Biology subjects

Dhar, P. K.

Publications and source records attributed to Dhar, P. K..

6 recordsLinked to original sources

bioRxiv2023

Repurposing the dark genome. IV - noncoding proteins

The dark genome comprising of non-expressing, non-translating, and extinct DNA sequences has remained a largely unexplored genomic space. Using computational and experimental approaches, novel insights into the dark matter genome have recently been gained, revealing the presence of a vast and unexplored resource. Non-coding RNA (ncRNA) refers to a class of RNA molecules that do not encode proteins but play important regulatory roles in the cell. We asked if it was possible to make functional peptides and proteins from ncRNA leading to a new biological insight and applications? Here we present initial computational data in support of making functional noncoding proteins (NCP) from ncRNA sequences. Different types of non-coding genomic sequences originating from Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens were studied to understand sequence composition, secondary structure, and physiochemical properties of NCPs. This work builds the foundation for experimentally characterizing the first-in-the-class non-coding proteins leading to a novel insights and applications.

synthetic biology↗

bioRxiv2023

Repurposing The Dark Genome. III - Intronic Proteins

Based on the expression patterns, genomes are viewed as a collection of protein-coding, RNA-coding, and non-expressing DNA sequences. Unlike most prokaryotes, eukaryotic gene expression comes with an additional step called alternative splicing. During the maturation process, different combinations of exons are spliced out and joined together resulting in the formation of mRNA isoforms. After removal from pre-mRNA, introns may be degraded by cellular exonucleases or form long non-coding RNAs (lncRNAs), or temporarily retained in the nucleus for regulating gene expression. We asked: Do introns have an unutilized potential for encoding proteins? If introns had an opportunity of getting translated, what kind of peptides or proteins, would they make? This study is based on the hypothesis of making functional proteins from leftover introns and is an extension of the original work of making functional proteins from the E. coli intergenic sequences (Dhar et al., 2009). Here full-length introns were computationally translated into proteins to study their potential structural, physicochemical, functional, and cellular location properties. Experimental validation is underway for a detailed understanding of the biology of intronic proteins. A synthetic intronic protein repository would provide an opportunity to design first-in-the-class molecules toward functional endpoints.

synthetic biology↗

bioRxiv2023

ClearX9-Stem™: an efficient, affordable & sustainable stem cell culture medium for biotech and cell-cultivated meat industries

Stem cells are extraordinary cells with a unique ability of self-renewal and differentiation into various cell types such as muscle, nerve, bone, and blood cells. Historically, they have found significant applications in the biotech and pharma sectors. To grow and maintain stem cells artificially, researchers use basal media formulations supplemented with nutrients and growth factors, with Fetal bovine serum (FBS) as the key component of the culture medium. However, to maintain this supply every year, millions of pregnant cows are slaughtered for preparing FBS. The process of harvesting FBS also raises concerns about contamination with pathogens, animal proteins that may interfere with cellular behavior and ethical considerations regarding animal welfare. To overcome these limitations, here we report ClearX9-Stem - an affordable, sustainable, effective, and ethical replacement for an FBS-enriched stem cell culture medium. A specialized ClearX9-Stem cell culture medium formulation was designed to grow chicken embryonic fibroblast (SL-29) in the absence of FBS. Based on the results obtained, ClearX9-Stem is undergoing further refinement to meet the growing academic and industrial demand for serum-free culture media formulations. In the future, there is a need to customize and optimize ClearX9-Stem for the scalable growth of cells in bioreactors. HIGHLIGHTSO_LIClearX9-Stem provides good nutritional support for the growth of chicken embryonic fibroblast cells. C_LIO_LIClearX9-Stem cell growth performance is comparable to the serum-enriched culture medium C_LIO_LIClearX9-Stem maintains a healthy morphological profile of cells during division C_LIO_LIClearX9-Stem generates a stress-free environment within cells C_LIO_LIClearX9-Stem does not require animal slaughter and reduces the environmental footprint C_LIO_LIClearX9-Stem has applications in the biotechnology, pharma, and cell-cultivated meat industries C_LI

synthetic biology↗

bioRxiv2023

ClearX9™: an efficient alternative to fetal bovine serum for growing animal cells in vitro

Fetal Bovine Serum (FBS) is a nutrient-rich fluid that contains nutritional and macromolecular factors essential for cell growth. Every year millions of pregnant cows are slaughtered in search of FBS leading to huge environmental consequences. Here we report ClearX9 - an affordable, sustainable, ethical, and effective replacement for FBS. ClearX9 cell culture medium was used to grow HeLa (cervical cancer cells), HEK293T (embryonic kidney transformed cells) and Nthy Ori-3-1 (primary thyroid follicular transformed epithelial cells) and showed encouraging growth patterns and good cellular health. Compared with the FBS-enriched cell culture medium, ClearX9 scored positive on all the parameters suggesting ClearX9 as a credible alternative to FBS. In future, more work is required to establish the efficacy of ClearX9 in toxicology testing, bio-manufacturing, regenerative medicine, and vaccine research. HIGHLIGHTSO_LIClearX9 provides good nutritional support for the growth of animal cells C_LIO_LIClearX9 cell growth performance is comparable to the serum-enriched medium C_LIO_LIClearX9 maintains a healthy morphological profile of cells during division C_LIO_LIClearX9 generates a stress-free environment within cells C_LIO_LIClearX9 does not require animal slaughter and reduces carbon footprint C_LIO_LIClearX9 has applications in biotechnology and cell cultivated meat industry C_LI

cell biology↗

bioRxiv2023

Repurposing the dark genome. II - Reverse Proteins

Based on the expression blueprint encoded in the genome, three groups of sequences have been identified - protein encoding, RNA encoding, and non-expressing. We asked: Why did nature choose a particular DNA sequence for expression? Did she sample every possibility, approving some for RNA synthesis, some for protein synthesis, and retiring/ignoring the rest. If evolution randomly selected sequences for metabolic trials, how much non-utilized (not-expressing) and under-utilized (only RNA encoding) information is currently available for innovations? These questions lead us to experimentally synthesizing functional proteins from intergenic sequences of E.coli (Dhar et al 2009). The current work is an extension of this original report and takes into consideration natural protein-coding sequences read backward to generate a new possibility. Reverse proteins are full-length translation equivalents of the existing protein-coding genes read in the -1 frame. The structural, functional and interaction predictions of reverse proteins in E.coli, S.cerevisiae and D.melanogaster, open up a new opportunity of producing first-in-the-class proteins towards functional endpoints. This study points to a large untapped genomic space from the fundamental biology and applications perspectives.

synthetic biology↗

bioRxiv2023

Repurposing the dark genome. I - Antisense Proteins

From the functional standpoint, the genome may be considered a collection of three types of sequences: protein encoding, RNA encoding, and non-expressing. Based on the standard sequencing and annotation work, it is now well accepted that a small proportion of the genome has been allocated the job of encoding proteins, while most of the genome encodes RNA, and some DNA sequences are not used for expression. The exact ratio among these three types of sequences varies based on the organism. We asked: Is it possible to artificially encode protein and peptide sequences from naturally non-expressing (dark genome) sequences? This led to proof of the concept of making functional proteins from the intergenic sequences of E.coli (Dhar et al 2009). This study is an extension of the original concept and has been organized around antisense DNA sequences. The full-length antisense gene equivalents in forward and reverse orientations were computationally studied for their structural, cellular location, and functional properties, leading to many interesting observations. The current study points to a huge untapped genomic space that needs to be examined from cell physiology, evolutionary, and application perspectives.

synthetic biology↗