential solution to dissect the heterogeneity of the disease and enables the study of similar cell types distant from the site of renal injury to identify novel biomarkers. We applied scRNA-seq to human renal and skin biopsy tissues and demonstrated that scRNA-seq can be performed on samples obtained during routine care. Chronicity index, IgG deposition, and quantity of proteinuria correlated with a transcriptomic-based score composed of IFN-inducible genes in renal tubular cells. Furthermore, analysis of cumulative expression profiles of single cell keratinocytes dissociated from nonlesional, non-sun-exposed skin of patients with lupus nephritis also revealed upregulation of IFN-inducible genes compared with keratinocytes isolated from healthy controls. This indicates the possible use of scRNA-seq analysis of skin biopsies as a biomarker of renal disease. These data support the potential utility of scRNA-seq to provide new insights into the pathogenesis of lupus nephritis and pave the way for exploiting a readily accessible tissue to reflect injury in the kidney.

Transplant-related experience in pharyngo-oesophageal reconstruction: use of organ preservation techniques to improve ischaemic tolerance in free jejunal flaps.

Reconstruction of pharyngo-oesophageal defects remains a surgical challenge. Free jejunal flaps, first described by Seidenberg in 1959, are used to reconstruct circumferential defects, but their main disadvantage is sensitivity to ischaemia. Others are secretions, an unpleasant smell, and problems at the donor site. To improve the tolerance of the jejunal segment to ischaemia and to give the surgeon more time, we cool it after harvest and flush it with organ preservation fluid. We describe the technique in a small case series of seven patients.

Biopsy of parotid masses: Review of current techniques.

Definitive diagnosis of parotid gland masses is required optimal management planning and for prognosis. There is controversy over whether fine needle aspiration cytology (FNAC) or ultrasound guided core biopsy (USCB) should be the standard for obtaining a biopsy. The aim of this review is to assess the current evidence available to assess the benefits of each technique and also to assess the use of intra-operative frozen section (IOFS). Literature searches were performed using pubmed and google scholar. The literature has been reviewed and the evidence is presented. FNAC is an accepted and widely used technique. It has been shown to have variable diagnostic capabilities depending on centres and experience of staff. USCB has a highly consistent diagnostic accuracy and can help with tumour grading and staging. However, the technique is more invasive and there is a question regarding potential for seeding. Furthermore, USCB is less likely to be offered as part of a one-stop clinic. IOFS has no role as a first line diagnostic technique but may be reserved as an adjunct or for lesions not amenable to percutaneous biopsy. On balance, USCB seems to be the method of choice. The current evidence suggests it has superior diagnostic potential and is safe. With time, USCB is likely to supplant FNAC as the biopsy technique of choice, replicating that which has occurred already in other areas of medicine such a breast practice.

Genome-wide identification and expression analysis of SWI1 genes in Boechera species.

As a mode of reproduction in plants, apomixis leads to the generation of clones via seeds. Apomictic plants form viable diploid female gametes without meiosis (apomeiosis) and produce embryos without fertilization (parthenogenesis). Apomeiosis, as a major component of apomixis, has recently been reported in some Arabidopsis thaliana mutants; dyad mutants of SWI1 showed developmental processes common to apomeiosis, such as producing functional diploid gametes. However, the orthologs of SWI1 genes in natural apomicts has not been previously reported. To identify the relationship between the SWI1 gene and the apomeiosis process, we isolated and sequenced SWI1 orthologs from Boechera species, including apomictic and sexual species. Boechera species are close relatives of A. thaliana and thus are advantageous model species for apomixis research. The SWI1 cDNAs were obtained by RT-PCR from apomictic and sexual Boechera young flower buds. We sequenced partial SWI1 transcripts that were 650bp for B. holboellii and 684bp for B. stricta. These SWI1-like sequences showed 86% similarity for B. holboellii and 92% for B. stricta to the A. thaliana SWI1 transcript. We also used available genome data and amplified genomic sequences for SWI1 orthologs in B. holboellii and B. stricta. The predicted proteins contain a phospholipase C domain and a nuclear localization signal. Sequence analysis did not show significant mutations related to apomixis, and phylogenetic analysis showed that SWI1-like sequences were common across plant families, regardless of the presence of a sexual or apomictic reproduction system. We also investigated the expression levels of SWI1 mRNA in the B. holboellii and B. stricta young unopened flower buds and found that relatively high levels of expression occurred in apomicts.

Unique microRNAs appear at different times during the course of a delayed-type hypersensitivity reaction in human skin.

Diphencyprone (DPCP) is a hapten that induces delayed-type hypersensitivity (DTH) reactions. MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate gene expression and have been implicated in various inflammatory skin diseases, but their role in DTH reactions is not well understood. We generated global miRNA expression profiles (using next-generation sequencing) of DPCP reactions in skin of seven healthy volunteers at 3, 14 and 120 days after challenge. Compared to placebo-treated sites, DPCP-challenged skin at 3 days (peak inflammation) had 127 miRNAs significantly deregulated. At 14 days (during resolution of inflammation), 43 miRNAs were deregulated and, at 120 days (when inflammation had completely resolved), six miRNAs were upregulated. While some miRNAs have been observed in psoriasis or atopic dermatitis, most of the deregulated miRNAs have not yet been studied in the context of skin biology or immunology. Across the three time points studied, many but not all miRNAs were uniquely expressed. As various miRNAs may influence T cell activation, this may indicate that the miRNAs exclusively expressed at different time points function to promote or resolve skin inflammation, and therefore, may inform on the paradoxical ability of DPCP to treat both autoimmune conditions (alopecia areata) and conditions of ineffective immunity (melanoma).

Kruppel-like factor 15 is a critical regulator of cardiac lipid metabolism.

The mammalian heart, the body's largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism.