TMBIM5 is the Ca2+ /H+ antiporter of mammalian mitochondria.

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.

Predictive ability of multi-population genomic prediction methods of phenotypes for reproduction traits in Chinese and Austrian pigs.

BACKGROUND: Multi-population genomic prediction can rapidly expand the size of the reference population and improve genomic prediction ability. Machine learning (ML) algorithms have shown advantages in single-population genomic prediction of phenotypes. However, few studies have explored the effectiveness of ML methods for multi-population genomic prediction. RESULTS: In this study, 3720 Yorkshire pigs from Austria and four breeding farms in China were used, and single-trait genomic best linear unbiased prediction (ST-GBLUP), multitrait GBLUP (MT-GBLUP), Bayesian Horseshoe (BayesHE), and three ML methods (support vector regression (SVR), kernel ridge regression (KRR) and AdaBoost.R2) were compared to explore the optimal method for joint genomic prediction of phenotypes of Chinese and Austrian pigs through 10 replicates of fivefold cross-validation. In this study, we tested the performance of different methods in two scenarios: (i) including only one Austrian population and one Chinese pig population that were genetically linked based on principal component analysis (PCA) (designated as the "two-population scenario") and (ii) adding reference populations that are unrelated based on PCA to the above two populations (designated as the "multi-population scenario"). Our results show that, the use of MT-GBLUP in the two-population scenario resulted in an improvement of 7.1% in predictive ability compared to ST-GBLUP, while the use of SVR and KKR yielded improvements in predictive ability of 4.5 and 5.3%, respectively, compared to MT-GBLUP. SVR and KRR also yielded lower mean square errors (MSE) in most population and trait combinations. In the multi-population scenario, improvements in predictive ability of 29.7, 24.4 and 11.1% were obtained compared to ST-GBLUP when using, respectively, SVR, KRR, and AdaBoost.R2. However, compared to MT-GBLUP, the potential of ML methods to improve predictive ability was not demonstrated. CONCLUSIONS: Our study demonstrates that ML algorithms can achieve better prediction performance than multitrait GBLUP models in multi-population genomic prediction of phenotypes when the populations have similar genetic backgrounds; however, when reference populations that are unrelated based on PCA are added, the ML methods did not show a benefit. When the number of populations increased, only MT-GBLUP improved predictive ability in both validation populations, while the other methods showed improvement in only one population.

Inbreeding depression and its effect on sperm quality traits in Pietrain pigs.

In most cases, inbreeding is expected to have unfavourable effects on traits in livestock. The consequences of inbreeding depression could be substantial, primarily in reproductive and sperm quality traits, and thus lead to decreased fertility. Therefore, the objectives of this study were (i) to compute inbreeding coefficients using pedigree (FPED ) and genomic data based on runs of homozygosity (ROH) in the genome (FROH ) of Austrian Pietrain pigs, and (ii) to assess inbreeding depression on four sperm quality traits. In total, 74,734 ejaculate records from 1034 Pietrain boars were used for inbreeding depression analyses. Traits were regressed on inbreeding coefficients using repeatability animal models. Pedigree-based inbreeding coefficients were lower than ROH-based inbreeding values. The correlations between pedigree and ROH-based inbreeding coefficients ranged from 0.186 to 0.357. Pedigree-based inbreeding affected only sperm motility while ROH-based inbreeding affected semen volume, number of spermatozoa, and motility. For example, a 1% increase in pedigree inbreeding considering 10 ancestor generations (FPED10 ) was significantly (p < 0.05) associated with a 0.231% decrease in sperm motility. Almost all estimated effects of inbreeding on the traits studied were unfavourable. It is advisable to properly manage the level of inbreeding to avoid high inbreeding depression in the future. Further, analysis of effects of inbreeding depression for other traits, including growth and litter size for the Austrian Pietrain population is strongly advised.

Prehospital benzodiazepine use and need for respiratory support in paediatric seizures.

BACKGROUND: Paramedics are frequently called to attend seizures in children. High-quality evidence on second-line treatment of benzodiazepine (BZD)-refractory convulsions with parenteral long-acting antiepileptic drugs in children has become available from the ED. In order to address the potential need for an alternative agent, we set out to determine the association of BZD use prehospital and the need for respiratory support. METHODS: We conducted a retrospective observational study of state-wide ambulance service data (Ambulance Victoria in Victoria, Australia, population: 6.5 million). Children aged 0-17 years assessed for seizures by paramedics were analysed for demographics, process factors, treatment and airway management. We calculated adjusted ORs (AOR) of the requirement for respiratory support in relation to the number of BZD doses administered. RESULTS: Paramedics attended 5112 children with suspected seizures over 1 year (1 July 2018 to 30 June 2019). Overall, need for respiratory support was low (n=166; 3.2%). Before ambulance arrival, 509 (10.0%) had already received a BZD and 420 (8.2%) were treated with midazolam by paramedics. Of the 846 (16.5%) patients treated with BZD, 597 (70.6%) received 1 BZD dose, 156 (18.4%) 2 doses and 93 (11.0%) >2 doses of BZD. Patients who were administered 1, 2 and >2 doses of BZD received respiratory support in 8.9%, 32.1% (AOR 4.6 vs 1 dose, 95% CI 2.9 to 7.4) and 49.5% (AOR 10.3 vs 1 dose, 95% CI 6.0 to 17.9), respectively. CONCLUSIONS: Increasing administration of BZD doses was associated with higher use of respiratory support. Alternative prehospital antiepileptic drugs to minimise respiratory depression should be investigated in future research.

The anti-mitotic agents PTC-028 and PTC596 display potent activity in pre-clinical models of multiple myeloma but challenge the role of BMI-1 as an essential tumour gene.

Future progress in the treatment of multiple myeloma (MM) requires both the characterisation of key drivers of the disease and novel, innovative approaches to tackle these vulnerabilities. The present study focussed on the pre-clinical evaluation of a novel drug class, BMI-1 modulators, in MM. We demonstrate potent activity of PTC-028 and PTC596 in a comprehensive set of in vitro and in vivo models, including models of drug resistance and stromal support. Treatment of MM cells with PTC-028 and PTC596 downregulated BMI-1 protein levels, which was found to correlate with drug activity. Surprisingly, BMI-1 was dispensable for the activity of BMI-1 modulators and MM cell growth. Our data rather point to mitotic arrest accompanied by myeloid cell leukaemia-1 (MCL-1) loss as key anti-MM mechanisms and reveal impaired MYC and AKT signalling activity due to BMI-1 modulator treatment. Moreover, we observed a complete eradication of MM after PTC596 treatment in the 5TGM.1 in vivo model and define epigenetic compounds and B cell leukaemia/lymphoma 2 homology domain 3 (BH3) mimetics as promising combination partners. These results bring into question the postulated role of BMI-1 as an essential MM gene and confirm BMI-1 modulators as potent anti-mitotic agents with encouraging pre-clinical activity that supports their rapid translation into clinical trials.

An oligosaccharyltransferase from Leishmania major increases the N-glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana.

N-glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central-protein complex facilitating the N-glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N-glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single-subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well-established production platform for recombinant proteins. A fluorescent protein-tagged LmSTT3D variant was predominately found in the ER and co-located with plant oligosaccharyltransferase subunits. Co-expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N-glycosylation site occupancy on all N-glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N-glycosylation efficiency in plants.

A comparison of methods to calculate a total merit index using stochastic simulation.

BACKGROUND: Modern dairy cattle breeding goals include several production and more and more functional traits. Estimated breeding values (EBV) that are combined in the total merit index usually come from single-trait models or from multivariate models for groups of traits. In most cases, a multivariate animal model based on phenotypic data for all traits is not feasible and approximate methods based on selection index theory are applied to derive the total merit index. Therefore, the objective of this study was to compare a full multitrait animal model with two approximate multitrait models and a selection index approach based on simulated data. METHODS: Three production and two functional traits were simulated to mimic the national Austrian Brown Swiss population. The reference method for derivation of the total merit index was a multitrait evaluation based on all phenotypic data. Two of the approximate methods were variations of an approximate multitrait model that used either yield deviations or de-regressed breeding values. The final method was an adaptation of the selection index method that is used in routine evaluations in Austria and Germany. Three scenarios with respect to residual covariances were set up: residual covariances were equal to zero, or half of or equal to the genetic covariances. RESULTS: Results of both approximate multitrait models were very close to those of the reference method, with rank correlations of 1. Both methods were nearly unbiased. Rank correlations for the selection index method showed good results when residual covariances were zero but correlations with the reference method decreased when residual covariances were large. Furthermore, EBV were biased when residual covariances were high. CONCLUSIONS: We applied an approximate multitrait two-step procedure to yield deviations and de-regressed breeding values, which led to nearly unbiased results. De-regressed breeding values gave even slightly better results. Our results confirmed that ignoring residual covariances when a selection index approach is applied leads to remarkable bias. This could be relevant in terms of selection accuracy. Our findings suggest that the approximate multitrait approach applied to de-regressed breeding values can be used in routine genetic evaluation.

CTPS1 is a novel therapeutic target in multiple myeloma which synergizes with inhibition of CHEK1, ATR or WEE1.

Targeting nucleotide biosynthesis is a proven strategy for the treatment of cancer but is limited by toxicity, reflecting the fundamental nucleotide requirement of dividing cells. The rate limiting step in de novo pyrimidine synthesis is of interest, being catalyzed by two homologous enzymes, CTP synthase 1 (CTPS1) and CTPS2, that could be differentially targeted. Herein, analyses of publicly available datasets identified an essential role for CTPS1 in multiple myeloma (MM), linking high expression of CTPS1 (but not CTPS2) with advanced disease and poor outcomes. In cellular experiments, CTPS1 knockout induced apoptosis of MM cell lines. Exposure of MM cells to STP-B, a novel and highly selective pharmacological inhibitor of CTPS1, inhibited proliferation, induced S phase arrest and led to cell death by apoptosis. Mechanistically, CTPS1 inhibition by STP-B activated DNA damage response (DDR) pathways and induced double-strand DNA breaks which accumulated in early S phase. Combination of STP-B with pharmacological inhibitors of key components of the DDR pathway (ATR, CHEK1 or WEE1) resulted in synergistic growth inhibition and early apoptosis. Taken together, these findings identify CTPS1 as a promising new target in MM, either alone or in combination with DDR pathway inhibition.

The effect of the proportion of Modern European ancestry on grower and sow performance of pigs in smallholder systems in Uganda.

Several factors, including breed, lead to divergent performance of pigs for production and reproduction traits in different environments. A recent genomics study showed that Modern European (ME) pig breeds contribute to the ancestry of smallholder pigs in the Hoima and Kamuli districts, Uganda. These pigs were also involved in a longitudinal study with several traits recorded, including 540 body weights (WT) of 374 growing pigs, 195 records of total number of piglets born alive (TBA) of 157 sows, and 110 total number weaned (TNW) records of 94 sows. Linear mixed-effects models were used to test for the significance of environmental effects, including housing system, geographic location, and the season when the events occurred as well as animal-specific effects like age, sex, parity, and farrow-to-weaning interval. Stepwise model reduction starting from models with all main effects and pairwise interactions was applied. The final models were then expanded to include proportions of Modern European (ME) ancestry for the subset of animals genotyped, following genomic ancestry analysis based on a Porcine 50K SNP Chip. ME ancestry proportions ranged from 0.02 to 0.50 and were categorized into three classes (low/medium/high ME) based on 33.3% quantiles. The effects of ME classes on WT and TBA were not significant. ME showed a significant effect on TNW. Sows with a high proportion of ME weaned 2.4 piglets more than the low group, the medium ME group being intermediate. This study used genomic data to investigate the effects of genetic ancestry on the performance of smallholder pigs in Uganda. The proportion of Modern European ancestry did not exceed 0.50, therefore not allowing for the comparison of local versus pure "exotic" types of pigs. For the range of ancestries observed, which is the relevant one for current smallholder systems in Uganda, differences were small for the body weight of growing pigs and the number of piglets born alive, while higher proportions of ME ancestry resulted in significantly more piglets weaned. The availability of genotypes of a higher number of growing pigs would have been beneficial for drawing conclusions on the effect of ME ancestry on the growth rates of smallholder pigs in Uganda.

CTP Synthase 1 Is a Novel Therapeutic Target in Lymphoma.

Lymphoma is the most common hematological malignancy and is among the 10 most prevalent cancers worldwide. Although survival has been improved by modern immunochemotherapeutic regimens, there remains a significant need for novel targeted agents to treat both B-cell and T-cell malignancies. Cytidine triphosphate synthase 1 (CTPS1), which catalyzes the rate-limiting step in pyrimidine synthesis, plays an essential and nonredundant role in B-cell and T-cell proliferation but is complemented by the homologous CTPS2 isoform outside the hemopoietic system. This report describes the identification and characterization of CTPS1 as a novel target in B- and T-cell cancers. A series of small molecules have been developed which show potent and highly selective inhibition of CTPS1. Site-directed mutagenesis studies identified the adenosine triphosphate pocket of CTPS1 as the binding site for this small molecule series. In preclinical studies, a potent and highly selective small molecule inhibitor of CTPS1 blocked the in vitro proliferation of human neoplastic cells, showing the highest potency against lymphoid neoplasms. Importantly, pharmacological CTPS1 inhibition induced cell death by apoptosis in the majority of lymphoid cell lines tested, thus demonstrating a cytotoxic mechanism of action. Selective CTPS1 inhibition also inhibited the growth of neoplastic human B- and T- cells in vivo. These findings identify CTPS1 as a novel therapeutic target in lymphoid malignancy. A compound from this series is in phase 1/2 clinical studies for the treatment of relapsed/refractory B- and T-cell lymphoma (NCT05463263).

Parenteral Long-Acting Antiseizure Medications Are Used More Often to Treat Seizure Clusters Than Convulsive Status Epilepticus in the Pediatric Emergency Department.

Objective:Recent trials provide high-quality evidence for second-line treatment of convulsive status epilepticus (CSE) in children. However, the most effective medications for other seizure emergencies are poorly understood without established treatment algorithms. We investigated children presenting to the emergency department with repetitive or prolonged convulsions who required intravenous long-acting antiseizure medications, to determine the relative importance and treatment responsiveness of status epilepticus and seizure clusters. Methods: Retrospective observational study in the emergency department, Royal Children's Hospital, Melbourne, Australia (annual census 90 000) using hospital electronic medical records data of patients presenting with seizures in 2018. For patients receiving parenteral long-acting antiseizure medications, seizures were categorized as convulsive status epilepticus, nonconvulsive status epilepticus, and seizure clusters. Results: 1468 patients (2% of all visits) presented with seizures to the emergency department in 2018. Long-acting antiseizure medications were administered to 97 (7%) children for the emergency management of seizures. The majority presented with seizure clusters (n = 69; 71%). Only 11 (11%) were in convulsive status epilepticus and 17 (18%) in nonconvulsive status epilepticus. In convulsive status epilepticus, nonconvulsive status epilepticus, and seizure clusters, phenytoin was used in 27%, 53%, and 58% and levetiracetam in 73%, 47%, and 32%, respectively. Conclusions:Convulsive status epilepticus represents a small portion of patients requiring parenteral long-acting antiseizure medications. Seizure clusters accounted for >6 times the number of convulsive status epilepticus, yet evidence and treatment algorithms are lacking.

A Mix of Old British and Modern European Breeds: Genomic Prediction of Breed Composition of Smallholder Pigs in Uganda.

Pig herds in Africa comprise genotypes ranging from local ecotypes to commercial breeds. Many animals are composites of these two types and the best levels of crossbreeding for particular production systems are largely unknown. These pigs are managed without structured breeding programs and inbreeding is potentially limiting. The objective of this study was to quantify ancestry contributions and inbreeding levels in a population of smallholder pigs in Uganda. The study was set in the districts of Hoima and Kamuli in Uganda and involved 422 pigs. Pig hair samples were taken from adult and growing pigs in the framework of a longitudinal study investigating productivity and profitability of smallholder pig production. The samples were genotyped using the porcine GeneSeek Genomic Profiler (GGP) 50K SNP Chip. The SNP data was analyzed to infer breed ancestry and autozygosity of the Uganda pigs. The results showed that exotic breeds (modern European and old British) contributed an average of 22.8% with a range of 2-50% while "local" blood contributed 69.2% (36.9-95.2%) to the ancestry of the pigs. Runs of homozygosity (ROH) greater than 2 megabase (Mb) quantified the average genomic inbreeding coefficient of the pigs as 0.043. The scarcity of long ROH indicated low recent inbreeding. We conclude that the genomic background of the pig population in the study is a mix of old British and modern pig ancestries. Best levels of admixture for smallholder pigs are yet to be determined, by linking genotypes and phenotypic records.

Sparse Convolutional Neural Networks for Genome-Wide Prediction.

Genome-wide prediction (GWP) has become the state-of-the art method in artificial selection. Data sets often comprise number of genomic markers and individuals in ranges from a few thousands to millions. Hence, computational efficiency is important and various machine learning methods have successfully been used in GWP. Neural networks (NN) and deep learning (DL) are very flexible methods that usually show outstanding prediction properties on complex structured data, but their use in GWP is nevertheless rare and debated. This study describes a powerful NN method for genomic marker data that can easily be extended. It is shown that a one-dimensional convolutional neural network (CNN) can be used to incorporate the ordinal information between markers and, together with pooling and ℓ 1-norm regularization, provides a sparse and computationally efficient approach for GWP. The method, denoted CNNGWP, is implemented in the deep learning software Keras, and hyper-parameters of the NN are tuned with Bayesian optimization. Model averaged ensemble predictions further reduce prediction error. Evaluations show that CNNGWP improves prediction error by more than 25% on simulated data and around 3% on real pig data compared with results obtained with GBLUP and the LASSO. In conclusion, the CNNGWP provides a promising approach for GWP, but the magnitude of improvement depends on the genetic architecture and the heritability.

Assessment of Piglet Vitality by Farmers-Validation of A Scoring Scheme and Estimation of Associated Genetic Parameters.

Maternal breeds for sows have been bred for high prolificacy during recent decades. Although large litters may be beneficial for economic efficiency, pre-weaning mortality is increased. Thus, focus should instead be put on new traits such as piglet vitality (PV). Until now, no validated scoring scheme for piglet vitality exists, which is feasible to be applied for routine on-farm trait recording. The objective of this study was to validate a four-point vitality scoring scheme (1 = low vitality to 4 = high vitality) applied by farmers based on pre-weaning mortality and to estimate genetic parameters. A linear mixed model was fitted for piglet vitality for 3172 litters from Large White and Landrace sows on 23 farms and correlations were calculated for vitality score and piglet mortality. A subsample of 2900 records was used for genetic analysis. Pre-weaning mortality differed significantly between all vitality score categories except for 1 and 2, ranging between 7.98% (category 4) and 29.1% (category 1). PV was genetically negatively correlated to litter size (-0.68) and mortality rate (-0.65), whereas litter size was positively correlated with mortality rate (0.59). Including PV into breeding programs may, thus, improve animal welfare.

LETM1-Mediated K+ and Na+ Homeostasis Regulates Mitochondrial Ca2+ Efflux.

Ca2+ transport across the inner membrane of mitochondria (IMM) is of major importance for their functions in bioenergetics, cell death and signaling. It is therefore tightly regulated. It has been recently proposed that LETM1­an IMM protein with a crucial role in mitochondrial K+/H+ exchange and volume homeostasis­also acts as a Ca2+/H+ exchanger. Here we show for the first time that lowering LETM1 gene expression by shRNA hampers mitochondrial K+/H+ and Na+/H+ exchange. Decreased exchange activity resulted in matrix K+ accumulation in these mitochondria. Furthermore, LETM1 depletion selectively decreased Na+/Ca2+ exchange mediated by NCLX, as observed in the presence of ruthenium red, a blocker of the Mitochondrial Ca2+ Uniporter (MCU). These data confirm a key role of LETM1 in monovalent cation homeostasis, and suggest that the effects of its modulation on mitochondrial transmembrane Ca2+ fluxes may reflect those on Na+/H+ exchange activity.

Radical cystectomy for urothelial carcinoma of the bladder without neoadjuvant or adjuvant therapy: long-term results in 1100 patients.

BACKGROUND: The optimal treatment strategy for muscle-invasive bladder cancer (BCa) remains controversial. OBJECTIVE: Better define the long-term outcomes of radical cystectomy (RC) alone for BCa and determine the impact of pathologic downstaging after transurethral resection in a large and homogeneous single-center series. DESIGN, SETTING, AND PARTICIPANTS: A cohort of 1100 patients undergoing RC with pelvic lymph node dissection (PLND) without neoadjuvant therapy for urothelial carcinoma of the bladder between January 1, 1986, and December 2009 was evaluated. Patients with other than metastases to the pelvic lymph nodes were excluded. Median age was 65 yr. Clinical course, pathologic characteristics, and long-term outcomes were evaluated. Follow-up was obtained until December 2009 with a median of 38 mo and a completeness of 96.5%. INTERVENTION: RC with PLND; urinary diversion with ileal neobladder whenever possible. MEASUREMENTS: Primary end points were disease-specific survival (DSS), recurrence-free survival (RFS), and overall survival (OS) according to the tumor stage of the RC specimen versus the maximum tumor stage. The log-rank test was used to compare subgroups. RESULTS AND LIMITATIONS: The 30-d (90-d) mortality rate was 3.2% (5.2%). The 10-yr OS, DSS, and RFS rates were 44.3%, 66.8%, and 65.5%, respectively. Based on the tumor stage of the RC specimen, the 10-yr DSS rate was pT0/a/is/1 pN0: 90.5%, pT2a/b pN0: 66.8%, pT3a/b pN0: 59.7%, pT4a/b pN0: 36.6%, and pTall pN+: 16.7%. Downstaging by transurethral resection of the prostate was observed in 382 patients. Patients with maximum tumor stage pT2a/b pN0 had distinctly better 10-yr DSS rates than those with pT2a/b pN0 in the RC specimen: pT2a pN0: 92.2% versus 73.8%; pT2b: 75.0% versus 62.0%. A total of 49% female and 80% male patients received an ileal neobladder. CONCLUSIONS: This contemporary and homogeneous single-center series found acceptable OS, DFS, and RFS for patients undergoing RC. Pathologic downstaging had a significant impact on survival.