In silico, in vitro and in vivo characterization of host-associated Latilactobacillus curvatus strains for potential probiotic applications in farmed Atlantic salmon (Salmo salar).

Salmon aquaculture is the fastest growing animal protein production system in the world; however, intensive farming leads to poor weight gain, stress, and disease outbreaks. Probiotics offer the potential to enhance growth performance and feed efficiency in Atlantic salmon, as well as immunostimulate fish against common pathogens, benefitting farmers and consumers with more efficient production. Here, we isolated and identified 900 native microbial isolates including 18 Lactobacilli from the farmed salmon intestines. Based on whole-genome sequencing and phylogenetic analysis, the Lactobacillus candidates belonged to Latilactobacillus curvatus (L. curvatus) species and formed two distinct phylogenetic groups. Using bioinformatics and in vitro analyses, we selected two candidates L. curvatus ATCC PTA-127116 and L. curvatus ATCC PTA-127117, which showed desirable safety and probiotic properties. The two L. curvatus candidates were evaluated for safety and efficacy (higher final weight) in Atlantic salmon alongside spore-forming Bacilli isolated from salmon, poultry, and swine. All the tested candidates were safe to salmon with no adverse effects. While we did not see efficacy in any Bacillus supplemented groups, compared to untreated group, the group administered with the two L. curvatus strains consortium in feed for seven weeks in freshwater showed indicators of improvement in final body weight by 4.2%. Similarly, the two L. curvatus candidates were also evaluated for safety and efficacy in Atlantic salmon in saltwater; the group administered with the two L. curvatus strains consortium in feed for 11 weeks showed indicators of improvement in final body weight by 4.7%. Comprehensive metabolomics analyses in the presence of different prebiotics and/or additives identified galactooligosaccharide as a potential prebiotic to enhance the efficacy of two L. curvatus candidates. All together, these data provide comprehensive genomic, phenotypic and metabolomic evidence of safety and desirable probiotic properties as well as indicators of in vivo efficacy of two novel endogenous L. curvatus candidates for potential probiotic applications in Atlantic salmon. The in vivo findings need to be confirmed in larger performance studies, including field trials.

In silico, in vitro and in vivo safety evaluation of Limosilactobacillus reuteri strains ATCC PTA-126787 & ATCC PTA-126788 for potential probiotic applications.

The last two decades have witnessed a tremendous growth in probiotics and in the numbers of publications on their potential health benefits. Owing to their distinguishing beneficial effects and long history of safe use, species belonging to the Lactobacillus genus are among the most widely used probiotic species in human food and dietary supplements and are finding increased use in animal feed. Here, we isolated, identified, and evaluated the safety of two novel Limosilactobacillus reuteri (L. reuteri) isolates, ATCC PTA-126787 & ATCC PTA-126788. More specifically, we sequenced the genomes of these two L. reuteri strains using the PacBio sequencing platform. Using a combination of biochemical and genetic methods, we identified the two strains as belonging to L. reuteri species. Detailed in silico analyses showed that the two strains do not encode for any known genetic sequences of concern for human or animal health. In vitro assays confirmed that the strains are susceptible to clinically relevant antibiotics and do not produce potentially harmful by-products such as biogenic amines. In vitro bile and acid tolerance studies demonstrated that the two strains have similar survival profiles as the commercial L. reuteri probiotic strain DSM 17938. Most importantly, daily administration of the two probiotic strains to broiler chickens in drinking water for 26 days did not induce any adverse effect, clinical disease, or histopathological lesions, supporting the safety of the strains in an in vivo avian model. All together, these data provide in silico, in vitro and in vivo evidence of the safety of the two novel candidates for potential probiotic applications in humans as well as animals.

Singapore Undiagnosed Disease Program: Genomic Analysis aids Diagnosis and Clinical Management.

OBJECTIVE: Use next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting. DESIGN: A diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019. SETTING: Inpatient and outpatient genetics service at two large academic centres in Singapore. PATIENTS: Inclusion criteria: patients suspected of genetic disorders, based on abnormal antenatal ultrasound, multiple congenital anomalies and developmental delay. EXCLUSION CRITERIA: patients with known genetic disorders, either after clinical assessment or investigations (such as karyotype or chromosomal microarray). INTERVENTIONS: Use of NGS technology-whole exome sequencing (WES) or whole genome sequencing (WGS). MAIN OUTCOME MEASURES: (1) Diagnostic yield by sequencing type, (2) diagnostic yield by phenotypical categories, (3) reduction in time to diagnosis and (4) change in clinical outcomes and management. RESULTS: We demonstrate a 37.8% diagnostic yield for WES (n=172) and a 33.3% yield for WGS (n=24). The yield was higher when sequencing was conducted on trios (40.2%), as well as for certain phenotypes (neuromuscular, 54%, and skeletal dysplasia, 50%). In addition to aiding genetic counselling in 100% of the families, a positive result led to a change in treatment in 27% of patients. CONCLUSION: Genomic sequencing is an effective method for diagnosing rare disease or previous 'undiagnosed' disease. The clinical utility of WES/WGS is seen in the shortened time to diagnosis and the discovery of novel variants. Additionally, reaching a diagnosis significantly impacts families and leads to alteration in management of these patients.

The genetic basis of inbreeding depression in potato.

Inbreeding depression confers reduced fitness among the offspring of genetic relatives. As a clonally propagated crop, potato (Solanum tuberosum L.) suffers from severe inbreeding depression; however, the genetic basis of inbreeding depression in potato is largely unknown. To gain insight into inbreeding depression in potato, we evaluated the mutation burden in 151 diploid potatoes and obtained 344,831 predicted deleterious substitutions. The deleterious mutations in potato are enriched in the pericentromeric regions and are line specific. Using three F2 populations, we identified 15 genomic regions with severe segregation distortions due to selection at the gametic and zygotic stages. Most of the deleterious recessive alleles affecting survival and growth vigor were located in regions with high recombination rates. One of these deleterious alleles is derived from a rare mutation that disrupts a gene required for embryo development. This study provides the basis for genome design of potato inbred lines.

Population genomic analyses of the chocolate tree, Theobroma cacao L., provide insights into its domestication process.

Domestication has had a strong impact on the development of modern societies. We sequenced 200 genomes of the chocolate plant Theobroma cacao L. to show for the first time to our knowledge that a single population, the Criollo population, underwent strong domestication ~3600 years ago (95% CI: 2481-13,806 years ago). We also show that during the process of domestication, there was strong selection for genes involved in the metabolism of the colored protectants anthocyanins and the stimulant theobromine, as well as disease resistance genes. Our analyses show that domesticated populations of T. cacao (Criollo) maintain a higher proportion of high-frequency deleterious mutations. We also show for the first time the negative consequences of the increased accumulation of deleterious mutations during domestication on the fitness of individuals (significant reduction in kilograms of beans per hectare per year as Criollo ancestry increases, as estimated from a GLM, P = 0.000425).

ChronQC: a quality control monitoring system for clinical next generation sequencing.

Summary: ChronQC is a quality control (QC) tracking system for clinical implementation of next-generation sequencing (NGS). ChronQC generates time series plots for various QC metrics to allow comparison of current runs to historical runs. ChronQC has multiple features for tracking QC data including Westgard rules for clinical validity, laboratory-defined thresholds and historical observations within a specified time period. Users can record their notes and corrective actions directly onto the plots for long-term recordkeeping. ChronQC facilitates regular monitoring of clinical NGS to enable adherence to high quality clinical standards. Availability and implementation: ChronQC is freely available on GitHub (https://github.com/nilesh-tawari/ChronQC), Docker (https://hub.docker.com/r/nileshtawari/chronqc/) and the Python Package Index. ChronQC is implemented in Python and runs on all common operating systems (Windows, Linux and Mac OS X). Contact: tawari.nilesh@gmail.com or pauline.c.ng@gmail.com. Supplementary information: Supplementary data are available at Bioinformatics online.

High throughput sequencing reveals Drosophila suzukii responses to insecticides.

Global climate change and acquired resistance to insecticides are threats to world food security. Drosophila suzukii, a devastating invasive pest in many parts of the world, causes substantial economic losses to fruit production industries, forcing farmers to apply broad-spectrum insecticides frequently. This could lead to the development of insecticide resistance. We determined the Lethal Concentration 50 (median lethal concentration, LC50 ) values of zeta-cypermethrin, spinosad, and malathion insecticides against D. suzukii colonies established from Clarke and Pierce county Georgia, United States. The LC50 values were 3 fold higher in the Pierce county population for all insecticide treatments. We then used RNA sequencing to analyze the responses of Pierce and Clarke population flies surviving a LC50 treatment of the 3 insecticides. We identified a high number of differentially expressed genes that are likely involved in detoxification and reduced cuticular penetration, especially in the Pierce population, with extensive overlap in differentially expressed genes between the 3 insecticide treatments. Finally, we predicted fewer nonsynonymous single nucleotide variants having deleterious effects on protein function among detoxification, insecticide target, and cuticular protein encoding genes in Pierce flies. Thus a combination of increased gene expression and fewer deleterious single nucleotide variants highlights molecular mechanisms underlying the higher LC50 values for Pierce population flies.

Whole-Genome Resequencing Identifies the Molecular Genetic Cause for the Absence of a Gy5 Glycinin Protein in Soybean PI 603408.

During ongoing proteomic analysis of the soybean (Glycine max (L.) Merr) germplasm collection, PI 603408 was identified as a landrace whose seeds lack accumulation of one of the major seed storage glycinin protein subunits. Whole genomic resequencing was used to identify a two-base deletion affecting glycinin 5 The newly discovered deletion was confirmed to be causative through immunological, genetic, and proteomic analysis, and no significant differences in total seed protein content were found to be due to the glycinin 5 loss-of-function mutation per se In addition to focused studies on this one specific glycinin subunit-encoding gene, a total of 1,858,185 nucleotide variants were identified, of which 39,344 were predicted to affect protein coding regions. In order to semiautomate analysis of a large number of soybean gene variants, a new SIFT 4G (Sorting Intolerant From Tolerated 4 Genomes) database was designed to predict the impact of nonsynonymous single nucleotide soybean gene variants, potentially enabling more rapid analysis of soybean resequencing data in the future.

Rational drug design, synthesis and biological evaluation of dihydrofolate reductase inhibitors as antituberculosis agents.

BACKGROUND: A series of 2,4-diamino-s-triazines was designed, with potential for activity against Mycobacterium tuberculosis (Mtb) dihydrofolate reductase enzyme, on the basis of virtual screening results and structure-based drug design. RESULTS: The compounds were evaluated against Mtb (H37Rv) and their cytotoxicity was assessed using VERO cell lines. Of particular note, two compounds were found to have the most promising antituberculosis activity (6b minimum inhibitory concentration: 1.76 µM and 6i minimum inhibitory concentration: 1.57 µM) along with low cytotoxicity (CC50: >300 µM). The enzyme assay results of these two indicated significant inhibition of Mtb dihydrofolate reductase along with selectivity. Selected derivatives were tested against dormant tubercle bacilli in vivo and ex vivo indicating potential inhibition. CONCLUSION: This study provides promising antituberculosis dihydrofolate reductase inhibitors that can act as potential leads for further development.

Novel 2-hydrazino-pyrimidin-4(3H)-one derivatives with pseudofunctional- similarity to siderophores as potential antimycobacterial agents.

Siderophores are small molecules produced by bacteria under iron-scarcity conditions faced by bacteria inside host. Sideophores bind iron with high affinity (Kd < 10-25 M) and are required for iron transport into the bacterial cell. Small molecules interfering with siderophore functioning can be promising anti-mycobacterial agents. Several molecules with hydrazone as a structural feature are known to have metal chelating property. This prompted us to investigate the metal chelating ability of 2-hydrazino-pyrimidin-4(3H)-one derivatives. In this light, a library of 22 novel molecules with 2- hydrazino-pyrimidin-4(3H)-one moiety was synthesized and the compounds were evaluated against M. tuberculosis under iron-limiting and iron-rich conditions. Interestingly, several molecules showed promising (MIC: < 10 µM) selective activity under iron scarcity conditions. Furthermore, compounds were found to be nontoxic at lower concentration in VERO cell lines using MTT assay. Taken together, we have discovered novel 2-hydrazino-pyrimidin-4(3H)-one molecules active against M. tuberculosis which can be developed as potent antimycobacterial agents.

Pharmacophore modeling and density functional theory analysis for a series of nitroimidazole compounds with antitubercular activity.

In an attempt to highlight structural features required for potent antitubercular activity, five pharmacophoric features were developed for PA-824 and its analogs. The generated pharmacophore indicated importance of a nitro group, three hydrogen bond acceptor features, and a distal aromatic ring for potent activity. The model based on pharmacophore alignment has good correlation coefficient for the training set (r(2) = 0.81, SD = 0.31, F = 122.9, N = 152), which was evaluated using a test set (Q(2) = 0.77, root-mean-square error = 0.35, Pearson-R = 0.88, N = 49). Structure-activity relationship investigation further revealed that hydrophobic substitutions at the para-position of distal aromatic ring could lead to more potent analogs. The most active and inactive compounds were further studied using density functional theory at B3LYP/3-21*G level. The calculated electrostatic profile indicated that these compounds possess maximum negative potential in the vicinity of nitro group extending laterally to the imidazole ring. Furthermore, the calculated electron affinity values indicate the stability of radical anions, which could form upon one electron reduction in the biological system, thus, indicating the electron acceptor capacity of these compounds. Results of this study are expected to be useful in the design of novel potent nitroimidazoles as antitubercular agents.

A review of molecular modelling studies of dihydrofolate reductase inhibitors against opportunistic microorganisms and comprehensive evaluation of new models.

Dihydrofolate reductase (DHFR) has been used as a target for antimicrobial drug discovery against a variety of pathogenic microorganisms, including opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii (tg) and Mycobacterium avium complex (ma). In this regard, several DHFR inhibitors are reported against pc and tg and ma. However, selectivity issue of these inhibitors over human DHFR often preclude their development and clinical use. In the first part of this work, various computational approaches including available crystallographic structures, binding affinity prediction, pharmacophore mapping, QSAR, homology modelling used for design of DHFR inhibitors against opportunistic microorganisms are reviewed, to understand specific interactions required for inhibition of microbial DHFR. Secondly, comprehensive molecular modelling techniques were used, to establish structure-chemical-feature-based pharmacophore models for pcDHFR, tgDHFR and mammalian DHFR. The results show that, the information encoded by ligand based approaches like pharmacophore mapping and 3D-QSAR methods are in well agreement with the information coded in the receptor structure. A combination of ligand and structure based approaches provides understanding of ligand-receptor interactions. The study indicated that the value of small alkyl moieties at position 5 of the bicyclic nitrogen containing nucleus along with a bulky group attached at the C-6 via suitable linker could optimize activity, with regard to both potency and selectivity.

Predictive models for nucleoside bisubstrate analogs as inhibitors of siderophore biosynthesis in Mycobacterium tuberculosis: pharmacophore mapping and chemometric QSAR study.

Inhibitors of aryl acid adenylating enzymes (AAAE), known as MbtA, involved in siderophore biosynthesis in Mycobacterium tuberculosis, are being explored as potential antitubercular agents. In this article, we report the development of a robust pharmacophore model and investigation of structure-activity relationship of several nucleoside bisubstrate analogs reported as MbtA inhibitors. The developed pharmacophore model revealed the importance of two hydrogen bond donors and one hydrogen bond acceptor features. Furthermore, it was found that an aromatic ring at the distal part of molecule away from the two aromatic rings of adenyl moiety is a critical requirement for the tight binding of inhibitor. The generated pharmacophore-based alignment was used to derive a predictive atom-based 3D-QSAR model for training set (r (2) = 0.97, SD = 0.23, F = 310.6, N = 48) and test set (Q (2) = 0.71, RMSE = 0.65, Pearson-R = 0.85, N = 15). Structure-activity relationship investigation further revealed that bulky substitutions at the C-6 position of adenyl moiety is detrimental to activity, while hydrophobic substitutions can be tolerated at C-2 position. Taken together, the PLS-generated QSAR regression cubes along with developed pharmacophore model provide a qualitative picture of the active site and can be used as a powerful tool for the rational modification of bisubstrate inhibitors of MbtA in search of better antitubercular agents. Furthermore, a three-class classification chemometric QSAR model was developed using molecular descriptors for the prediction of whole-cell activity which could be used in the predictive layer for screening of compounds before synthesis.

Design, synthesis, and biological evaluation of 4-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives as potent antitubercular agents.

Based on stereoelectronic feature analysis using density functional theory (DFT) at B3LYP/3-21∗G level, a series of 4-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives with low LUMO energies (<-0.10eV); concentrated over the nitro group, furan moiety and α,ß-unsaturated carbonyl bridge were envisaged as potential antitubercular agents. The target compounds were prepared by condensation of 5-nitro-2-furaldehyde with various ketones under acidic condition. The compounds were evaluated for antitubercular activity against Mycobacterium tuberculosis H37Rv and their cytotoxicity in VERO cell line. Several synthesized compounds showed good antitubercular activity of <5µM along with low cytotoxicity. In particular, compound ((E)-3-(5-nitrofuran-2-yl)-1-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one) (3v) was found to be very potent (MIC: 0.19µM) with good selectivity index (MIC(90)/CC(50): >1800). Thus, this study shows the potential of stereoelectronic property analysis in developing improved nitroaromatics as antitubercular agents.

Design, synthesis, biological evaluation and computational investigation of novel inhibitors of dihydrofolate reductase of opportunistic pathogens.

The present work deals with design, synthesis and biological evaluation of novel, diverse compounds as potential inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii (tg) and Mycobacterium avium (ma). A set of 14 structurally diverse compounds were designed with varying key pharmacophoric features of DHFR inhibitors, bulky distal substitutions and different bridges joining the distal part and 2,4-diaminopyrimidine nucleus. The designed compounds were synthesized and evaluated in enzyme assay against pc, tg and ma DHFR. The rat liver (rl) DHFR was used as mammalian standard. As the next logical step of the project, flexible molecular docking studies were carried out to predict the binding modes of these compounds in pcDHFR active site and the obtained docked poses were post processed using MM-GBSA protocol for prediction of relative binding affinity. The predicted binding modes were able to rationalize the experimental results in most cases. Of particular interest, both the docking scores and MM-GBSA predicted Delta G(bind) were able to distinguish between the active and low active compounds. Furthermore, good correlation coefficient of 0.797 was obtained between the IC(50) values and MM-GBSA predicted Delta G(bind). Taken together, the current work provides not only a novel scaffold for further optimization of DHFR inhibitors but also an understanding of the specific interactions of inhibitors with DHFR and structural modifications that improve selectivity.

Novel molecular hybrids of cinnamic acids and guanylhydrazones as potential antitubercular agents.

In an attempt to identify potential new agents active against tuberculosis, 20 novel phenylacrylamide derivatives incorporating cinnamic acids and guanylhydrazones were synthesized using microwave assisted synthesis. Activity of the synthesized compounds was evaluated using resazurin microtitre plate assay (REMA) against Mycobacterium tuberculosis H37Rv. Based on empirical structure-activity relationship data it was observed that both steric and electronic parameters play major role in the activity of this series of compounds. Compound 7s (2E)-N-((-2-(3,4-dimethoxybenzylidene) hydrazinyl) (imino) methyl)-3-(4-methoxyphenyl) acrylamide showed MIC of 6.49microM along with good safety profile of >50-fold in VERO cell line. Thus, this compound could act as a potential lead for further antitubercular studies.

In vitro biological evaluation of biguanides and dihydrotriazines against Brugia malayi and folate reversal studies.

Dihydrofolate reductase (DHFR) is a well-known target for antibacterial and anticancer therapy. DHFR inhibitors are useful for protozoan parasites, but are yet to be explored against metazoan species; hence the present work was designed to evaluate the efficacy of DHFR inhibitors against filariasis, one of the major neglected tropical diseases. Molecules from our in-house library of synthetic antifolate agents (biguanide and dihydrotriazine derivatives) were evaluated along with the antimalarial drug pyrimethamine and the antibacterial drug trimethoprim in an in vitro model against Brugia malayi microfilariae (Mf). Three biguanides and two dihydrotriazines were more potent than trimethoprim and pyrimethamine against B. malayi Mf. Trimethoprim, pyrimethamine and four of the five compounds active against Mf were also active against adult worms. To probe the mechanism of action of the compounds, reversal of activity of active compounds by folic acid and folinic acid was studied. In conclusion, DHFR inhibitors could be used as leads for new antifilarial drugs.

Synthesis and biological evaluation of biguanide and dihydrotriazine derivatives as potential inhibitors of dihydrofolate reductase of opportunistic microorganisms.

Twenty-one biguanide and dihydrotriazine derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms: Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium (ma), and rat liver (rl). The most potent compound in the series was B2-07 with 12 nM activity against tgDHFR. The most striking observation was that B2-07 showed similar potency to trimetrexate, approximately 233-fold improved potency over trimethoprim and approximately 7-fold increased selectivity as compared to trimetrexate against tgDHFR. Molecular docking studies in the developed homology model of tgDHFR rationalized the observed potency of B2-07. This molecule can act as a good lead for further design of molecules with better selectivity and improved potency.

Pharmacophore mapping and electronic feature analysis for a series of nitroaromatic compounds with antitubercular activity.

A five point pharmacophore was generated using PHASE for a series of nitroaromatic compounds and their congeners as antitubercular agents. The generated pharmacophore yielded significant 3D-QSAR model with r(2) of 0.890 for a training set of 92 molecules. The model also showed excellent predictive power with correlation coefficient Q(2) of 0.857 for a test set of 31 compounds. The pharmacophore indicated that presence of a nitro group, a piperazine moiety, one aromatic ring feature and two acceptor features are necessary for potent antitubercular activity. The pharmacophore was supported by electronic property analysis using density functional theory (DFT) at B3LYP/3-21*G level. Molecular electrostatic profile of the compounds was consistent with the generated pharmacophore model, particularly appearance of localized negative potential regions near both the oxygen atoms of nitro group extending laterally to the isoxazole ring system/amide bond in the most active compounds. Calculated data further revealed that all active compounds have smaller LUMO energies located over the nitro group, furan ring, and isoxazole ring/amide bond attached to it. Higher negative values of LUMO energies concentrated over the nitro group are indicative of the electron acceptor capacity of the compounds, suggesting that these compounds are prodrugs and must be activated by TB-nitroreductase. The results obtained from this study should aid in efficient design and development of nitroaromatic compounds as antitubercular agents.

Pharmacophore mapping of a series of pyrrolopyrimidines, indolopyrimidines and their congeners as multidrug-resistance-associated protein (MRP1) modulators.

Pharmacophore mapping studies were undertaken for a series of molecules belonging to pyrrolopyrimidines, indolopyrimidines and their congeners as multidrug resistance-associated protein (MRP1) modulators. A five-point pharmacophore with two hydrogen bond acceptors (A), one lipophilic/hydrophobic group (H), one positive ionic feature (P) and one aromatic ring (R) as pharmacophoric features was developed. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a correlation coefficient of r2 = 0.799 for training set molecules. The model generated showed excellent predictive power, with a correlation coefficient Q2 = 0.679 for an external test set of 20 molecules. The pharmacophore was further validated using four structurally diverse compounds with MRP1 modulatory activity. These compounds mapped well onto four of the five features of the pharmacophore. The pharmacophore proposed here was then utilised for the successful retrieval of active molecules with diverse chemotypes from database search. The geometry and features of pharmacophore are expected to be useful for the design of selective MRP1 inhibitors.