Biology subjects

Subramanian, S.

Publications and source records attributed to Subramanian, S..

102 records · Page 6Linked to original sources

bioRxiv2019

Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution

Glycogen phosphorylase (GlyP) was the first allosteric enzyme to be described. Yet, the precise dynamic changes in solution phase structure and stability that underpin functional regulation have remained elusive. We have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. This enabled measurements of the solution phase local structural dynamics involved in allosteric regulation of GlyP. Here, we quantify GlyP structural dynamics in solution, describing correlated changes in structure in the activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of the enzyme. The sensitivity of these measurements discerned that the 250s loop is natively disordered in the apo T-state, adopting a more ordered conformation in the active state. The quantitative change in stability of the 280s loop is identified, providing the first direct evidence of the entropic switch that sterically regulates substrate access to the active site.\n\nSignificance StatementWe have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. Measurements of glycogen phosphorylase quantify the solution phase stability of local structure at near-amino acid structural resolution and with no appreciable lower limit of stability. This uncovered the highly-resolved local alterations in stability which provides direct evidence of the entropic mechanism by which access to the active site is gated by the 280s loop.\n\nFootnotesAuthor contributions: M.K., V.S., S.S., N.L., F.V., N.B., L.C. and J.J.P. designed research; M.K., V.S., S.S., L.C. and J.J.P. performed research; M.K., V.S., S.S., L.C. and J.J.P. analyzed data; and M.K. and J.J.P. wrote the manuscript.

biophysics↗

bioRxiv2019

A simple and sensitive SYBR Gold-based assay to quantify DNA-protein interactions

A simple, accessible, and inexpensive assay to quantify the strength of DNA-protein interactions was developed. The assay relies on capturing DNA-protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA-protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA-protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA-protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different Auxin Response Factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose-response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.

plant biology↗

bioRxiv2019

Mitogenomic Diversity in Sacred Ibis Mummies sheds light on early Egyptian practices

The ancient catacombs of Egypt harbor millions of well-preserved mummified Sacred Ibis (Threskiornis aethiopicus) dating from [~]600BC. Although it is known that a very large number of these votive mummies were sacrificed to the Egyptian God Thoth, how the ancient Egyptians obtained millions of these birds for mummification remains unresolved. Ancient Egyptian textual evidences suggest they may have been raised in dedicated large-scale farms. To investigate the most likely method used by the priests to secure birds for mummification, we report the first study of complete mitochondrial genomes of 14 Sacred Ibis mummies interred [~]2500 years ago. We analysed and compared the mitogenomic diversity among Sacred Ibis mummies to that found in modern Sacred Ibis populations from throughout Africa. The ancient birds show a high level of genetic variation comparable to that identified in modern African populations, contrary to the suggestion in ancient hieroglyphics (or ancient writings) of centralized industrial scale farming of sacrificial birds. This suggests a sustained short-term taming of the wild Sacred Ibis for the ritual yearly demand.

evolutionary biology↗

bioRxiv2019

Increasing global heterogeneity in the life expectancy of older populations

With overall global improvements in life expectancy, one important concern is whether there is cross-country convergence in life expectancy at various ages. Insights in convergence patterns can help realign research priorities help governments better structure health investments across various age groups. We reveal global patterns in life expectancy improvements and identify convergent clubs in life expectancy at various ages for 201 countries / areas between 1950 and 2015. In the case of life expectancy at younger ages, most countries are moving in the same direction, but we observe significant cross-country variation for older adults and the elderly. Further, we observe increasing variance in life expectancy for older adults and elderly across countries. Increasing global heterogeneity in survival experience of older adults and the elderly population thus has remained a neglected aspect in the discussions on global life expectancy improvements. Data, research and policy focus beyond life-expectancy at birth is therefore critical to accelerate survival gains among older adults and elderly, particularly from the developing world.

epidemiology↗

bioRxiv2018

Fishing for Mitochondrial DNA in The Egyptian Sacred Ibis Mummies

The long-term preservation of DNA requires a number of optimal conditions, including consistent exposure to cool, dry, and dark environments. As a result, the successful recovery of ancient DNA from material from warmer climates such as those in Egypt has often been met with scepticism. Egypt has an abundance of ancient mummified animals and humans, whose genetic analyses would offer important insights into ancient cultural practices. To date, the retrieval of complete genomes from ancient Egyptian remains of humans or other animals has been largely unsuccessful. To test for the presence of even short DNA sequences in Egyptian material, we performed second-generation shotgun sequencing of DNA libraries constructed from ancient Sacred Ibis mummies. Since most of the resulting Illumina libraries were shown to contain extremely low levels (less than 0.06%) of endogenous mitochondrial DNA, we aimed to enrich these samples using targeted in-solution hybridisation methods. Using biotinylated RNA baits designed to Sacred Ibis complete mitochondrial sequences, we trialled a number of conditions and parameters and achieved up to 4705-fold enrichment. We also found that a combination of hybridisation temperature and the use of the polymerase KAPA HiFi significantly increased both the efficiency of targeted hybridisation and post-hybridisation amplification respectively. Furthermore, improved enrichment was accompanied with only minor increases in clonality. Our method enabled us to reconstruct the first complete mitochondrial genomes from ancient Egyptian sub-fossil material.

genomics↗

bioRxiv2018

Microbiota-metabolites interactions in non-human primate gastrointestinal tract

BackgroundThe microbiota has been recognised as an important part for maintaining human health. Perturbation to its structure has been implicated in many diseases, such as obesity and cancers. The microbiota is highly metabolically active and fills in many niche metabolic pathways absent from the human host. Diseases such as obesity, cardiovascular disease and colorectal cancer has been linked to altered microbiota metabolism. However, there is a gap in the current knowledge on how mucosal-associated microbiota and colon mucosa interact. Here we performed an integrated analysis between the mucosal-associated microbiota and the mucosal tissue metabolites in healthy non-human primates.\n\nResultsWe found that the overall microbiota composition is influenced by both the tissue location as well as the host. We also identified bacteria signatures for different intestinal locations. The distal colon bacterial signature includes Ruminococcaceae, Bacteroidales, Christensenellaceae, Clostridiales, Sphaerochaeta, Victivallaceae, GMD14H09, CF231, ML615J-28, RF39 and R4-45B taxa. In the cecum, the signatures include Prevotella, Anaerovibrio, Roseburia, and Anaerostipes. Desulfovibrionaceae family is the only taxon that may be a signature for the duodenum. We also found an intricate global relationship between the microbiota and the host tissue metabolome that is mainly driven by the distal colon. Most importantly, we found microbial-centric tissue metabolites clusters that may have potential implications to studying host-microbiota metabolic interactions.

genomics↗

bioRxiv2018

OMCD: OncoMir Cancer Database

MicroRNAs (miRNAs) are crucially important in the development of cancer. Their dysregulation, commonly observed in various types of cancer, is largely cancer-dependent. Thus, to understand the tumor biology and to develop accurate and sensitive biomarkers, we need to understand pan-cancer miRNA expression. At the University of Minnesota, we developed the OncoMir Cancer Database (OMCD), hosted on a web server, which allows easy and systematic comparative genomic analyses of miRNA sequencing data derived from more than 9,500 cancer patients tissue samples available in the Cancer Genome Atlas (TCGA). OMCD includes associated clinical information and is searchable by organ-specific terms common to the TCGA. Freely available to all users (www.oncomir.umn.edu/omcd/), OMCD enables (1) simple visualization of TCGA miRNA sequencing data, (2) statistical analysis of differentially expressed miRNAs for each cancer type, and (3) exploration of miRNA clusters across cancer types.\n\nDatabase URLwww.oncomir.umn.edu/omcd

bioinformatics↗

bioRxiv2018

Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are robustly suppressed by deletion of either SIR2 or SIR3. While this and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using both MCM ChIP-Seq and MNase-H4K16ac ChIP-Seq data, we show that a SIR2 deletion rescues MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibits a pervasive effect at the majority of euchromatic origins. Importantly, in wild type (i.e. CDC6) cells, origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner. In addition, both Sir2 and Sir3 directly bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin - SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 - correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly bind euchromatic DNA replication origins and modify their local chromatin environment.

genetics↗

bioRxiv2018

Using Network Analysis to Localize the Epileptogenic Zone from Invasive EEG Recordings in Intractable Focal Epilepsy

Treatment of medically intractable focal epilepsy (MIFE) by surgical resection of the epileptogenic zone (EZ) is often effective provided the EZ can be reliably identified. Even with the use of invasive recordings, the clinical differentiation between the EZ and normal brain areas can be quite challenging, mainly in patients without MRI detectable lesions. Consequently, despite relatively large brain regions being removed, surgical success rates barely reach 60-65%. Such variable and unfavorable outcomes associated with high morbidity rates are often caused by imprecise and/or inaccurate EZ localization. We developed a localization algorithm that uses network-based data analytics to process invasive EEG recordings. This network algorithm analyzes the centrality signatures of every contact electrode within the recording network and characterizes contacts into susceptible EZ based on the centrality trends over time. The algorithm was tested in a retrospective study that included 42 patients from four epilepsy centers. Our algorithm had higher agreement with EZ regions identified by clinicians for patients with successful surgical outcomes and less agreement for patients with failed outcomes. These findings suggest that network analytics and a network systems perspective of epilepsy may be useful in assisting clinicians in more accurately localizing the EZ.\n\nAUTHOR SUMMARYEpilepsy is a disease that results in abnormal firing patterns in parts of the brain that comprise the epileptogenic network, known as the epileptogenic zone (EZ). Current methods to localize the EZ for surgical treatment often requires observations of hundreds of thousands of EEG data points measured from many electrodes implanted in a patients brain. In this paper, we used network science to show that EZ regions may exhibit specific network signatures before, during and after seizure events. Our algorithm computes the likelihood of each electrode being in the EZ and tends to agree more with clinicians during successful resections and less during failed surgeries. These results suggest that a networked analysis approach to EZ localization may be valuable in a clinical setting.

neuroscience↗

bioRxiv2017

A tangled tale of convergence and divergence: archaeal chromosomal proteins and Chromo-like domains in bacteria and eukaryotes

The Chromo-like superfamily of SH3-fold {beta}-barrel domains recognize epigenetic marks in eukaryotic proteins. Their provenance has been placed either in archaea, based on apparent structural similarity to chromatin-compacting Sul7d and Cren7 proteins, or in bacteria based on the presence of sequence homologs. Using sequence and structural evidence we establish that the archaeal Cren7/Sul7 proteins emerged from a zinc ribbon (ZnR) ancestor. Further, we show that the ancestral eukaryotic Chromo-like domains evolved from bacterial precursors acquired from early endosymbioses, which already possessed an aromatic cage for recognition of modified amino-groups. These bacterial versions are part of a radiation of secreted SH3-fold domains, which spawned both chromo-like domains and classical SH3 domains in the context of peptide-recognition in the peptidoglycan. This establishes that Cren7/Sul7 converged to a \"SH3\"-like state from a ZnR precursor via the loss of metal-chelation and acquisition of stronger hydrophobic interactions; it is unlikely to have participated in the evolution of the chromo-like domains. We show that archaea possess several Cren7/Sul7-related proteins with intact Zn-chelating ligands, which we predict to play previously unstudied roles in cell-division comparable to the PRC barrel.

bioinformatics↗

bioRxiv2017

Interaction Between Host MicroRNAs and the Gut Microbiota in Colorectal Cancer

BackgroundAlthough variation in gut microbiome composition has been linked with colorectal cancer (CRC), the factors that mediate the interactions between CRC tumors and the microbiome are poorly understood. MicroRNAs (miRNAs) are known to regulate CRC progression and patient survival outcomes. In addition, recent studies suggested that host miRNAs can also regulate bacterial growth and influence the composition of the gut microbiome. Here, we investigated the association between miRNAs expression in human CRC tumor and normal tissues and the microbiome composition associated with these same tissues.\n\nMethodWe sequenced the small RNAs from patient-matched tumor and normal tissue samples collected from 44 human CRC patients performed an integrated analysis with microbiome taxonomic composition data from these same samples. We then interrogated the functions of the bacteria correlated with miRNAs that were differentially expressed (DE) between tumor and matched normal tissues, as well as the functions of miRNAs correlated with bacterial taxa that have been previously associated with CRC, including Fusobacterium, Providencia, Bacteroides, Akkermansia, Roseburia, Porphyromonas, and Peptostreptococcus.\n\nResultsWe identified 76 miRNAs as DE between CRC and normal tissue, including known oncogenic miRNAs miR-182, miR-503, and miR-17[~]92. These DE miRNAs were correlated with the relative abundance of several bacterial taxa, including Firmicutes, Bacteroidetes, and Proteobacteria. Bacteria correlated with DE miRNAs were enriched with distinct predicted metabolic categories. Additionally, we found that miRNAs correlated with CRC-associated bacteria are predicted to regulate targets that are relevant for host-microbiome interactions, and highlight a possible role for miRNA-driven glycan production in the recruitment of pathogenic microbial taxa.\n\nConclusionsOur work characterized a global relationship between microbial community composition and miRNA expression in human CRC tissues. Our results support a role for miRNAs in mediating a bi-directional host-microbiome interaction in CRC. In addition, we highlight sets of potentially interacting microbes and host miRNAs, suggesting several pathways that can be targeted via future therapies.

systems biology↗

bioRxiv2017

Population size determines the type of nucleotide variations in humans

It is well known that the effective size of a population (Ne) is one of the major determinants of the amount of genetic variation within the population. Here, we examined whether the types of genetic variations are dictated by the effective population size. Our results revealed that for low frequency variants, the ratio of AT[->]GC to GC[->]AT variants ({beta}) was similar across populations, suggesting the similarity of the pattern of mutation in various populations. However, for high frequency variants, {beta} showed a positive correlation with the effective population size of the populations. This suggests a much higher proportion of high frequency AT[->]GC variants in large populations (e.g. Africans) compared to those with small population sizes (e.g. Asians). These results imply that the substitution patterns vary significantly between populations. These findings could be explained by the effect of GC-biased gene conversion (gBGC), which favors the fixation of G/C over A/T variants in populations. In large population, gBGC causes high {beta}. However, in small populations, genetic drift reduces the effect of gBGC resulting in reduced {beta}. This was further confirmed by a positive relationship between Ne and {beta} for homozygous variants. Our results highlight the huge variation in the types of homozygous and high frequency polymorphisms between world populations. We observed the same pattern for deleterious variants, implying that the homozygous polymorphisms associated with recessive genetic diseases will be more enriched with G or C in populations with large Ne (e.g. Africans) than in populations with small Ne (e.g. Europeans).

genomics↗