A molecular single-cell lung atlas of lethal COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.

The Evf2 Ultraconserved Enhancer lncRNA Functionally and Spatially Organizes Megabase Distant Genes in the Developing Forebrain.

Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5'UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3' end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3'-activator and 5'UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility.

The genome-wide association study of serum IgE levels demonstrated a shared genetic background in allergic diseases.

Immunoglobulin E (IgE) synthessis is highly related to a variety of atopic diseases, and several genome-wide association studies (GWASs) have demonstrated the association between genes and IgE level. In this study, we conducted the largest genome-wide association study of IgE involving a Taiwanese Han population. Eight independent variants exhibited genome-wide significance. Among them, an intronic SNP of CD28, rs1181388, and an intergenic SNP, rs1002957030, on 11q23.2 were identified as novel signals for IgE. Seven of the loci were replicated successfully in a meta-analysis using data on Japanese population. Among all the human leukocyte antigen (HLA) regions, HLA-DQA1*03:02 - HLA-DQB1*03:03 was the most significant haplotype (OR = 1.25, SE = 0.02, FDR = 1.6 × 10-14), corresponding to HLA-DQA1 Asp160 and HLA-DQB1 Leu87 amino acid residues. The genetic correlation showed significance between IgE and allergic diseases including asthma, atopic dermatitis, and pollinosis. IgE PRS was significantly correlated with total IgE levels. Furthermore, the top decile IgE polygenic risk score (PRS) group had the highest risk of asthma for the Taiwan Biobank and Biobank Japan cohorts. IgE PRS may be used to aid in predicting the occurrence of allergic reactions before symptoms occur and biomarkers are detectable. Our study provided a more comprehensive understanding of the impact of genomic variants, including complex HLA alleles, on serum IgE levels.

Shear Strain-Induced Two-Dimensional Slip Avalanches in Rhombohedral MoS2.

Slip avalanches are ubiquitous phenomena occurring in three-dimensional materials under shear strain, and their study contributes immensely to our understanding of plastic deformation, fragmentation, and earthquakes. So far, little is known about the role of shear strain in two-dimensional (2D) materials. Here we show some evidence of 2D slip avalanches in exfoliated rhombohedral MoS2, triggered by shear strain near the threshold level. Utilizing interfacial polarization in 3R-MoS2, we directly probe the stacking order in multilayer flakes and discover a wide variety of polarization domains with sizes following a power-law distribution. These findings suggest that slip avalanches can occur during the exfoliation of 2D materials, and the stacking orders can be changed via shear strain. Our observation has far-reaching implications for the development of new materials and technologies, where precise control over the atomic structure of these materials is essential for optimizing their properties as well as for our understanding of fundamental physical phenomena.

Perceptions of COVID-19 Vaccination in Patients with Genitourinary Cancers: A Survey Study.

Since the approval of the COVID-19 vaccines, their safety and efficacy has been widely demonstrated in patients with cancer. However, there remain patients with reservations regarding vaccination. We aimed to assess genitourinary cancer patients' perceptions of the vaccines as well as barriers and influencers of decision-making through the completion of a questionnaire. While vaccine-associated concerns were observed, most patients with genitourinary cancers were willing to receive the vaccine. Moving forward, differing strategies could be considered to enhance patient education on the utility of vaccination in the setting of cancer and beyond.

Surgical interventions for bilateral congenital cataract in children aged two years and under.

BACKGROUND: Congenital cataracts are lens opacities in one or both eyes of babies or children present at birth. These may cause a reduction in vision severe enough to require surgery. Cataracts are proportionally the most treatable cause of visual loss in childhood, and are a particular problem in low-income countries, where early intervention may not be possible. Paediatric cataracts provide different challenges to those in adults. Intense inflammation, amblyopia (vision is obstructed by cataract from birth which prevents normal development of the visual system), posterior capsule opacification and uncertainty about the final trajectory of ocular growth parameters can affect results of treatment. Two options currently considered for children under 2 years of age with bilateral congenital cataracts are: (i) intraocular lens (IOL) implantation; or (ii) leaving a child with primary aphakia (no lens in the eye), necessitating the need for contact lenses or aphakic glasses. Other important considerations regarding surgery include the prevention of visual axis opacification (VAO), glaucoma and the route used to perform lensectomy. OBJECTIVES: To assess the effectiveness of infant cataract surgery or lensectomy to no surgery for bilateral congenital cataracts in children aged 2 years and under. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL; which contains the Cochrane Eyes and Vision Trials Register; 2022, Issue 1); Ovid MEDLINE; Ovid Embase; the ISRCTN registry; ClinicalTrials.gov and the WHO ICTRP. The date of the search was 25 January 2022. SELECTION CRITERIA: We included all randomised controlled trials (RCTs) that compared infant cataract surgery or lensectomy to no surgery, in children with bilateral congenital cataracts aged 2 years and younger. This update (of a review published in 2001 and updated in 2006) does not include children over 2 years of age because they have a wider variety of aetiologies, and are therefore managed differently, and have contrasting outcomes. DATA COLLECTION AND ANALYSIS: We used standard methods expected by Cochrane. Two review authors extracted data independently. We assessed the risk of bias of included studies using RoB 1 and assessed the certainty of the evidence using GRADE. MAIN RESULTS: We identified three RCTs that met our inclusion criteria with each trial comparing a different aspect of surgical intervention for this condition. The trials included a total of 79 participants under 2 years of age, were conducted in India and follow-up ranged from 1 to 5 years. Study participants and outcome assessors were not masked in these trials. One study (60 children) compared primary IOL implantation with primary aphakia. The results from this study suggest that there may be little or no difference in visual acuity at 5 years comparing children with pseudophakia (mean logMAR 0.50) and aphakia (mean logMAR 0.59) (mean difference (MD) -0.09 logMAR, 95% confidence intervals (CIs) -0.24 to 0.06; 54 participants; very low-certainty evidence), but the evidence is very uncertain. The evidence is very uncertain as to the effect of IOL implantation compared with aphakia on visual axis opacification (VAO) (risk ratio (RR) 1.29, 95% CI 0.23 to 7.13; 54 participants; very low-certainty evidence). The trial investigators did not report on the cases of amblyopia. There was little evidence of a difference betwen the two groups in cases of glaucoma at 5 years follow-up (RR 0.86, 95% CI 0.24 to 3.10; 54 participants; very low-certainty evidence). Cases of retinal detachment and reoperation rates were not reported. The impact of IOL implantation on adverse effects is very uncertain because of the sparse data available: of the children who were pseudophakic, 1/29  needed a trabeculectomy and 8/29 developed posterior synechiae. In comparison, no trabeculectomies were needed in the aphakic group and 2/25 children had posterior synechiae (54 participants; very low-certainty evidence).  The second study (14 eyes of 7 children under 2 years of age) compared posterior optic capture of IOL without vitrectomy versus endocapsular implantations with anterior vitrectomy (commonly called 'in-the-bag surgery'). The authors did not report on visual acuity, amblyopia, glaucoma and reoperation rate. They had no cases of VAO in either group. The evidence is very uncertain as to the effect of in-the-bag implantation in children aged under 1 year. There was a higher incidence of inflammatory sequelae: 4/7 in-the-bag implantation eyes and 1/7 in optic capture eyes (P = 0.04, 7 participants; very low-certainty evidence). We graded the certainty of evidence as low or very low for imprecision in all outcomes because their statistical analysis reported that a sample size of 13 was needed in each group to achieve a power of 80%, whereas their subset of children under the age of 1 year had only 7 eyes in each group. The third study (24 eyes of 12 children) compared a transcorneal versus pars plana route using a 25-gauge transconjunctival sutureless vitrectomy system. The evidence is very uncertain as to the effect of the route chosen on the incidence of VAO, with no cases reported at 1 year follow-up in either group. The investigators did not report on visual acuity, amblyopia, glaucoma, retinal detachment and reoperation rate. The pars plana route had the adverse effects of posterior capsule rupture in 2/12 eyes, and 1/12 eyes needing sutures. Conversely, 1/12 eyes operated on by the transcorneal route needed sutures. We graded the outcomes with very low-certainty because of the small sample size and the absence of a priori sample size calculation. AUTHORS' CONCLUSIONS: There is no high level evidence for the effectiveness of one type of surgery for bilateral congenital cataracts over another, or whether surgery itself is better than primary aphakia. Further RCTs are required to inform modern practice about concerns, including the timing of surgery, age at which surgery should be undertaken, age for implantation of an IOL and development of complications, such as reoperations, glaucoma and retinal detachment. Standardising the methods used to measure visual function, along with objective monitoring of compliance with the use of aphakic glasses/contact lenses would greatly improve the quality of study data and enable more reliable interpretation of outcomes.

Novel, abundant Drosha isoforms are deficient in miRNA processing in cancer cells.

MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22-nucleotide (nt) in length that collectively regulate more than 60% of coding genes. Aberrant miRNA expression is associated with numerous diseases, including cancer. miRNA biogenesis is licenced by the ribonuclease (RNase) III enzyme Drosha, the regulation of which is critical in determining miRNA levels. We and others have previously revealed that alternative splicing regulates the subcellular localization of Drosha. To further investigate the alternative splicing landscape of Drosha transcripts, we performed PacBio sequencing in different human cell lines. We identified two novel isoforms resulting from partial intron-retention in the region encoding the Drosha catalytic domain. One isoform (AS27a) generates a truncated protein that is unstable in cells. The other (AS32a) produces a full-length Drosha with a 14 amino acid insertion in the RIIID domain. By taking advantage of Drosha knockout cells in combination with a previously established reporter assay, we demonstrated that Drosha-AS32a lacks cleavage activity. Furthermore, neither Drosha-27a nor Drosha-32a were able to rescue miRNA expression in the Drosha knockout cells. Interestingly, both isoforms were abundantly detected in a wide range of cancer cell lines (up to 15% of all Drosha isoforms). Analysis of the RNA-seq data from over 1000 breast cancer patient samples revealed that the AS32a is relatively more abundant in tumours than in normal tissue, suggesting that AS32a may play a role in cancer development.

RNA editing-based classification of diffuse gliomas: predicting isocitrate dehydrogenase mutation and chromosome 1p/19q codeletion.

BACKGROUND: Accurate classification of diffuse gliomas, the most common tumors of the central nervous system in adults, is important for appropriate treatment. However, detection of isocitrate dehydrogenase (IDH) mutation and chromosome1p/19q codeletion, biomarkers to classify gliomas, is time- and cost-intensive and diagnostic discordance remains an issue. Adenosine to inosine (A-to-I) RNA editing has emerged as a novel cancer prognostic marker, but its value for glioma classification remains largely unexplored. We aim to (1) unravel the relationship between RNA editing and IDH mutation and 1p/19q codeletion and (2) predict IDH mutation and 1p/19q codeletion status using machine learning algorithms. RESULTS: By characterizing genome-wide A-to-I RNA editing signatures of 638 gliomas, we found that tumors without IDH mutation exhibited higher total editing level compared with those carrying it (Kolmogorov-Smirnov test, p < 0.0001). When tumor grade was considered, however, only grade IV tumors without IDH mutation exhibited higher total editing level. According to 10-fold cross-validation, support vector machines (SVM) outperformed random forest and AdaBoost (DeLong test, p < 0.05). The area under the receiver operating characteristic curve (AUC) of SVM in predicting IDH mutation and 1p/19q codeletion were 0.989 and 0.990, respectively. After performing feature selection, AUCs of SVM and AdaBoost in predicting IDH mutation were higher than that of random forest (0.985 and 0.983 vs. 0.977; DeLong test, p < 0.05), but AUCs of the three algorithms in predicting 1p/19q codeletion were similar (0.976-0.982). Furthermore, 67% of the six continuously misclassified samples by our 1p/19q codeletion prediction models were misclassifications in the original labelling after inspection of 1p/19q status and/or pathology report, highlighting the accuracy and clinical utility of our models. CONCLUSIONS: The study represents the first genome-wide analysis of glioma editome and identifies RNA editing as a novel prognostic biomarker for glioma. Our prediction models provide standardized, accurate, reproducible and objective classification of gliomas. Our models are not only useful in clinical decision-making, but also able to identify editing events that have the potential to serve as biomarkers and therapeutic targets in glioma management and treatment.

The Evolution of Trauma in Los Angeles County Over More Than a Decade.

OBJECTIVE: Explore trends in trauma incidence and mortality rates in Los Angeles County. DESIGN: Data for patients treated at Los Angeles County trauma centers from 2000 to 2011 were analyzed for this study. Age-adjusted incidence and mortality rates were calculated by gender, race, injury type, injury severity, and mechanism of injury. Trends were assessed using linear regression to determine the annual percentage change (APC). RESULTS: There were 223 773 patients included. The trauma incidence rate increased by 14.6% driven by an increase in blunt injury of 5.4% annually (P < .05). Penetrating injury decreased at -6.9% APC (P < .01). Mortality rate decreased at -11.5% APC (P < .01), with reduction in both blunt (-6.8% APC [P < .01]) and penetrating injuries (-16.7% APC [P < .01]). The trends in mortality persisted with stratification by age, gender, race, and injury severity score. CONCLUSION: In this mature trauma system, the trauma incidence increased slightly from 2000 to 2011, while the mortality steadily declined. Public health officials in other areas could perform a similar self-evaluation to describe and monitor injury events and trends in their jurisdictions, a reassessment of priority and trauma system resource allocation, which will directly benefit the regional population.

Evf2 lncRNA/BRG1/DLX1 interactions reveal RNA-dependent inhibition of chromatin remodeling.

Transcription-regulating long non-coding RNAs (lncRNAs) have the potential to control the site-specific expression of thousands of target genes. Previously, we showed that Evf2, the first described ultraconserved lncRNA, increases the association of transcriptional activators (DLX homeodomain proteins) with key DNA enhancers but represses gene expression. In this report, mass spectrometry shows that the Evf2-DLX1 ribonucleoprotein (RNP) contains the SWI/SNF-related chromatin remodelers Brahma-related gene 1 (BRG1, SMARCA4) and Brahma-associated factor (BAF170, SMARCC2) in the developing mouse forebrain. Evf2 RNA colocalizes with BRG1 in nuclear clouds and increases BRG1 association with key DNA regulatory enhancers in the developing forebrain. While BRG1 directly interacts with DLX1 and Evf2 through distinct binding sites, Evf2 directly inhibits BRG1 ATPase and chromatin remodeling activities. In vitro studies show that both RNA-BRG1 binding and RNA inhibition of BRG1 ATPase/remodeling activity are promiscuous, suggesting that context is a crucial factor in RNA-dependent chromatin remodeling inhibition. Together, these experiments support a model in which RNAs convert an active enhancer to a repressed enhancer by directly inhibiting chromatin remodeling activity, and address the apparent paradox of RNA-mediated stabilization of transcriptional activators at enhancers with a repressive outcome. The importance of BRG1/RNA and BRG1/homeodomain interactions in neurodevelopmental disorders is underscored by the finding that mutations in Coffin-Siris syndrome, a human intellectual disability disorder, localize to the BRG1 RNA-binding and DLX1-binding domains.

Impact of polyvascular disease on patients with atrial fibrillation: Insights from ROCKET AF.

BACKGROUND: We investigated the impact of polyvascular disease in patients enrolled in ROCKET AF. METHODS: Cox regression models were used to assess clinical outcomes and treatment effects of rivaroxaban compared with warfarin in patients with atrial fibrillation and coronary, peripheral, or carotid artery disease, or any combination of the 3. RESULTS: A total of 655 (4.6%) patients had polyvascular disease (≥2 disease locations), and 3,391 (23.8%) had single-arterial bed disease. Patients with polyvascular disease had similar rates of stroke/systemic embolism but higher rates of cardiovascular and bleeding events when compared with those without vascular disease. Use of rivaroxaban compared with warfarin was associated with higher rates of stroke in patients with polyvascular disease (hazard ratio [HR] 2.41, 95% CI 1.05-5.54); however, this was not seen in patients with single-bed (HR 0.90, 95% CI 0.64-1.28) or no vascular disease (HR 0.85, 95% CI 0.69-1.04; interaction P = .058). There was a significant interaction for major or nonmajor clinically relevant bleeding in patients with polyvascular (HR 1.23, 95% CI 0.91-1.65) and single-bed vascular disease (HR 1.30, 95% CI 1.13-1.49) treated with rivaroxaban compared with warfarin when compared with those without vascular disease (HR 0.95, 95% CI 0.87-1.04; interaction P = .0006). Additional antiplatelet therapy in this population did not improve stroke or cardiovascular outcomes. CONCLUSION: The use of rivaroxaban compared with warfarin was associated with a higher risk of stroke and bleeding in patients with polyvascular disease enrolled in ROCKET AF. Further studies are needed to understand the optimal management of this high-risk population.

Clinical and metabolomic risk factors associated with rapid renal function decline in sickle cell disease.

Sickle cell disease (SCD) nephropathy and lower estimated glomerular filtration rate (eGFR) are risk factors for early mortality. Furthermore, rate of eGFR decline predicts progression to end-stage renal disease in many clinical settings. However, factors predicting renal function decline in SCD are poorly documented. Using clinical, laboratory, genetic, and metabolomic data, we evaluated predictors of renal function decline in a longitudinal cohort of 288 adults (mean age 33.0 years). In 193 subjects with 5-year follow-up data, mean rate of eGFR decline was 2.35 mL/min/1.73 m2 /year, nearly twice that of African American adults overall. Hyperfiltration was prevalent at baseline (61.1%), and 36.8% of subjects experienced rapid eGFR decline (≥3 mL/min/1.73 m2 /year). Severe Hb genotype; proteinuria; higher platelet and reticulocyte counts, and systolic BP; and lower Hb level and BMI were associated with rapid decline. A risk scoring system was created using these 7 variables and was highly predictive of rapid eGFR decline, with odds of rapid decline increasing 1.635-fold for every point increment (P < 0.0001). Rapid eGFR decline was also associated with higher organ system severity score and peak creatinine. Additionally, two metabolites (asymmetric dimethylarginine and quinolinic acid) were associated with rapid decline. Further investigation into longitudinal SCD nephropathy (SCDN) trajectory, early markers of SCDN, and tools for risk stratification should inform interventional studies targeted to slowing GFR decline and improving SCD outcomes.

Transcriptome dynamics of developing maize leaves and genomewide prediction of cis elements and their cognate transcription factors.

Maize is a major crop and a model plant for studying C4 photosynthesis and leaf development. However, a genomewide regulatory network of leaf development is not yet available. This knowledge is useful for developing C3 crops to perform C4 photosynthesis for enhanced yields. Here, using 22 transcriptomes of developing maize leaves from dry seeds to 192 h post imbibition, we studied gene up- and down-regulation and functional transition during leaf development and inferred sets of strongly coexpressed genes. More significantly, we developed a method to predict transcription factor binding sites (TFBSs) and their cognate transcription factors (TFs) using genomic sequence and transcriptomic data. The method requires not only evolutionary conservation of candidate TFBSs and sets of strongly coexpressed genes but also that the genes in a gene set share the same Gene Ontology term so that they are involved in the same biological function. In addition, we developed another method to predict maize TF-TFBS pairs using known TF-TFBS pairs in Arabidopsis or rice. From these efforts, we predicted 1,340 novel TFBSs and 253 new TF-TFBS pairs in the maize genome, far exceeding the 30 TF-TFBS pairs currently known in maize. In most cases studied by both methods, the two methods gave similar predictions. In vitro tests of 12 predicted TF-TFBS interactions showed that our methods perform well. Our study has significantly expanded our knowledge on the regulatory network involved in maize leaf development.

Endothelial specific SIRT3 deletion impairs glycolysis and angiogenesis and causes diastolic dysfunction.

Endothelial glycolysis plays a critical role in the regulation of angiogenesis. We investigated the role of Sirtuin 3 (SIRT3) on endothelial cell (EC) glycolytic metabolism, angiogenesis, and diastolic function. Our aim was to test the hypothesis that loss of SIRT3 in ECs impairs endothelial glycolytic metabolism and angiogenesis and contributes to myocardial capillary rarefaction and the development of diastolic dysfunction. Using SIRT3 deficient ECs, SIRT3 was found to regulate a metabolic switch between mitochondrial respiration and glycolysis. SIRT3 knockout (KO)-ECs exhibited higher mitochondrial respiration and reactive oxygen species (ROS) formation. SIRT3 knockout (KO)-ECs exhibited a reduction in the expression of glycolytic enzyme, PFKFB3, and a fall in glycolysis and angiogenesis. Blockade of PFKFB3 reduced glycolysis and downregulated expression of VEGF and Angiopoietin-1 (Ang-1) in ECs. Deletion of SIRT3 in ECs also impaired hypoxia-induced expression of HIF-2α, VEGF, and Ang-1, as well as reduced angiogenesis. In vivo, endothelial-specific SIRT3 KO (ECKO) mice exhibited a myocardial capillary rarefaction together with a reduced coronary flow reserve (CFR) and diastolic dysfunction. Histologic study further demonstrated that knockout of SIRT3 in ECs significantly increased perivascular fibrosis in the coronary artery. These results implicate a role of SIRT3 in modulating endothelial function and cardiac function. Ablation of SIRT3 leads to impairment of EC glycolytic metabolism and angiogenic signaling, which may contribute to coronary microvascular rarefaction and diastolic dysfunction in SIRT3 ECKO mice.

Ablation of endothelial prolyl hydroxylase domain protein-2 promotes renal vascular remodelling and fibrosis in mice.

Accumulating evidence demonstrates that hypoxia-inducible factor (HIF-α) hydroxylase system has a critical role in vascular remodelling. Using an endothelial-specific prolyl hydroxylase domain protein-2 (PHD2) knockout (PHD2EC KO) mouse model, this study investigates the regulatory role of endothelial HIF-α hydroxylase system in the development of renal fibrosis. Knockout of PHD2 in EC up-regulated the expression of HIF-1α and HIF-2α, resulting in a significant decline of renal function as evidenced by elevated levels of serum creatinine. Deletion of PHD2 increased the expression of Notch3 and transforming growth factor (TGF-ß1) in EC, thus further causing glomerular arteriolar remodelling with an increased pericyte and pericyte coverage. This was accompanied by a significant elevation of renal resistive index (RI). Moreover, knockout of PHD2 in EC up-regulated the expression of fibroblast-specific protein-1 (FSP-1) and increased interstitial fibrosis in the kidney. These alterations were strongly associated with up-regulation of Notch3 and TGF-ß1. We concluded that the expression of PHD2 in endothelial cells plays a critical role in renal fibrosis and vascular remodelling in adult mice. Furthermore, these changes were strongly associated with up-regulation of Notch3/TGF-ß1 signalling and excessive pericyte coverage.

Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.

The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.

Identification of genes and pathways related to lymphovascular invasion in breast cancer patients: A bioinformatics analysis of gene expression profiles.

Surgery is the most effective treatment for breast cancer patients. However, some patients developed recurrence and distant metastasis after surgery. Adjuvant therapy is considered for high-risk patients depending on several prognostic markers, and lymphovascular invasion has become one of such prognostic markers that help physicians to identify the risk for distant metastasis and recurrence. However, the mechanism of lymphovascular invasion in breast cancer remains unknown. This study aims to unveil the genes and pathways that may involve in lymphovascular invasion in breast cancer. In total, 108 breast cancer samples were collected during surgery and microarray analysis was performed. Significance analysis of the microarrays and limma package for R were used to examine differentially expressed genes between lymphovascular invasion-positive and lymphovascular invasion-negative cases. Network and pathway analyses were mapped using the Ingenuity Pathway Analysis and the Database for Annotation, Visualization and Integrated Discovery. In total, 86 differentially expressed genes, including 37 downregulated genes and 49 upregulated genes were identified in lymphovascular invasion-positive patients. Among these genes, TNFSF11, IL6ST, and EPAS1 play important roles in cytokine-receptor interaction, which is the most enriched pathway related to lymphovascular invasion. Moreover, the results also suggested that an imbalance between extracellular matrix components and tumor micro-environment could induce lymphovascular invasion. Our study evaluated the underlying mechanisms of lymphovascular invasion, which may further help to assess the risk of breast cancer progression and identify potential targets of adjuvant treatment.

Evf2 (Dlx6as) lncRNA regulates ultraconserved enhancer methylation and the differential transcriptional control of adjacent genes.

Several lines of evidence suggest that long non-coding RNA (lncRNA)-dependent mechanisms regulate transcription and CpG DNA methylation. Whereas CpG island methylation has been studied in detail, the significance of enhancer DNA methylation and its relationship with lncRNAs is relatively unexplored. Previous experiments proposed that the ultraconserved lncRNA Evf2 represses transcription through Dlx6 antisense (Dlx6as) transcription and methyl-CpG binding protein (MECP2) recruitment to the Dlx5/6 ultraconserved DNA regulatory enhancer (Dlx5/6ei) in embryonic day 13.5 medial ganglionic eminence (E13.5 MGE). Here, genetic epistasis experiments show that MECP2 transcriptional repression of Evf2 and Dlx5, but not Dlx6, occurs through antagonism of DLX1/2 in E13.5 MGE. Analysis of E13.5 MGE from mice lacking Evf2 and of partially rescued Evf2 transgenic mice shows that Evf2 prevents site-specific CpG DNA methylation of Dlx5/6ei in trans, without altering Dlx5/6 expression. Dlx1/2 loss increases CpG DNA methylation, whereas Mecp2 loss does not affect Dlx5/6ei methylation. Based on these studies, we propose a model in which Evf2 inhibits enhancer DNA methylation, effectively modulating competition between the DLX1/2 activator and MECP2 repressor. Evf2 antisense transcription and Evf2-dependent balanced recruitment of activator and repressor proteins enables differential transcriptional control of adjacent genes with shared DNA regulatory elements.

Anatomical and transcriptional dynamics of maize embryonic leaves during seed germination.

Our anatomical analysis revealed that a dry maize seed contains four to five embryonic leaves at different developmental stages. Rudimentary kranz structure (KS) is apparent in the first leaf with a substantial density, but its density decreases toward younger leaves. Upon imbibition, leaf expansion occurs rapidly with new KSs initiated from the palisade-like ground meristem cells in the middle of the leaf. In parallel to the anatomical analysis, we obtained the time course transcriptomes for the embryonic leaves in dry and imbibed seeds every 6 h up to hour 72. Over this time course, the embryonic leaves exhibit transcripts of 30,255 genes at a level that can be regarded as "expressed." In dry seeds, ∼25,500 genes are expressed, showing functional enrichment in transcription, RNA processing, protein synthesis, primary metabolic pathways, and calcium transport. During the 72-h time course, ∼13,900 genes, including 590 transcription factor genes, are differentially expressed. Indeed, by 30 h postimbibition, ∼2,200 genes expressed in dry seeds are already down-regulated, and ∼2,000 are up-regulated. Moreover, the top 1% expressed genes at 54 h or later are very different from those before 30 h, reflecting important developmental and physiological transitions. Interestingly, clusters of genes involved in hormone metabolism, signaling, and responses are differentially expressed at various time points and TF gene expression is also modular and stage specific. Our dataset provides an opportunity for hypothesizing the timing of regulatory actions, particularly in the context of KS development.