SummaryGenome editing in induced pluripotent stem cells is currently hampered by the laborious and expensive nature of identifying homology-directed repair (HDR)-modified cells. We present an approach where isolation of cells bearing a selectable, HDR-mediated editing event at one locus enriches for HDR-mediated edits at additional loci. This strategy, called co-targeting with selection, improves the probability of isolating cells bearing HDR-mediated variants and accelerates the production of disease models.

Triclosan, an antimicrobial chemical found in consumer personal care products, has been shown to stimulate antibiotic resistance in pathogenic bacteria. Although many studies focus on antibiotic resistance pertinent to medical scenarios, resistance developed in natural and engineered environments is less studied and has become an emerging concern for human health. In this study, the impacts of chronic triclosan (TCS) exposure on antibiotic resistance genes (ARGs) and microbial community structure were assessed in lab-scale anaerobic digesters. TCS concentrations from below detection to 2500 mg kg(-1) dry solids were amended into anaerobic digesters over 110 days and acclimated for >3 solid retention time values. Four steady state TCS concentrations were chosen (30-2500 mg kg(-1)). Relative abundance of mexB, a gene coding for a component of a multidrug efflux pump, was significantly higher in all TCS-amended digesters (30 mg kg(-1) or higher) relative to the control. TCS selected for bacteria carrying tet(L) and against those carrying erm(F) at concentrations which inhibited digester function; the pH decrease associated with digester failure was suspected to cause this selection. Little to no impact of TCS was observed on intI1 relative abundance. Microbial communities were also surveyed by high-throughput 16S rRNA gene sequencing. Compared to the control digesters, significant shifts in community structure towards clades containing commensal and pathogenic bacteria were observed in digesters containing TCS. Based on these results, TCS should be included in studies and risk assessments that attempt to elucidate relationships between chemical stressors (e.g. antibiotics), antibiotic resistance genes, and public health.

Anaerobic membrane bioreactors (AnMBR) play a key role in future plans for sustainable wastewater treatment and resource recovery because they have no energy-intensive oxygen transfer requirements and can produce biomethane for renewable energy. Recent research results show that they can meet relatively stringent discharge limits with respect to BOD 5 and TSS when treating municipal wastewater primary effluent. Sustainable used water recovery plans should also consider removal of unregulated pollutants. Antibiotic resistance genes (ARGs) represent an important emerging contaminant due to public health concerns surrounding the spread of infections resistant to common antibiotics. Conventional activated sludge processes have demonstrated mixed results regarding ARG removal. The objective of this research was to determine the impact of an AnMBR on ARG removal when treating municipal primary clarifier effluent at 20°C. AnMBR treatment resulted in 3.3 to 3.6 log reduction of ARG and the horizontal gene transfer determinate, intI1, copies in filtrate. Membrane treatment significantly decreased the total biomass as indicated by a decrease in 16S rRNA gene concentration. Microbial community analysis via Illumina sequencing revealed that the relative abundance of putative pathogens was higher in membrane filtrate compared to primary effluent although the overall bacterial 16S rRNA gene concentrations was lower in filtrate. Membrane treatment also substantially reduced microbial diversity in filtrate compared to anaerobic reactor contents.Mainstream anaerobic wastewater treatment is a promising technology for sustainable water resource recovery. Anaerobic membrane bioreactors (AnMBRs) can meet relatively stringent BOD 5 and TSS regulatory standards (BOD 5 <10 mg L −1 , TSS <10 mg L −1 ) for municipal wastewater at temperatures as low as 10°C. The research reported herein demonstrated that AnMBR technology can also significantly reduce antibiotic resistance gene copies.

Mutations of HSPB5 (also known as CRYAB or ␣B-crystallin), a bona fide heat shock protein and molecular chaperone encoded by the HSPB5 (crystallin, alpha B) gene, are linked to multisystem disorders featuring variable combinations of cataracts, cardiomyopathy, and skeletal myopathy. This study aimed to investigate the pathological mechanisms involved in an earlyonset myofibrillar myopathy manifesting in a child harboring a homozygous recessive mutation in HSPB5, 343delT. To study HSPB5 343delT protein dynamics, we utilize model cell culture systems including induced pluripotent stem cells derived from the 343delT patient (343delT/343delT) along with isogenic, heterozygous, gene-corrected control cells (WT KI/ 343delT) and BHK21 cells, a cell line lacking endogenous HSPB5 expression. 343delT/343delT and WT KI/343delT-induced pluripotent stem cell-derived skeletal myotubes and cardiomyocytes did not express detectable levels of 343delT protein, contributable to the extreme insolubility of the mutant protein. Overexpression of HSPB5 343delT resulted in insoluble mutant protein aggregates and induction of a cellular stress response. Co-expression of 343delT with WT prevented visible aggregation of 343delT and improved its solubility. Additionally, in vitro refolding of 343delT in the presence of WT rescued its solubility. We demonstrate an interaction between WT and 343delT both in vitro and within cells. These data support a loss-of-function model for the myopathy observed in the patient because the insoluble mutant would be unavailable to perform normal functions of HSPB5, although additional gain-of-function effects of the mutant protein cannot be excluded. Additionally, our data highlight the solubilization of 343delT by WT, concordant with the recessive inheritance of the disease and absence of symptoms in carrier individuals.HSPB5 (also known as CRYAB or ␣B-crystallin) is a small molecular weight heat shock protein encoded by the HSPB5 (crystallin, alpha B) gene and functions as a molecular chaperone. Its promoter contains a heat shock element, a stress-responsive binding site of heat shock transcription factor 1 (HSF1), that functionally up-regulates the expression of HSPB5. Increased levels of HSPB5 can then go on to provide