Rapid Generation of Somatic Mouse Mosaics with Locus-Specific, Stably Integrated Transgenic Elements.

In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.

Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies.

The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.

Effect of buffer pH on methane production and fermentation characteristics of three forages tested in vitro.

BACKGROUND: Low rumen pH is proposed to be a major mechanism for low methane (CH4) emissions from sheep fed forage rape. However, it is difficult to separate this from other in vivo factors, such as rumen passage rate. The objective of this study was to determine the effect of pH alone on CH4 production in vitro using different pH buffers. Ryegrass, white clover and forage rape were incubated in vitro using three different incubation buffers with starting pH values of 5.5, 6.2 and 6.8. RESULTS: Decreasing pH reduced overall in vitro CH4 emission relative to fermented hexoses (CH4/FHex) by up to 54% and overall fermentation by 40%. pH also changed fermentation profiles where the acetate + butyrate to propionate + valerate ratio decreased when pH decreased. Within the three forages, forage rape led to the lowest CH4/FHex, but only in pH 5.5 and 6.2 buffer, and this was enhanced when the pH fell below 6. CONCLUSION: Reducing pH in vitro decreased CH4 production and overall fermentation across all forages. The lower pH reached by forage rape compared to ryegrass and white clover appears to drive the lower CH4 production relative to the extent of fermentation from forage rape compared to the other forages. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Abdominal normothermic regional perfusion in controlled donation after circulatory determination of death liver transplantation: Outcomes and risk factors for graft loss.

Postmortem normothermic regional perfusion (NRP) is a rising preservation strategy in controlled donation after circulatory determination of death (cDCD). Herein, we present results for cDCD liver transplants performed in Spain 2012-2019, with outcomes evaluated through December 31, 2020. Results were analyzed retrospectively and according to recovery technique (abdominal NRP [A-NRP] or standard rapid recovery [SRR]). During the study period, 545 cDCD liver transplants were performed with A-NRP and 258 with SRR. Median donor age was 59 years (interquartile range 49-67 years). Adjusted risk estimates were improved with A-NRP for overall biliary complications (OR 0.300, 95% CI 0.197-0.459, p < .001), ischemic type biliary lesions (OR 0.112, 95% CI 0.042-0.299, p < .001), graft loss (HR 0.371, 95% CI 0.267-0.516, p < .001), and patient death (HR 0.540, 95% CI 0.373-0.781, p = .001). Cold ischemia time (HR 1.004, 95% CI 1.001-1.007, p = .021) and re-transplantation indication (HR 9.552, 95% CI 3.519-25.930, p < .001) were significant independent predictors for graft loss among cDCD livers with A-NRP. While use of A-NRP helps overcome traditional limitations in cDCD liver transplantation, opportunity for improvement remains for cases with prolonged cold ischemia and/or technically complex recipients, indicating a potential role for complimentary ex situ perfusion preservation techniques.

Effect of dietary inclusion of winter brassica crops on milk production, feeding behavior, rumen fermentation, and plasma fatty acid profile in dairy cows.

This study determined feeding behavior, dry matter (DM) intake (DMI), rumen fermentation, and milk production responses of lactating dairy cows fed with kale (Brassica oleracea) or swede (Brassica napus ssp. napobrassica). Twelve multiparous lactating dairy cows (560 ± 22 kg of body weight, 30 ± 4 kg of milk/d, and 60 ± 11 d in milk at the beginning of the experiment; mean ± standard deviation) were randomly allocated to 3 dietary treatments in a replicated 3 × 3 Latin square design. The control diet comprised 10 kg of grass silage DM/d, 4 kg of ryegrass herbage DM/d, and 8.8 kg of concentrate DM/d. Then, 25% of herbage, silage, and concentrate (DM basis) was replaced with either kale or swede. Cows offered kale had decreased total DMI compared with cows fed the control and swede diets, whereas inclusion of swede increased eating time. Milk production, composition, and energy-corrected milk:DMI ratio were not affected. Cows fed with kale had a greater rumen acetate:propionate ratio, whereas swede inclusion increased the relative percentage of butyrate. Estimated microbial N was not affected by dietary treatments, but N excretion was reduced with inclusion of kale, improving N utilization. Cows fed kale tended to have increased nonesterified fatty acids and showed presence of Heinz-Ehrlich bodies, whereas hepatic enzymes such as aspartate aminotransferase, γ-glutamyl transferase, and glutamate dehydrogenase were not affected by dietary treatments. In plasma, compared with the control, swede and kale reduced total saturated fatty acids and increased total polyunsaturated fatty acids and total n-3 fatty acids. Overall, feeding cows with winter brassicas had no negative effect on production responses. However, mechanisms to maintain milk production were different. Inclusion of swede increased the time spent eating and maintained DMI with a greater relative rumen percentage of butyrate and propionate, whereas kale reduced DMI but increased triacylglycerides mobilization, which can negatively affect reproductive performance. Thus, the inclusion of swede may be more suitable for feeding early-lactating dairy cows during winter.

Preoperative intragastric balloon in morbid obesity is unable to decrease early postoperative morbidity of bariatric surgery (sleeve gastrectomy and gastric bypass): a clinical assay.

INTRODUCTION: There are few studies that investigate the usefulness of the preoperative intragastric balloon (IGB). This study will evaluate if pre-surgical weight loss with IGB reduces morbidity and mortality after surgery. METHOD: Prospective randomised study of patients with morbid obesity treated with gastric bypass or vertical gastrectomy, with two arms: the balloon arm (B-arm), where an IGB was inserted within the 6 months before surgery, and the control arm (C-arm). RESULTS: The study included 66 patients: 65.6% women, 69.6% with bypass. Age: 43 years (SD 10.2) B-arm and 42.6 years (SD 9.2) in the C-arm. We found 34.4% therapeutic failures in IGB. The mean body weight loss, %EWL and BMI reduction before surgery was 16.2 kg (SD 9.84) B-arm versus 4.7 (SD 8.70) in the C-arm, 23.6% versus 4.7% (p < 0.001) and 6.04 versus 1 (p < 0.001), respectively. The hospital stay was 7 days (p25-75: 5-8) B-arm and 7 days (p25-75: 5-9) in the C-arm (p = 0.937). Post-surgical morbidity with IGB was 25% versus 29.5% in the C-arm, p = 0.689. The number needed to treat (NNT) to prevent of post-surgical morbidity was 23 patients. The B-arm presented 54.5% moderate-severe post-surgical adverse events (12.5%) versus 82.6% in the C-arm (23.5%), p = 0.111. The cost of placing a balloon was more than 4000 Euros each. CONCLUSIONS: The preoperative balloon does not achieve a reduction in the post-surgical morbidity, nor does it reduce the hospital stay or rate of re-operations. The balloon achieves a higher weight loss result when compared to a diet programme, its added cost must also be given due consideration. TRAIL REGISTRY: This study has been registered on ClinicalTrials.gov with the Identifier: NCT01998243 (November 28, 2013).

Normothermic regional perfusion vs. super-rapid recovery in controlled donation after circulatory death liver transplantation.

BACKGROUND & AIMS: Although there is increasing interest in its use, definitive evidence demonstrating a benefit for postmortem normothermic regional perfusion (NRP) in controlled donation after circulatory death (cDCD) liver transplantation is lacking. The aim of this study was to compare results of cDCD liver transplants performed with postmortem NRP vs. super-rapid recovery (SRR), the current standard for cDCD. METHODS: This was an observational cohort study including all cDCD liver transplants performed in Spain between June 2012 and December 2016, with follow-up ending in December 2017. Each donor hospital determined whether organ recovery was performed using NRP or SRR. The propensity scores technique based on the inverse probability of treatment weighting (IPTW) was used to balance covariates across study groups; logistic and Cox regression models were used for binary and time-to-event outcomes. RESULTS: During the study period, there were 95 cDCD liver transplants performed with postmortem NRP and 117 with SRR. The median donor age was 56 years (interquartile range 45-65 years). After IPTW analysis, baseline covariates were balanced, with all absolute standardised differences <0.15. IPTW-adjusted risks were significantly improved among NRP livers for overall biliary complications (odds ratio 0.14; 95% CI 0.06-0.35, p <0.001), ischaemic type biliary lesions (odds ratio 0.11; 95% CI 0.02-0.57; p = 0.008), and graft loss (hazard ratio 0.39; 95% CI 0.20-0.78; p = 0.008). CONCLUSIONS: The use of postmortem NRP in cDCD liver transplantation appears to reduce postoperative biliary complications, ischaemic type biliary lesions and graft loss, and allows for the transplantation of livers even from cDCD donors of advanced age. LAY SUMMARY: This is a propensity-matched nationwide observational cohort study performed using livers recovered from donors undergoing cardiac arrest provoked by the intentional withdrawal of life support (controlled donation after circulatory death, cDCD). Approximately half of the livers were recovered after a period of postmortem in situ normothermic regional perfusion, which restored warm oxygenated blood to the abdominal organs, whereas the remainder were recovered after rapid preservation with a cold solution. The study results suggest that the use of postmortem normothermic regional perfusion helps reduce rates of post-transplant biliary complications and graft loss and allows for the successful transplantation of livers from older cDCD donors.

Parenteral administration of l-arginine to twin-bearing Romney ewes during late pregnancy is associated with reduced milk somatic cell count during early lactation.

Maternal milk is the primary source of nutrition for suckling mammals, and its yield and composition are important determinants of survival during the early neonatal period. The objective of this study was to examine whether parenteral administration of l-Arg to twin-bearing ewes, during mid to late pregnancy, influenced prepartum maternal mammary gland development and subsequent lactation performance in the early postpartum period (14 d). At 80 d of pregnancy, multiparous Romney ewes were housed indoors in group pens, split into 2 cohorts, and fed a lucerne-based pellet diet, formulated to meet 100% of National Research Council-recommended requirements for twin-bearing pregnant ewes, once a day. Cohort 1 was administered l-Arg (72.7 mg/kg of live weight via i.v, 3 times a day) from d 100 of pregnancy until d 140. At d 140, ewes were euthanized and maternal mammary tissues were collected for analysis of the biochemical indices total DNA, RNA, protein, protein synthetic efficiency (protein:RNA), cell size (protein:DNA), transcriptional efficiency (RNA:DNA), and the abundance of mammalian target of rapamycin (mTOR) and mTORSer2448 protein. Cohort 2 was administered an identical l-Arg regimen as cohort 1, but from d 100 until parturition. Milk was collected over a 14-d period (d 1, 4, 7, 10, and 14) to assess milk yield and composition. In cohort 1, total mammary DNA (cell number) tended to be higher in l-Arg ewes, with no change in total mammary RNA or protein content, biochemical indices of protein synthetic efficiency, cell size or transcriptional efficiency, or mTOR protein abundance or phosphorylation. In cohort 2, milk composition analysis from l-Arg ewes showed lower (d 7-14) milk somatic cell counts, greater crude protein percentage from d 7 to 10 but lower at d 14, and altered absolute concentrations of some free AA (d 7 and 14) compared with controls. We propose that parenteral administration of l-Arg during late pregnancy is associated with increased mammary gland cellular content and decreased somatic cell counts during early lactation.

Omentin-1 Changes following Biliopancreatic Diversion and Relationship with Cardiovascular Risk Factors.

INTRODUCTION: Omentin-1 might play a role in the pathogenesis of insulin resistance and obesity. The aim of the present study was to evaluate the influence of weight loss after biliopancreatic diversion on serum omentin-1 concentrations. Material and Methods A Caucasian population of 24 morbid obese patients was analyzed before and after 12 months of a biliopancreatic diversion surgery. Biochemical and anthropometric evaluation were realized at basal visit and at 12 months. Body weight, fat mass, waist circumferences, blood pressure, fasting blood glucose, fasting insulin, insulin resistance (HOMA-IR), lipid concentrations and omentin-1 were measured. RESULTS: After bariatric surgery and in both gender groups (males vs. females); BMI, weight, fat mass, waist circumference, blood pressure, glucose , total cholesterol, LDL cholesterol, triglycerides, HOMA-IR and fasting insulin decreased in a statistical manner from basal values. Omentin-1 levels increased after bariatric surgery and in both gender the improvement was similar (males vs. females); (delta: -87.1 ± 19.0 ng/dL; p = 0.02 vs. -93.8 ± 28.1 ng/dL; p = 0.03). In the multiple regression analysis adjusted by age and sex; BMI kg/m2 (Beta -0.32: 95% CI -3.98 to -0.12) and insulin UI/L (Beta -0.41: 95% CI -8.38 to -0.16) remained in the model with basal omentin-1 levels as dependent variable. The regression model with post-surgery omentin-1 levels as dependent variable showed as independent variables BMI kg/m2 (Beta -0.13: 95% CI -7.69 to -0.09) and insulin UI/L (Beta -0.24: 95% CI -5.69 to -0.08), too. CONCLUSION: This study showed a significant increase in omentin-1 levels after weight loss secondary biliopancreatic diversion surgery. A weak negative correlation with BMI and basal insulin levels was detected.

A mechanistic model of hydrogen-methanogen dynamics in the rumen.

Existing mathematical models to estimate methane production in the rumen are based on calculation of hydrogen balances without considering the presence of methanogens. In this study, a mechanistic model of methane production is proposed that depicts the interaction between hydrogen concentration and methanogens in the rumen. Analytical results show that it meets biological expectations, namely increased fractional passage rate leads to a greater growth rate of methanogens, and a greater steady state hydrogen concentration. This model provides a basis on which to develop a more comprehensive model of methane production in the rumen that includes thermodynamics and feed fermentation pathways.

Effect on Liver Enzymes of Biliopancreatic Diversion: 4 Years of Follow-Up.

BACKGROUND: Elevated serum aminotransferase levels are commonly associated with obesity and with a progression to chronic liver disease. Bariatric surgery is the most effective strategy to achieve weight loss. METHODS: We conducted the present study with the aim of evaluating the influence of biliopancreatic diversion (BPD) on liver enzymes levels during 4 years in morbid obese patients with normal aminotransferase (n = 65) and in morbid obese patients with high aminotransferase basal levels (n = 50). RESULTS: A decrease in alanine aminotransferase and aspartate aminotransferase activities was significant after biliopancreatic diversion. The basal percentage of high aminotransferase levels and percentage of ratio ALT/AST <1 also decreased significantly at 1-, 2-, 3- and 4-years of follow-up in both groups. ALT (52 to 20%), AST (42 to 10%) and ALT/AST (80 to 22%) in patients with normal aminotransferase. ALT (82 to 20%), AST (70 to 6%) and ALT/AST (90 to 20%) in patients with elevated transaminase basal levels. Bariatric surgery was associated with a significant and sustained decrease in body weight in both groups. Serum trasnaminases level changes were positively correlated to body weight changes during follow-up. CONCLUSION: BPD is an effective method of achieving sustainable weight loss and reduced aminotransferase levels and enzyme ratios of liver damage.

Role of the rs6923761 gene variant in glucagon-like peptide 1 receptor gene on cardiovascular risk factors and weight loss after biliopancreatic diversion surgery.

BACKGROUND: Studies of the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) have been directed at identifying polymorphisms in the GLP-1R gene that may be a contributing factor in the pathogenesis of obesity. OBJECTIVE: We decided to investigate the role of the rs6923761 GLP-1R polymorphism on outcomes after biliopancreatic diversion. DESIGN: A sample of 137 morbidly obese patients was operated. Weight, blood pressure, basal glucose and lipid profile were measured at the basal visit and at each following visit (basal, 3, 9, 12 and 18 months). RESULTS: Body mass index, weight and waist circumference were lower in non-A allele carriers than A allele carriers 12 and 18 months after surgery. The initial weight percent loss at 12 months (45.6 vs. 39.8%; p < 0.05) or 18 months (49.6 vs. 41.3%; p < 0.05) was higher in patients with GG genotype than A allele carriers. CONCLUSION: Our study showed a higher weight loss 12 and 18 months after bariatric surgery in GG variant than A allele carriers. The biochemical parameters and cardiovascular comorbidity rates improved similarly in both genotypes.

Role of rs2282679 in 25-hydroxyvitamin D levels and insulin resistance after a sleeve gastrectomy.

BACKGROUND & AIMS: Some studies have reported links between 25-hydroxyvitamin D levels and the presence of obesity and some genetic variants. The aim of our design was to evaluate the effects of rs2282679 genetic variant of CG gene on 25-hydroxyvitamin D levels, weight loss and metabolic parameters after a robotic sleeve gastrectomy in premenopausal females with obesity. METHODS: 76 participants were enrolled. 25-hydroxyvitamin D levels, biochemical evaluation and anthropometric parameters were registered before surgery and after 3, 6 and 12 months follow up. Genotype of rs2282679 CG gene was evaluated. RESULTS: The improvements in anthropometric parameters, blood pressure and lipid profile were similar in both genotypes (TT vs TG + GG). Basal insulin levels and HOMA-IR were greater in G allele carriers than non-carriers (Delta: 6.7 ± 1.2 mUI/L; p = 0.01) and (Delta: 1.3 ± 0.1 units; p = 0.02). 25-hydroxyvitamin D levels were lower in G allele carriers than non-carriers (Delta: 8.1 ± 1.1 ng/dl; p = 0.03). The levels of insulin and HOMA-IR remained greater in G allele carriers than non-carriers throughout all the visits. The levels of 25-hydroxyvitamin D remained lower in G allele carriers than non-G allele. The average level of 25-hydroxyvitamin D at 12 months in non-G allele carriers were above 30 ng/dl (36.0 ± 3.1 ng/dl) and the level in G allele carriers were below (24.9 ± 4.9 ng/dl). CONCLUSIONS: rs 2282679 (GC) was related with low 25 hydroxyvitamin D levels and insulin resistance. In addition, the presence of G allele produced a decrease in the improvement of 25-hydroxyvitamin D levels and insulin resistance after weight loss during 12 months.

SOX9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys.

The steps governing healing with or without fibrosis within the same microenvironment are unclear. After acute kidney injury (AKI), injured proximal tubular epithelial cells activate SOX9 for self-restoration. Using a multimodal approach for a head-to-head comparison of injury-induced SOX9 lineages, we identified a dynamic SOX9 switch in repairing epithelia. Lineages that regenerated epithelia silenced SOX9 and healed without fibrosis (SOX9on-off). By contrast, lineages with unrestored apicobasal polarity maintained SOX9 activity in sustained efforts to regenerate, which were identified as a SOX9on-on Cadherin6pos cell state. These reprogrammed cells generated substantial single-cell WNT activity to provoke a fibroproliferative response in adjacent fibroblasts, driving AKI to chronic kidney disease. Transplanted human kidneys displayed similar SOX9/CDH6/WNT2B responses. Thus, we have uncovered a sensor of epithelial repair status, the activity of which determines regeneration with or without fibrosis.

Internal and external validation of indocyanine green plasma disappearance rate to discard liver grafts before procurement.

Backgrounds/Aims: Thirty percent of liver grafts in donors after brain death (DBD) in Spain are rejected by procurement surgeons owing to marginal graft quality. Poor donor indocyanine green (ICG) clearance has been associated with graft discard and malfunction. This study aimed to internally and externally validate the predictive value of ICG-plasma disappearance rate (ICG-PDR) to reject grafts before donation and set a cut-off to avoid missing any potential effective donors. Methods: Between March 2017 and August 2023, ICG clearance test was performed immediately before procurement in 71 DBD. The surgeon was blinded to test results. Univariate and multivariate analyses were performed to detect independent predictors of graft discard. Discrimination and calibration of predictors were assessed and a cut-off with 100% specificity was set. External validation was performed on 17 donors evaluated by three other transplantation teams. Results: In the training cohort, 30 of 71 grafts were discarded for transplantation. ICG-PDR was the only donor variable independently associated with graft discard. The area under receiver operating characteristic curve for ICG-PDR was 0.875 (95% confidence interval: 0.768-0.947) and good calibration was observed. Below a PDR of 13.5%/min, no graft was accepted for transplantation. These results were successfully validated using the external cohort of donors. Conclusions: ICG clearance test performed in DBD was internally and externally validated to predict liver graft discard. It could be used as a screening tool before donation to avoid unnecessary costs of travel and human resources.