Broad anti-pathogen potential of DEAD box RNA helicase eIF4A-targeting rocaglates.

Inhibition of eukaryotic initiation factor 4A has been proposed as a strategy to fight pathogens. Rocaglates exhibit the highest specificities among eIF4A inhibitors, but their anti-pathogenic potential has not been comprehensively assessed across eukaryotes. In silico analysis of the substitution patterns of six eIF4A1 aa residues critical to rocaglate binding, uncovered 35 variants. Molecular docking of eIF4A:RNA:rocaglate complexes, and in vitro thermal shift assays with select recombinantly expressed eIF4A variants, revealed that sensitivity correlated with low inferred binding energies and high melting temperature shifts. In vitro testing with silvestrol validated predicted resistance in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. Our analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens with rocaglates. Finally, our findings might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.

Multi-level analysis of the gut-brain axis shows autism spectrum disorder-associated molecular and microbial profiles.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by heterogeneous cognitive, behavioral and communication impairments. Disruption of the gut-brain axis (GBA) has been implicated in ASD although with limited reproducibility across studies. In this study, we developed a Bayesian differential ranking algorithm to identify ASD-associated molecular and taxa profiles across 10 cross-sectional microbiome datasets and 15 other datasets, including dietary patterns, metabolomics, cytokine profiles and human brain gene expression profiles. We found a functional architecture along the GBA that correlates with heterogeneity of ASD phenotypes, and it is characterized by ASD-associated amino acid, carbohydrate and lipid profiles predominantly encoded by microbial species in the genera Prevotella, Bifidobacterium, Desulfovibrio and Bacteroides and correlates with brain gene expression changes, restrictive dietary patterns and pro-inflammatory cytokine profiles. The functional architecture revealed in age-matched and sex-matched cohorts is not present in sibling-matched cohorts. We also show a strong association between temporal changes in microbiome composition and ASD phenotypes. In summary, we propose a framework to leverage multi-omic datasets from well-defined cohorts and investigate how the GBA influences ASD.

Targeting the DEAD-Box RNA Helicase eIF4A with Rocaglates-A Pan-Antiviral Strategy for Minimizing the Impact of Future RNA Virus Pandemics.

The increase in pandemics caused by RNA viruses of zoonotic origin highlights the urgent need for broad-spectrum antivirals against novel and re-emerging RNA viruses. Broad-spectrum antivirals could be deployed as first-line interventions during an outbreak while virus-specific drugs and vaccines are developed and rolled out. Viruses depend on the host's protein synthesis machinery for replication. Several natural compounds that target the cellular DEAD-box RNA helicase eIF4A, a key component of the eukaryotic translation initiation complex eIF4F, have emerged as potential broad-spectrum antivirals. Rocaglates, a group of flavaglines of plant origin that clamp mRNAs with highly structured 5' untranslated regions (5'UTRs) onto the surface of eIF4A through specific stacking interactions, exhibit the largest selectivity and potential therapeutic indices among all known eIF4A inhibitors. Their unique mechanism of action limits the inhibitory effect of rocaglates to the translation of eIF4A-dependent viral mRNAs and a minor fraction of host mRNAs exhibiting stable RNA secondary structures and/or polypurine sequence stretches in their 5'UTRs, resulting in minimal potential toxic side effects. Maintaining a favorable safety profile while inducing efficient inhibition of a broad spectrum of RNA viruses makes rocaglates into primary candidates for further development as pan-antiviral therapeutics.

The rocaglate CR-31-B (-) inhibits SARS-CoV-2 replication at non-cytotoxic, low nanomolar concentrations in vitro and ex vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, a severe respiratory disease with varying clinical presentations and outcomes, and responsible for a major pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against multiple RNA viruses including coronaviruses. Specifically, rocaglates inhibit eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. Here, we assessed the antiviral activity of the synthetic rocaglate CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In primary human airway epithelial cells, CR-31-B (-) reduced viral titers to undetectable levels at a concentration of 100 nM. Reduced virus reproduction was accompanied by substantially reduced viral protein accumulation and replication/transcription complex formation. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.

Integrating Systems Biology and an Ex Vivo Human Tumor Model Elucidates PD-1 Blockade Response Dynamics.

Ex vivo human tumor models have emerged as promising, yet complex tools to study cancer immunotherapy response dynamics. Here, we present a strategy that integrates empirical data from an ex vivo human system with computational models to interpret the response dynamics of a clinically prescribed PD-1 inhibitor, nivolumab, in head and neck squamous cell carcinoma (HNSCC) biopsies (N = 50). Using biological assays, we show that drug-induced variance stratifies samples by T helper type 1 (Th1)-related pathways. We then built a systems biology network and mathematical framework of local and global sensitivity analyses to simulate and estimate antitumor phenotypes, which implicate a dynamic role for the induction of Th1-related cytokines and T cell proliferation patterns. Together, we describe a multi-disciplinary strategy to analyze and interpret the response dynamics of PD-1 blockade using heterogeneous ex vivo data and in silico simulations, which could provide researchers a powerful toolset to interrogate immune checkpoint inhibitors.

Metabolic Surgery in the Treatment Algorithm for Type 2 Diabetes: a Joint Statement by International Diabetes Organizations.

BACKGROUND: Despite growing evidence that bariatric/metabolic surgery powerfully improves type 2 diabetes (T2D), existing diabetes treatment algorithms do not include surgical options. AIM: The 2nd Diabetes Surgery Summit (DSS-II), an international consensus conference, was convened in collaboration with leading diabetes organizations to develop global guidelines to inform clinicians and policymakers about benefits and limitations of metabolic surgery for T2D. METHODS: A multidisciplinary group of 48 international clinicians/scholars (75% nonsurgeons), including representatives of leading diabetes organizations, participated in DSS-II. After evidence appraisal (MEDLINE [1 January 2005-30 September 2015]), three rounds of Delphi-like questionnaires were used to measure consensus for 32 data-based conclusions. These drafts were presented at the combined DSS-II and 3rd World Congress on Interventional Therapies for Type 2 Diabetes (London, U.K., 28-30 September 2015), where they were open to public comment by other professionals and amended face-to-face by the Expert Committee. RESULTS: Given its role in metabolic regulation, the gastrointestinal tract constitutes a meaningful target to manage T2D. Numerous randomized clinical trials, albeit mostly short/midterm, demonstrate that metabolic surgery achieves excellent glycemic control and reduces cardiovascular risk factors. On the basis of such evidence, metabolic surgery should be recommended to treat T2D in patients with class III obesity (BMI ≥40 kg/m2) and in those with class II obesity (BMI 35.0-39.9 kg/m2) when hyperglycemia is inadequately controlled by lifestyle and optimal medical therapy. Surgery should also be considered for patients with T2D and BMI 30.0-34.9 kg/m2 if hyperglycemia is inadequately controlled despite optimal treatment with either oral or injectable medications. These BMI thresholds should be reduced by 2.5 kg/m2 for Asian patients. CONCLUSIONS: Although additional studies are needed to further demonstrate long-term benefits, there is sufficient clinical and mechanistic evidence to support inclusion of metabolic surgery among antidiabetes interventions for people with T2D and obesity. To date, the DSS-II guidelines have been formally endorsed by 45 worldwide medical and scientific societies. Health care regulators should introduce appropriate reimbursement policies.

Metabolic Surgery in the Treatment Algorithm for Type 2 Diabetes: A Joint Statement by International Diabetes Organizations.

BACKGROUND: Despite growing evidence that bariatric/metabolic surgery powerfully improves type 2 diabetes (T2D), existing diabetes treatment algorithms do not include surgical options. AIM: The 2nd Diabetes Surgery Summit (DSS-II), an international consensus conference, was convened in collaboration with leading diabetes organizations to develop global guidelines to inform clinicians and policymakers about benefits and limitations of metabolic surgery for T2D. METHODS: A multidisciplinary group of 48 international clinicians/scholars (75% nonsurgeons), including representatives of leading diabetes organizations, participated in DSS-II. After evidence appraisal (MEDLINE [1 January 2005-30 September 2015]), three rounds of Delphi-like questionnaires were used to measure consensus for 32 data-based conclusions. These drafts were presented at the combined DSS-II and 3rd World Congress on Interventional Therapies for Type 2 Diabetes (London, U.K., 28-30 September 2015), where they were open to public comment by other professionals and amended face-to-face by the Expert Committee. RESULTS: Given its role in metabolic regulation, the gastrointestinal tract constitutes a meaningful target to manage T2D. Numerous randomized clinical trials, albeit mostly short/midterm, demonstrate that metabolic surgery achieves excellent glycemic control and reduces cardiovascular risk factors. On the basis of such evidence, metabolic surgery should be recommended to treat T2D in patients with class III obesity (BMI≥40 kg/m(2)) and in those with class II obesity (BMI 35.0-39.9 kg/m(2)) when hyperglycemia is inadequately controlled by lifestyle and optimal medical therapy. Surgery should also be considered for patients with T2D and BMI 30.0-34.9 kg/m(2) if hyperglycemia is inadequately controlled despite optimal treatment with either oral or injectable medications. These BMI thresholds should be reduced by 2.5 kg/m(2) for Asian patients. CONCLUSIONS: Although additional studies are needed to further demonstrate long-term benefits, there is sufficient clinical and mechanistic evidence to support inclusion of metabolic surgery among antidiabetes interventions for people with T2D and obesity. To date, the DSS-II guidelines have been formally endorsed by 45 worldwide medical and scientific societies. Health care regulators should introduce appropriate reimbursement policies.

Metabolic Surgery in the Treatment Algorithm for Type 2 Diabetes: A Joint Statement by International Diabetes Organizations.

BACKGROUND: Despite growing evidence that bariatric/metabolic surgery powerfully improves type 2 diabetes (T2D), existing diabetes treatment algorithms do not include surgical options. AIM: The 2nd Diabetes Surgery Summit (DSS-II), an international consensus conference, was convened in collaboration with leading diabetes organizations to develop global guidelines to inform clinicians and policymakers about benefits and limitations of metabolic surgery for T2D. METHODS: A multidisciplinary group of 48 international clinicians/scholars (75% nonsurgeons), including representatives of leading diabetes organizations, participated in DSS-II. After evidence appraisal (MEDLINE [1 January 2005-30 September 2015]), three rounds of Delphi-like questionnaires were used to measure consensus for 32 data-based conclusions. These drafts were presented at the combined DSS-II and 3rd World Congress on Interventional Therapies for Type 2 Diabetes (London, U.K., 28-30 September 2015), where they were open to public comment by other professionals and amended face-to-face by the Expert Committee. RESULTS: Given its role in metabolic regulation, the gastrointestinal tract constitutes a meaningful target to manage T2D. Numerous randomized clinical trials, albeit mostly short/midterm, demonstrate that metabolic surgery achieves excellent glycemic control and reduces cardiovascular risk factors. On the basis of such evidence, metabolic surgery should be recommended to treat T2D in patients with class III obesity (BMI ≥40 kg/m(2)) and in those with class II obesity (BMI 35.0-39.9 kg/m(2)) when hyperglycemia is inadequately controlled by lifestyle and optimal medical therapy. Surgery should also be considered for patients with T2D and BMI 30.0-34.9 kg/m(2) if hyperglycemia is inadequately controlled despite optimal treatment with either oral or injectable medications. These BMI thresholds should be reduced by 2.5 kg/m(2) for Asian patients. CONCLUSIONS: Although additional studies are needed to further demonstrate long-term benefits, there is sufficient clinical and mechanistic evidence to support inclusion of metabolic surgery among antidiabetes interventions for people with T2D and obesity. To date, the DSS-II guidelines have been formally endorsed by 45 worldwide medical and scientific societies. Health care regulators should introduce appropriate reimbursement policies.

Oligonucleotide microarrays for the study of coastal microbial communities.

DNA microarrays are well suited as a tool for analyzing functional gene diversity as well as community composition in aquatic environments. Microarrays allow for the semi-quantitative characterization of target genes by means of specific hybridization of labeled target gene sequences, amplified from the environment, to the corresponding oligonucleotide probes on the slide. Specificity and sensitivity are determined by the probe design. In their current implementation, environmental DNA microarrays are useful for analyzing microbial communities as well as for analyzing the presence of functional genes involved in larger biogeochemical processes, such as nitrogen cycling. Here, we lay out a basic protocol to analyze genes in the environment, which can be applied to most target genes of interest.

Oligonucleotide microarray for the study of functional gene diversity in the nitrogen cycle in the environment.

The analysis of functional diversity and its dynamics in the environment is essential for understanding the microbial ecology and biogeochemistry of aquatic systems. Here we describe the development and optimization of a DNA microarray method for the detection and quantification of functional genes in the environment and report on their preliminary application to the study of the denitrification gene nirS in the Choptank River-Chesapeake Bay system. Intergenic and intragenic resolution constraints were determined by an oligonucleotide (70-mer) microarray approach. Complete signal separation was achieved when comparing unrelated genes within the nitrogen cycle (amoA, nifH, nirK, and nirS) and detecting different variants of the same gene, nirK, corresponding to organisms with two different physiological modes, ammonia oxidizers and denitrifying halobenzoate degraders. The limits of intragenic resolution were investigated with a microarray containing 64 nirS sequences comprising 14 cultured organisms and 50 clones obtained from the Choptank River in Maryland. The nirS oligonucleotides covered a range of sequence identities from approximately 40 to 100%. The threshold values for specificity were determined to be 87% sequence identity and a target-to-probe perfect match-to-mismatch binding free-energy ratio of 0.56. The lower detection limit was 10 pg of DNA (equivalent to approximately 10(7) copies) per target per microarray. Hybridization patterns on the microarray differed between sediment samples from two stations in the Choptank River, implying important differences in the composition of the denitirifer community along an environmental gradient of salinity, inorganic nitrogen, and dissolved organic carbon. This work establishes a useful set of design constraints (independent of the target gene) for the implementation of functional gene microarrays for environmental applications.

Genome-wide dynamic transcriptional profiling of the light-to-dark transition in Synechocystis sp. strain PCC 6803.

We report the results of whole-genome transcriptional profiling of the light-to-dark transition with the model photosynthetic prokaryote Synechocystis sp. strain PCC 6803 (Synechocystis). Experiments were conducted by growing Synechocystis cultures to mid-exponential phase and then exposing them to two cycles of light/dark conditions, during which RNA samples were obtained. These samples were probed with a full-genome DNA microarray (3,169 genes, 20 samples) as well as a partial-genome microarray (88 genes, 29 samples). We concluded that (i) 30-min sampling intervals accurately captured transcriptional dynamics throughout the light/dark transition, (ii) 25% of the Synechocystis genes (783 genes) responded positively to the presence of light, and (iii) the response dynamics varied greatly for individual genes, with a delay of up to 120 to 150 min for some genes. Four classes of genes were identified on the basis of their dynamic gene expression profiles: class I (108 genes, 30-min response time), class II (279 genes, 60 to 90 min), class III (258 genes, 120 to 150 min), and class IV (138 genes, 180 min). The dynamics of several transcripts from genes involved in photosynthesis and primary energy generation are discussed. Finally, we applied Fisher discriminant analysis to better visualize the progression of the overall transcriptional program throughout the light/dark transition and to determine those genes most indicative of the lighting conditions during growth.