Control of mammalian locomotion by ventral spinocerebellar tract neurons.

Locomotion is a complex behavior required for animal survival. Vertebrate locomotion depends on spinal interneurons termed the central pattern generator (CPG), which generates activity responsible for the alternation of flexor and extensor muscles and the left and right side of the body. It is unknown whether multiple or a single neuronal type is responsible for the control of mammalian locomotion. Here, we show that ventral spinocerebellar tract neurons (VSCTs) drive generation and maintenance of locomotor behavior in neonatal and adult mice. Using mouse genetics, physiological, anatomical, and behavioral assays, we demonstrate that VSCTs exhibit rhythmogenic properties and neuronal circuit connectivity consistent with their essential role in the locomotor CPG. Importantly, optogenetic activation and chemogenetic silencing reveals that VSCTs are necessary and sufficient for locomotion. These findings identify VSCTs as critical components for mammalian locomotion and provide a paradigm shift in our understanding of neural control of complex behaviors.

Zinc-finger protein Zpr1 is a bespoke chaperone essential for eEF1A biogenesis.

The conserved regulon of heat shock factor 1 in budding yeast contains chaperones for general protein folding as well as zinc-finger protein Zpr1, whose essential role in archaea and eukaryotes remains unknown. Here, we show that Zpr1 depletion causes acute proteotoxicity driven by biosynthesis of misfolded eukaryotic translation elongation factor 1A (eEF1A). Prolonged Zpr1 depletion leads to eEF1A insufficiency, thereby inducing the integrated stress response and inhibiting protein synthesis. Strikingly, we show by using two distinct biochemical reconstitution approaches that Zpr1 enables eEF1A to achieve a conformational state resistant to protease digestion. Lastly, we use a ColabFold model of the Zpr1-eEF1A complex to reveal a folding mechanism mediated by the Zpr1's zinc-finger and alpha-helical hairpin structures. Our work uncovers the long-sought-after function of Zpr1 as a bespoke chaperone tailored to the biogenesis of one of the most abundant proteins in the cell.

Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

Local circuit amplification of spatial selectivity in the hippocampus.

Local circuit architecture facilitates the emergence of feature selectivity in the cerebral cortex1. In the hippocampus, it remains unknown whether local computations supported by specific connectivity motifs2 regulate the spatial receptive fields of pyramidal cells3. Here we developed an in vivo electroporation method for monosynaptic retrograde tracing4 and optogenetics manipulation at single-cell resolution to interrogate the dynamic interaction of place cells with their microcircuitry during navigation. We found a local circuit mechanism in CA1 whereby the spatial tuning of an individual place cell can propagate to a functionally recurrent subnetwork5 to which it belongs. The emergence of place fields in individual neurons led to the development of inverse selectivity in a subset of their presynaptic interneurons, and recruited functionally coupled place cells at that location. Thus, the spatial selectivity of single CA1 neurons is amplified through local circuit plasticity to enable effective multi-neuronal representations that can flexibly scale environmental features locally without degrading the feedforward input structure.

Neuronal programming by microbiota regulates intestinal physiology.

Neural control of the function of visceral organs is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence, and is often dysregulated in gastrointestinal disorders1. Luminal factors, such as diet and microbiota, regulate neurogenic programs of gut motility2-5, but the underlying molecular mechanisms remain unclear. Here we show that the transcription factor aryl hydrocarbon receptor (AHR) functions as a biosensor in intestinal neural circuits, linking their functional output to the microbial environment of the gut lumen. Using nuclear RNA sequencing of mouse enteric neurons that represent distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles that are controlled by the combined effects of host genetic programs and microbial colonization. Microbiota-induced expression of AHR in neurons of the distal gastrointestinal tract enables these neurons to respond to the luminal environment and to induce expression of neuron-specific effector mechanisms. Neuron-specific deletion of Ahr, or constitutive overexpression of its negative feedback regulator CYP1A1, results in reduced peristaltic activity of the colon, similar to that observed in microbiota-depleted mice. Finally, expression of Ahr in the enteric neurons of mice treated with antibiotics partially restores intestinal motility. Together, our experiments identify AHR signalling in enteric neurons as a regulatory node that integrates the luminal environment with the physiological output of intestinal neural circuits to maintain gut homeostasis and health.

Correction: Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress.

[This corrects the article DOI: 10.1371/journal.pgen.1009875.].

Heterozygous mutations cause genetic instability in a yeast model of cancer evolution.

Genetic instability, a heritable increase in the rate of genetic mutation, accelerates evolutionary adaptation1 and is widespread in cancer2,3. In mammals, instability can arise from damage to both copies of genes involved in DNA metabolism and cell cycle regulation4 or from inactivation of one copy of a gene whose product is present in limiting amounts (haploinsufficiency5); however, it has proved difficult to determine the relative importance of these two mechanisms. In Escherichia coli6, the application of repeated, strong selection enriches for genetic instability. Here we have used this approach to evolve genetic instability in diploid cells of the budding yeast Saccharomyces cerevisiae, and have isolated clones with increased rates of point mutation, mitotic recombination, and chromosome loss. We identified candidate, heterozygous, instability-causing mutations; engineering these mutations, as heterozygotes, into the ancestral diploid strain caused genetic instability. Mutations that inactivated one copy of haploinsufficient genes were more common than those that dominantly altered the function of the mutated gene copy. The mutated genes were enriched for genes functioning in transport, protein quality control, and DNA metabolism, and have revealed new targets for genetic instability7-11, including essential genes. Although only a minority (10 out of 57 genes with orthologues or close homologues) of the targets we identified have homologous human genes that have been implicated in cancer2, the remainder are candidates to contribute to human genetic instability. To test this hypothesis, we inactivated six examples in a near-haploid human cell line; five of these mutations increased instability. We conclude that single genetic events cause genetic instability in diploid yeast cells, and propose that similar, heterozygous mutations in mammalian homologues initiate genetic instability in cancer.

Impact of nocturnal hypoxia on glycaemic control, appetite, gut microbiota and inflammation in adults with type 2 diabetes mellitus: A single-blind cross-over trial.

High altitude residents have a lower incidence of type 2 diabetes mellitus (T2DM). Therefore, we examined the effect of repeated overnight normobaric hypoxic exposure on glycaemic control, appetite, gut microbiota and inflammation in adults with T2DM. Thirteen adults with T2DM [glycated haemoglobin (HbA1c): 61.1 ± 14.1 mmol mol-1; aged 64.2 ± 9.4 years; four female] completed a single-blind, randomised, sham-controlled, cross-over study for 10 nights, sleeping when exposed to hypoxia (fractional inspired O2 [ F I O 2 ${{F}_{{\mathrm{I}}{{{\mathrm{O}}}_{\mathrm{2}}}}}$ ] = 0.155; ∼2500 m simulated altitude) or normoxic conditions ( F I O 2 ${{F}_{{\mathrm{I}}{{{\mathrm{O}}}_{\mathrm{2}}}}}$  = 0.209) in a randomised order. Outcome measures included: fasted plasma [glucose]; [hypoxia inducible factor-1α]; [interleukin-6]; [tumour necrosis factor-α]; [interleukin-10]; [heat shock protein 70]; [butyric acid]; peak plasma [glucose] and insulin sensitivity following a 2 h oral glucose tolerance test; body composition; appetite indices ([leptin], [acyl ghrelin], [peptide YY], [glucagon-like peptide-1]); and gut microbiota diversity and abundance [16S rRNA amplicon sequencing]. During intervention periods, accelerometers measured physical activity, sleep duration and efficiency, whereas continuous glucose monitors were used to assess estimated HbA1c and glucose management indicator and time in target range. Overnight hypoxia was not associated with changes in any outcome measure (P > 0.05 with small effect sizes) except fasting insulin sensitivity and gut microbiota alpha diversity, which exhibited trends (P = 0.10; P = 0.08 respectively) for a medium beneficial effect (d = 0.49; d = 0.59 respectively). Ten nights of overnight moderate hypoxic exposure did not significantly affect glycaemic control, gut microbiome, appetite, or inflammation in adults with T2DM. However, the intervention was well tolerated and a medium effect-size for improved insulin sensitivity and reduced alpha diversity warrants further investigation. KEY POINTS: Living at altitude lowers the incidence of type 2 diabetes mellitus (T2DM). Animal studies suggest that exposure to hypoxia may lead to weight loss and suppressed appetite. In a single-blind, randomised sham-controlled, cross-over trial, we assessed the effects of 10 nights of hypoxia (fractional inspired O2 ∼0.155) on glucose homeostasis, appetite, gut microbiota, inflammatory stress ([interleukin-6]; [tumour necrosis factor-α]; [interleukin-10]) and hypoxic stress ([hypoxia inducible factor 1α]; heat shock protein 70]) in 13 adults with T2DM. Appetite and inflammatory markers were unchanged following hypoxic exposure, but an increased insulin sensitivity and reduced gut microbiota alpha diversity were associated with a medium effect-size and statistical trends, which warrant further investigation using a definitive large randomised controlled trial. Hypoxic exposure may represent a viable therapeutic intervention in people with T2DM and particularly those unable or unwilling to exercise because barriers to uptake and adherence may be lower than for other lifestyle interventions (e.g. diet and exercise).

Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress.

In haploid budding yeast, evolutionary adaptation to constitutive DNA replication stress alters three genome maintenance modules: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on genomic features by comparing the adaptation in three strains: haploids, diploids, and recombination deficient haploids. In all three, adaptation happens within 1000 generations at rates that are correlated with the initial fitness defect of the ancestors. Mutations in individual genes are selected at different frequencies in populations with different genomic features, but the benefits these mutations confer are similar in the three strains, and combinations of these mutations reproduce the fitness gains of evolved populations. Despite the differences in the selected mutations, adaptation targets the same three functional modules in strains with different genomic features, revealing a common evolutionary response to constitutive DNA replication stress.

Epidemiology of musculoskeletal injury in professional and amateur golfers: a systematic review and meta-analysis.

OBJECTIVE: To determine the prevalence and incidence of musculoskeletal injury in amateur and professional golfers, and to identify common injury sites and factors associated with increased injury frequency. DESIGN: Systematic epidemiological review and meta-analysis. DATA SOURCES: PubMed (Medline), Embase, the Cochrane Library and SPORTDiscus were searched in September 2023. ELIGIBILITY CRITERIA: Studies published in the English language reporting the incidence or prevalence of musculoskeletal injuries in golfers at all anatomical sites. RESULTS: 20 studies (9221 golfers, 71.9% male, 28.1% female) were included, with mean age 46.8 years. Lifetime injury prevalence was significantly greater in professional golfers (73.5% (95% CI: 47.3% to 93.0%)) than amateur golfers (56.6% (95% CI: 47.4% to 65.5%); relative risk (RR)=1.50, p<0.001). Professional golfers had a significantly greater lifetime prevalence of hand and wrist (RR=3.33, p<0.001) and lower back injury (RR=3.05, p<0.001). Soft tissue injuries were most common, and diagnoses were typically non-specific. Injury frequency was not associated with age or sex. Two studies reported a greater injury risk in amateur golfers playing more than three and four rounds per week. CONCLUSION: Over half of golfers are at risk of sustaining a musculoskeletal injury during their lifetime. Risks and patterns of injury differ between professional and amateur golfers, with professionals significantly more likely to develop lower back, and hand and wrist injuries. A recent international consensus statement on the reporting of injury and illness in golf should aid consistency in future research assessing the epidemiology of specific diagnoses, informing golf injury prevention and management strategies. PROSPERO REGISTRATION NUMBER: CRD42023408738.

Inequities in the Training Environment and Health of Female Golfers Participating in the 2022 International Golf Federation World Amateur Team Championships.

OBJECTIVE: To assess health problems and training environment of female golfers participating in the 2022 World Amateur Team Championships (WATC) and to compare golfers (a) with and without health problems prior the WATC and (b) living and training in countries ranking in the upper versus lower 50% of the team results at the 2022 WATC. DESIGN: Cross-sectional cohort study using an anonymous questionnaire. SETTING: International Golf Federation WATC. PARTICIPANTS: One hundred sixty-two female golfers from 56 countries. INTERVENTIONS: N/A. MAIN OUTCOME MEASURES: Golfers' answers on the presence and characteristics of health problems, their training environment, and to the Oslo Sport Trauma Research Centre Questionnaire. RESULTS: Almost all golfers (n = 162; 96%) answered the questionnaire. In the 4 weeks before the WATC, 101 golfers (63.1%) experienced 186 musculoskeletal complaints, mainly at the lumbar spine/lower back, wrist, or shoulder. Just half of the golfers (50.6%) performed injury prevention exercises always or often. More than a third (37.4%) of the golfers reported illness complaints and 32.5% mental health problems in the 4 weeks preceding the WATC. General anxiety, performance anxiety, and low mood/depression were the most frequent mental health problems. Golfers with injury complaints rated their daily training environment poorer. Golfers ranking in the lower 50% at the WATC had significantly less support staff, rated their training environment poorer, and had a higher prevalence of illness complaints and mental health problems. CONCLUSIONS: Effective illness and injury prevention programs should be implemented and better access to education and health support in the daily training environment provided.

Practices and perceptions of strength and conditioning training in female golf: A cross-sectional survey study of high-level amateur players.

This study aims to ascertain an in-depth understanding of current practices and perceptions of S&C training in high-level amateur female golfers. A cross-sectional, explorative survey study was constructed which asked questions relating to four key areas: i) general participant information, ii) current strength and conditioning (S&C) practices, iii) the perceived influence of S&C training on golf performance, and iv) knowledge and awareness of S&C. Results showed that the majority of female players had participated in some form of S&C training in the past, with the majority believing that clubhead speed and carry distance were the primary golfing metrics which could be positively impacted. More specifically, 91-97% of the players "Strongly agreed" or "Agreed" that the key physical characteristics for golf were strength and power for the lower and upper body, and flexibility. Interestingly, 58% of the players believed that S&C training should mimic the movement of the golf swing, which based off current evidence, is not how drive metrics and ultimately shots gained, can be maximised. This survey study provides useful information relating to the practices and perceptions of S&C training in high-level female amateur players and areas where education may be able to further advance player understanding of physical preparation.

Longitudinal Internal Training Load and Exposure in a High-Performance Basketball Academy.

ABSTRACT: Lever, JR, Duffield, R, Murray, A, Bartlett, JD, and Fullagar, HHK. Longitudinal internal training load and exposure in a high-performance basketball academy. J Strength Cond Res XX(X): 000-000, 2024-This study describes the longitudinal training exposure (session counts) and internal training load (Rating of Perceived Exertion [RPE] and Session Rating of Perceived Exertion [sRPE]) of youth basketball players at a high-performance academy, based on the training year, training term, and playing position. Historical internal training load and training exposure data were collated from 45 male high-performance youth basketball athletes between 2015 and 2019. Data included session duration, RPE, sRPE, training type, and date. Linear mixed models and pairwise comparisons were performed on the weekly means and categorized by training year (year 1, year 2, year 3), term (term 1, term 2, term 3, term 4), and playing position (Backcourt, Frontcourt). Linear mixed models indicate that the individual athlete had the greatest influence on variance in training load and exposure. Significant differences were observed for increased session count, duration, and sRPE (p < 0.001) in year 2 compared with year 1. These measures also increased within each year whereby term 3 and term 4 (p < 0.001) were significantly greater than term 1 and term 2. No significant differences were observed between playing position (p > 0.05). Training exposure and internal training load increase in year 2 from year 1 for high-performance youth basketball academy athletes. Differences between training load and exposure for terms (i.e., training blocks) suggest the phase of season influences training prescription, while playing position has limited effect.

Effects of Physical Training and Associations Between Physical Performance Characteristics and Golf Performance in Female Players: A Systematic Review With Meta-Analysis.

ABSTRACT: Robinson, L, Murray, A, Ehlert, A, Wells, J, Jarvis, P, Turner, A, Glover, D, Coughlan, D, Hembrough, R, and Bishop, C. Effects of physical training and associations between physical performance characteristics and golf performance in female players: A systematic review with meta-analysis. J Strength Cond Res 37(12): e646-e655, 2023-The aims of this systematic review were to assess the association between physical performance and measures of golf performance, and the effects of physical training on measures of golf performance, in female golfers. A systematic literature search was conducted in PubMed, SPORTDiscus, Medline, and CINAHL. Inclusion criteria required studies to (a) have conducted a physical training intervention of any duration in female players and determine the effects on measures of golf performance, (b) determine the association between physical performance in at least one test and golf performance in female players, and (c) be peer-reviewed and published in English language. Methodological quality was assessed using a modified version of the Downs and Black Quality Index tool, and heterogeneity was examined through the Q statistic and I2 . Pooled effect sizes were calculated using standardized mean differences (SMDs) (with 95% confidence interval [CI]s) within a random-effects model, with Egger's regression test used to assess small study bias (inclusive of publication bias). Of the 2,378 articles screened, only 9 were included in the final review, with 3 of these being associative by design and 6 being training interventions. From an associative standpoint, clubhead speed (CHS) was reported in all 3 studies and was associated with measures of strength ( r = 0.54), lower-body power ( r = 0.60), upper-body power ( r = 0.56-0.57), and flexibility ( r = 0.52-0.71). When assessing the effects of physical training interventions, CHS was again the most commonly reported golf outcome measure ( n = 5). The random-effect model indicated that CHS significantly improves within each training group following training interventions (SMD = 0.73 [95% CIs: 0.32-1.14], Z = 3.50, p < 0.001), with trivial heterogeneity ( I2 = 0.00%, Q = 0.18; p = 0.9963) and no prevalence of small study bias depicted through the Egger's regression test ( z = -0.28, p = 0.78). From the available research, it seems that CHS can be positively affected from strength, power, and flexibility training interventions. From an associative standpoint, only 3 studies have been conducted solely in female players, with one showcasing questionable methodology. Future research should aim to carefully select test measures which better represent the physical capacities needed for the sport when determining the effects of and relationships with golf performance.

Validity and Reliability of the FlightScope Mevo+ Launch Monitor for Assessing Golf Performance.

ABSTRACT: Brennan, A, Murray, A, Coughlan, D, Mountjoy, M, Wells, J, Ehlert, A, Xu, J, Broadie, M, Turner, A, and Bishop, C. Validity and reliability of the FlightScope Mevo+ launch monitor for assessing golf performance. J Strength Cond Res 38(4): e174-e181, 2024-The purpose of this study was to (a) assess the validity of the FlightScope Mevo+ against the TrackMan 4 and (b) determine the within-session reliability of both launch monitor systems when using a driver and a 6-iron. Twenty-nine youth golfers, with a minimum of 3 years of playing experience, volunteered for this study. All golfers completed 10 shots with a 6-iron and a driver, with 8 metrics concurrently monitored from both launch monitor systems in an indoor biomechanics laboratory. For both clubs, Pearson's r values ranged from small to near perfect ( r range = 0.254-0.985), with the strongest relationships evident for clubhead speed (CHS) and ball speed ( r ≥ 0.92). Bland-Altman plots showed almost perfect levels of agreement between devices for smash factor (mean bias ≤-0.016; 95% CI: -0.112, 0.079), whereas the poorest levels of agreement was for spin rate (mean bias ≤1,238; 95% CI: -2,628, 5,103). From a reliability standpoint, the TrackMan showed intraclass correlation coefficients (ICCs) ranging from moderate to excellent (ICC = 0.60-0.99) and coefficient of variation (CV) values ranged from good to poor (CV = 1.31-230.22%). For the Mevo+ device, ICC data ranged from poor to excellent (ICC = -0.22 to 0.99) and CV values ranged from good to poor (CV = 1.46-72.70%). Importantly, both devices showed similar trends, with the strongest reliability consistently evident for CHS, ball speed, carry distance, and smash factor. Finally, statistically significant differences ( p < 0.05) were evident between devices for spin rate (driver: d = 1.27; 6-iron: d = 0.90), launch angle (driver: d = 0.54), and attack angle (driver: d = -0.51). Collectively, these findings suggest that the FlightScope Mevo+ launch monitor is both valid and reliable when monitoring CHS, ball speed, carry distance, and smash factor. However, additional variables such as spin rate, launch angle, attack angle, and spin axis exhibit substantially greater variation compared with the TrackMan 4, suggesting that practitioners may wish to be cautious when providing golfers with feedback relating to these metrics.

A Scoping Review of the Epidemiology, Management, and Outcomes of Golf-Related Fractures.

ABSTRACT: Golf is a popular sport; however, there is a paucity of data in relation to golf-associated fractures, and the rate and timing of returning to golf. The aim of this review is to describe golf-associated fractures, including epidemiology, management, and timing of returning to golf following treatment. A literature search was performed using MEDLINE/PubMed, Embase, and Web of Science. Data were extracted and summarized in a narrative synthesis. A total of 436 articles were identified with an initial search of which 58 met the inclusion criteria. Twelve anatomical sites of golf swing-related fractures were identified, of which 10 sites were specific for stress fractures. The most common sites of golf swing-related stress fractures were the ribs followed by the hook of hamate. There was a common theme of delay to diagnosis, being initially assigned to a soft tissue injury. Most golfers with swing-related stress fractures were able to return to golf with the exception of osteoporotic associated vertebral stress fractures. Timing of returning to golf was between 4 and 12 months for most of the golfers with stress fractures following conservative management. Operative intervention was an option of hook of hamate nonunion, following a stress fracture, and tibial shaft stress fractures. Golf equipment-related fractures were not rare and were associated with major trauma and in some cases associated with significant persistent morbidity. Golf-related stress fractures commonly involve the ribs and hook of hamate; knowledge of this may aid in early diagnosis and appropriate treatment when symptomatic golfers are encountered. Although golf is a noncontact sport, fractures associated with golf equipment can be life changing, and safety training guidelines should be established.

The metabolic response of human trophoblasts derived from term placentas to metformin.

AIMS/HYPOTHESIS: Metformin is increasingly used therapeutically during pregnancy worldwide, particularly in the treatment of gestational diabetes, which affects a substantial proportion of pregnant women globally. However, the impact on placental metabolism remains unclear. In view of the association between metformin use in pregnancy and decreased birthweight, it is essential to understand how metformin modulates the bioenergetic and anabolic functions of the placenta. METHODS: A cohort of 55 placentas delivered by elective Caesarean section at term was collected from consenting participants. Trophoblasts were isolated from the placental samples and treated in vitro with clinically relevant doses of metformin (0.01 mmol/l or 0.1 mmol/l) or vehicle. Respiratory function was assayed using high-resolution respirometry to measure oxygen concentration and calculated [Formula: see text]. Glycolytic rate and glycolytic stress assays were performed using Agilent Seahorse XF assays. Fatty acid uptake and oxidation measurements were conducted using radioisotope-labelled assays. Lipidomic analysis was conducted using LC-MS. Gene expression and protein analysis were performed using RT-PCR and western blotting, respectively. RESULTS: Complex I-supported oxidative phosphorylation was lower in metformin-treated trophoblasts (0.01 mmol/l metformin, 61.7% of control, p<0.05; 0.1 mmol/l metformin, 43.1% of control, p<0.001). The proton efflux rate arising from glycolysis under physiological conditions was increased following metformin treatment, up to 23±5% above control conditions following treatment with 0.1 mmol/l metformin (p<0.01). There was a significant increase in triglyceride concentrations in trophoblasts treated with 0.1 mmol/l metformin (p<0.05), particularly those of esters of long-chain polyunsaturated fatty acids. Fatty acid oxidation was reduced by ~50% in trophoblasts treated with 0.1 mmol/l metformin compared with controls (p<0.001), with no difference in uptake between treatment groups. CONCLUSIONS/INTERPRETATION: In primary trophoblasts derived from term placentas metformin treatment caused a reduction in oxidative phosphorylation through partial inactivation of complex I and potentially by other mechanisms. Metformin-treated trophoblasts accumulate lipids, particularly long- and very-long-chain polyunsaturated fatty acids. Our findings raise clinically important questions about the balance of risk of metformin use during pregnancy, particularly in situations where the benefits are not clear-cut and alternative therapies are available.

Simple and accurate transcriptional start site identification using Smar2C2 and examination of conserved promoter features.

The precise and accurate identification and quantification of transcriptional start sites (TSSs) is key to understanding the control of transcription. The core promoter consists of the TSS and proximal non-coding sequences, which are critical in transcriptional regulation. Therefore, the accurate identification of TSSs is important for understanding the molecular regulation of transcription. Existing protocols for TSS identification are challenging and expensive, leaving high-quality data available for a small subset of organisms. This sparsity of data impairs study of TSS usage across tissues or in an evolutionary context. To address these shortcomings, we developed Smart-Seq2 Rolling Circle to Concatemeric Consensus (Smar2C2), which identifies and quantifies TSSs and transcription termination sites. Smar2C2 incorporates unique molecular identifiers that allowed for the identification of as many as 70 million sites, with no known upper limit. We have also generated TSS data sets from as little as 40 pg of total RNA, which was the smallest input tested. In this study, we used Smar2C2 to identify TSSs in Glycine max (soybean), Oryza sativa (rice), Sorghum bicolor (sorghum), Triticum aestivum (wheat) and Zea mays (maize) across multiple tissues. This wide panel of plant TSSs facilitated the identification of evolutionarily conserved features, such as novel patterns in the dinucleotides that compose the initiator element (Inr), that correlated with promoter expression levels across all species examined. We also discovered sequence variations in known promoter motifs that are positioned reliably close to the TSS, such as differences in the TATA box and in the Inr that may prove significant to our understanding and control of transcription initiation. Smar2C2 allows for the easy study of these critical sequences, providing a tool to facilitate discovery.

Effects of Germline VHL Deficiency on Growth, Metabolism, and Mitochondria.

Mutations in VHL, which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222C→A, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598C→T) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.).

Many, but not all, lineage-specific genes can be explained by homology detection failure.

Genes for which homologs can be detected only in a limited group of evolutionarily related species, called "lineage-specific genes," are pervasive: Essentially every lineage has them, and they often comprise a sizable fraction of the group's total genes. Lineage-specific genes are often interpreted as "novel" genes, representing genetic novelty born anew within that lineage. Here, we develop a simple method to test an alternative null hypothesis: that lineage-specific genes do have homologs outside of the lineage that, even while evolving at a constant rate in a novelty-free manner, have merely become undetectable by search algorithms used to infer homology. We show that this null hypothesis is sufficient to explain the lack of detected homologs of a large number of lineage-specific genes in fungi and insects. However, we also find that a minority of lineage-specific genes in both clades are not well explained by this novelty-free model. The method provides a simple way of identifying which lineage-specific genes call for special explanations beyond homology detection failure, highlighting them as interesting candidates for further study.