Engineered tRNAs suppress nonsense mutations in cells and in vivo.

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2-7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into  efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP-sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

Mechanisms by which the cystic fibrosis transmembrane conductance regulator may influence SARS-CoV-2 infection and COVID-19 disease severity.

Patients with cystic fibrosis (CF) exhibit pronounced respiratory damage and were initially considered among those at highest risk for serious harm from SARS-CoV-2 infection. Numerous clinical studies have subsequently reported that individuals with CF in North America and Europe-while susceptible to severe COVID-19-are often spared from the highest levels of virus-associated mortality. To understand features that might influence COVID-19 among patients with cystic fibrosis, we studied relationships between SARS-CoV-2 and the gene responsible for CF (i.e., the cystic fibrosis transmembrane conductance regulator, CFTR). In contrast to previous reports, we found no association between CFTR carrier status (mutation heterozygosity) and more severe COVID-19 clinical outcomes. We did observe an unexpected trend toward higher mortality among control individuals compared with silent carriers of the common F508del CFTR variant-a finding that will require further study. We next performed experiments to test the influence of homozygous CFTR deficiency on viral propagation and showed that SARS-CoV-2 production in primary airway cells was not altered by the absence of functional CFTR using two independent protocols. On the contrary, experiments performed in vitro strongly indicated that virus proliferation depended on features of the mucosal fluid layer known to be disrupted by absent CFTR in patients with CF, including both low pH and increased viscosity. These results point to the acidic, viscous, and mucus-obstructed airways in patients with cystic fibrosis as unfavorable for the establishment of coronaviral infection. Our findings provide new and important information concerning relationships between the CF clinical phenotype and severity of COVID-19.

Discovery of New Isotope

The new isotope ^{241}U was synthesized and systematic atomic mass measurements of nineteen neutron-rich Pa-Pu isotopes were performed in the multinucleon transfer reactions of the ^{238}U+^{198}Pt system at the KISS facility. The present experimental results demonstrate the crucial role of the multinucleon transfer reactions for accessing unexplored neutron-rich actinide isotopes toward the N=152 shell gap in this region of nuclides.

Diagnostic test accuracy of D-dimer with or without a clinical decision rule in peripartum patients with suspected venous thromboembolism: A systematic review and meta-analysis.

BACKGROUND: Pregnancy and the peripartum period is a hypercoagulable state increasing the risk of venous thromboembolism (VTE). There may be a role in utilising D-dimer in the peripartum setting. AIMS: The purpose of this review was to summarise the latest evidence regarding the diagnostic accuracy of D-dimer in the peripartum setting with or without the addition of clinical decision rules. METHODS: We searched PubMed and CENTRAL databases to identify articles that included studies of women who had suspected VTE, underwent a D-dimer index test to rule out VTE and where radiological imaging or clinical follow-up, to a minimum of 30 days, was used as the reference standard. RESULTS: We included 11 studies in the systematic review and meta-analysis. The log diagnostic odds ratio (DOR) for identifying VTE using D-dimer was 1.56 (95% confidence interval (CI) 0.59-2.52). The pooled sensitivity was 87% (95% CI 76.8-93%), specificity was 63.2% (95% CI 47.1-76.7%), and the area under receiver operator characteristic (ROC) curves was 0.76. We included four studies evaluating D-dimer combined with YEARS to detect VTE. The log DOR for identifying VTE using D-dimer combined with YEARS was 1.13 (95% CI 0.005-2.25). The pooled sensitivity was 89.8% (95% CI 60.2-98.1%), specificity was 65.7% (95% CI 54.7-75.2%) and the area under ROC for studies included with the YEARS clinical decision rule was 0.49. CONCLUSION: This review highlighted that D-dimer use in the peripartum period for detection of VTE had a high sensitivity and high DOR but a poor area under ROC, which may limit its use in clinical practice.

Impact of tuberculosis on the incidence of osteoporosis and osteoporotic fractures: a nationwide population-based cohort study.

OBJECTIVES: Despite the high prevalence of tuberculosis (TB) and the disease burden of osteoporosis and osteoporotic fractures, there is still a lack of well-designed, large-scale studies demonstrating associations among them. We aimed to investigate the effect of TB on the incidence of osteoporosis and osteoporotic fractures. STUDY DESIGN: This was a nationwide population-based cohort study. METHODS: This study was conducted using the National Health Insurance Service Database of South Korea. We included patients with newly diagnosed TB aged >40 years from January 2006 to December 2017. An uninfected control for each TB patient was randomly extracted by frequency matching for sex, age, income level, residence, and registration date at a 2:1 ratio. The primary outcome was the incidence of osteoporosis and osteoporotic fractures between the two groups, adjusted for sex, age, income level, residence, comorbidities, body mass index, blood pressure, laboratory tests, alcohol drinking, and smoking. The risk factors associated with osteoporosis or osteoporotic fractures were also investigated. RESULTS: A total of 164,389 patients with TB and 328,778 matched controls were included (71.9% males). The mean duration of follow-up was 7.00 ± 3.49 years. The incidence of osteoporosis in patients with TB was 6.1 cases per 1000 person-years, which was significantly higher than that in matched controls (adjusted hazard ratio [aHR] 1.349, 95% confidence interval [CI] 1.302-1.398, P < 0.001). The incidence of osteoporotic fractures was also higher in patients with TB than in controls (aHR 1.392, 95% CI 1.357-1.428, P < 0.001). Among fractures, the risk of hip fracture was the highest (aHR 1.703, 95% CI 1.612-1.798, P < 0.001). CONCLUSIONS: TB independently contributes to the incidence of osteoporosis and osteoporotic fractures, particularly hip fractures.

The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue.

Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (non-native) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the "lasso" helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair.

Correlation of polyp grading scales with patient symptom scores and olfaction in chronic rhinosinusitis: a systematic review and meta-analysis.

BACKGROUND: Various nasal polyp (NP) scoring systems have been proposed and used in the literature. However, no single system has been identified as superior. Correlations between NP scoring systems and patient symptoms, quality of life (QOL) or olfaction vary widely. METHODS: A systematic search of PubMed, CINAHL, Scopus, and Cochrane Library was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-analyses guideline. Any study examining endoscopy scores and symptom, QOL or olfaction measures in cross sectional manner or after therapeutic intervention were included. RESULTS: This review identified 55 studies for a pooled meta-analysis of Lund-Kennedy (LK-NP) polyp scores (N = 6), Meltzer scores (N = 6), Nasal polyp scores (NPS; N = 19), Total polyp score (TPS; N=8) Lilholdt scores (N = 8), Olfactory cleft endoscopy score (OCES; N =4), Discharge, inflammation, polyp/edema score (DIP; N = 2), and Perioperative sinus endoscopy score (POSE; N = 2). Meta-regression assessed correlations between NP grading systems and SNOT-22, nasal congestion scores, total nasal symptom scores (TNSS), and Smell Identification Test-40 (SIT40). None of the NP grading systems correlated significantly with any symptom, QOL or olfactory metric. In intervention studies of surgery or monoclonal antibody treatment, changes in NPS scores did not correlate with any patient reported outcome measure (PROM) or olfactory outcomes. CONCLUSION: Current NP endoscopic scoring systems are not associated with PROMs such as SNOT-22, nasal congestion scores, and TNSS as well as objective measures of olfaction. NP grading systems with improved clinical utility are needed.

Correlation of polyp grading scales with patient symptom scores and olfaction in chronic rhinosinusitis: a systematic review and meta-analysis.

BACKGROUND: Various nasal polyp (NP) scoring systems have been proposed and used in the literature. However, no single system has been identified as superior. Correlations between NP scoring systems and patient symptoms, quality of life (QOL) or olfaction vary widely. METHODS: A systematic search of PubMed, CINAHL, Scopus, and Cochrane Library was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-analyses guideline. Any study examining endoscopy scores and symptom, QOL or olfaction measures in cross sectional manner or after therapeutic intervention were included. RESULTS: This review identified 55 studies for a pooled meta-analysis of Lund-Kennedy (LK-NP) polyp scores (N = 6), Meltzer scores (N = 6), Nasal polyp scores (NPS; N = 19), Total polyp score (TPS; N=8) Lilholdt scores (N = 8), Olfactory cleft endoscopy score (OCES; N =4), Discharge, inflammation, polyp/edema score (DIP; N = 2), and Perioperative sinus endoscopy score (POSE; N = 2). Meta-regression assessed correlations between NP grading systems and SNOT-22, nasal congestion scores, total nasal symptom scores (TNSS), and Smell Identification Test-40 (SIT40). None of the NP grading systems correlated significantly with any symptom, QOL or olfactory metric. In intervention studies of surgery or monoclonal antibody treatment, changes in NPS scores did not correlate with any patient reported outcome measure (PROM) or olfactory outcomes. CONCLUSION: Current NP endoscopic scoring systems are not associated with PROMs such as SNOT-22, nasal congestion scores, and TNSS as well as objective measures of olfaction. NP grading systems with improved clinical utility are needed.

Anterior resection syndrome: a randomized clinical trial of a 5-HT3 receptor antagonist (ramosetron) in male patients with rectal cancer.

BACKGROUND: No effective treatment exists for anterior resection syndrome (ARS) following sphincter-saving surgery for rectal cancer. This RCT assessed the safety and efficacy of a 5-HT3 receptor antagonist, ramosetron, for ARS. METHODS: A single-centre, randomized, controlled, open-label, parallel group trial was conducted. Male patients with ARS 1 month after rectal cancer surgery or ileostomy reversal were enrolled and randomly assigned (1 : 1) to 5 µg of ramosetron (Irribow®) daily or conservative treatment for 4 weeks. Low ARS (LARS) score was calculated after randomization and 4 weeks after treatment. The study was designed as a superiority test with a primary endpoint of the proportion of patients with major LARS between the groups. Primary outcome analysis was based on the modified intention-to-treat population. Safety was assessed by monitoring adverse events during the study. RESULTS: : A total of 100 patients were randomized to the ramosetron (49 patients) or conservative treatment group (51 patients). Two patients were excluded, and 48 and 50 patients were analysed in the ramosetron and control groups, respectively. The proportion of major LARS after 4 weeks was 58 per cent (28 of 48 patients) in the ramosetron group versus 82 per cent (41 of 50 patients) in the control group, with a difference of 23.7 per cent (95 per cent c.i. 5.58 to 39.98, P = 0.011). There were minor adverse events in five patients, which were hard stool, frequent stool or anal pain. These were not different between the two groups. There were no serious adverse events. CONCLUSION: : Ramosetron could be safe and feasible for male patients with ARS. TRIAL REGISTRATION NUMBER: NCT02869984 (http://www.clinicaltrials.gov).

Subtrochanteric atypical femoral fracture induced solely by glucocorticoid without bisphosphonate treatment: a case report.

An AFF is a form of stress fracture induced by excessive physiologic repetitive stress over the bone remodeling capacity. Although glucocorticoid administration is a known risk factor for AFF, no case of AFF with glucocorticoid administration as the only risk factor has been previously reported. In this report, we aimed to highlight the risk of AFF associated with long-term administration of glucocorticoids, and the importance of surveillance and correction of risk factors in patients undergoing long-term glucocorticoid therapy. A 58-year-old male patient was diagnosed with subtrochanteric AFF. He had no medical history of any condition that might disrupt bone metabolism and no known risk factors for AFF, except for long-term administration of glucocorticoid. After fixation of the fracture, the glucocorticoid was replaced with an alternative medication. Although AFF is notorious for delayed union or nonunion, complete union of the fracture was obtained at 14 months postoperatively. This case brought to our attention the possibility that glucocorticoids alone may be responsible for inducing AFF and highlighted the importance of regular assessments in case of necessity of glucocorticoid administration. Additionally, correction of risk factors might expedite the union of AFF.

Stability Prediction for Mutations in the Cytosolic Domains of Cystic Fibrosis Transmembrane Conductance Regulator.

Cystic Fibrosis (CF) is caused by mutations to the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. CFTR is composed of two membrane spanning domains, two cytosolic nucleotide-binding domains (NBD1 and NBD2) and a largely unstructured R-domain. Multiple CF-causing mutations reside in the NBDs and some are known to compromise the stability of these domains. The ability to predict the effect of mutations on the stability of the cytosolic domains of CFTR and to shed light on the mechanisms by which they exert their effect is therefore important in CF research. With this in mind, we have predicted the effect on domain stability of 59 mutations in NBD1 and NBD2 using 15 different algorithms and evaluated their performances via comparison to experimental data using several metrics including the correct classification rate (CCR), and the squared Pearson correlation (R2) and Spearman's correlation (ρ) calculated between the experimental ΔTm values and the computationally predicted ΔΔG values. Overall, the best results were obtained with FoldX and Rosetta. For NBD1 (35 mutations), FoldX provided R2 and ρ values of 0.64 and -0.71, respectively, with an 86% correct classification rate (CCR). For NBD2 (24 mutations), FoldX R2, ρ, and CCR were 0.51, -0.73, and 75%, respectively. Application of the Rosetta high-resolution protocol (Rosetta_hrp) to NBD1 yielded R2, ρ, and CCR of 0.64, -0.75, and 69%, respectively, and for NBD2 yielded R2, ρ, and CCR of 0.29, -0.27, and 50%, respectively. The corresponding numbers for the Rosetta's low-resolution protocol (Rosetta_lrp) were R2 = 0.47, ρ = -0.69, and CCR = 69% for NBD1 and R2 = 0.27, ρ = -0.24, and CCR = 63% for NBD2. For NBD1, both algorithms suggest that destabilizing mutations suffer from destabilizing vdW clashes, whereas stabilizing mutations benefit from favorable H-bond interactions. Two triple consensus approaches based on FoldX, Rosetta_lpr, and Rosetta_hpr were attempted using either "majority-voting" or "all-voting". The all-voting consensus outperformed the individual predictors, albeit on a smaller data set. In summary, our results suggest that the effect of mutations on the stability of CFTR's NBDs could be largely predicted. Since NBDs are common to all ABC transporters, these results may find use in predicting the effect and mechanism of the action of multiple disease-causing mutations in other proteins.

Comparison of five membrane filters to collect bioaerosols for airborne microbiome analysis.

AIMS: Recovering DNA of airborne micro-organisms (AM) from air is a challenging task. We compared five membrane filters for bioaerosol sampling-mixed cellulose ester (MCE), polyethersulfone (PES), polyamide (PA), polytetrafluorethylene (PTFE) and polyvinylidene fluoride (PVDF)-based on their bacterial, fungal and eukaryotic DNA recoveries. METHODS AND RESULTS: Bacterial, fungal and eukaryotic populations were quantified using quantitative PCR. With a bacterial consortium, PTFE exhibited the best recovery efficiency (113%), followed by PA (92%), PES (86%), MCE (48%) and PVDF (1%). When filters were compared with air, PA was used as a control to normalize results from the others. The bacterial, fungal and eukaryotic DNA recovery ratios were markedly greater in PES (9·3, 11·5 and 10·3 respectively) than in the remaining. Eukaryotic MiSeq sequencing revealed that PES recovered a more diverse and considerably richer assemblage (richness ratios, 4·97 vs ≤ 1·16 for PES vs the others). Rank abundance distribution analysis showed that distribution tails were longer (>4 times) in PES, but these did not differ between the remaining and PA. Community comparison showed that PES exhibited a lower variation across trials than the PA, while the remaining did not. CONCLUSIONS: PES filter markedly outperformed the other filters in quantitative and qualitative recovery of AM. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings demonstrated the importance of filter selection for sampling AM.

All-cause mortality, cardiovascular mortality, and incidence of cardiovascular disease according to a screening program of cardiovascular risk in South Korea among young adults: a nationwide cohort study.

OBJECTIVES: We aimed to determine whether there are any differences in all-cause and cause-specific mortality with cardiovascular disease (CVD) risk between health screening attenders and non-attenders among young adults. STUDY DESIGN: We performed a retrospective cohort study using claim data from the Korean National Health Insurance Service database. METHODS: Individuals aged 20-39 years who had received health screening at least once between 2002 and 2005 were classified as attenders, and the others were classified as non-attenders. After propensity score matching according to attendance of health screening, 2,060,409 attenders and 2,060,409 non-attenders were included. We estimated adjusted hazard ratios (HRs) and 95% confidence interval (CI) for all-cause mortality, cause-specific mortality, and hospitalization of CVD from 2006 to 2015. RESULTS: Survival from all-cause mortality was greater among attenders than among non-attenders (log rank P < 0.001). Similarly, death from CVD (log rank P = 0.007) and CVD events (log rank P < 0.001) were less likely among attenders. The risk for all-cause mortality in attenders was significantly lower than that in non-attenders (HR = 0.83, 95% CI = 0.81 to 0.84). The risk for CVD mortality (HR = 0.80, 95% CI = 0.73 to 0.87) and hospitalization of CVD (HR = 0.92, 95% CI = 0.91 to 0.94) were lower in attenders. In stratified analyses, the risk for all-cause and cause-specific mortalities was lower among attenders regardless of insurance type. CONCLUSIONS: Among young adults, the risk for all-cause mortality, CVD mortality, and hospitalization of CVD were lower for those who underwent health screenings. Future studies that evaluate the cost-effectiveness of health screening with additional consideration of psychosocial aspects are needed.

G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations.

G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (NBD2) and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (WT) background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double charge-reversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity, and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations.

Treatment with K6PC-5, a selective stimulator of SPHK1, ameliorates intestinal homeostasis in an animal model of Huntington's disease.

Emerging evidence indicates that Huntington's disease (HD) may be described as multi-organ pathology. In this context, we and others have contributed to demonstrate that the disease is characterized by an impairment of the homeostasis of gastro-intestinal (GI) tract. Sphingolipids represent a class of molecules involved in the regulation and maintenance of different tissues and organs including GI system. In this study, we investigated whether the alteration of Sphingosine-1-phosphate (S1P) metabolism, previously described in human HD brains and animal models, is also detectable peripherally in R6/2 HD mice. Our findings indicate, for the first time, that sphingolipid metabolism is perturbed early in the disease in the intestinal tract of HD mice and, its modulation by K6PC-5, a selective activator of S1P synthesis, preserved intestinal integrity and homeostasis. These results further support the evidence that modulation of sphingolipid pathways may represent a potential therapeutic option in HD and suggest that it has also the potential to counteract the peripheral disturbances which may usually complicate the management of the disease and affect patient's quality of life.

Laparoscopic sentinel node navigation surgery versus laparoscopic gastrectomy with lymph node dissection for early gastric cancer: short-term outcomes of a multicentre randomized controlled trial (SENORITA).

BACKGROUND: Sentinel node navigation surgery reduces the extent of gastric and lymph node dissection, and may improve quality of life. The benefit and harm of laparoscopic sentinel node navigation surgery (LSNNS) for early gastric cancer is unknown. The SENORITA (SEntinel Node ORIented Tailored Approach) trial investigated the pathological and surgical outcomes of LSNNS compared with laparoscopic standard gastrectomy (LSG) with lymph node dissection. METHODS: The SENORITA trial was an investigator-initiated, open-label, parallel-assigned, non-inferiority, multicentre RCT conducted in Korea. The primary endpoint was 3-year disease-free survival. The secondary endpoints, morbidity and mortality within 30 days of surgery, are reported in the present study. RESULTS: A total of 580 patients were randomized to LSG (292) or LSNNS (288). Surgery was undertaken in 527 patients (LSG 269, LSNNS 258). LSNNS could be performed according to the protocol in 245 of 258 patients, and a sentinel node basin was detected in 237 (96·7 per cent) Stomach-preserving surgery was carried out in 210 of 258 patients (81·4 per cent). Postoperative complications occurred in 51 patients in the LSG group (19·0 per cent) and 40 (15·5 per cent) in the LSNNS group (P = 0·294). Complications with a Clavien-Dindo grade of III or higher occurred in 16 (5·9 per cent) and 13 (5·0 per cent) patients in the LSG and LSNNS groups respectively (P = 0·647). CONCLUSION: The rate and severity of complications following LSNNS for early gastric cancer are comparable to those after LSG with lymph node dissection. Registration number: NCT01804998 ( http://www.clinicaltrials.gov).

ANTECEDENTES: La cirugía de navegación del ganglio centinela (sentinel node navigation surgery, SNNS) reduce la extensión de la resección gástrica y ganglionar, y puede mejorar la calidad de vida. Se desconoce el beneficio y el daño de la cirugía de navegación del ganglio centinela por vía laparoscópica (laparoscopic sentinel node navigation surgery, LSNNS) para el cáncer gástrico precoz. El ensayo clínico SENORITA investigó los resultados patológicos y quirúrgicos de LSNNS en comparación con la gastrectomía laparoscópica estándar (laparoscopic gastrectomy, LSG) con disección ganglionar (lymph node dissection, LND). MÉTODOS: El ensayo SENORITA fue un ensayo multicéntrico aleatorizado y controlado, iniciado por investigadores, abierto, con asignación a grupos paralelos y de no inferioridad llevado a cabo en Corea. El resultado primario fue la supervivencia libre de enfermedad a los 3 años. En el presente estudio, se describen los resultados secundarios correspondientes a morbilidad y mortalidad a los 30 días del postoperatorio. RESULTADOS: Un total de 580 pacientes fueron aleatorizados a LG (n = 292) o LSNNS (n = 288). La cirugía se realizó en 527 pacientes (LG 269, LSNNS 258). LSNNS pudo ser realizada de acuerdo con el protocolo en 245 de 258 pacientes y en 237 de 245 pacientes (96,7%) se detectó un ganglio centinela. La cirugía con preservación del estómago se realizó en 210 de 258 pacientes (81,4%). Las complicaciones postoperatorias se presentaron en 51 pacientes del grupo LSG (19,0%) y en 40 pacientes (15,5%) del grupo LSNNS (P = 0,294). Las complicaciones grado III o mayor de Clavien-Dindo se detectaron en 16 (5,9%) y 13 pacientes (5,0%) de los grupos LSG y LSNNS, respectivamente (P = 0,647). CONCLUSIÓN: El porcentaje y la gravedad de las complicaciones tras LSNNS para cancer gástrico precoz son comparables a la LSG con LND.

Pooled cohort risk equation and subclinical cerebrovascular diseases.

BACKGROUND AND PURPOSE: In 2013, the American College of Cardiology/American Heart Association (ACC/AHA) introduced a novel pooled cohort risk (PCR) model for atherosclerotic cardiovascular disease. In this study, we evaluated the relationship between the PCR score and cerebral large- and small-vessel diseases (cLVD and cSVD) in a healthy population, METHODS: We assessed consecutive health check-up volunteers from 2006 to 2013. We calculated the estimated 10-year atherosclerotic cardiovascular disease risk as the PCR score based on the 2013 ACC/AHA guidelines. We evaluated both cSVD/cLVD, including the prevalence of cLVD, lacunes and cerebral microbleed (CMB), and the volume of white matter hyperintensity (WMH). In addition to PCR score, the risk factors that were associated with outcome variables at P < 0.10 in univariate analysis were included for further multivariable linear or regression analyses. RESULTS: A total of 2720 participants were evaluated (mean age, 57 years, male sex, 54%). In multivariable analysis, PCR score was associated with WMH volume [ß = 0.361; 95% confidence interval (CI), 0.320-0.402, P < 0.001], cLVD [adjusted odds ratio (aOR), 1.66; 95% CI, 1.29-2.16, P < 0.001], lacunes (aOR, 1.80; 95% CI, 1.52-2.14, P < 0.001) and CMBs (aOR, 1.75; 95% CI, 1.40-2.19, P < 0.001). Furthermore, PCR score also showed dose-response tendencies according to the burden of cLVD, WMH, lacunes and CMB. CONCLUSIONS: A higher PCR score based on the ACC/AHA guidelines is closely associated with a higher prevalence and burden of cLVD and cSVD.

High-throughput analysis of genes involved in biocontrol performance of Pseudomonas fluorescens NBC275 against Gray mold.

AIMS: Many physiological and microbial characteristics influence the biocontrol performance of the biological control agents (BCAs) in agricultural fields. To implement effective biocontrol, the contribution of specific genes, mechanisms and traits to the biocontrol performance of BCAs need to be characterized and explored in greater detail. METHODS AND RESULTS: In this study, a transposon (Tn) mutant library using the BCA Pseudomonas fluorescens NBC275 (Pf275) was generated to explore genes and bacterial characteristics involved in antifungal activity and biocontrol performance. Among the Tn mutants, 205 strains showing variations in antifungal activity compared to wild-type (WT) were selected and further analysed for biocontrol efficacy against gray mold in pepper fruits. The genes involved in pyoverdine biosynthesis (pvdI and pvdD) and chitin-binding protein (gbpA) played essential roles in the antifungal activity and biocontrol capacity of Pf275. In addition, a mutation in phlD completely abolished the antifungal activity and significantly suppressed the biocontrol ability of the strain. Genes affecting antifungal activity of Pf275 significantly influenced swimming motility, which was identified as an important trait for the biocontrol ability of the bacterial strain. CONCLUSIONS: Overall, our results suggest that antifungal compound production, siderophore biosynthesis and swimming motility synergistically contribute to Pf275 biocontrol performance. The utility of this library was demonstrated by identifying genes for antagonism and biocontrol ability in this BCA strain. The functional roles of many genes identified as contributing to antagonism and in vivo biocontrol activity require further study. SIGNIFICANCE AND IMPACT OF THIS STUDY: Genes contributing to antifungal activity and biocontrol performance of P. fluorescens were identified and highlighted by Tn mutagenesis, which will give insight to improve the biocontrol performance of this BCA.

Effects of quorum quenching on temporal succession of activated sludge microbial community in a membrane bioreactor.

AIMS: Quorum quenching (QQ) is an attractive strategy for mitigating biofouling in membrane bioreactors (MBRs). However, the effects of QQ on the activated sludge (AS) process have not been adequately evaluated. This study investigated the long-term effects of QQ on a laboratory-scale anoxic-oxic MBR, focusing on AS performance and microbial community. METHODS AND RESULTS: Anoxic-oxic MBRs with and without QQ were operated for 91 days. QQ did not affect COD and TN removal efficiencies over the experimental period, during which its activity remained >90%. QQ reduced floc size by approximately 8% but had no effect on biomass concentration. AS microbial communities were regularly analysed using massively parallel sequencing. AS bacterial communities were temporally dynamic irrespective of QQ presence, for example, a temporal increase in bacterial diversity and a temporal decay of community similarity. QQ counteracted the temporal change in diversity and the temporal distance-community decay. Community comparison revealed that QQ changed the successional trajectory of the AS community at a late period, because it decelerated temporal changes of specific members, such as Thiothrix and Sphingomonadaceae*. Correlation networks revealed that QQ increased network clustering, complexity and density. The combined results suggest that the tighter microbial association by QQ increased the community resistance. CONCLUSIONS: QQ can enhance the diversity and stability of the AS community in MBR by counteracting the innate temporal change in community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings are useful for the further advancement of QQ-based strategies in engineered microbial environments.