Glycine homeostasis requires reverse SHMT flux.

The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Using rodent models, here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable-isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered, but SHMT2- and serine-dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis is largely insensitive to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 a major glycine-consuming enzyme.

Glycine homeostasis requires reverse SHMT flux.

The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered but SHMT2- and serine dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis does not respond to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 as a major glycine-consuming enzyme.

SID-4/NCK-1 is important for dsRNA import in Caenorhabditis elegans.

RNA interference is sequence-specific gene silencing triggered by double-stranded RNA. Systemic RNA interference is where double-stranded RNA, expressed or introduced into 1 cell, is transported to and initiates RNA interference in other cells. Systemic RNA interference is very efficient in Caenorhabditis elegans and genetic screens for systemic RNA interference-defective mutants have identified RNA transporters (SID-1, SID-2, and SID-5) and a signaling protein (SID-3). Here, we report that SID-4 is nck-1, a C. elegans NCK-like adaptor protein. sid-4 null mutations cause a weak, dose-sensitive, systemic RNA interference defect and can be effectively rescued by SID-4 expression in target tissues only, implying a role in double-stranded RNA import. SID-4 and SID-3 (ACK-1 kinase) homologs interact in mammals and insects, suggesting that they may function in a common signaling pathway; however, a sid-3; sid-4 double mutants showed additive resistance to RNA interference, suggesting that these proteins likely interact with other signaling pathways as well. A bioinformatic screen coupled to RNA interference sensitivity tests identified 23 additional signaling components with weak RNA interference-defective phenotypes. These observations suggest that environmental conditions may modulate systemic RNA interference efficacy, and indeed, sid-3 and sid-4 are required for growth temperature effects on systemic RNA interference silencing efficiency.

Multi-institutional review of non-hypospadiac penile urethral stricture management and outcomes.

OBJECTIVES: Penile urethral stricture disease not associated with hypospadias is rare, and there is a wide range of commonly used surgical repair techniques for this disease. We sought to compile a multi-institutional database of patients who had surgical correction of strictures in the penile urethra not limited to the meatus, and who had no history of hypospadias, for analysis using the Trauma and Urologic Reconstructive Network of Surgeons length, urethral segment and etiology classification system. METHODS: A retrospective database from 13 institutions was compiled of patients who had undergone surgical correction of Trauma and Urologic Reconstructive Network of Surgeons length, urethral segment and etiology urethral stricture segments S2b/S2c and excluding E5, with a minimum follow-up time of 4 months. Failure was defined as cystoscopically confirmed recurrence of a stricture measuring less than 16-Fr. RESULTS: We analyzed 222 patients with a median age of 57 years and a follow-up of 49 months. The overall surgical success rate was 80.2%. On multivariate analysis, the two variables identified that were predictive of surgical success were stricture length ≤2 cm as well as use of a buccal mucosa graft as compared to use of a fasciocutaneous flap, which had success rates of 83% and 52%, respectively (P = 0.0004). No statistically significant differences were found based on incisional approach or surgical technique, nor were outcomes different based on etiology or preoperative patient demographics. CONCLUSIONS: Surgical repair of penile urethral strictures of non-hypospadiac origin have a favorable overall success rate, at 80.2%. Regardless of incisional approach or surgical technique, all operations appear to have similar outcomes other than repairs using fasciocutaneous flap, which were statistically less successful than those using buccal mucosa graft.

Transurethral ventral buccal mucosa graft inlay for treatment of distal urethral strictures: international multi-institutional experience.

PURPOSE: To critically evaluate a multi-institutional patient cohort undergoing single-stage distal urethral repair using a novel transurethral buccal mucosa graft inlay urethroplasty technique (TBMGI). METHODS: A retrospective multi-institutional review of consecutive patients with fossa navicularis (FN) strictures treated with a single-stage TBMGI technique at 12 institutions from March 2014-March 2018 was performed. Patient demographics, stricture characteristics, clinical and patient-reported outcomes were analyzed. The primary outcomes were stricture recurrence and complications. Secondary outcomes were change in maximum urinary flow rate (Qmax), PVR, and changes in IPSS, SHIM and global response assessment (GRA) questionnaire responses. Descriptive statistical analysis was used for evaluation of outcomes. RESULTS: Sixty-eight men met inclusion criteria. Median age and stricture length were 60 years (IQR 48-69) and 2 cm (IQR 2-3), respectively. Most common stricture etiology was lichen sclerosus (34%). Median operative time and EBL were 72 min (IQR 50-120) and 20 mL (IQR 10-43), respectively. Fifty-seven men completed ≥ 12-month follow-up. At a median follow-up of 17 months (IQR 13-22), 54 patients (95%) remained stricture-free. Median Qmax improved from 5 to 18 mL/s (p < 0.0001), PVR 76-21 mL (p < 0.0001), and IPSS 15-5 (p < 0.0001); IPSS-QOL score: 5-1 (p < 0.0001). SHIM score did not significantly change following repair (median 22-21 p = 0.85). On GRA assessment, a majority of men reported "marked" (64%) or "moderate" (28%) overall improvement. No patient developed fistula, glanular dehiscence, graft necrosis or chordee. CONCLUSIONS: This novel minimally invasive transurethral urethroplasty technique is feasible and has demonstrated generalizable outcomes in a multi-institutional cohort with varying etiologies.

Efficient Homologous Recombination in Mice Using Long Single Stranded DNA and CRISPR Cas9 Nickase.

The CRISPR/Cas9 nickase mutant is less prone to off-target double-strand (ds)DNA breaks than wild-type Cas9 because to produce dsDNA cleavage it requires two guide RNAs to target the nickase to nearby opposing strands. Like wild-type Cas9 lesions, these staggered lesions are repaired by either non-homologous end joining or, if a repair template is provided, by homologous recombination (HR). Here, we report very efficient (up to 100%) recovery of heterozygous insertions in Mus musculus produced by long (>300 nt), single-stranded DNA donor template-guided repair of paired-nickase lesions.

The Benefits of Using a Small Caliber Ureteroscope in Evaluation and Management of Urethral Stricture.

INTRODUCTION AND OBJECTIVE: The proper evaluation of urethral strictures is an essential part of the surgical planning in urethral reconstruction. The proper evaluation of the stricture can be challenging in certain situations, especially when the meatus is involved. We propose that the use of a small caliber ureteroscope (4.5 Fr and 6.5 Fr) can offer additional help and use for the surgical planning in urethroplasty. METHODS: We prospectively collected data on 76 patients who underwent urethroplasties in Kulkarni Reconstructive Urology Center, Pune, India and Thunder Bay Regional Health Sciences Center, Thunder Bay, Canada. Patients had retrograde and micturition urethrograms performed preoperatively. The stricture was assessed visually using a 6.5 Fr ureteroscope. If the stricture was smaller than 6.5 Fr, we attempted using the 4.5 Fr ureteroscope. In nonobliterated strictures, we attempted bypassing the stricture making sure not to dilate the stricture. A glide wire would be passed to the bladder under vision. Stricture length, tissue quality, presence of other proximal strictures, false passages, and bladder tumors or stones would be assessed visually. If the penile stricture was near obliterative (smaller than 4.5 Fr caliber), a two-staged procedure is elected to be performed. For proximal bulbar strictures, if the urethral caliber is less than 4.5 Fr and the stricture length is less than 1 cm, we perform a nontransecting anastomotic urethroplasty (NTAU). If the stricture length is >1 cm, we perform a double-face augmented urethroplasty (DFAU). If the urethral caliber is >4.5 Fr and particularly those who are sexually active, ventral inlay with buccal mucosal grafts (BMGs) is performed. In mid to distal bulbar strictures, if the urethral caliber is >4.5 Fr, our procedure of choice is dorsal onlay with BMG. For those with urethral caliber <4.5 Fr and a stricture less than 1 cm, we perform a NTAU. For strictures longer than 1 cm, we perform a DFAU. With the exception of trauma, we very rarely transect the urethra. For panurethral strictures, we almost exclusively perform Kulkarni one-sided dissection. RESULTS: Urethroscopy was performed in 76 patients who presented for urethroplasty from July 2014 to September 2014 (in Pune) and between April 2016 and September 2017 (in Thunder Bay). Bypassing the stricture was achieved in 68 patients (89%) while it was unsuccessful in 8 patients (11%). In all unsuccessful urethroscopies, the stricture was near obliterative <4.5 Fr. Our surgical planning changed in (13) 17% of the cases. Out of 43 bulbar strictures, the decision was changed in (9) 21% where we performed 4 DFAU, 3 AAU (augmented anastomotic urethroplasty), and 2 EAU (end anastomotic urethroplasty). In 13 penile strictures, we opted for staged urethroplasty including 3 Johansons and 1 first-stage Asopa in 30.7%. In 20 panurethral urethroplasties, 1 patient (5%) had a urethral stone found in a proximal portion of the bulbar urethra distal to a stricture ring that was removed using an endoscopic grasper. CONCLUSION: The use of the small caliber ureteroscope can help in evaluation of the stricture caliber, length, and tissue quality. The scope can also aid in placing a guide wire, evaluating the posterior urethra, and screening for urethral or bladder stones. It can also improve the preoperative patient counselling and avoid unwanted surprises.

Corrigendum: Commensal bacteria make GPCR ligands that mimic human signalling molecules.

This corrects the article DOI: 10.1038/nature23874.

Integrating new approaches to atrial fibrillation management: the 6th AFNET/EHRA Consensus Conference.

There are major challenges ahead for clinicians treating patients with atrial fibrillation (AF). The population with AF is expected to expand considerably and yet, apart from anticoagulation, therapies used in AF have not been shown to consistently impact on mortality or reduce adverse cardiovascular events. New approaches to AF management, including the use of novel technologies and structured, integrated care, have the potential to enhance clinical phenotyping or result in better treatment selection and stratified therapy. Here, we report the outcomes of the 6th Consensus Conference of the Atrial Fibrillation Network (AFNET) and the European Heart Rhythm Association (EHRA), held at the European Society of Cardiology Heart House in Sophia Antipolis, France, 17-19 January 2017. Sixty-two global specialists in AF and 13 industry partners met to develop innovative solutions based on new approaches to screening and diagnosis, enhancing integration of AF care, developing clinical pathways for treating complex patients, improving stroke prevention strategies, and better patient selection for heart rate and rhythm control. Ultimately, these approaches can lead to better outcomes for patients with AF.

Intergenerational Transmission of Gene Regulatory Information in Caenorhabditis elegans.

Epigenetic mechanisms can stably maintain gene expression states even after the initiating conditions have changed. Often epigenetic information is transmitted only to daughter cells, but evidence is emerging, in both vertebrate and invertebrate systems, for transgenerational epigenetic inheritance (TEI), the transmission of epigenetic gene regulatory information across generations. Each new description of TEI helps uncover the properties, molecular mechanisms and biological roles for TEI. The nematode Caenorhabditis elegans has been particularly instrumental in the effort to understand TEI, as multiple environmental and genetic triggers can initiate an epigenetic signal that can alter the expression of both transgenes and endogenous loci. Here, we review recent studies of TEI in C. elegans.

SID-1 Domains Important for dsRNA Import in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) spreads systemically to cause gene silencing throughout the organism and its progeny. We confirm that Caenorhabditis nematode SID-1 orthologs have dsRNA transport activity and demonstrate that the SID-1 paralog CHUP-1 does not transport dsRNA. Sequence comparison of these similar proteins, in conjunction with analysis of loss-of-function missense alleles, identifies several conserved 2-7 amino acid microdomains within the extracellular domain (ECD) that are important for dsRNA transport. Among these missense alleles, we identify and characterize a sid-1 allele, qt95, which causes tissue-specific silencing defects most easily explained as a systemic RNAi export defect. However, we conclude from genetic and biochemical analyses that sid-1(qt95) disrupts only import, and speculate that the apparent export defect is caused by the cumulative effect of sequentially impaired dsRNA import steps. Thus, consistent with previous studies, we fail to detect a requirement for sid-1 in dsRNA export, but demonstrate for the first time that SID-1 functions in the intestine to support environmental RNAi (eRNAi).

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

Commensal bacteria make GPCR ligands that mimic human signalling molecules.

Commensal bacteria are believed to have important roles in human health. The mechanisms by which they affect mammalian physiology remain poorly understood, but bacterial metabolites are likely to be key components of host interactions. Here we use bioinformatics and synthetic biology to mine the human microbiota for N-acyl amides that interact with G-protein-coupled receptors (GPCRs). We found that N-acyl amide synthase genes are enriched in gastrointestinal bacteria and the lipids that they encode interact with GPCRs that regulate gastrointestinal tract physiology. Mouse and cell-based models demonstrate that commensal GPR119 agonists regulate metabolic hormones and glucose homeostasis as efficiently as human ligands, although future studies are needed to define their potential physiological role in humans. Our results suggest that chemical mimicry of eukaryotic signalling molecules may be common among commensal bacteria and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a possible small-molecule therapeutic modality (microbiome-biosynthetic gene therapy).

SID-1 Functions in Multiple Roles To Support Parental RNAi in Caenorhabditis elegans.

Systemic RNA interference (RNAi) in Caenorhbaditis elegans requires sid-1, sid-3, and sid-5 Injected, expressed, or ingested double-stranded RNA (dsRNA) is transported between cells, enabling RNAi in most tissues, including the germline and progeny (parental RNAi). A recent report claims that parental RNAi also requires the yolk receptor rme-2 Here, we characterize the role of the sid genes and rme-2 in parental RNAi. We identify multiple independent paths for maternal dsRNA to reach embryos and initiate RNAi. We showed previously that maternal and embryonic sid-1 contribute independently to parental RNAi. Here we demonstrate a role for embryonic sid-5, but not sid-2 or sid-3 in parental RNAi. We also find that maternal rme-2 contributes to but is not required for parental RNAi. We determine that parental RNAi by feeding occurs nearly exclusively in adults. We also introduce 5-ethynyluridine to densely internally label dsRNA, avoiding complications associated with other labeling strategies such as inhibition of normal dsRNA trafficking and separation of label and RNA. Labeling shows that yolk and dsRNA do not colocalize following endocytosis, suggesting independent uptake, and, furthermore, dsRNA appears to rapidly progress through the RAB-7 endocytosis pathway independently of sid-1 activity. Our results support the premise that although sid-1 functions in multiple roles, it alone is central and absolutely required for inheritance of silencing RNAs.

A novel composite two-stage urethroplasty for complex penile strictures: A multicenter experience.

INTRODUCTION: Complex penile strictures are usually repaired using a two-stage urethroplasty. Buccal mucosal graft (BMG) placed in the first stage can have a significant contraction rate, which may require a subsequent revision surgery. We describe a composite two-stage penile urethroplasty using BMG for patients of complex penile strictures who have some salvageable urethral plate. METHODS: Within a multi-institutional cohort, 82 patients underwent a two-stage urethroplasty for complex stricture of the penile urethra. Of these 42 patients who underwent our composite two-stage penile urethroplasty using BMG implanted at the second-stage were included. Patients with genital lichen sclerosus or incomplete clinical records were excluded from this study. The primary outcome of the study was to evaluate stricture-free success rate. RESULTS: Of total 42, 4 patients were lost to follow-up. 42% of stricture etiology was failed hypospadias repair. Mean stricture length was 4.5 cm (range 3-8 cm). Seventeen (44.7%) patients had undergone the previous urethroplasty. At a median follow-up of 44 months, of 38 patients, 34 (89.5%) were successful, and 4 (10.5%) had a recurrence. No patient required revision surgery before the second-stage and required redo buccal graft harvesting for subsequent urethroplasty. CONCLUSIONS: The composite two-stage technique in repairing complex penile urethral strictures is a valid and reproducible surgical treatment for complex penile stricture and it may reduce the rate of contraction of the transplanted BMG.

Early Developmental Exposure to dsRNA Is Critical for Initiating Efficient Nuclear RNAi in C. elegans.

RNAi has enabled researchers to study the function of many genes. However, it is not understood why some RNAi experiments succeed while others do not. Here, we show in C. elegans that pharyngeal muscle is resistant to RNAi when initially exposed to double-stranded RNA (dsRNA) by feeding but sensitive to RNAi in the next generation. Investigating this observation, we find that pharyngeal muscle cells as well as vulval muscle cells require nuclear rather than cytoplasmic RNAi. Further, we find in these cell types that nuclear RNAi silencing is most efficiently triggered during early development, defining a critical period for initiating nuclear RNAi. Finally, using heat-shock-induced dsRNA expression, we show that synMuv B class mutants act in part to extend this critical window. The synMuv-B-dependent early-development-associated critical period for initiating nuclear RNAi suggests that mechanisms that restrict developmental plasticity may also restrict the initiation of nuclear RNAi.

Stable Heritable Germline Silencing Directs Somatic Silencing at an Endogenous Locus.

The importance of transgenerationally inherited epigenetic states to organismal fitness remains unknown as well-documented examples are often not amenable to mechanistic analysis or rely on artificial reporter loci. Here we describe an induced silenced state at an endogenous locus that persists, at 100% transmission without selection, for up to 13 generations. This unusually persistent silencing enables a detailed molecular genetic analysis of an inherited epigenetic state. We find that silencing is dependent on germline nuclear RNAi factors and post-transcriptional mechanisms. Consistent with these later observations, inheritance does not require the silenced locus, and we provide genetic evidence that small RNAs embody the inherited silencing signal. Notably, heritable germline silencing directs somatic epigenetic silencing. Somatic silencing does not require somatic nuclear RNAi but instead requires both maternal germline nuclear RNAi and chromatin-modifying activity. Coupling inherited germline silencing to somatic silencing may enable selection for physiologically important traits.

Biogenesis, delivery, and function of extracellular RNA.

The Extracellular RNA (exRNA) Communication Consortium was launched by the National Institutes of Health to focus on the extent to which RNA might function in a non-cell-autonomous manner. With the availability of increasingly sensitive tools, small amounts of RNA can be detected in serum, plasma, and other bodily fluids. The exact mechanism(s) by which RNA can be secreted from cells and the mechanisms for the delivery and uptake by recipient cells remain to be determined. This review will summarize current knowledge about the biogenesis and delivery of exRNA and outline projects seeking to understand the functional impact of exRNA.

Medical costs of Alzheimer's disease misdiagnosis among US Medicare beneficiaries.

INTRODUCTION: Recent developments in diagnostic technology can support earlier, more accurate diagnosis of non-Alzheimer's disease (AD) dementias. METHODS: To evaluate potential economic benefits of early rule-out of AD, annual medical resource use and costs for Medicare beneficiaries potentially misdiagnosed with AD prior to their diagnosis of vascular dementia (VD) or Parkinson's disease (PD) were compared with that of similar patients never diagnosed with AD. RESULTS: Patients with prior AD diagnosis used substantially more medical services every year until their VD/PD diagnosis, resulting in incremental annual medical costs of approximately $9,500-$14,000. However, following their corrected diagnosis, medical costs converged with those of patients never diagnosed with AD. DISCUSSION: The observed correlation between timing of correct diagnosis and subsequent reversal in excess costs is strongly suggestive of the role of misdiagnosis of AD - rather than AD comorbidity - in this patient population. Our findings suggest potential benefits from earlier, accurate diagnosis.