Lrh1 can help reprogram sexual cell fate and is required for Sertoli cell development and spermatogenesis in the mouse testis.

The mammalian nuclear hormone receptors LRH1 (NR5A2) and SF1 (NR5A1) are close paralogs that can bind the same DNA motif and play crucial roles in gonadal development and function. Lrh1 is essential for follicle development in the ovary and has been proposed to regulate steroidogenesis in the testis. Lrh1 expression in the testis is highly elevated by loss of the sex regulator Dmrt1, which triggers male-to-female transdifferentiation of Sertoli cells. While Sf1 has a well-defined and crucial role in testis development, no function for Lrh1 in the male gonad has been reported. Here we use conditional genetics to examine Lrh1 requirements both in gonadal cell fate reprogramming and in normal development of the three major cell lineages of the mouse testis. We find that loss of Lrh1 suppresses sexual transdifferentiation, confirming that Lrh1 can act as a key driver in reprogramming sexual cell fate. In otherwise wild-type testes, we find that Lrh1 is dispensable in Leydig cells but is required in Sertoli cells for their proliferation, for seminiferous tubule morphogenesis, for maintenance of the blood-testis barrier, for feedback regulation of androgen production, and for support of spermatogenesis. Expression profiling identified misexpressed genes likely underlying most aspects of the Sertoli cell phenotype. In the germ line we found that Lrh1 is required for maintenance of functional spermatogonia, and hence mutants progressively lose spermatogenesis. Reduced expression of the RNA binding factor Nxf2 likely contributes to the SSC defect. Unexpectedly, however, over time the Lrh1 mutant germ line recovered abundant spermatogenesis and fertility. This finding indicates that severe germ line depletion triggers a response allowing mutant spermatogonia to recover the ability to undergo complete spermatogenesis. Our results demonstrate that Lrh1, like Sf1, is an essential regulator of testis development and function but has a very distinct repertoire of functions.

Relationship between serum ß-hydroxybutyrate and hepatic fatty acid oxidation in individuals with obesity and NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation that can progress to inflammation (nonalcoholic steatohepatitis, NASH), and fibrosis. Serum ß-hydroxybutyrate (ß-HB), a product of the ketogenic pathway, is commonly used as a surrogate marker for hepatic fatty acid oxidation (FAO). However, it remains uncertain whether this relationship holds true in the context of NAFLD in humans. We compared fasting serum ß-HB levels with direct measurement of liver mitochondrial palmitate oxidation in humans stratified based on NAFLD severity (n = 142). Patients were stratified based on NAFLD activity score (NAS): NAS = 0 (no disease), NAS = 1-2 (mild), NAS = 3-4 (moderate), and NAS ≥ 5 (advanced). Moderate and advanced NAFLD is associated with reductions in liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), serum ß-HB, but not 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) mRNA, relative to no disease. Worsening liver mitochondrial complete palmitate oxidation corresponded with lower HMGCS2 mRNA but not total (complete + incomplete) palmitate oxidation. Interestingly, we found that liver HMGCS2 mRNA and serum ß-HB correlated with liver mitochondrial ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activity and CPT1A mRNA. Also, lower mitochondrial mass and markers of mitochondrial turnover positively correlated with lower HMGCS2 in the liver. These data suggest that liver ketogenesis and FAO occur at comparable rates in individuals with NAFLD. Our findings support the utility of serum ß-HB to serve as a marker of liver injury and hepatic FAO in the context of NAFLD.NEW & NOTEWORTHY Serum ß-hydroxybutyrate (ß-HB) is frequently utilized as a surrogate marker for hepatic fatty acid oxidation; however, few studies have investigated this relationship during states of liver disease. We found that the progression of nonalcoholic fatty liver disease (NAFLD) is associated with reductions in circulating ß-HB and liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). As well, decreased rates of hepatic fatty acid oxidation correlated with liver HMGCS2 mRNA and serum ß-HB. Our work supports serum ß-HB as a potential marker for hepatic fatty acid oxidation and liver injury during NAFLD.

Neuraminidase-induced externalization of phosphatidylserine activates ADAM17 and impairs insulin signaling in endothelial cells.

Endothelial insulin resistance represents a causal factor in the pathogenesis of type 2 diabetes (T2D) and vascular disease, thus the need to identify molecular mechanisms underlying defects in endothelial insulin signaling. We previously have shown that a disintegrin and metalloproteinase-17 (ADAM17) is increased while insulin receptor α-subunit (IRα) is decreased in the vasculature of patients with T2D, leading to impaired insulin-induced vasodilation. We have also demonstrated that ADAM17 sheddase activity targets IRα; however, the mechanisms driving endothelial ADAM17 activity in T2D are largely unknown. Herein, we report that externalization of phosphatidylserine (PS) to the outer leaflet of the plasma membrane causes ADAM17-mediated shedding of IRα and blunting of insulin signaling in endothelial cells. Furthermore, we demonstrate that endothelial PS externalization is mediated by the phospholipid scramblase anoctamin-6 (ANO6) and that this process can be stimulated by neuraminidase, a soluble enzyme that cleaves sialic acid residues. Of note, we demonstrate that men and women with T2D display increased levels of neuraminidase activity in plasma, relative to age-matched healthy individuals, and this occurs in conjunction with increased ADAM17 activity and impaired leg blood flow responses to endogenous insulin. Collectively, this work reveals the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.NEW & NOTEWORTHY This work provides the first evidence that neuraminidase, an enzyme increased in the circulation of men and women with type 2 diabetes (T2D), promotes anoctamin-6 (ANO6)-dependent externalization of phosphatidylserine in endothelial cells, which in turn leads to activation of a disintegrin and metalloproteinase-17 (ADAM17) and consequent shedding of the insulin receptor-α from the cell surface. Hence, this work supports that consideration should be given to the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.

Propensity score matched analysis of laparoscopic revisional and conversional sleeve gastrectomy with concurrent hiatal hernia repair.

INTRODUCTION: The primary aim of this study was to evaluate outcomes associated with concurrent hiatal hernia repair (CHHR) when performing a conversional or revisional vertical sleeve gastrectomy (VSG). CHHR is often necessary during VSG due to potential gastroesophageal reflux disease (GERD) development or obstructive symptoms. METHODS: The Metabolic and Bariatric Surgery Accreditation and Quality Improvement (MBSAQIP) participant use file was assessed for the years 2015-2020 for revisional/conversional VSG procedures. The presence of CHHR was used to create two groups. Propensity score matching (PSM) was performed with E-analysis. RESULTS: There were 33,909 patients available, with 5986 undergoing the VSG procedure with CHHR. In the unmatched analysis, there was an increased frequency of patients being female (85.72 vs 83.30%; p < 0.001), having a history of GERD (38.01 vs 31.25%; p < 0.001), and being of older age (49.59 ± 10.97 vs 48.70 ± 10.83; p < 0.001). Patients undergoing VSG with CHHR experienced decreased sleep apnea (25.00 vs 28.84%; p < 0.001) and diabetes (14.27 vs 17.80%; p < 0.001). PSM yielded 5986 patient pairs. Matched patients with CHHR experienced increased operative time (115 min ± 53 vs 103 min ± 51; p < 0.001), increased risk of postoperative pneumonia (0.45 vs 0.15%; p = 0.005) and readmission (4.69 vs 3.58%; p = 0.002) within thirty days. However, patients undergoing CHHR with revisional or conversional VSG did not experience increased risk of death, postoperative bleeding, postoperative leak, or reoperations. CONCLUSION: Despite a small association with increased postoperative pneumonia, the rate of complications in patients undergoing laparoscopic revisional/conversional VSG and CHHR are low. CHHR is a safe option when combined with the laparoscopic revisional/conversional VSG procedure in the early postoperative period.

Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD.

BACKGROUND AND AIMS: NAFLD and its more-advanced form, steatohepatitis (NASH), is associated with obesity and is an independent risk factor for cardiovascular, liver-related, and all-cause mortality. Available human data examining hepatic mitochondrial fatty acid oxidation (FAO) and hepatic mitochondrial turnover in NAFLD and NASH are scant. APPROACH AND RESULTS: To investigate this relationship, liver biopsies were obtained from patients with obesity undergoing bariatric surgery and data clustered into four groups based on hepatic histopathological classification: Control (CTRL; no disease); NAFL (steatosis only); Borderline-NASH (steatosis with lobular inflammation or hepatocellular ballooning); and Definite-NASH (D-NASH; steatosis, lobular inflammation, and hepatocellular ballooning). Hepatic mitochondrial complete FAO to CO2 and the rate-limiting enzyme in ß-oxidation (ß-hydroxyacyl-CoA dehydrogenase activity) were reduced by ~40%-50% with D-NASH compared with CTRL. This corresponded with increased hepatic mitochondrial reactive oxygen species production, as well as dramatic reductions in markers of mitochondrial biogenesis, autophagy, mitophagy, fission, and fusion in NAFL and NASH. CONCLUSIONS: These findings suggest that compromised hepatic FAO and mitochondrial turnover are intimately linked to increasing NAFLD severity in patients with obesity.

Evolution of mitochondrial and nuclear genomes in Pennatulacea.

We examine the phylogeny of sea pens using sequences of whole mitochondrial genomes and the nuclear ribosomal cluster generated through low coverage Illumina sequencing. Taxon sampling includes 30 species in 19 genera representing 13 families. Ancestral state reconstruction shows that most sea pen mitochondrial genomes have the ancestral gene order, and that Pennatulacea with diverse gene orders are found in a single clade. The monophyly of Pennatulidae and Protoptilidae are rejected by both the mitochondrial and nuclear dataset, while the mitochondrial dataset further rejects monophyly of Virgulariidae, and the nuclear dataset rejects monophyly of Kophobelemnidae. We show discordance between nuclear ribosomal gene cluster phylogenies and whole mitochondrial genome phylogenies and highlight key Pennatulacea taxa that could be included in cnidarian genome-wide studies to better resolve the sea pen tree of life. We further illustrate how well frequently sequenced markers capture the overall diversity of the mitochondrial genome and the nuclear ribosomal genes in sea pens.

Isotope Labeling and Biochemical Assessment of Liver-Triacylglycerol in Patients with Different Levels of Histologically-Graded Liver Disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) prevalence is rapidly growing, and fatty liver has been found in a quarter of the US population. Increased liver lipids, particularly those derived from the pathway of de novo lipogenesis (DNL), have been identified as a hallmark feature in individuals with high liver fat. This has led to much activity in basic science and drug development in this area. No studies to date have investigated the contribution of DNL across a spectrum of disease, although it is clear that inhibition of DNL has been shown to reduce liver fat. OBJECTIVES: The purpose of this study was to determine whether liver lipid synthesis increases across the continuum of liver injury. METHODS: Individuals (n = 49) consumed deuterated water for 10 d before their scheduled bariatric surgeries to label DNL; blood and liver tissue samples were obtained on the day of the surgery. Liver lipid concentrations were quantitated, and levels of protein and gene expression assessed. RESULTS: Increased liver DNL, measured isotopically, was significantly associated with liver fatty acid synthase protein content (R = 0.470, P = 0.003), total steatosis assessed by histology (R = 0.526, P = 0.0008), and the fraction of DNL fatty acids in plasma very low-density lipoprotein-triacylglycerol (R = 0.747, P < 0.001). Regression analysis revealed a parabolic relationship between fractional liver DNL (percent) and NAFLD activity score (R = 0.538, P = 0.0004). CONCLUSION: These data demonstrate that higher DNL is associated with early to mid stages of liver disease, and this pathway may be an effective target for the treatment of NAFLD and nonalcoholic steatohepatitis. This study was registered at clinicaltrials.gov as NCT03683589.

Concurrent paraesophageal hernia repair in revisional/conversional laparoscopic Roux-en-Y gastric bypass: propensity score-matched analysis of the MBSAQIP database.

BACKGROUND: Patients requiring concurrent paraesophageal hernia repair (CPHR) have been shown to have favorable outcomes in primary bariatric surgery. However, patients requiring revisional or conversional surgery represent a group of patients with higher perioperative risk. Currently, few reports on concurrent paraesophageal hernia repair utilizing the Metabolic and Bariatric Surgery Accreditation and Quality Improvement Program (MBSAQIP) database are available. The primary aim of this study was to determine perioperative complications associated with CPHR and the Roux-en-Y gastric bypass (RYGB) as a revisional/conversional operation. METHODS: In this study, patients undergoing revisional/conversional RYGB between 2015 and 2020 were accessed via the MBSAQIP database. Patients were categorized based on the presence of a paraesophageal hernia as a concurrent procedure. Patients who underwent revisional/conversional surgery without additional procedures were utilized for controls. A propensity score-matched cohort was generated and E-analysis utilized to assess unmeasured confounding. RESULTS: After exclusions, 35,698 patients were available. Patients receiving CPHR were more likely to be female (90.79% vs 87.37%; p < 0.001) and have increased frequency of gastroesophageal reflux disease (69.20% vs 51.69%; p < 0.001). However, these patients had lower frequencies of sleep apnea (24.12% vs 30.13%; p < 0.001), hypertension requiring medication (38.51% vs 42.59%; p < 0.001), and decreased frequency of hyperlipidemia (19.44% vs 21.60%;p < 0.001). After matching, 6,231 patient pairs were developed and showed that patients undergoing CPHR were at increased risk of readmission (9.44% vs 7.58%; p < 0.001), intervention (3.56% vs 2.79%; p = 0.018), increased requirement for outpatient dehydration treatment (5.87% vs 4.67%;p = 0.004), and overall increased operation time (169.3 min ± 76.0 vs 153.5 ± 73.3; p < 0.001). However, there were no significant increases in the rates of reoperation, death, postoperative leak complications, or bleeding complications after CPHR. CONCLUSION: Patients undergoing revisional/conversional RYGB with CPHR may be at higher risk for a small number of rare postoperative complications. CPHR is a safe procedure in patients undergoing revisional/conversional RYGB in the short-term postoperative period.

Role of hospital strain in determining outcomes for people hospitalised with COVID-19 in England.

BACKGROUND: In England, reported COVID-19 mortality rates increased during winter 2020/21 relative to earlier summer and autumn months. This study aimed to examine the association between COVID-19-related hospital bed-strain during this time and patient outcomes. METHODS: This was a retrospective observational study using Hospital Episode Statistics data for England. All unique patients aged ≥18 years in England with a diagnosis of COVID-19 who had a completed (discharged alive or died in hospital) hospital stay with an admission date between 1 July 2020 and 28 February 2021 were included. Bed-strain was calculated as the number of beds occupied by patients with COVID-19 divided by the maximum COVID-19 bed occupancy during the study period. Bed-strain was categorised into quartiles for modelling. In-hospital mortality was the primary outcome of interest and length of stay a secondary outcome. RESULTS: There were 253 768 unique hospitalised patients with a diagnosis of COVID-19 during a hospital stay. Patient admissions peaked in January 2021 (n=89 047), although the crude mortality rate peaked slightly earlier in December 2020 (26.4%). After adjustment for covariates, the mortality rate in the lowest and highest quartile of bed-strain was 23.6% and 25.3%, respectively (OR 1.13, 95% CI 1.09 to 1.17). For the lowest and the highest quartile of bed-strain, adjusted mean length of stay was 13.2 days and 11.6 days, respectively in survivors and was 16.5 days and 12.6 days, respectively in patients who died in hospital. CONCLUSIONS: High levels of bed-strain were associated with higher in-hospital mortality rates, although the effect was relatively modest and may not fully explain increased mortality rates during winter 2020/21 compared with earlier months. Shorter hospital stay during periods of greater strain may partly reflect changes in patient management over time.

ADAM17 cleaves the insulin receptor ectodomain on endothelial cells and causes vascular insulin resistance.

Inflammation and vascular insulin resistance are hallmarks of type 2 diabetes (T2D). However, several potential mechanisms causing abnormal endothelial insulin signaling in T2D need further investigation. Evidence indicates that the activity of ADAM17 (a disintegrin and metalloproteinase-17) and the presence of insulin receptor (IR) in plasma are increased in subjects with T2D. Accordingly, we hypothesized that in T2D, increased ADAM17 activity sheds the IR ectodomain from endothelial cells and impairs insulin-induced vasodilation. We used small visceral arteries isolated from a cross-sectional study of subjects with and without T2D undergoing bariatric surgery, human cultured endothelial cells, and recombinant proteins to test our hypothesis. Here, we demonstrate that arteries from subjects with T2D had increased ADAM17 expression, reduced presence of tissue inhibitor of metalloproteinase-3 (TIMP3), decreased extracellular IRα, and impaired insulin-induced vasodilation versus those from subjects without T2D. In vitro, active ADAM17 cleaved the ectodomain of the IRß subunit. Endothelial cells with ADAM17 overexpression or exposed to the protein kinase-C activator, PMA, had increased ADAM17 activity, decreased IRα presence on the cell surface, and increased IR shedding. Moreover, pharmacological inhibition of ADAM17 with TAPI-0 rescued PMA-induced IR shedding and insulin-signaling impairments in endothelial cells and insulin-stimulated vasodilation in human arteries. In aggregate, our findings suggest that ADAM17-mediated shedding of IR from the endothelial surface impairs insulin-mediated vasodilation. Thus, we propose that inhibition of ADAM17 sheddase activity should be considered a strategy to restore vascular insulin sensitivity in T2D.NEW & NOTEWORTHY To our knowledge, this is the first study to investigate the involvement of ADAM17 in causing impaired insulin-induced vasodilation in T2D. We provide evidence that ADAM17 activity is increased in the vasculature of patients with T2D and support the notion that ADAM17-mediated shedding of endothelial IRα ectodomains is a novel mechanism causing vascular insulin resistance. Our results highlight that targeting ADAM17 activity may be a potential therapeutic strategy to correct vascular insulin resistance in T2D.

The impact of COVID-19 on the spatial distribution of shooting violence in Buffalo, NY.

OBJECTIVES: This paper examines the extent to which hotspots of shooting violence changed following the emergence of COVID-19. METHODS: This analysis uses Andresen's Spatial Point Pattern test on 1500 by 1500 foot grid cells, correcting for multiple comparisons, on a 10-year sample of geocoded shooting data from Buffalo New York. RESULTS: This work finds zero micro-grid cells are not statistically different from pre to post COVID stay at home orders and instead that the observed rise in shootings in the sample appears to be a consistent proportional increase across the city. CONCLUSIONS: These findings provide law enforcement with useful information about how to respond to the recent rise in shooting violence, but additional work is needed to better understand what, among a number of competing theories, is driving the increase. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11292-021-09497-4.

Reducing rates of discharge against medical advice in the neonatal intensive care unit in a tertiary care hospital in South India: a mixed-methods study.

OBJECTIVE: To elucidate characteristics among neonates and their mothers who were discharged against medical advice (DAMA), providers' perspectives on DAMA and the effect of an intervention to reduce DAMA in a tertiary care hospital in South India. METHODS: We conducted a mixed-methods study to identify neonates at risk of DAMA. We reviewed charts of neonates and their mothers who were DAMA and conducted logit regression analysis to calculate adjusted odds ratios (aOR) and 95% confidence intervals (CI) to determine associations with DAMA. We conducted focus group discussions with nurses and doctors. We developed an intervention that included family counselling, supplemental funds for hospital bills and involving family members to reduce DAMA. RESULTS: Of 10 834 neonates, 179 (1.7%) were DAMA over the study period. Maternal characteristics associated with DAMA included higher previous parity (aOR 1.9, 95% CI 1.1-2.3, P = 0.001). Mothers who received antenatal care had lower odds of DAMA (aOR 0.2, 95% CI 0.1-0.7, P = 0.039). Neonates with lower birth weight (aOR 2.1, 95% CI 1.7-9.4, P = 0.002) and congenital malformations (aOR 3.3, 95% CI 1.1-5.3, P = 0.005) also had higher odds of DAMA. The most commonly cited reasons for DAMA were financial constraints, inadequate counselling and perceived poor prognosis. The average monthly number of neonates who were DAMA decreased from 3.6 (1.6%) to 1.5 (0.6%) after our multi-pronged intervention. CONCLUSIONS: Neonates with severe illness and poor prognosis had higher odds of DAMA. A multi-pronged intervention demonstrated reductions in the rates of DAMA. This intervention may be trialled in similar settings to reduce DAMA.

The "Little MonSta" Deep-Sea Benthic, Precision Deployable, Multi-Sensor and Sampling Lander Array.

The "Little MonSta" benthic lander array consists of 8 ROV-deployable (remotely operated vehicle) instrumented lander platforms for monitoring physical and chemical oceanographic properties and particle sampling developed as part of the MMMonKey_Pro program (mapping, modeling, and monitoring key processes and controls in cold-water coral habitats in submarine canyons). The Little MonStas offer flexible solutions to meet the need to monitor marine benthic environments during a historically unprecedented time of climate-driven oceanic change, develop an understanding of meso-scale benthic processes (natural and man-made), and to calibrate geological environmental archives. Equipped with acoustic Doppler current profilers (ADCPs), sediment traps, nylon settlement plates and homing beacons, the compact and upgradable lander platforms can be deployed by ROVs to precise locations in extreme terrains to a water depth of 3000 m. The array allows cluster-monitoring in heterogeneous environments or simultaneous monitoring over wider areas. A proof-of-concept case study was presented from the cold-water coral habitable zone in the upper Porcupine Bank Canyon, where the Little MonStas collected 868.8 h of current speed, direction, temperature, and benthic particulate flux records, as well as 192 particle samples subsequently analyzed for particular organic carbon (POC), lithic sediment, live foraminifera, and microplastics. The potential to upgrade the Little MonStas with additional sensors and acoustic releases offers greater and more flexible operational capabilities.

Overproduction of endothelin-1 impairs glucose tolerance but does not promote visceral adipose tissue inflammation or limit metabolic adaptations to exercise.

Endothelin-1 (ET-1) is a potent vasoconstrictor and proinflammatory peptide that is upregulated in obesity. Herein, we tested the hypothesis that ET-1 signaling promotes visceral adipose tissue (AT) inflammation and disrupts glucose homeostasis. We also tested if reduced ET-1 is a required mechanism by which exercise ameliorates AT inflammation and improves glycemic control in obesity. We found that 1) diet-induced obesity, AT inflammation, and glycemic dysregulation were not accompanied by significantly increased levels of ET-1 in AT or circulation in wild-type mice and that endothelial overexpression of ET-1 and consequently increased ET-1 levels did not cause AT inflammation yet impaired glucose tolerance; 2) reduced AT inflammation and improved glucose tolerance with voluntary wheel running was not associated with decreased levels of ET-1 in AT or circulation in obese mice nor did endothelial overexpression of ET-1 impede such exercise-induced metabolic adaptations; 3) chronic pharmacological blockade of ET-1 receptors did not suppress AT inflammation in obese mice but improved glucose tolerance; and 4) in a cohort of human subjects with a wide range of body mass indexes, ET-1 levels in AT, or circulation were not correlated with markers of inflammation in AT. In aggregate, we conclude that ET-1 signaling is not implicated in the development of visceral AT inflammation but promotes glucose intolerance, thus representing an important therapeutic target for glycemic dysregulation in conditions characterized by hyperendothelinemia. Furthermore, we show that the salutary effects of exercise on AT and systemic metabolic function are not contingent on the suppression of ET-1 signaling.

Engineering Nanodisc Scaffold Proteins for Native Mass Spectrometry.

Lipoprotein nanodiscs are ideally suited for native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane proteins into the gas phase. However, native mass spectrometry of nanodiscs produces complex spectra that can be challenging to assign unambiguously. To simplify interpretation of nanodisc spectra, we engineered a series of mutant membrane scaffold proteins (MSP) that do not affect nanodisc formation but shift the masses of nanodiscs in a controllable way, eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Finally, we demonstrate the use of mixed belt nanodiscs with embedded membrane proteins to confirm the dissociation of MSP prior to desolvation.

Repeat PEG placement is safe for head and neck cancer patients.

PURPOSE: Percutaneous endoscopic gastrostomy (PEG) provides durable nutritional access for head and neck (HNC) patients as they undergo treatment. Continuing treatment of HNC may necessitate repeat PEG placement. We report our outcomes with repeat PEG compared to first-time PEG in HNC patients. MATERIALS AND METHODS: A retrospective chart review identified morbidity, mortality, and possible risk factors for complications. RESULTS: Repeat PEG tubes constituted 17% of PEG procedures. Morbidity was rare and similar complication rates were found between the initial PEG and repeat PEG groups (2% vs. 11%, p=0.131). There were no mortalities. CONCLUSIONS: Repeat PEG plays an important role in the care of HNC patients and can be considered a safe means to establish durable enteric feeding access for patients with recurrent cancer or treatment complications.

Laparoscopic Conversion of Gastric Bypass to Distal Gastric Bypass for Inadequate Weight Loss.

BACKGROUND: Although uncommon, significant weight recurrence after Roux-en-Y gastric bypass (RYGB) can occur. Options are limited to help patients achieve additional weight loss, and improved techniques for revisional/conversional surgery are needed to achieve optimal outcomes while avoiding significant side effects. Although limited data exist regarding distalization of the Roux limb to achieve a longer biliopancreatic limb leading to some level of malabsorption, we have seen adequate weight loss with minimal significant side effects in patients undergoing this procedure with our approach. An appropriate technical approach to this procedure is important to avoid immediate and long-term complications. METHODS AND RESULTS: We present a video describing our approach to Roux limb distalization for weight gain after gastric bypass, describing our approach for work-up, operative technical pearls, and postoperative monitoring in these patients. A 61 year-old female who initially had good weight loss after RYGB with a body mass index (BMI) nadir of 33, from a preoperative BMI of 53, experienced weight recurrence with her BMI increasing to 48. After preoperative nutritional optimization, dietary counseling, and behavioral counseling, she underwent conversion of RYGB to distalization of Roux limb to create a distal RYGB. She tolerated the procedure well and was discharged on postoperative day 2. At 1-year follow-up, her BMI had decreased to 37 with improvement in dyslipidemia, elevation of liver transaminases, and improvement in hemoglobin A1C. This reflects the impact of this procedure on not only weight loss but also concurrent metabolic diseases associated with obesity. CONCLUSION: We present a case of distalization of a RYGB for weight recurrence, highlighting the technical pearls when performing the procedure. Accurate, intraoperative measurement of the total alimentary limb length is essential to achieve weight loss while minimizing malnutrition and vitamin deficiencies. Assessment of preoperative nutritional levels for evidence of any protein calorie malnutrition is important during surgical decision-making when this conversional metabolic operation is considered. Frequent, postoperative nutritional monitoring is important and occurs with a full bariatric nutritional panel at 3, 6, 9, and 12 months then yearly thereafter. Using our approach, we feel that conversion of RYGB to distalization of Roux limb can lead to improved weight loss without significant side effects.

The effectiveness of selection in a species affects the direction of amino acid frequency evolution.

Nearly neutral theory predicts that species with higher effective population size (N e ) are better able to purge slightly deleterious mutations. We compare evolution in high-N e vs. low-N e vertebrates to reveal which amino acid frequencies are subject to subtle selective preferences. We take three complementary approaches, two measuring flux and one measuring outcomes. First, we fit non-stationary substitution models of amino acid flux using maximum likelihood, comparing the high-N e clade of rodents and lagomorphs to its low-N e sister clade of primates and colugos. Second, we compare evolutionary outcomes across a wider range of vertebrates, via correlations between amino acid frequencies and N e . Third, we dissect the details of flux in human, chimpanzee, mouse, and rat, as scored by parsimony - this also enables comparison to a historical paper. All three methods agree on which amino acids are preferred under more effective selection. Preferred amino acids tend to be smaller, less costly to synthesize, and to promote intrinsic structural disorder. Parsimony-induced bias in the historical study produces an apparent reduction in structural disorder, perhaps driven by slightly deleterious substitutions. Within highly exchangeable pairs of amino acids, arginine is strongly preferred over lysine, and valine over isoleucine, consistent with more effective selection preferring a marginally larger free energy of folding. These two preferences match differences between thermophiles and mesophilic relatives. These results reveal the biophysical consequences of mutation-selection-drift balance, and demonstrate the utility of nearly neutral theory for understanding protein evolution.

Order of amino acid recruitment into the genetic code resolved by Last Universal Common Ancestor's protein domains.

The current "consensus" order in which amino acids were added to the genetic code is based on potentially biased criteria such as absence of sulfur-containing amino acids from the Urey-Miller experiment which lacked sulfur. Even if inferred perfectly, abiotic abundance might not reflect abundance in the organisms in which the genetic code evolved. Here, we instead exploit the fact that proteins that emerged prior to the genetic code's completion are likely enriched in early amino acids and depleted in late amino acids. We identify the most ancient protein-coding sequences born prior to the archaeal-bacterial split. Amino acid usage in protein sequences whose ancestors date back to a single homolog in the Last Universal Common Ancestor (LUCA) largely matches the consensus order. However, our findings indicate that metal-binding (cysteine and histidine) and sulfur-containing (cysteine and methionine) amino acids were added to the genetic code much earlier than previously thought. Surprisingly, even more ancient protein sequences - those that had already diversified into multiple distinct copies in LUCA - show a different pattern to single copy LUCA sequences: significantly less depleted in the late amino acids tryptophan and tyrosine, and enriched rather than depleted in phenylalanine. This is compatible with at least some of these sequences predating the current genetic code. Their distinct enrichment patterns thus provide hints about earlier, alternative genetic codes.

The protein domains of vertebrate species in which selection is more effective have greater intrinsic structural disorder.

The nearly neutral theory of molecular evolution posits variation among species in the effectiveness of selection. In an idealized model, the census population size determines both this minimum magnitude of the selection coefficient required for deleterious variants to be reliably purged, and the amount of neutral diversity. Empirically, an "effective population size" is often estimated from the amount of putatively neutral genetic diversity and is assumed to also capture a species' effectiveness of selection. A potentially more direct measure of the effectiveness of selection is the degree to which selection maintains preferred codons. However, past metrics that compare codon bias across species are confounded by among-species variation in %GC content and/or amino acid composition. Here we propose a new Codon Adaptation Index of Species (CAIS), based on Kullback-Leibler divergence, that corrects for both confounders. We demonstrate the use of CAIS correlations, as well as the Effective Number of Codons, to show that the protein domains of more highly adapted vertebrate species evolve higher intrinsic structural disorder.