Enrichment of intrinsically disordered residues in ohnologs facilitates abiotic stress resilience in Brassica rapa.

Arabidopsis thaliana and Brassica rapa are in the same evolutionary lineage, although the latter experienced an additional whole genome triplication event. Therefore, it would be intriguing to investigate the traits that gene duplication imposes to mediate plant stress tolerance. Here, we noticed that B. rapa abiotic stress resistance (ASR) genes which code at least one stress responsive domain have a significantly higher number of paralogs than A. thaliana. Analysing the disordered content of the ASR genes in both species, we found that intrinsically disordered residues (IDR) are specifically enriched in whole genome duplication (WGD) derived paralogs. Subsequently, domain similarity analysis between WGD pairs of both species has revealed that majority of WGD pairs in B. rapa did not share domains with each other. Furthermore, domain enrichment analysis has shown that B. rapa paralogs contain 36 distinct stress responsive enriched domains, significantly higher than A. thaliana paralogs. Next, we performed MSA to investigate the domain conservation between orthologs and ohnologs pairs, we found that 80.13% of B. rapa ohnologs acquire new domains, depicting the fact that ohnologs play a significant role in stress-related behaviours. The average IDR content of the ohnologs enriching new domains after gene duplication in B. rapa (0.19), is also significantly higher than A. thaliana (0.04). Interestingly, we also found that all of these attributes i.e., exhibiting higher number of WGD paralogs and enhancement of IDR in ASR genes of B. rapa compared to A. thaliana is exclusive for ASR genes only. No such significant differences were observed in randomly selected non-ASR genes between the two species. Together these results provide strong support for the hypothesis that augmentation of IDR content followed by a whole genome duplication event imposes the stress resistance potentiality in B. rapa. This research will shed light on the mechanism of how B. rapa is able to successfully adapt to stress over the evolutionary timescale.

Interplay between gene expression and gene architecture as a consequence of gene and genome duplications: evidence from metabolic genes of Arabidopsis thaliana.

Gene and genome duplications have been widespread during the evolution of flowering plant which resulted in the increment of biological complexity as well as creation of plasticity of a genome helping the species to adapt to changing environments. Duplicated genes with higher evolutionary rates can act as a mechanism of generating novel functions in secondary metabolism. In this study, we explored duplication as a potential factor governing the expression heterogeneity and gene architecture of Primary Metabolic Genes (PMGs) and Secondary Metabolic Genes (SMGs) of Arabidopsis thaliana. It is remarkable that different types of duplication processes controlled gene expression and tissue specificity differently in PMGs and SMGs. A complex relationship exists between gene architecture and expression patterns of primary and secondary metabolic genes. Our study reflects, expression heterogeneity and gene structure variation of primary and secondary metabolism in Arabidopsis thaliana are partly results of duplication events of different origins. Our study suggests that duplication has differential effect on PMGs and SMGs regarding expression pattern by controlling gene structure, epigenetic modifications, multifunctionality and subcellular compartmentalization. This study provides an insight into the evolution of metabolism in plants in the light of gene and genome scale duplication. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01188-2.

Mutations in membrane-fusion subunit of spike glycoprotein play crucial role in the recent outbreak of COVID-19.

Coronavirus disease-2019 (COVID-19), the ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major threat to the entire human race. It is reported that SARS-CoV-2 seems to have relatively low pathogenicity and higher transmissibility than previously outbroke SARS-CoV. To explore the reason of the increased transmissibility of SARS-CoV-2 compared with SARS-CoV, we have performed a comparative analysis on the structural proteins (spike, envelope, membrane, and nucleoprotein) of two viruses. Our analysis revealed that extensive substitutions of hydrophobic to polar and charged amino acids in spike glycoproteins of SARS-CoV2 creates an intrinsically disordered region (IDR) at the beginning of membrane-fusion subunit and intrinsically disordered residues in fusion peptide. IDR provides a potential site for proteolysis by furin and enriched disordered residues facilitate prompt fusion of the SARS-CoV2 with host membrane by recruiting molecular recognition features. Here, we have hypothesized that mutation-driven accumulation of intrinsically disordered residues in spike glycoproteins play dual role in enhancing viral transmissibility than previous SARS-coronavirus. These analyses may help in epidemic surveillance and preventive measures against COVID-19.

Chaperone client proteins evolve slower than non-client proteins.

Chaperones are important molecular machinery that assists proteins to attain their native three-dimensional structure crucial for function. Earlier studies using experimental evolution showed that chaperones impose a relaxation of sequence constraints on their "client" proteins, which may lead to the fixation of slightly deleterious mutations on the latter. However, we hypothesized that such a phenomenon might be harmful to the organism in a natural physiological condition. In this study, we investigated the evolutionary rates of chaperone client and non-client proteins in five model organisms from both prokaryotic and eukaryotic lineages. Our study reveals a slower evolutionary rate of chaperone client proteins in all five organisms. Additionally, the slower folding rate and lower aggregation propensity of chaperone client proteins reveal that the chaperone may play an essential role in rescuing the slightly disadvantageous effects due to random mutations and subsequent protein misfolding. However, the fixation of such mutations is less likely to be selected in the natural population.

The optimization of mRNA expression level by its intrinsic properties-Insights from codon usage pattern and structural stability of mRNA.

Codon usage bias (CUB) and mRNA structural stability are important intrinsic features of mRNA that correlate positively with mRNA expression level. However, it remains unclear whether the mRNA expression level can be regulated by adjusting these two parameters, influencing the mRNAs' structure. Here we explored the influence of CUB and mRNA structural stability on mRNA expression levels in Saccharomyces cerevisiae, using both wild type and computationally mutated mRNAs. Although in wild type, both CUB and mRNA stability positively regulate the mRNA expression level, any deviation from natural situation breaks such equilibrium. The naturally occurring codon composition is responsible for optimizing the mRNA expression, and under such composition, the mRNA structure having highest stability is selected by nature.

Evolutionary rate heterogeneity between multi- and single-interface hubs across human housekeeping and tissue-specific protein interaction network: Insights from proteins' and its partners' properties.

Integrating gene expression into protein-protein interaction network (PPIN) leads to the construction of tissue-specific (TS) and housekeeping (HK) sub-networks, with distinctive TS- and HK-hubs. All such hub proteins are divided into multi-interface (MI) hubs and single-interface (SI) hubs, where MI hubs evolve slower than SI hubs. Here we explored the evolutionary rate difference between MI and SI proteins within TS- and HK-PPIN and observed that this difference is present only in TS, but not in HK-class. Next, we explored whether proteins' own properties or its partners' properties are more influential in such evolutionary discrepancy. Statistical analyses revealed that this evolutionary rate correlates negatively with protein's own properties like expression level, miRNA count, conformational diversity and functional properties and with its partners' properties like protein disorder and tissue expression similarity. Moreover, partial correlation and regression analysis revealed that both proteins' and its partners' properties have independent effects on protein evolutionary rate.

The role of introns in the conservation of the metabolic genes of Arabidopsis thaliana.

In Arabidopsis thaliana, primary metabolic genes (PMGs) are more evolutionarily conserved and intron-rich than secondary metabolic genes. We observed that PMGs are more primitive and pan-taxonomically persistent as compared to secondary (SMGs) and non-metabolic genes (NMGs). This difference in primitiveness and persistence is primarily correlated with intron number and is independent of gene expression level. We propose a twofold explanation behind higher intron enrichment in PMGs. Firstly, introns might increase protein versatility amongst PMGs through alternative splicing, providing selective advantage of PMGs and making them more persistent across diverse plant taxa. Also, multifunctional PMGs may acquire functional domains by increasing the intronic burden. Additionally, single nucleotide polymorphisms (SNPs) accumulate at a higher rate in introns as compared to exons. Moreover, a strong negative correlation between cumulative exonic SNPs density and intron number indicates that introns may protect the exonic regions against the deleterious effect of these mutations, making them more conserved.

Codon degeneracy and amino acid abundance influence the measures of codon usage bias: improved Nc (N̂c ) and ENCprime (N̂'c ) measures.

Effective number of codons (N^c) and its variant N^'c (effective number of codons prime) are the two widely used methods for measuring unequal usage of synonymous codons in coding sequences, known as the codon usage bias (CUB). The mathematical formula used in calculating N^c and N^'c values is giving inappropriate measures of CUB in case of low abundance of amino acids. In addition, the magnitude of error also varies according to codon degeneracy. In this study, a modified formula for N^c and N^'c has been developed to measure the CUB more accurately. Online implementations of the modified formula are available in the web portal at http://agnigarh.tezu.ernet.in/~ssankar/cub.php.

Exploring the major cross-talking edges of competitive endogenous RNA networks in human Chronic and Acute Myeloid Leukemia.

BACKGROUND: Human Chronic and Acute Myeloid Leukemia are myeloproliferative disorders in myeloid lineage of blood cells characterized by accumulation of aberrant white blood cells. In cancer, the anomalous transcriptome includes deregulated expression of non-coding RNAs in conjunction with protein-coding mRNAs in human genome. The coding or non-coding RNA transcripts harboring miRNA-binding sites can converse with and regulate each other by explicitly contending for a limited pool of shared miRNAs and act as competitive endogenous RNAs (ceRNAs). An unifying hypothesis attributing 'modulation of expression of transcripts' in this fashion had been defined as 'competitive endogenous RNA hypothesis'. Network built with ceRNAs evidently offers a platform to elucidate complex regulatory interactions at post-transcriptional level in human cancers. METHODS: Contemplating cancers of human myeloid lineage we constructed ceRNA networks for CML and AML coding and non-coding repertoire utilizing patient sample data. Through functional enrichment analysis we selected the significant functional modules for transcripts being differentially expressed in Blastic phases of each cancer types with respect to Normal. After retrieving free energy of binding and duplex formation of shared miRNAs on ceRNAs, we performed statistical averaging of energy values over the ensemble of populations considering cellular system as in canonical (Iso-thermal) situation. RESULTS AND CONCLUSIONS: We aimed to shed light on 'Sibling Rivalry' in ceRNA partners from the perspective of statistical thermodynamics, identified major cross-talking tracks and ceRNAs influencing transcripts concerned in myeloid cancer systems. GENERAL SIGNIFICANCE: Insights into ceRNA-regulation will shed light on progression and prognosis of human Chronic and Acute Myeloid Leukemia.

Impact of the US FDA "Biopharmaceutics Classification System" (BCS) Guidance on Global Drug Development.

The FDA guidance on application of the biopharmaceutics classification system (BCS) for waiver of in vivo bioequivalence (BE) studies was issued in August 2000. Since then, this guidance has created worldwide interest among biopharmaceutical scientists in regulatory agencies, academia, and industry toward its implementation and further expansion. This article describes how the review implementation of this guidance was undertaken at the FDA and results of these efforts over last dozen years or so across the new, and the generic, drug domains are provided. Results show that greater than 160 applications were approved, or tentatively approved, based on the BCS approach across multiple therapeutic areas; an additional significant finding was that at least 50% of these approvals were in the central nervous system (CNS) area. These findings indicate a robust utilization of the BCS approach toward reducing unnecessary in vivo BE studies and speeding up availability of high quality pharmaceutical products. The article concludes with a look at the adoption of this framework by regulatory and health policy organizations across the globe, and FDA's current thinking on areas of improvement of this guidance.

Exploring the evolutionary rate differences between human disease and non-disease genes.

Comparisons of evolutionary features between human disease and non-disease genes have a wide implication to understand the genetic basis of human disease genes. However, it has not yet been resolved whether disease genes evolve at slower or faster rate than the non-disease genes. To resolve this controversy, here we integrated human disease genes from several databases and compared their protein evolutionary rates with non-disease genes in both housekeeping and tissue-specific group. We noticed that in tissue specific group, disease genes evolve significantly at a slower rate than non-disease genes. However, we found no significant difference in evolutionary rates between disease and non-disease genes in housekeeping group. Tissue specific disease genes have a higher protein complex number, elevated gene expression level and are also associated with conserve biological processes. Finally, our regression analysis suggested that protein complex number followed by protein multifunctionality independently modulates the evolutionary rate of human disease genes.

Insights into the dN/dS ratio heterogeneity between brain specific genes and widely expressed genes in species of different complexity.

In mammals, it has long been suggested that brain-specific genes (BSGs) and widely expressed genes (WEGs) have seemingly lower dN/dS ratio than any other gene sets. However, to what extent these genes differ in their dN/dS ratio has still remained controversial. Here, we have revealed lower dN/dS ratio of BSGs than WEGs in human-mouse, human-orangutan, human-chimpanzee and mouse-rat orthologous pair. The significance level of dN/dS ratio difference indicates a trend of decreasing difference as complexity of compared pairs increases. Further studies with the human-mouse pair revealed that, removal of the duplicated genes from both the dataset has nullified this difference which dictates a vital role of duplicated genes in governing the selection pressure. Conclusively, higher paralog number, expression level, and longer regulatory region length of BSGs allow fewer nucleotide substitutions within them. Our results show for the first time to our knowledge lower dN/dS ratio of BSGs than WEGs.

Global analysis of human duplicated genes reveals the relative importance of whole-genome duplicates originated in the early vertebrate evolution.

BACKGROUND: Gene duplication is a genetic mutation that creates functionally redundant gene copies that are initially relieved from selective pressures and may adapt themselves to new functions with time. The levels of gene duplication may vary from small-scale duplication (SSD) to whole genome duplication (WGD). Studies with yeast revealed ample differences between these duplicates: Yeast WGD pairs were functionally more similar, less divergent in subcellular localization and contained a lesser proportion of essential genes. In this study, we explored the differences in evolutionary genomic properties of human SSD and WGD genes, with the identifiable human duplicates coming from the two rounds of whole genome duplication occurred early in vertebrate evolution. RESULTS: We observed that these two groups of duplicates were also dissimilar in terms of their evolutionary and genomic properties. But interestingly, this is not like the same observed in yeast. The human WGDs were found to be functionally less similar, diverge more in subcellular level and contain a higher proportion of essential genes than the SSDs, all of which are opposite from yeast. Additionally, we explored that human WGDs were more divergent in their gene expression profile, have higher multifunctionality and are more often associated with disease, and are evolutionarily more conserved than human SSDs. CONCLUSIONS: Our study suggests that human WGD duplicates are more divergent and entails the adaptation of WGDs to novel and important functions that consequently lead to their evolutionary conservation in the course of evolution.

Overlapping genes: a new strategy of thermophilic stress tolerance in prokaryotes.

Overlapping genes (OGs) draw the focus of recent day's research. However, the significance of OGs in prokaryotic genomes remained unexplored. As an adaptation to high temperature, thermophiles were shown to eliminate their intergenic regions. Therefore, it could be possible that prokaryotes would increase their OG content to adapt to high temperature. To test this hypothesis, we carried out a comparative study on OG frequency of 256 prokaryotic genomes comprising both thermophiles and non-thermophiles. It was found that thermophiles exhibit higher frequency of overlapping genes than non-thermophiles. Moreover, overlap frequency was found to correlate with optimal growth temperature (OGT) in prokaryotes. Long overlap frequency was found to hold a positive correlation with OGT resulting in an abundance of long overlaps in thermophiles compared to non-thermophiles. On the other hand, short overlap (1-4 nucleotides) frequency (SOF) did not yield any direct correlation with OGT. However, the correlation of SOF with CAIavg (extent of variation of codon usage bias measured as the mean of codon adaptation index of all genes in a given genome) and IG% (proportion of intergenic regions) indicate that they might upregulate the aforementioned factors (CAIavg and IG%) which are already known to be vital forces for thermophilic adaptation. From these evidences, we propose that the OG content bears a strong link to thermophily. Long overlaps are important for their genome compaction and short overlaps are important to uphold high CAIavg. Our findings will surely help in better understanding of the significance of overlapping gene content in prokaryotic genomes.

GC-made protein disorder sheds new light on vertebrate evolution.

At the emergence of endothermic vertebrates, GC rich regions of the ectothermic ancestral genomes underwent a significant GC increase. Such an increase was previously postulated to increase thermodynamic and structural stability of proteins through selective increase of protein hydrophobicity. Here, we found that, increase in GC content promotes a higher content of disorder promoting amino acid in endothermic vertebrates proteins and that the increase in hydrophobicity is mainly due to a higher content of the small disorder promoting amino acid alanine. In endothermic vertebrates, prevalence of disordered residues was found to promote functional diversity of proteins encoded by GC rich genes. Higher fraction of disordered residues in this group of proteins was also found to minimize their aggregation tendency. Thus, we propose that the GC transition has favored disordered residues to promote functional diversity in GC rich genes, and to protect them against functional loss by protein misfolding.

Insights into the miRNA regulations in human disease genes.

BACKGROUND: MicroRNAs are a class of short non-coding RNAs derived from either cellular or viral transcripts that act post-transcriptionally to regulate mRNA stability and translation. In recent days, increasing numbers of miRNAs have been shown to be involved in the development and progression of a variety of diseases. We, therefore, intend to enumerate miRNA targets in several known disease classes to explore the degree of miRNA regulations on them which is unexplored till date. RESULTS: Here, we noticed that miRNA hits in cancer genes are remarkably higher than other diseases in human. Our observation suggests that UTRs and the transcript length of cancer related genes have a significant contribution in higher susceptibility to miRNA regulation. Moreover, gene duplication, mRNA stability, AREScores and evolutionary rate were likely to have implications for more miRNA targeting on cancer genes. Consequently, the regression analysis have confirmed that the AREScores plays most important role in detecting miRNA targets on disease genes. Interestingly, we observed that epigenetic modifications like CpG methylation and histone modification are less effective than miRNA regulations in controlling the gene expression of cancer genes. CONCLUSIONS: The intrinsic properties of cancer genes studied here, for higher miRNA targeting will enhance the knowledge on cancer gene regulation.

Current challenges in bioequivalence, quality, and novel assessment technologies for topical products.

This paper summarises the proceedings of a recent workshop which brought together pharmaceutical scientists and dermatologists from academia, industry and regulatory agencies to discuss current regulatory issues and industry practices for establishing therapeutic bioequivalence (BE) of dermatologic topical products. The methods currently available for assessment of BE were reviewed as well as alternatives and the advantages and disadvantages of each method were considered. Guidance on quality and performance of topical products was reviewed and a framework to categorise existing and alternative methods for evaluation of BE was discussed. The outcome of the workshop emphasized both a need for greater attention to quality, possibly, via a Quality-By-Design (QBD) approach and a need to develop a "whole toolkit" approach towards the problem of determination of rate and extent in the assessment of topical bioavailability. The discussion on the BE and clinical equivalence of topical products revealed considerable concerns about the variability present in the current methodologies utilized by the industry and regulatory agencies. It was proposed that academicians, researchers, the pharmaceutical industry and regulators work together to evaluate and validate alternative methods that are based on both the underlying science and are adapted to the drug product itself instead of single "universal" method.

Best practices for the development, scale-up, and post-approval change control of IR and MR dosage forms in the current quality-by-design paradigm.

In this whitepaper, the Manufacturing Technical Committee of the Product Quality Research Institute provides information on the common, best practices in use today in the development of high-quality chemistry, manufacturing and controls documentation. Important topics reviewed include International Conference on Harmonization, in vitro-in vivo correlation considerations, quality-by-design approaches, process analytical technologies and current scale-up, and process control and validation practices. It is the hope and intent that this whitepaper will engender expanded dialog on this important subject by the pharmaceutical industry and its regulatory bodies.

Drug Dissolution in Oral Drug Absorption: Workshop Report.

The in-person workshop "Drug Dissolution in Oral Drug Absorption" was held on May 23-24, 2023, in Baltimore, MD, USA. The workshop was organized into lectures and breakout sessions. Three common topics that were re-visited by various lecturers were amorphous solid dispersions (ASDs), dissolution/permeation interplay, and in vitro methods to predict in vivo biopharmaceutics performance and risk. Topics that repeatedly surfaced across breakout sessions were the following: (1) meaning and assessment of "dissolved drug," particularly of poorly water soluble drug in colloidal environments (e.g., fed conditions, ASDs); (2) potential limitations of a test that employs sink conditions for a poorly water soluble drug; (3) non-compendial methods (e.g., two-stage or multi-stage method, dissolution/permeation methods); (4) non-compendial conditions (e.g., apex vessels, non-sink conditions); and (5) potential benefit of having both a quality control method for batch release and a biopredictive/biorelevant method for biowaiver or bridging scenarios. An identified obstacle to non-compendial methods is the uncertainty of global regulatory acceptance of such methods.

Evolutionary dynamics of human autoimmune disease genes and malfunctioned immunological genes.

BACKGROUND: One of the main issues of molecular evolution is to divulge the principles in dictating the evolutionary rate differences among various gene classes. Immunological genes have received considerable attention in evolutionary biology as candidates for local adaptation and for studying functionally important polymorphisms. The normal structure and function of immunological genes will be distorted when they experience mutations leading to immunological dysfunctions. RESULTS: Here, we examined the fundamental differences between the genes which on mutation give rise to autoimmune or other immune system related diseases and the immunological genes that do not cause any disease phenotypes. Although the disease genes examined are analogous to non-disease genes in product, expression, function, and pathway affiliation, a statistically significant decrease in evolutionary rate has been found in autoimmune disease genes relative to all other immune related diseases and non-disease genes. Possible ways of accumulation of mutation in the three steps of the central dogma (DNA-mRNA-Protein) have been studied to trace the mutational effects predisposed to disease consequence and acquiring higher selection pressure. Principal Component Analysis and Multivariate Regression Analysis have established the predominant role of single nucleotide polymorphisms in guiding the evolutionary rate of immunological disease and non-disease genes followed by m-RNA abundance, paralogs number, fraction of phosphorylation residue, alternatively spliced exon, protein residue burial and protein disorder. CONCLUSIONS: Our study provides an empirical insight into the etiology of autoimmune disease genes and other immunological diseases. The immediate utility of our study is to help in disease gene identification and may also help in medicinal improvement of immune related disease.