In Silico Analyses on the Comparative Potential of Therapeutic Human Monoclonal Antibodies Against Newly Emerged SARS-CoV-2 Variants Bearing Mutant Spike Protein

Since the start of the pandemic, SARS-CoV-2 has already infected more than 250 million people globally, with more than five million fatal cases and huge socio-economic losses. In addition to corticosteroids, and antiviral drugs like remdesivir, various immunotherapies including monoclonal antibodies (mAbs) to S protein of SARS-CoV-2 have been investigated to treat COVID-19 patients. These mAbs were initially developed against the wild-type SARS-CoV-2; however, emergence of variant forms of SARS-CoV-2 having mutations in the spike protein in several countries including India raised serious questions on the potential use of these mAbs against SARS-CoV-2 variants. In this study, using an in silico approach, we have examined the binding abilities of eight mAbs against several SARS-CoV-2 variants of Alpha (B.1.1.7) and Delta (B.1.617.2) lineages. The structure of the Fab region of each mAb was designed in silico and subjected to molecular docking against each mutant protein. mAbs were subjected to two levels of selection based on their binding energy, stability, and conformational flexibility. Our data reveal that tixagevimab, regdanvimab, and cilgavimab can efficiently neutralize most of the SARS-CoV-2 Alpha strains while tixagevimab, bamlanivimab, and sotrovimab can form a stable complex with the Delta variants. Based on these data, we have designed, by in silico, a chimeric antibody by conjugating the CDRH3 of regdanivimab with a sotrovimab framework to combat the variants that could potentially escape from the mAb-mediated neutralization. Our finding suggests that though currently available mAbs could be used to treat COVID-19 caused by the variants of SARS-CoV-2, better results could be expected with the chimeric antibodies.

Genome-Guided Discovery of the First Myxobacterial Biarylitide Myxarylin Reveals Distinct C–N Biaryl Crosslinking in RiPP Biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products. They feature a wide range of intriguing post-translational modifications, as exemplified by the biarylitides. These are a family of cyclic tripeptides found in Planomonospora, carrying a biaryl linkage between two aromatic amino acids. Recent genomic analyses revealed that the minimal biosynthetic prerequisite of biarylitide biosynthesis consists of only one ribosomally synthesized pentapeptide precursor as the substrate and a modifying cytochrome-P450-dependent enzyme. In silico analyses revealed that minimal biarylitide RiPP clusters are widespread among natural product producers across phylogenetic borders, including myxobacteria. We report here the genome-guided discovery of the first myxobacterial biarylitide MeYLH, termed Myxarylin, from Pyxidicoccus fallax An d48. Myxarylin was found to be an N-methylated tripeptide that surprisingly exhibits a C–N biaryl crosslink. In contrast to Myxarylin, previously isolated biarylitides are N-acetylated tripeptides that feature a C–C biaryl crosslink. Furthermore, the formation of Myxarylin was confirmed by the heterologous expression of the identified biosynthetic genes in Myxococcus xanthus DK1622. These findings expand the structural and biosynthetic scope of biarylitide-type RiPPs and emphasize the distinct biochemistry found in the myxobacterial realm.

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant

Background Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains. Results Cobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains. Conclusions Cobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

Genome-guided Discovery of the First Myxobacterial Biarylitide Myxarylin Reveals Distinct C–N Biaryl Crosslinking in RiPP Biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products. They feature a wide range of intriguing posttranslational modifications as exemplified by the biarylitides. These are a family of cyclic tripeptides found in Planomonospora, carrying a biaryl-linkage between two aromatic amino acids. Recent genomic analyses revealed the minimal biosynthetic prerequisite of biarylitide biosynthesis consisting of only one ribosomally synthesized pentapeptide precursor as substrate and a modifying cytochrome P450 dependent enzyme. In silico analyses revealed that the minimal biarylitide RiPP clusters are widespread among natural product producers across phylogenetic borders including myxobacteria. We report here the genome-guided discovery of the first myxobacterial biarylitide MeYLH termed Myxarylin from Pyxidicoccus fallax An d48. Myxarylin was found to be an N-methylated tripeptide surprisingly exhibiting a C–N biaryl crosslink. In contrast to Myxarylin, previously isolated biarylitides are N-acetylated tripeptides featuring a C–C biaryl crosslink. Furthermore, the formation of Myxarylin was confirmed by heterologous expression of the identified biosynthetic genes in Myxococcus xanthus DK1622. These findings expand the structural and biosynthetic scope of biarylitide type RiPPs and emphasize the distinct biochemistry found in the myxobacterial realm.

Targeted CRISPR/Cas9-Based Knock-Out of the Rice Orthologs TILLER ANGLE CONTROL 1 (TAC1) in Poplar Induces Erect Leaf Habit and Shoot Growth

Pyramidal-, erect- or upright-growing plant forms are characterized by narrow branch angles of shoots and leaves. The putative advantage of upright-leaf and shoot habit could be a more efficient penetration of light into lower canopy layers. Pyramidal genotypes have already been reported for various tree genotypes including peach. The paralogous rice ortholog TILLER ANGLE CONTROL 1 (TAC1) has been proposed to be the responsible gene for upright growth. However, it has not really been demonstrated for any of the pyramidal tree genotypes that a knock-out mutation of the TAC1 gene is causing pyramidal plant growth. By in silico analyses, we have identified a putative rice TAC1 ortholog (Potri.014G102600, “TAC-14”) and its paralog (Potri.002G175300, “TAC-2”) in the genome of P. trichocarpa. Two putative PcTAC1 orthologs in the P. × canescens clone INRA 717-1B4 were successfully knocked-out by applying a transgenic CRISPR/Cas9-approach. The mutants were molecularly analyzed and phenotyped over a period of three years in a glasshouse. Our results indicate that the homozygous knock-out of “TAC-14” is sufficient to induce pyramidal plant growth in P. × canescens. If up to twice as many pyramidal individuals were planted on short rotation coppices (SRCs), this could lead to higher wood yield, without any breeding, simply by increasing the number of trees on a default field size.

Sexually dimorphic patterns in maternal circulating microRNAs in pregnancies complicated by fetal growth restriction

Background Current methods fail to accurately predict women at greatest risk of developing fetal growth restriction (FGR) or related adverse outcomes, including stillbirth. Sexual dimorphism in these adverse pregnancy outcomes is well documented as are sex-specific differences in gene and protein expression in the placenta. Circulating maternal serum microRNAs (miRNAs) offer potential as biomarkers that may also be informative of underlying pathology. We hypothesised that FGR would be associated with an altered miRNA profile and would differ depending on fetal sex. Methods miRNA expression profiles were assessed in maternal serum (> 36 weeks’ gestation) from women delivering a severely FGR infant (defined as an individualised birthweight centile (IBC) < 3rd) and matched control participants (AGA; IBC = 20–80th), using miRNA arrays. qPCR was performed using specific miRNA primers in an expanded cohort of patients with IBC < 5th (n = 15 males, n = 16 females/group). Maternal serum human placental lactogen (hPL) was used as a proxy to determine if serum miRNAs were related to placental dysfunction. In silico analyses were performed to predict the potential functions of altered miRNAs. Results Initial analyses revealed 11 miRNAs were altered in maternal serum from FGR pregnancies. In silico analyses revealed all 11 altered miRNAs were located in a network of genes that regulate placental function. Subsequent analysis demonstrated four miRNAs showed sexually dimorphic patterns. miR-28-5p was reduced in FGR pregnancies (p < 0.01) only when there was a female offspring and miR-301a-3p was only reduced in FGR pregnancies with a male fetus (p < 0.05). miR-454-3p was decreased in FGR pregnancies (p < 0.05) regardless of fetal sex but was only positively correlated to hPL when the fetus was female. Conversely, miR-29c-3p was correlated to maternal hPL only when the fetus was male. Target genes for sexually dimorphic miRNAs reveal potential functional roles in the placenta including angiogenesis, placental growth, nutrient transport and apoptosis. Conclusions These studies have identified sexually dimorphic patterns for miRNAs in maternal serum in FGR. These miRNAs may have potential as non-invasive biomarkers for FGR and associated placental dysfunction. Further studies to determine if these miRNAs have potential functional roles in the placenta may provide greater understanding of the pathogenesis of placental dysfunction and the differing susceptibility of male and female fetuses to adverse in utero conditions.