SPLASH: A statistical, reference-free genomic algorithm unifies biological discovery.

Today's genomics workflows typically require alignment to a reference sequence, which limits discovery. We introduce a unifying paradigm, SPLASH (Statistically Primary aLignment Agnostic Sequence Homing), which directly analyzes raw sequencing data, using a statistical test to detect a signature of regulation: sample-specific sequence variation. SPLASH detects many types of variation and can be efficiently run at scale. We show that SPLASH identifies complex mutation patterns in SARS-CoV-2, discovers regulated RNA isoforms at the single-cell level, detects the vast sequence diversity of adaptive immune receptors, and uncovers biology in non-model organisms undocumented in their reference genomes: geographic and seasonal variation and diatom association in eelgrass, an oceanic plant impacted by climate change, and tissue-specific transcripts in octopus. SPLASH is a unifying approach to genomic analysis that enables expansive discovery without metadata or references.

Selenium supplementation may improve COVID-19 survival in sickle cell disease.

Sickle cell disease is associated with lower selenium levels, and the serum selenium level is inversely associated with haemolysis in SCD. The SCD population is more vulnerable to adverse COVID-19 outcomes. SARS-CoV-2 infection lowers the serum selenium level and this is associated with severity of COVID-19. Selenium supplementation is proposed to improve COVID-19 outcomes in the sickle cell disease population.

Meat, eggs, full-fat dairy, and nutritional boogeymen: Does the way in which animals are raised affect health differently in humans?

Background: Food recommendations to improve cancer prevention are generally based on epidemiologic data and remain inconsistent. These epidemiologic studies, while controversial, have generally produced results that caution against the consumption of high-fat foods, including eggs, red meat, and full-fat dairy, such as butter and cheese. Yet, limited data exist assessing the quality of individual sources of these foods and the effect each has after its consumption. This study set out to assess the impact sources of food within the same groups from animals raised differently on variables associated with health in human studies. Methods and Materials: A search was conducted through MEDLINE, Embase, and PubMed. In total, twenty-nine studies met inclusion criteria, measuring physiologic changes in humans after consuming animal products following animal diet manipulation. A meta-analysis was attempted to assess the differences between the cohorts in these studies, but was aborted due to poor study quality, vast differences in study design, and a limited number of studies. Results: Studies varied by animal, animal diet manipulation, food product, and overall design. Significant differences were present between groups eating the same food (cheese, beef, eggs, and butter) from animals raised differently, including levels of: conjugated linoleic acid, omega-3 fatty acids (alpha linoleic acid [ALA], docosahexaenoic acid [DHA], and eicosapentaenoic acid [EPA]), and inflammatory factors (triacyl glycerol [TAG], interleukin-6 [IL-6], interleukin-8 [IL-8], tumor necrosis factor [TNF], and C-reactive protein [CRP]). Lipid levels were minimally affected. Conclusions: This work highlights differences in human health markers after consumption of the same foods from animals raised differently. Overall, lipid levels remained relatively neutral, but significant changes in inflammatory and other serum markers and phospholipids were present. Future studies and dietary recommendations should consider how animals are raised, as this can produce different effects on health markers.

How reliable is the statistical evidence for limiting saturated fat intake? A fresh look at the influential Hooper meta-analysis.

BACKGROUND: Evidence from meta-analyses has been influential in deciding whether or not limiting saturated fat intake reduces the incidence of cardiovascular disease. Recently, random effects analyses have been criticised for exaggerating the influence of publication bias and an alternative proposed which obviates this issue: 'inverse-variance heterogeneity'. AIMS: We re-analysed the influential Hooper meta-analysis that supports limiting saturated fat intake to decide whether or not the results of the study were sensitive to the method used. METHODS: Inverse-variance heterogeneity analysis of this summary study was carried out, and the results contrasted with standard methods. Publication bias was also considered. RESULTS: Inverse variance heterogeneity analysis of the Hooper combined cardiovascular disease end point results returned a pooled relative risk of 0.93 (95% confidence interval: 0.74-1.16). This finding contrasts with the traditional random effects analysis with the corresponding statistic of 0.83 (95% confidence interval: 0.72-0.96). Egger tests, funnel and Doi plots along with recently published suppressed trial results suggest that publication bias is present. CONCLUSIONS: This study questions the use of the Hooper study as evidence to support limiting saturated fat intake. Our re-analysis, together with concordant results from other meta-analyses of trials indicate that routine advice to reduce saturated fat intake in people with (or at risk for) cardiovascular disease be reconsidered.

Role for Cystathionine γ Lyase (CSE) in an Ethanol (E)-Induced Lesion in Fetal Brain GSH Homeostasis.

Earlier, we reported that gestational ethanol (E) can dysregulate neuron glutathione (GSH) homeostasis partially via impairing the EAAC1-mediated inward transport of Cysteine (Cys) and this can affect fetal brain development. In this study, we investigated if there is a role for the transulfuration pathway (TSP), a critical bio-synthetic point to supply Cys in E-induced dysregulation of GSH homeostasis. These studies utilized an in utero E binge model where the pregnant Sprague⁻Dawley (SD) rat dams received five doses of E at 3.5 g/kg by gastric intubation beginning embryonic day (ED) 17 until ED19 separated by 12 h. The postnatal day 7 (PN7) alcohol model employed an oral dosing of 4 g/kg body weight split into 2 feedings at 2 h interval and an iso-caloric and iso-volumic equivalent maltose-dextrin milk solution served as controls. The in vitro model consisted of cerebral cortical neuron cultures from embryonic day (ED) 16⁻17 fetus from SD rats and differentiated neurons from ED18 rat cerebral cortical neuroblasts. E concentrations were 4 mg/mL. E induced an accumulation of cystathionine in primary cortical neurons (PCNs), 2nd trimester equivalent in utero binge, and 3rd trimester equivalent PN7 model suggesting that breakdown of cystathionine, a required process for Cys supply is impaired. This was associated with a significant reduction in cystathionine γ-lyase (CSE) protein expression in PCN (p < 0.05) and in fetal cerebral cortex in utero (53%, p < 0.05) without a change in the expression of cystathionine ß-synthase (CBS). Concomitantly, E decreased Cse mRNA expression in PCNs (by 32% within 6 h of exposure, p < 0.05) and in fetal brain (33%, p < 0.05). In parallel, knock down of CSE in differentiated rat cortical neuroblasts exaggerated the E-induced ROS, GSH loss with a pronounced caspase-3 activation and cell death. These studies illustrate the importance of TSP in CSE-related maintenance of GSH and the downstream events via Cys synthesis in neurons and fetal brain.

Ethanol (E) Impairs Fetal Brain GSH Homeostasis by Inhibiting Excitatory Amino-Acid Carrier 1 (EAAC1)-Mediated Cysteine Transport.

Central among the fetotoxic responses to in utero ethanol (E) exposure is redox-shift related glutathione (GSH) loss and apoptosis. Previously, we reported that despite an E-generated Nrf2 upregulation, fetal neurons still succumb. In this study, we investigate if the compromised GSH results from an impaired inward transport of cysteine (Cys), a precursor of GSH in association with dysregulated excitatory amino acid carrier1 (EAAC1), a cysteine transporter. In utero binge model involves administration of isocaloric dextrose or 20% E (3.5 g/kg)/ by gavage at 12 h intervals to pregnant Sprague Dawley (SD) rats, starting gestation day (gd) 17 with a final dose on gd19, 2 h prior to sacrifice. Primary cerebral cortical neurons (PCNs) from embryonic day 16-17 fetal SD rats were the in vitro model. E reduced both PCN and cerebral cortical GSH and Cys up to 50% and the abridged GSH could be blocked by administration of N-acetylcysteine. E reduced EAAC1 protein expression in utero and in PCNs (p < 0.05). This was accompanied by a 60-70% decrease in neuron surface expression of EAAC1 along with significant reductions of EAAC1/Slc1a1 mRNA (p < 0.05). In PCNs, EAAC1 knockdown significantly decreased GSH but not oxidized glutathione (GSSG) illustrating that while not the sole provider of Cys, EAAC1 plays an important role in neuron GSH homeostasis. These studies strongly support the concept that in both E exposed intact fetal brain and cultured PCNs a mechanism underlying E impairment of GSH homeostasis is reduction of import of external Cys which is mediated by perturbations of EAAC1 expression/function.

Ethanol induces cytostasis of cortical basal progenitors.

BACKGROUND: Developing brain is a major target for alcohol's actions and neurological/functional abnormalities include microencephaly, reduced frontal cortex, mental retardation and attention-deficits. Previous studies have shown that ethanol altered the lateral ventricular neuroepithelial cell proliferation. However, the effect of ethanol on subventricular basal progenitors which generate majority of the cortical layers is not known. METHODS: We utilized spontaneously immortalized rat brain neuroblasts obtained from cultures of 18-day-old fetal rat cerebral cortices using in vitro ethanol exposures and an in utero binge model. In the in vitro acute model, cells were exposed to 86 mM ethanol for 8, 12 and 24 h. The second in vitro model comprised of chronic intermittent ethanol (CIE) exposure which consisted of 14 h of ethanol treatment followed by 10 h of withdrawal with three repetitions. RESULTS: E18 neuroblasts expressing Tbr2 representing immature basal progenitors displayed significant reduction of proliferation in response to ethanol in both the models. The decreased proliferation was accompanied by absence of apoptosis or autophagy as illustrated by FACS analysis and expression of apoptotic and autophagic markers. The BrdU incorporation assay indicated that ethanol enhanced the accumulation of cells at G1 with reduced cell number in S phase. In addition, the ethanol-inhibited basal neuroblasts proliferation was connected to decrease in cyclin D1 and Rb phosphorylation indicating cell cycle arrest. Further, in utero ethanol exposure in pregnant rats during E15-E18 significantly decreased Tbr2 and cyclin D1 positive cell number in cerebral cortex of embryos as assessed by cell sorting analysis by flow cytometry. CONCLUSIONS: Altogether, the current findings demonstrate that ethanol impacts the expansion of basal progenitors by inducing cytostasis that might explain the anomalies of cortico-cerebral development associated with fetal alcohol syndrome.

Alcohol-induced one-carbon metabolism impairment promotes dysfunction of DNA base excision repair in adult brain.

The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr(+/-) mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.

Programmed cell death 4 (PDCD4): a novel player in ethanol-mediated suppression of protein translation in primary cortical neurons and developing cerebral cortex.

BACKGROUND: Prenatal exposure to ethanol (EtOH) elicits a range of neuro-developmental abnormalities, microcephaly to behavioral deficits. Impaired protein synthesis has been connected to pathogenesis of EtOH-induced brain damage and abnormal neuron development. However, mechanisms underlying these impairments of protein synthesis are not known. In this study, we illustrate the effects of EtOH on programmed cell death protein 4 (PDCD4), a tumor and translation repressor. METHODS: Primary cortical neurons (PCNs) were treated with 2.5 and 4 mg/ml EtOH for different time points (4 to 24 hours), and PDCD4 expression was detected by Western blotting. Protein synthesis was determined using [(35) S] methionine incorporation assay. Methyl cap pull-down assay was performed to establish the effect of EtOH on association of eukaryotic initiation factor 4A (eIF4A) with capped mRNA. Luciferase assay was performed to determine the in vivo translation. A 2-day acute 5-dose binge model with EtOH (4 g/kg body wt, 25% v/v) was performed in Sprague-Dawley rats at 12-hour intervals and analyzed for PDCD4, eIF4A, and eIF4A-methyl cap association. RESULTS: EtOH increased PDCD4 expression in a time- and dose-dependent manner in PCNs, which inhibited the association of eIF4A with methyl cap. EtOH and ectopic PDCD4 expression suppressed in vivo translation in PCNs and RNAi targeting of PDCD4 blocked the inhibitory effect of EtOH on protein synthesis. In utero exposure of pregnant rats to EtOH resulted in a significant increase in PDCD4 in fetal cerebral cortex along with the inhibition of methyl cap-associated eIF4A, compared with isocaloric controls. Increased PDCD4 also occurred in pooled fractions of remaining brain regions. CONCLUSIONS: Our data, for the first time, illustrate that PDCD4 mediates inhibitory effects of EtOH on protein synthesis in PCNs and developing brain.

Cognitive symptoms and welding fume exposure.

BACKGROUND: Prevalence of moderate to severe cognitive symptoms is markedly higher in UK professional divers who have also worked as a welder (28%) than in either divers who have not welded (18%) or offshore workers who have worked neither as a diver nor as a welder (6%). OBJECTIVES: To determine whether cognitive symptoms are related to welding fume exposure or diving. METHODS: Three age-matched groups of male workers were studied using postal questionnaire: professional divers who had worked as a welder (PDW, n = 361), professional welders who had not dived (NDW, n = 352), and offshore oil field workers who had neither dived nor welded (NDNW, n =503). Health-related quality of life was assessed by the Short Form 12 questionnaire (SF12). Cognitive symptomatology was assessed using the Cognitive Failures Questionnaire (CFQ). A single variable for welding fume exposure (mg m(-3) days) was calculated, incorporating welding experience in different environments and using different welding techniques and respiratory protective equipment. The level of fume exposure during hyperbaric welding operations was measured during such work as ambient PM(10) (particles of 10 µm or less). Diving exposure was assessed as the number of dives performed plus the number of days spent working during saturation diving. RESULTS: Questionnaires were returned by 153 PDW, 108 NDW, and 252 NDNW. SF12 scores were the same in all groups and fell within normative values. Mean (95% CI) CFQ scores were higher in PDW [40.3 (37.7-42.9)] than in both NDW [34.6 (31.6-37.7)] and NDNW [32.1 (30.4-33.9)], but the scores in no groups fell outside the normative range. The mean PM(10) exposure during hyperbaric welding operations was 2.58 mg m(-3). The geometric mean mg m(-3) days (95% CI) for welding fume exposure in NDW [33 128 (24 625-44 567) n = 85] was higher than for that in PDW [10 904 (8103-14 673) n = 112]. For PDW the geometric mean (95% CI) diving exposure was 1491 [(1192-1866) n = 94] dives and days in saturation. In the general linear model regression analyses adjusted for age, alcohol consumption, and somatization, there was no signification association of CFQ score with either welding fume exposure (F = 0.072, P = 0.79, n = 152) or diving exposure (F = 0.042, P = 0.84, n = 74). CONCLUSIONS: In conclusion, cognitive sympomatology was not related to retrospectively assessed measures of welding fume exposure or diving experience. In addition, the levels of cognitive symptomatology, even in PDW, did not exceed normative values.

SPLASH: a statistical, reference-free genomic algorithm unifies biological discovery.

Today's genomics workflows typically require alignment to a reference sequence, which limits discovery. We introduce a new unifying paradigm, SPLASH (Statistically Primary aLignment Agnostic Sequence Homing), an approach that directly analyzes raw sequencing data to detect a signature of regulation: sample-specific sequence variation. The approach, which includes a new statistical test, is computationally efficient and can be run at scale. SPLASH unifies detection of myriad forms of sequence variation. We demonstrate that SPLASH identifies complex mutation patterns in SARS-CoV-2 strains, discovers regulated RNA isoforms at the single cell level, documents the vast sequence diversity of adaptive immune receptors, and uncovers biology in non-model organisms undocumented in their reference genomes: geographic and seasonal variation and diatom association in eelgrass, an oceanic plant impacted by climate change, and tissue-specific transcripts in octopus. SPLASH is a new unifying approach to genomic analysis that enables an expansive scope of discovery without metadata or references.

[WITHDRAWN] SPLASH: a statistical, reference-free genomic algorithm unifies biological discovery.

The authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2022/497555. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: https://doi.org/10.1101/2022.06.24.497555.

Ethanol inhibition of undifferentiated rat neural progenitor cell replication can be prevented by chlorogenic acid via the NFATc4/CSE signaling pathway.

BACKGROUND: Prenatal ethanol exposure hinders oxidative stress-mediated neuroblast/neural progenitor cell proliferation by inhibiting G1-S transition, a process vital to neocortical development. We previously showed that ethanol elicits this redox imbalance by repressing cystathionine γ-lyase (CSE), the rate-limiting enzyme in the transsulfuration pathway in fetal brain and cultured cerebral cortical neurons. However, the mechanism by which ethanol impacts the CSE pathway in proliferating neuroblasts is not known. We conducted experiments to define the effects of ethanol on CSE regulation and the molecular signaling events that control this vital pathway. This enabled us to develop an intervention to prevent the ethanol-associated cytostasis. METHODS: Spontaneously immortalized undifferentiated E18 rat neuroblasts from brain cerebral cortex were exposed to ethanol to mimic an acute consumption pattern in humans. We performed loss- and gain-of-function studies to evaluate whether NFATc4 is a transcriptional regulator of CSE. The neuroprotective effects of chlorogenic acid (CGA) against the effects of ethanol were assessed using ROS and GSH/GSSG assays as measures of oxidative stress, transcriptional activation of NFATc4, and expression of NFATc4 and CSE by qRT-PCR and immunoblotting. RESULTS: Ethanol treatment of E18-neuroblast cells elicited oxidative stress and significantly reduced CSE expression with a concomitant decrease in NFATc4 transcriptional activation and expression. In parallel, inhibition of the calcineurin/NFAT pathway by FK506 exaggerated ethanol-induced CSE loss. In contrast, NFATc4 overexpression prevented loss of ethanol-induced CSE. CGA increased and activated NFATc4, amplified CSE expression, rescued ethanol-induced oxidative stress, and averted the cytostasis of neuroblasts by rescuing cyclin D1 expression. CONCLUSIONS: These findings demonstrate that ethanol can perturb CSE-dependent redox homeostasis by impairing the NFATc4 signaling pathway in neuroblasts. Notably, ethanol-associated impairments were rescued by genetic or pharmacological activation of NFATc4. Furthermore, we found a potential role for CGA in mitigating the ethanol-related neuroblast toxicity with a compelling connection to the NFATc4/CSE pathway.

Overexpression of Nrf2 protects cerebral cortical neurons from ethanol-induced apoptotic death.

Ethanol (ETOH) can cause apoptotic death of neurons by depleting GSH with an associated increase in oxidative stress. The current study illustrates a means to overcome this ETOH-induced neurotoxicity by enhancing GSH through boosting Nrf2, a transcription factor that controls GSH homeostasis. ETOH treatment caused a significant increase in Nrf2 protein, transcript expression, Nrf2-DNA binding activity, and expression of its transcriptional target, NQO1, in primary cortical neuron (PCNs). However, this increase in Nrf2 did not maintain GSH levels in response to ETOH, and apoptotic death still occurred. To elucidate this phenomenon, we silenced Nrf2 in neurons and found that ETOH-induced GSH depletion and the increase in superoxide levels were exacerbated. Furthermore, Nrf2 knockdown resulted in significantly increased (P < 0.05) caspase 3 activity and apoptosis. Adenovirus-mediated overexpression of Nrf2 prevented ETOH-induced depletion of GSH from the medium and high GSH subpopulations and prevented ETOH-related apoptotic death. These studies illustrate the importance of Nrf2-dependent maintenance of GSH homeostasis in cerebral cortical neurons in the defense against oxidative stress and apoptotic death elicited by ETOH exposure.

Ketohexokinase-dependent metabolism of fructose induces proinflammatory mediators in proximal tubular cells.

Increased consumption of fructose may play an important role in the epidemic of metabolic syndrome and may presage the development of diabetes, cardiovascular disease, and chronic kidney disease. Once in the cell, fructose is phosphorylated by ketohexokinase (KHK), leading to consumption of ATP, formation of AMP, and generation of uric acid through xanthine oxidoreductase (XOR). This study aimed to examine the direct effects of fructose in human kidney proximal tubular cells (HK-2) and whether they are mediated by the fructose metabolism via KHK. At a similar concentration to that observed in peripheral blood after a meal, fructose induced production of monocyte chemotactic protein 1 (MCP-1) and reactive oxygen species in HK-2 cells. Knockdown of KHK by stable transfection with small hairpin RNA demonstrated that these processes were KHK dependent. Several antioxidants, including specific inhibitors of NADPH oxidase and XOR, prevented MCP-1 secretion. We detected XOR mRNA in HK-2 cells and confirmed its activity by identifying uric acid by mass spectrometry. Fructose increased intracellular uric acid, and uric acid induced production of MCP-1 as well. In summary, postprandial concentrations of fructose stimulate redox- and urate-dependent inflammatory mediators in proximal tubular cells.