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.

[WITHDRAWN] SPLASH: a statistical, reference-free genomic algorithm unifies biological discovery.

The authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2022/497555.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.

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.

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.

Low-carbohydrate, healthy-fat eating: A cost comparison with national dietary guidelines.

AIM: A low-carbohydrate, healthy-fat (LCHF) dietary approach has been demonstrated as an effective strategy for improving metabolic health; however, it is often criticised for being more expensive than following a dietary approach guided by the national, Ministry of Health nutrition guidelines. This study compared the cost of these two nutritionally replete dietary approaches for one day for a family of four. METHODS: In this descriptive case study, one-day meal plans were designed for a hypothetical family of four representing the average New Zealand (NZ) male and female weight-stable adult and two adolescent children. National documented heights, a healthy body mass index range (18.5-25.0 kg/m2 ), and a 1.7-activity factor was used to estimate total energy requirements using the Schofield equation. Total daily costs were compared based on food prices from a popular Auckland supermarket. Meal plans were analysed for their nutritional adequacy using FoodWorks 8 dietary analysis software against national Australian and NZ nutrient reference value thresholds. RESULTS: The total daily costs were $43.42 (national guidelines) and $51.67 (LCHF) representing an $8.25 difference, or $2.06 per person, with the LCHF meal plan being the costlier option. CONCLUSIONS: We consider this increased cost for an LCHF approach to be negligible. In practice, less costly food items with similar nutrition qualities can be substituted to reduce costs further should this be a goal. The LCHF approach should therefore not be disregarded as a viable dietary approach for improving health outcomes, based on its perceived expense.

Ethanol Impairs NRF2/Antioxidant and Growth Signaling in the Intact Placenta In Vivo and in Human Trophoblasts.

NRF2 is a redox-sensitive transcription factor that depending on the duration or magnitude of the stress, either translocates to the nucleus (beneficial) or is degraded in the cytosol (harmful). However, the role of NRF2-based mechanism(s) under ethanol (E)-induced developmental toxicity in the placental context remains unknown. Here, we used a rat prenatal model of maternal alcohol stress consisting of intermittent ethanol vapor (IEV) daily from GD11 to GD20 with a 6 h ON/18 h OFF in a vapor chamber and in vitro placental model consisting of HTR-8 trophoblasts exposed to 86 mM of E for either 24 h or 48 h. The role of NRF2 was evaluated through the NRF2-transactivation reporter assay, qRT-PCR, and Western blotting for NRF2 and cell growth-promoting protein, and cell proliferation assay. In utero and in vitro E decreased the nuclear NRF2 content and diminished its transactivation ability along with dysregulation of the proliferation indices, PCNA, CYCLIN-D1, and p21. This was associated with a ~50% reduction in cell proliferation in vitro in trophoblasts. Interestingly, this was found to be partially rescued by ectopic Nrf2 overexpression. These results indicate that ethanol-induced dysregulation of NRF2 coordinately regulates PCNA/CYCLIN-D1/p21 involving growth network, at least partially to set a stage for placental perturbations.

The use of continuous spectral analysis for the assessment of postural stability changes after sports-related concussion.

Impaired postural stability is associated with a variety of pathologies including sports-related concussion (SRC). Quantification of centre of pressure (COP) movement is the most common focus of instrumented assessment. Frequency-domain COP analyses have focused primarily on summary measures or pre-defined frequency bands but continuous analysis may provide novel and complementary insight into pathological control mechanisms. Our aims were (i) to compare post-SRC COP trajectory changes identified using clinician scores (Modified Balance Error Scoring System (M-BESS)), time-domain COP variables and continuous frequency spectral comparison; and (ii) to characterise frequency spectra changes. Male rugby players aged 15-19 years (n = 135) completed a pre-season baseline assessment comprising vision-obscured double-leg, single-leg and tandem stances on a force platform. Participants diagnosed with SRC during the season (n = 15) underwent repeat testing (median 4 days post-SRC; IQR 2.5-6.5). Baseline and post-SRC COP trajectories were compared using common time-domain COP variables, M-BESS scores and continuous frequency spectra. Post-SRC changes were identified using all three approaches. Spectral analysis revealed the largest effect size (Cliff's delta 0.39) and was the only method to identify differences in all three stances and in double-leg stance. All post-SRC increases in spectral content were in the anteroposterior direction; all decreases were in the mediolateral direction. Changes were localised to higher frequencies (1.7-8 Hz) except for double-leg stance anteroposterior direction, for which increases were observed throughout the analysed range. Our findings suggest that this method of spectral comparison may provide a more responsive and meaningful measure of postural stability changes after SRC than other commonly-used variables.

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.

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.

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.