Transcriptomics reveal different metabolic strategies for acid resistance and gamma-aminobutyric acid (GABA) production in select Levilactobacillus brevis strains.

BACKGROUND: Of the many neurotransmitters in humans, gamma-aminobutyric acid (GABA) shows potential for improving several mental health indications such as stress and anxiety. The microbiota-gut-brain axis is an important pathway for GABAergic effects, as microbially-secreted GABA within the gut can affect host mental health outcomes. Understanding the molecular characteristics of GABA production by microbes within the gut can offer insight to novel therapies for mental health. RESULTS: Three strains of Levilactobacillus brevis with syntenous glutamate decarboxylase (GAD) operons were evaluated for overall growth, glutamate utilization, and GABA production in typical synthetic growth media supplemented with monosodium glutamate (MSG). Levilactobacillus brevis Lbr-6108™ (Lbr-6108), formerly known as L. brevis DPC 6108, and Levilactobacillus brevis Lbr-35 ™ (Lbr-35) had similar growth profiles but differed significantly in GABA secretion and acid resistance. Lbr-6108 produced GABA early within the growth phase and produced significantly more GABA than Lbr-35 and the type strain Levilactobacillus brevis ATCC 14869 after the stationary phase. The global gene expression during GABA production at several timepoints was determined by RNA sequencing. The GAD operon, responsible for GABA production and secretion, activated in Lbr-6108 after only 6 h of fermentation and continued throughout the stationary phase. Furthermore, Lbr-6108 activated many different acid resistance mechanisms concurrently, which contribute to acid tolerance and energy production. In contrast, Lbr-35, which has a genetically similar GAD operon, including two copies of the GAD gene, showed no upregulation of the GAD operon, even when cultured with MSG. CONCLUSIONS: This study is the first to evaluate whole transcriptome changes in Levilactobacillus brevis during GABA production in different growth phases. The concurrent expression of multiple acid-resistance mechanisms reveals niche-specific metabolic functionality between common human commensals and highlights the complex regulation of GABA metabolism in this important microbial species. Furthermore, the increased and rapid GABA production of Lbr-6108 highlights the strain's potential as a therapeutic and the overall value of screening microbes for effector molecule output.

Virus-induced genetics revealed by multidimensional precision medicine transcriptional workflow applicable to COVID-19.

Precision medicine requires the translation of basic biological understanding to medical insights, mainly applied to characterization of each unique patient. In many clinical settings, this requires tools that can be broadly used to identify pathology and risks. Patients often present to the intensive care unit with broad phenotypes, including multiple organ dysfunction syndrome (MODS) resulting from infection, trauma, or other disease processes. Etiology and outcomes are unique to individuals, making it difficult to cohort patients with MODS, but presenting a prime target for testing/developing tools for precision medicine. Using multitime point whole blood (cellular/acellular) total transcriptomics in 27 patients, we highlight the promise of simultaneously mapping viral/bacterial load, cell composition, tissue damage biomarkers, balance between syndromic biology versus environmental response, and unique biological insights in each patient using a single platform measurement. Integration of a transcriptome workflow yielded unexpected insights into the complex interplay between host genetics and viral/bacterial specific mechanisms, highlighted by a unique case of virally induced genetics (VIG) within one of these 27 patients. The power of RNA-Seq to study unique patient biology while investigating environmental contributions can be a critical tool moving forward for translational sciences applied to precision medicine.

Using large-scale tropical dry forest restoration to test successional theory.

Microclimatic conditions change dramatically as forests age and impose strong filters on community assembly during succession. Light availability is the most limiting environmental factor in tropical wet forest succession; by contrast, water availability is predicted to strongly influence tropical dry forest (TDF) successional dynamics. While mechanisms underlying TDF successional trajectories are not well understood, observational studies have demonstrated that TDF communities transition from being dominated by species with conservative traits to species with acquisitive traits, the opposite of tropical wet forest. Determining how functional traits predict TDF tree species' responses to changing environmental conditions could elucidate mechanisms underlying tree performance during TDF succession. We implemented a 6-ha restoration experiment on a degraded Vertisol in Costa Rica to determine (1) how TDF tree species with different resource-use strategies performed along a successional gradient and (2) how ecophysiological functional traits correlated with tree performance in simulated successional stages. We used two management treatments to simulate distinct successional stages including: clearing all remnant vegetation (early-succession), or interplanting seedlings with no clearing (mid-succession). We crossed these two management treatments (cleared/interplanted) with two species mixes with different resource-use strategies (acquisitive/conservative) to examine their interaction. Overall seedling survival after 2 yr was low, 15.1-26.4% in the four resource-use-strategy × management-treatment combinations, and did not differ between the management treatments or resource-use-strategy groups. However, seedling growth rates were dramatically higher for all species in the cleared treatment (year 1, 69.1% higher; year 2, 143.3% higher) and defined resource-use strategies had some capacity to explain seedling performance. Overall, ecophysiological traits were better predictors of species' growth and survival than resource-use strategies defined by leaf and stem traits such as specific leaf area. Moreover, ecophysiological traits related to water use had a stronger influence on seedling performance in the cleared, early-successional treatment, indicating that the influence of microclimatic conditions on tree survival and growth shifts predictably during TDF succession. Our findings suggest that ecophysiological traits should be explicitly considered to understand shifts in TDF functional composition during succession and that using these traits to design species mixes could greatly improve TDF restoration outcomes.

The effect of probiotic Bifidobacterium lactis Bl-04 on innate antiviral responses invitro.

Consumption of certain probiotic strains may be beneficial for reducing the risk of acute upper respiratory tract infections (URTIs), however, underlying immunological mechanisms are elusive. Bifidobacterium lactis Bl-04™ has been reported in humans to significantly reduce the risk of URTIs, affect the innate immunity in the nasal mucosa, and reduce nasal lavage virus titer after a rhinovirus (RV) challenge. To study the immunological mechanisms, we investigated the effect of Bl-04 on cytokine production and transcriptomes of human monocyte-derived macrophages (Mfs) and dendritic cells (DCs), and further on RV replication and cytokine production in MRC-5 fibroblasts. The results showed that Bl-04 modulates antiviral immune responses and potentiates cytokine production during viral challenge mimic in immune cells. However, effect of Bl-04 on RV replication and cytokine production in fibroblasts was negligible. Overall, the findings suggest that Bl-04 mildly stimulates antiviral immunity in Mfs and DCs, and potentially influences viral replication in fibroblasts that however warrants further investigations.

The Effect of Bifidobacterium animalis subsp. lactis Bl-04 on Influenza A Virus Infection in Mice.

Influenza A virus infection is a major global disease requiring annual vaccination. Clinical studies indicate that certain probiotics may support immune function against influenza and other respiratory viruses, but direct molecular evidence is scarce. Here, mice were treated with a placebo or Bifidobacterium animalis subsp. lactis Bl-04 (Bl-04) orally via food (cereal) and also by gavage and exposed to Influenza A virus H1N1 (H1N1). The symptoms of the infection were observed, and tissues and digesta were collected for viral load RT-qPCR, transcriptomics, and microbiomics. The treatment decreased the viral load by 48% at day 3 post-infection in lungs and symptoms of infection at day 4 compared to placebo. Tissue transcriptomics showed differences between the Bl-04 and placebo groups in the genes in the Influenza A pathway in the intestine, blood, and lungs prior to and post-infection, but the results were inconclusive. Moreover, 16S rRNA gene profiling and qPCR showed the presence of Bl-04 in the intestine, but without major shifts in the microbiome. In conclusion, Bl-04 treatment may influence the host response against H1N1 in a murine challenge model; however, further studies are required to elucidate the mechanism of action.

The effect of the probiotic consortia on SARS-CoV-2 infection in ferrets and on human immune cell response in vitro.

Probiotics have been suggested as one solution to counter detrimental health effects by SARS-CoV-2; however, data so far is scarce. We tested the effect of two probiotic consortia, OL-1 and OL-2, against SARS-CoV-2 in ferrets and assessed their effect on cytokine production and transcriptome in a human monocyte-derived macrophage (Mf) and dendritic cell (DC) model. The results showed that the consortia significantly reduced the viral load, modulated immune response, and regulated viral receptor expression in ferrets compared to placebo. In the human Mf and DC model, OL-1 and OL-2-induced cytokine production and genes related to SARS-CoV-2 antiviral immunity. The study results indicate that probiotic stimulation of the ferret immune system leads to improved antiviral immunity against SARS-COV-2, and the genes and cytokines associated with anti-SARS-CoV-2 immunity are stimulated in human immune cells in vitro. The effect of the consortia against SARS-CoV-2 warrants further investigations in human clinical trials.

Strain-specific strategies of 2'-fucosyllactose, 3-fucosyllactose, and difucosyllactose assimilation by Bifidobacterium longum subsp. infantis Bi-26 and ATCC 15697.

Human milk provides essential nutrients for infant nutrition. A large proportion of human milk is composed of human milk oligosaccharides (HMOs), which are resistant to digestion by the infant. Instead, HMOs act as a bioactive and prebiotic enriching HMO-utilizing bacteria and cause systematic changes in the host. Several species of Bifidobacterium have been shown to utilize HMOs by conserved, as well as species-specific pathways, but less work has been done to study variation within species or sub-species. B. longum subsp. infantis is a prevalent species in the breast-fed infant gut and the molecular mechanisms of HMO utilization for the type strain B. longum subsp. infantis ATCC 15697 (type strain) have been well characterized. We used growth, transcriptomic, and metabolite analysis to characterize key differences in the utilization of 2'FL, 3FL and DFL (FLs) between B. longum subsp. infantis Bi-26 (Bi-26) and the type strain. Bi-26 grows faster, produces unique metabolites, and has a distinct global gene transcription response to FLs compared to the type strain. Taken together the findings demonstrate major strain specific adaptations in Bi-26 to efficient utilization of FLs.

Gene expression signatures identify paediatric patients with multiple organ dysfunction who require advanced life support in the intensive care unit.

BACKGROUND: Multiple organ dysfunction syndrome (MODS) occurs in the setting of a variety of pathologies including infection and trauma. Some patients decompensate and require Veno-Arterial extra corporeal membrane oxygenation (ECMO) as a palliating manoeuvre for recovery of cardiopulmonary function. The molecular mechanisms driving progression from MODS to cardiopulmonary collapse remain incompletely understood, and no biomarkers have been defined to identify those MODS patients at highest risk for progression to requiring ECMO support. METHODS: Whole blood RNA-seq profiling was performed for 23 MODS patients at three time points during their ICU stay (at diagnosis of MODS, 72 hours after, and 8 days later), as well as four healthy controls undergoing routine sedation. Of the 23 MODS patients, six required ECMO support (ECMO patients). The predictive power of conventional demographic and clinical features was quantified for differentiating the MODS and ECMO patients. We then compared the performance of markers derived from transcriptomic profiling including [1] transcriptomically imputed leukocyte subtype distribution, [2] relevant published gene signatures and [3] a novel differential gene expression signature computed from our data set. The predictive power of our novel gene expression signature was then validated using independently published datasets. FINDING: None of the five demographic characteristics and 14 clinical features, including The Paediatric Logistic Organ Dysfunction (PELOD) score, could predict deterioration of MODS to ECMO at baseline. From previously published sepsis signatures, only the signatures positively associated with patient's mortality could differentiate ECMO patients from MODS patients, when applied to our transcriptomic dataset (P-value ranges from 0.01 to 0.04, Student's test). Deconvolution of bulk RNA-Seq samples suggested that lower neutrophil counts were associated with increased risk of progression from MODS to ECMO (P-value = 0.03, logistic regression, OR=2.82 [95% CI 0.63 - 12.45]). A total of 30 genes were differentially expressed between ECMO and MODS patients at baseline (log2 fold change ≥ 1 or ≤ -1 with false discovery rate ≤ 0.01). These genes are involved in protein maintenance and epigenetic-related processes. Further univariate analysis of these 30 genes suggested a signature of seven DE genes associated with ECMO (OR > 3.0, P-value ≤ 0.05, logistic regression). Notably, this contains a set of histone marker genes, including H1F0, HIST2H3C, HIST1H2AI, HIST1H4, HIST1H2BL and HIST1H1B, that were highly expressed in ECMO. A risk score derived from expression of these genes differentiated ECMO and MODS patients in our dataset (AUC = 0.91, 95% CI 0.79-1.00, P-value = 7e-04, logistic regression) as well as validation dataset (AUC= 0.73, 95% CI 0.53-0.93, P-value = 2e-02, logistic regression). INTERPRETATION: This study demonstrates that transcriptomic features can serve as indicators of severity that could be superior to traditional methods of ascertaining acuity in MODS patients. Analysis of expression of signatures identified in this study could help clinicians in the diagnosis and prognostication of MODS patients after arrival to the Hospital.

EditR: A Method to Quantify Base Editing from Sanger Sequencing.

CRISPR-Cas9-Cytidine deaminase fusion enzymes-termed "base editors"-allow targeted editing of genomic deoxycytidine to deoxythymidine (C:G→T:A) without the need for double-stranded break induction. Base editors represent a paradigm shift in gene editing technology due to their unprecedented efficiency to mediate targeted, single-base conversion. However, current analysis of base editing outcomes rely on methods that are either imprecise or expensive and time-consuming. To overcome these limitations, we developed a simple, cost-effective, and accurate program to measure base editing efficiency from fluorescence-based Sanger sequencing, termed "EditR." We provide EditR as a free online tool or downloadable desktop application requiring a single Sanger sequencing file and guide RNA sequence. EditR is more accurate than enzymatic assays, and provides added insight to the position, type, and efficiency of base editing. Furthermore, EditR is likely amenable to quantify base editing from the recently developed adenosine deaminase base editors that act on either DNA (adenosine deaminase base editors [ABEs]) or RNA (REPAIRs) (catalyzes A:T→G:C). Collectively, we demonstrate that EditR is a robust, inexpensive tool that will facilitate the broad application of base editing technology, thereby fostering further innovation in this burgeoning field.