Gene Editing: Powerful New Tools for Nephrology Research and Therapy.

Biologic research is experiencing a transformation brought about by the ability of programmable nucleases to manipulate the genome. In the recently developed CRISPR/Cas system, short RNA sequences guide the endonuclease Cas9 to any location in the genome, causing a DNA double-strand break (DSB). Repair of DSBs allows the introduction of targeted genetic manipulations with high precision. Cas9-mediated gene editing is simple, scalable, and rapid, and it can be applied to virtually any organism. Here, we summarize the development of modern gene editing techniques and the biology of DSB repair on which these techniques are based. We discuss technical points in applying this technology and review its use in model organisms. Finally, we describe prospects for the use of gene editing to treat human genetic diseases. This technology offers tremendous promise for equipping the nephrology research community to better model and ultimately, treat kidney diseases.

Rectal Staphylococcus aureus Carriage and Recurrence After Endoscopic Sinus Surgery for Chronic Rhinosinusitis With Nasal Polyps: A Prospective Cohort Study.

OBJECTIVE: Chronic rhinosinusitis with nasal polyps (CRSwNPs) remains a major challenge due to its high recurrence rate after endoscopic sinus surgery (ESS). We aimed to investigate the risk factors of recurrence among patients who underwent ESS for Chronic rhinosinusitis (CRS). METHODS: Prospective cohort study including 391 cases in a single institution receiving ESS were included for analysis from 2014 and 2017. Baseline characteristics including rectal Staphylococcus aureus (S aureus) carriage in patients receiving ESS for CRSwNPs. The primary outcome was the recurrence of CRSwNPs. Multivariate regression model was established to identify independently predictive factors for recurrence. RESULTS: Overall, 142 (36.3%) cases with recurrence within 2 years after ESS were observed in this study. After variable selection, multivariate regression model consisted of 4 variables including asthma (odds ratio [OR] = 3.41; P < .001), nonsteroidal anti-inflammatory drug allergy (OR = 2.27; P = .005), previous ESS (OR = 3.64; P < .001), and preoperative carriage of S aureus in rectum (OR = 2.34; P = .001). CONCLUSIONS: Based on our results, surgeons could predict certain groups of patients who are at high risk for recurrence after ESS. Rectal carriage of S aureus is more statistically related to the recurrence of CRSwNP after ESS compared with skin and nasal carriage.

Silencing miR-146a-5p Protects against Injury-Induced Osteoarthritis in Mice.

Osteoarthritis (OA), the most prevalent joint disease and the leading cause of disability, remains an incurable disease largely because the etiology and pathogenesis underlying this degenerative process are poorly understood. Low-grade inflammation within joints is a well-established factor that disturbs joint homeostasis and leads to an imbalance between anabolic and catabolic processes in articular cartilage; however, the complexity of the network between inflammatory factors that often involves positive and negative feedback loops makes current anti-cytokine therapy ineffective. MicroRNAs (miRNAs) have emerged as key regulators to control inflammation, and aberrant miRNAs expression has recently been linked to OA pathophysiology. In the present study, we characterized transcriptomic profiles of miRNAs in primary murine articular chondrocytes in response to a proinflammatory cytokine, IL-1ß, and identified miR-146a-5p as the most responsive miRNA to IL-1ß. miR-146a-5p was also found to be upregulated in human OA cartilage. We further demonstrated that knockdown of miR-146a-5p antagonized IL-1ß-mediated inflammatory responses and IL-1ß-induced catabolism in vitro, and silencing of miR-146a in chondrocytes ameliorated articular cartilage destruction and reduced OA-evoked pain in an injury-induced murine OA model. Moreover, parallel RNA sequencing revealed that differentially expressed genes in response to IL-1ß were enriched in pathways related to inflammatory processes, cartilage matrix homeostasis, and cell metabolism. Bioinformatic analyses of putative miR-146a-5p gene targets and following prediction of protein-protein interactions suggest a functional role of miR-146a-5p in mediating inflammatory processes and regulation of cartilage homeostasis. Our genetic and transcriptomic data define a crucial role of miR-146a-5p in OA pathogenesis and implicate modulation of miR-146a-5p in articular chondrocytes as a potential therapeutic strategy to alleviate OA.

DNA methylation-mediated Rbpjk suppression protects against fracture nonunion caused by systemic inflammation.

Challenging skeletal repairs are frequently seen in patients experiencing systemic inflammation. To tackle the complexity and heterogeneity of the skeletal repair process, we performed single-cell RNA sequencing and revealed that progenitor cells were one of the major lineages responsive to elevated inflammation and this response adversely affected progenitor differentiation by upregulation of Rbpjk in fracture nonunion. We then validated the interplay between inflammation (via constitutive activation of Ikk2, Ikk2ca) and Rbpjk specifically in progenitors by using genetic animal models. Focusing on epigenetic regulation, we identified Rbpjk as a direct target of Dnmt3b. Mechanistically, inflammation decreased Dnmt3b expression in progenitor cells, consequently leading to Rbpjk upregulation via hypomethylation within its promoter region. We also showed that Dnmt3b loss-of-function mice phenotypically recapitulated the fracture repair defects observed in Ikk2ca-transgenic mice, whereas Dnmt3b-transgenic mice alleviated fracture repair defects induced by Ikk2ca. Moreover, Rbpjk ablation restored fracture repair in both Ikk2ca mice and Dnmt3b loss-of-function mice. Altogether, this work elucidates a common mechanism involving a NF-κB/Dnmt3b/Rbpjk axis within the context of inflamed bone regeneration. Building on this mechanistic insight, we applied local treatment with epigenetically modified progenitor cells in a previously established mouse model of inflammation-mediated fracture nonunion and showed a functional restoration of bone regeneration under inflammatory conditions through an increase in progenitor differentiation potential.

Repair of the injured spinal cord and the potential of embryonic stem cell transplantation.

Traditionally, treatment of spinal cord injury seemed frustrating and hopeless because of the remarkable morbidity and mortality, and restricted therapeutic options. Recent advances in neural injury and repair, and the progress towards development of neuroprotective and regenerative interventions are basis for increased optimism. Neural stem cells have opened a new arena of discovery for the field of regenerative science and medicine. Embryonic stem (ES) cells can give rise to all neural progenitors and they represent an important scientific tool for approaching neural repair. The growing number of dedicated regeneration centers worldwide exemplifies the changing perception towards the do-ability of spinal cord repair and this review was born from a presentation at one such leading center, the Kentucky Spinal Cord Injury Research Center. Current concepts of the pathophysiology, repair, and restoration of function in the damaged spinal cord are presented with an overlay of how neural stem cells, particularly ES cells, fit into the picture as important scientific tools and therapeutic targets. We focus on the use of genetically tagged and selectable ES cell lines for neural induction and transplantation. Unique features of ES cells, including indefinite replication, pluripotency, and genetic flexibility, provide strong tools to address questions of neural repair. Selective marker expression in transplanted ES cell derived neural cells is providing new insights into transplantation and repair not possible previously. These features of ES cells will produce a predictable and explosive growth in scientific tools that will translate into discoveries and rapid progress in neural repair.

[Sterilizing effect and residual bacteria of four different methods for sterilizing simple breathing vesicles].

OBJECTIVE: To study the effects of 4 different methods for disinfection of simple breathing vesicles and microbial residue. METHODS: The disinfection tests were divided into 4 groups: G1 group (43 cases) with 500 mg/L chlorine dioxide spray, G2 group (28 cases) with alcohol spray, G3 group (47 cases) with 50 mg/L trichloroisocyanuric acid (TCCA) immersion, and G4 group (46 cases) with 50 mg/L chlorine dioxide solution immersion. After 30 min of disinfection, each group was examined by bacterial culture and colony count. The residual bacteria were identified and typed. RESULTS: The 4 methods showed significant differences in bacterial colony count (P<0.001). The rate of bacterial residue was 0% in G1 group, 53.6% in G2 group, 27.7% in G3 group, and 21.7% in G4 group, showing significant differences between the 4 groups (P<0.001). The residual bacteria included antibiotic-resistant common opportunistic pathogen such as Pseudomonas aeruginosa, Acinetobacter baumannii and Staphylococcus haemolytic. CONCLUSIONS: Disinfection with 500 mg/L chlorine dioxide spray is the best for simple breathing vesicles. Prolonged immersion in TCCA may lead to the growth of drug-resistant pathogens in the breathing vesicles.

Gap junctions and connexon hemichannels in human embryonic stem cells.

Intercellular communication via gap junctions is thought to play an important role in embryonic cell survival and differentiation. Classical studies demonstrated both dye and electrical coupling of cells in the inner cell mass of mouse embryos, as well as the development of restrictions against coupling between cells of the inner cell mass and surrounding trophectoderm. Here we demonstrate extensive gap junctional communication between human embryonic stem (ES) cells, the pluripotent cells isolated from the inner cell mass of preimplantation blastocysts. Human ES cells maintained in vitro expressed RNA for 18 of the 20 known connexins; only connexin 40.1 (Cx40.1) and Cx50 were not detected by reverse transcription-polymerase chain reaction. Cx40, Cx43, and Cx45 were visualized by immunofluorescence at points of contact between adjacent cells. Electron microscopy confirmed that neighboring cells formed zones of tight membrane apposition characteristic of gap junctions. Fluorescent dye injections demonstrated extensive coupling within human ES cell colonies growing on mouse embryonic fibroblast (MEF) feeder cells, whereas dye coupling between human ES cells and adjacent MEFs was extremely rare. Physiological recordings demonstrated electrical and dye coupling between human ES cells in feeder-free monolayers and between isolated human ES cell pairs. Octanol, 18-alpha-glycyrrhetinic acid, and arylaminobenzoates inhibited transjunctional currents. Dye uptake studies on human ES cell monolayers and recordings from solitary human ES cells gave evidence for the surface expression of connexon hemichannels. Human ES cells provide a unique system for the study of human connexin proteins and their potential functions in cellular differentiation and the maintenance of pluripotency.