Multiple pathological components in gliosarcoma

Pathogenesis and drug response of iPSC-derived cardiomyocytes from two Brugada syndrome patients with different Na_v1.5-subunit mutations

Brugada syndrome (BrS) is a complex genetic cardiac ion channel disease that causes a high predisposition to sudden cardiac death. Considering that its heterogeneity in clinical manifestations may result from genetic background, the application of patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) may help to reveal cell phenotype characteristics underlying different genetic variations. Here, to verify and compare the pathogenicity of mutations (SCN5A c.4213G>A and SCN1B c.590C>T) identified from two BrS patients, we generated two novel BrS iPS cell lines that carried missense mutations in SCN5A or SCN1B, compared their structures and electrophysiology, and evaluated the safety of quinidine in patient-specific iPSC-derived CMs. Compared to the control group, BrS-CMs showed a significant reduction in sodium current, prolonged action potential duration, and varying degrees of decreased V_max, but no structural difference. After applying different concentrations of quinidine, drug-induced cardiotoxicity was not observed within 3-fold unbound effective therapeutic plasma concentration (ETPC). The data presented proved that iPSC-CMs with variants in SCN5A c.4213G>A or SCN1B c.590C>T are able to recapitulate single-cell phenotype features of BrS and respond appropriately to quinidine without increasing incidence of arrhythmic events.

Dynamic regulation of anti-oxidation following donation repairing after circulatory determined death renal transplantation with prolonged non-heart-beating time

Donation after circulatory-determined death (DCD) is an important part of renal transplantation. Therefore, DCD renal transplantation animal model should be established to study the mechanism of organ injury. Here, we established a stable DCD rat renal transplantation model and investigated the dynamic regulation of graft self-repairing and antioxidant capacities with different non-heart-beating times (NHBTs). Male Sprague-Dawley rats were randomly divided into four groups with the NHBT of the donors from 0 to 15, 30, and 45 minutes. Recipients in long NHBT groups had a significantly lower survival rate and poorer graft function than those in short NHBT groups. Grafts from the 15-minute and 30-minute NHBT groups showed light and severe injury respectively at an early stage after transplantation and recovered within 7 days after transplantation, whereas the self-repairing of the grafts in the 45-minute NHBT group was delayed. The expressions of proliferating cell nuclear antigen (PCNA) and von Willebrand factor (vWF) were dependent on NHBT. The expression of antioxidant proteins paralleled graft recovery. In conclusion, the recipients can up-regulate antioxidant capacity to enhance graft self-repairing in DCD renal transplantation. Prolonged NHBT can delay the self-repairing and antioxidation of grafts.

PUM1 represses CDKN1B translation and contributes to prostate cancer progression

Posttranscriptional regulation of cancer gene expression programs plays a vital role in carcinogenesis; identifying the critical regulators of tumorigenesis and their molecular targets may provide novel strategies for cancer diagnosis and therapeutics. Highly conserved RNA-binding protein Pumilio-1 (PUM1) regulates mouse growth and cell proliferation, propelling us to examine its role in cancer. We found human PUM1 is highly expressed in a diverse group of cancer, including prostate cancer; enhanced PUM1 expression is also correlated with reduced survival among prostate cancer patients. Detailed expression analysis in twenty prostate cancer tissues showed enhanced expression of PUM1 at mRNA and protein levels. Knockdown of PUM1 reduced prostate cancer cell proliferation and colony formation, and subcutaneous injection of PUM1 knockdown cells led to reduced tumor size. Downregulation of PUM1 in prostate cancer cells consistently elevated cyclin-dependent kinase inhibitor 1B (CDKN1B) protein expression through increased translation but did not impact its mRNA level, while overexpression of PUM1 reduced CDKN1B protein level. Our finding established a critical role of PUM1 mediated translational control, particularly the PUM1-CDKN1B axis, in prostate cancer cell growth and tumorigenesis. We proposed that PUM1-CDKN1B regulatory axis may represent a novel mechanism for the loss of CDKN1B protein expression in diverse cancers and potential targets for therapeutics development.

A nomogram for predicting lymph node metastasis in superficial esophageal squamous cell carcinoma

Superficial esophageal squamous cell carcinoma (SESCC) is defined as carcinoma with mucosal or submucosal invasion, regardless of regional lymph node metastasis (LNM). The lymph node status is not only a key factor to determine the training strategy, but also the most important prognostic factor in esophageal cancer. In this study, we establish a clinical nomogram for predicting LNM in patients with SESCC. A predictive model was established based on the training cohort composed of 711 patients who underwent esophagectomy for SESCC from December 2009 to June 2018. A prospective cohort of 203 patients from June 2018 to January 2019 was used for validation. Favorable calibration and well-fitted decision curve analysis were conducted and good discrimination was observed (concordance index [C-index], 0.860; 95% confidence interval [CI], 0.825–0.894) through internal validation. The external validation cohort presented good discrimination (C-index, 0.916; 95% CI, 0.860–0.971). This model may facilitate the prediction of LNM in patients with SESCCs.

Differentiation of gestational diabetes mellitus by nuclear magnetic resonance-based metabolic plasma analysis

This study aimed to investigate the metabolic profile of gestational diabetes mellitus (GDM) at both antepartum and postpartum periods. Seventy pregnant women were divided into three groups: the normal glucose-tolerant group (NGT, n=35), the abnormal glucose-tolerant groups without insulin therapy (A1GDM, n=24) or with insulin therapy (A2GDM, n=11). Metabolic profiles of the plasma were acquired by proton nuclear magnetic resonance (¹H-NMR) spectroscopy and analyzed by multivariate statistical data analysis. The relationship between demographic parameters and the potential metabolite biomarkers was further explored. Group antepartum or postpartum showed similar metabolic trends. Compare with those of the NGT group, the levels of 2-hydroxybutyrate, lysine, acetate, glutamine, succinate, tyrosine, formate, and all three BCAAs (leucine, valine, isoleucine) in the A2GDM group were increased dramatically, and the levels of lysine, acetate, and formate in the A1GDM group were elevated significantly. The dramatically decreased levels of 3-methyl-2-oxovalerate and methanol were observed both in the A1GDM group and A2GDM group. Compare to the A1GDM group, the branched-chain amino acids (BCAAs) of leucine, valine, and isoleucine were increased dramatically in the A2GDM group. The levels of aromatic amino acids (AAAs), tyrosine and phenylalanine, were significantly increased in GDM women, consistent with the severity of GDM. Interference of amino acid metabolism and disturbance in energy metabolism occurred in women with different grades of GDM. Metabolic profiles could reflect the severity of GDM. Plasma BCAA concentrations showing strong positive correlations with weight and pre-delivery BMI. This study provides a new perspective to understand the pathogenesis and etiology of GDM, which may help the clinical management and treatment of GDM.

Regulatory role of sorting nexin 5 in protein stability and vesicular targeting of vesicular acetylcholine transporter to synaptic vesicle-like vesicles in PC12 cells

Accurate targeting of vesicular acetylcholine transporter (VAChT) to synaptic vesicles (SVs) is indispensable for efficient cholinergic transmission. Previous studies have suggested that the dileucine motif within the C-terminus of the transporter is sufficient for its targeting to SVs. However, the cytosolic machinery underlying specific regulation of VAChT trafficking and targeting to SVs is still unclear. Here we used the C-terminus of VAChT as a bait in a yeast two-hybrid screen to identify sorting nexin 5 (SNX5) as its novel interacting protein. SNX5 was detected in the SVs enriched LP2 subcellular fraction of rat brain homogenate and showed strong colocalization with VAChT in both brain sections and PC12 cells. Binding assays suggested that the C-terminal domain of VAChT can interact with both BAR and PX domain of SNX5. Depletion of SNX5 enhanced the degradation of VAChT and the process was mediated through the lysosomal pathway. More importantly, we found that, in PC12 cells, the depletion of SNX5 expression significantly decreased the synaptic vesicle-like vesicles (SVLVs) localization of VAChT. Therefore, the results suggest that SNX5 is a novel regulator for both stability and SV targeting of VAChT.

A transgenic pig model expressing a CMV-ZsGreen1 reporter across an extensive array of tissues

Since genetic engineering of pigs can benefit both biomedicine and agriculture, selecting a suitable gene promoter is critically important. The cytomegalovirus (CMV) promoter, which can robustly drive ubiquitous transgene expression, is commonly used at present, yet recent reports suggest tissue-specific activity in the pig. The objective of this study was to quantify ZsGreen1 protein (in lieu of CMV promoter activity) in tissues from pigs harboring a CMV-ZsGreen1 transgene with a single integration site. Tissue samples (n=35) were collected from neonatal hemizygous (n=3) and homozygous (n=3) piglets and ZsGreen1 abundance was determined via immunoblotting. ZsGreen1 was detected in all tissues, except hypothalamus, kidney cortex and oviduct. The expression patterns of homozygous and hemizygous piglets were similar (P>0.05). However, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within neural/endocrine tissues, ZsGreen1 abundance was highest in the anterior pituitary gland, intermediate in the cerebellum and lowest in the cerebrum, spinal cord and posterior pituitary (P<0.05). In the digestive system, ZsGreen1 was more abundant in the salivary gland than esophagus, stomach, pancreas, duodenum, jejunum, ileum, spleen, colon, gallbladder and liver (P<0.05). Interestingly, ZsGreen1 amounts also differed within an organ (i.e., the right ventricle had 3-fold higher levels than the other heart chambers; P<0.05). These results provide useful information for the use of the CMV promoter to drive transgene expression in the pig. Moreover, this swine model represents a novel resource of ZsGreen1-labeled organs and a valuable tool to advance genome editing research.

Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases

There are an estimated 10 000 monogenic diseases affecting tens of millions of individuals worldwide. The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients. CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA. The complexity of genomic insults resulting in heritable disease requires patient-specific genome editing strategies with consideration of DNA repair pathways, and CRISPR/Cas systems of different types, species, and those with additional enzymatic capacity and/or delivery methods. In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair, non-homologous end joining, microhomology-mediated end joining, and base editing to permanently correct diverse monogenic diseases.

Genome engineering technologies in rabbits

The rabbit has been recognized as a valuable model in various biomedical and biological research fields because of its intermediate size and phylogenetic proximity to primates. However, the technology for precise genome manipulations in rabbit has been stalled for decades, severely limiting its applications in biomedical research. Novel genome editing technologies, especially CRISPR/Cas9, have remarkably enhanced precise genome manipulation in rabbits, and shown their superiority and promise for generating rabbit models of human genetic diseases. In this review, we summarize the brief history of transgenic rabbit technology and the development of novel genome editing technologies in rabbits.

Therapeutic gene editing strategies using CRISPR-Cas9 for the β-hemoglobinopathies

With advancements in gene editing technologies, our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate, paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases. CRISPR-Cas9, short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9, is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells. This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic. The β-hemoglobinopathies are a group of monogenic diseases, which despite their high prevalence and chronic debilitating nature, continue to have few therapeutic options available. In this review, we will discuss our existing comprehension of the genetics and current state of treatment for β-hemoglobinopathies, consider potential genome editing therapeutic strategies, and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.

Base editing: a brief review and a practical example

Genome editing has undergone rapid development in recent years, yielding new approaches to make precise changes in genes. In this review, we discuss the development of various adenine and cytosine base-editing technologies, which share the ability to make specific base changes at specific sites in the genome. We also describe multiple applications of base editing in vitro and in vivo. Finally, as a practical example, we demonstrate the use of a cytosine base editor and an adenine base editor in human cells to introduce and then correct a prevalent mutation responsible for hereditary tyrosinemia type 1.

Harnessing CRISPR-Cas system diversity for gene editing technologies

The discovery and utilization of RNA-guided surveillance complexes, such as CRISPR-Cas9, for sequence-specific DNA or RNA cleavage, has revolutionised the process of gene modification or knockdown. To optimise the use of this technology, an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements, coupled with high cleavage efficacy and specificity. Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.

Designing and generating a mouse model: frequently asked questions

Genetically engineered mouse (GEM) models are commonly used in biomedical research. Generating GEMs involve complex set of experimental procedures requiring sophisticated equipment and highly skilled technical staff. Because of these reasons, most research institutes set up centralized core facilities where custom GEMs are created for research groups. Researchers, on the other hand, when they begin thinking about generating GEMs for their research, several questions arise in their minds. For example, what type of model(s) would be best useful for my research, how do I design them, what are the latest technologies and tools available for developing my model(s), and finally how to breed GEMs in my research. As there are several considerations and options in mouse designs, and as it is an expensive and time-consuming endeavor, careful planning upfront can ensure the highest chance of success. In this article, we provide brief answers to several frequently asked questions that arise when researchers begin thinking about generating mouse model(s) for their work.