COVID-19 Biomarkers in research: Extension of the OncoMX cancer biomarker data model to capture biomarker data from other diseases.

Scientists, medical researchers, and health care workers have mobilized worldwide in response to the outbreak of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; SCoV2). Preliminary data have captured a wide range of host responses, symptoms, and lingering problems post-recovery within the human population. These variable clinical manifestations suggest differences in influential factors, such as innate and adaptive host immunity, existing or underlying health conditions, co-morbidities, genetics, and other factors. As COVID-19-related data continue to accumulate from disparate groups, the heterogeneous nature of these datasets poses challenges for efficient extrapolation of meaningful observations, hindering translation of information into clinical applications. Attempts to utilize, analyze, or combine biomarker datasets from multiple sources have shown to be inefficient and complicated, without a unifying resource. As such, there is an urgent need within the research community for the rapid development of an integrated and harmonized COVID-19 Biomarker Knowledgebase. By leveraging data collection and integration methods, backed by a robust data model developed to capture cancer biomarker data we have rapidly crowdsourced the collection and harmonization of COVID-19 biomarkers. Our resource currently has 138 unique biomarkers. We found multiple instances of the same biomarker substance being suggested as multiple biomarker types during our extensive cross-validation and manual curation. As a result, our Knowledgebase currently has 265 biomarker type combinations. Every biomarker entry is made comprehensive by bringing in together ancillary data from multiple sources such as biomarker accessions (canonical UniProtKB accession, PubChem Compound ID, Cell Ontology ID, Protein Ontology ID, NCI Thesaurus Code, and Disease Ontology ID), BEST biomarker category, and specimen type (Uberon Anatomy Ontology) unified with ontology standards. Our preliminary observations show distinct trends in the collated biomarkers. Most biomarkers are related to the immune system (SAA,TNF-∝, and IP-10) or coagulopathies (D-dimer, antithrombin, and VWF) and a few have already been established as cancer biomarkers (ACE2, IL-6, IL-4 and IL-2). These trends align with proposed hypotheses of clinical manifestations compounding the complexity of COVID-19 pathobiology. We explore these trends as we put forth a COVID-19 biomarker resource that will help researchers and diagnosticians alike. All biomarker data are freely available from https://data.oncomx.org/covid19 .

A procedure to recruit members to enlarge protein family databases--the building of UECOG (UniRef-Enriched COG Database) as a model.

A procedure to recruit members to enlarge protein family databases is described here. The procedure makes use of UniRef50 clusters produced by UniProt. Current family entries are used to recruit additional members based on the UniRef50 clusters to which they belong. Only those additional UniRef50 members that are not fragments and whose length is within a restricted range relative to the original entry are recruited. The enriched dataset is then limited to contain only genomes from selected clades. We used the COG database - used for genome annotation and for studies of phylogenetics and gene evolution - as a model. To validate the method, a UniRef-Enriched COG0151 (UECOG) was tested with distinct procedures to compare recruited members with the recruiters: PSI-BLAST, secondary structure overlap (SOV), Seed Linkage, COGnitor, shared domain content, and neighbor-joining single-linkage, and observed that the former four agree in their validations. Presently, the UniRef50-based recruitment procedure enriches the COG database for Archaea, Bacteria and its subgroups Actinobacteria, Firmicutes, Proteobacteria, and other bacteria by 2.2-, 8.0-, 7.0-, 8.8-, 8.7-, and 4.2-fold, respectively, in terms of sequences, and also considerably increased the number of species.

Independent evolution of heavy metal-associated domains in copper chaperones and copper-transporting atpases.

Copper chaperones are small cytoplasmic proteins that bind intracellular copper (Cu) and deliver it to Cu-dependent enzymes such as cytochrome oxidase, superoxide dismutase, and amine oxidase. Copper chaperones are similar in sequence and structure to the Cu-binding heavy metal-associated (HMA) domains of Cu-transporting ATPases (Cu-ATPases), and the genes for copper chaperones and Cu-ATPases are often located in the same operon. Phylogenetic analysis shows that Cu chaperones and HMA domains of Cu-ATPases represent ancient and distinct lineages that have evolved largely independently since their initial separation. Copper chaperone-Cu-ATPase operons appear to have evolved independently in different prokaryotic lineages, probably due to a strong selective pressure for coexpression of these genes.

The COG database: new developments in phylogenetic classification of proteins from complete genomes.

The database of Clusters of Orthologous Groups of proteins (COGs), which represents an attempt on a phylogenetic classification of the proteins encoded in complete genomes, currently consists of 2791 COGs including 45 350 proteins from 30 genomes of bacteria, archaea and the yeast Saccharomyces cerevisiae (http://www.ncbi.nlm.nih. gov/COG). In addition, a supplement to the COGs is available, in which proteins encoded in the genomes of two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and shared with bacteria and/or archaea were included. The new features added to the COG database include information pages with structural and functional details on each COG and literature references, improvements of the COGNITOR program that is used to fit new proteins into the COGs, and classification of genomes and COGs constructed by using principal component analysis.

Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M-G(1) transition in mammals.

To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, were absent during mitosis and early G(1) phase, and reappeared as cells progressed through G(1) phase. Immunoblotting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 was stably bound to chromatin. Orc1 and Mcm3 were easily eluted from chromatin during mitosis and early G(1) phase, but became stably bound during mid-G(1) phase, concomitant with the appearance of a functional pre-replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre-replication complexes until mitosis is complete and a nuclear structure has formed.

Initiation of eukaryotic DNA replication: conservative or liberal?

The mechanism for initiation of eukaryotic DNA replication is highly conserved: the proteins required to initiate replication, the sequence of events leading to initiation, and the regulation of initiation are remarkably similar throughout the eukaryotic kingdom. Nevertheless, there is a liberal attitude when it comes to selecting initiation sites. Differences appear to exist in the composition of replication origins and in the way proteins recognize these origins. In fact, some multicellular eukaryotes (the metazoans) can change the number and locations of initiation sites during animal development, revealing that selection of initiation sites depends on epigenetic as well as genetic parameters. Here we have attempted to summarize our understanding of this process, to identify the similarities and differences between single cell and multicellular eukaryotes, and to examine the extent to which origin recognition proteins and replication origins have been conserved among eukaryotes. Published 2000 Wiley-Liss, Inc.

Selective activation of pre-replication complexes in vitro at specific sites in mammalian nuclei.

As the first step in determining whether or not pre-replication complexes are assembled at specific sites along mammalian chromosomes, nuclei from G(1)-phase hamster cells were incubated briefly in Xenopus egg extract in order to initiate DNA replication. Most of the nascent DNA consisted of RNA-primed DNA chains 0.5 to 2 kb in length, and its origins in the DHFR gene region were mapped using both the early labeled fragment assay and the nascent strand abundance assay. The results revealed three important features of mammalian replication origins. First, Xenopus egg extract can selectively activate the same origins of bi-directional replication (e.g. ori-beta) and (beta') that are used by hamster cells in vivo. Previous reports of a broad peak of nascent DNA centered at ori-(beta/(beta)' appeared to result from the use of aphidicolin to synchronize nuclei and from prolonged exposure of nuclei to egg extracts. Second, these sites were not present until late G(1)-phase of the cell division cycle, and their appearance did not depend on the presence of Xenopus Orc proteins. Therefore, hamster pre-replication complexes appear to be assembled at specific chromosomal sites during G(1)-phase. Third, selective activation of ori-(beta) in late G(1)-nuclei depended on the ratio of Xenopus egg extract to nuclei, revealing that epigenetic parameters such as the ratio of initiation factors to DNA substrate could determine the number of origins activated.

Using the COG database to improve gene recognition in complete genomes.

A complete understanding of the biology of an organism necessarily starts with knowledge of its genetic makeup. Proteins encoded in a genome must be identified and characterized, and the presence or absence of specific sets of proteins must be noted in order to determine the possible biochemical pathways or functional systems utilized by that organism. The COG database presents a set of tools suited to these purposes, including the ability to select protein families (COGs) that contain proteins from a specified set of species. The selection is based upon a phylogenetic pattern, which is a shorthand representation of the presence or absence of a particular species in a COG. Here we present the use of phylogenetic patterns as a means to perform targeted searches for undetected protein-coding genes in complete genomes.

Towards understanding the first genome sequence of a crenarchaeon by genome annotation using clusters of orthologous groups of proteins (COGs).

BACKGROUND: Standard archival sequence databases have not been designed as tools for genome annotation and are far from being optimal for this purpose. We used the database of Clusters of Orthologous Groups of proteins (COGs) to reannotate the genomes of two archaea, Aeropyrum pernix, the first member of the Crenarchaea to be sequenced, and Pyrococcus abyssi. RESULTS: A. pernix and P. abyssi proteins were assigned to COGs using the COGNITOR program; the results were verified on a case-by-case basis and augmented by additional database searches using the PSI-BLAST and TBLASTN programs. Functions were predicted for over 300 proteins from A. pernix, which could not be assigned a function using conventional methods with a conservative sequence similarity threshold, an approximately 50% increase compared to the original annotation. A. pernix shares most of the conserved core of proteins that were previously identified in the Euryarchaeota. Cluster analysis or distance matrix tree construction based on the co-occurrence of genomes in COGs showed that A. pernix forms a distinct group within the archaea, although grouping with the two species of Pyrococci, indicative of similar repertoires of conserved genes, was observed. No indication of a specific relationship between Crenarchaeota and eukaryotes was obtained in these analyses. Several proteins that are conserved in Euryarchaeota and most bacteria are unexpectedly missing in A. pernix, including the entire set of de novo purine biosynthesis enzymes, the GTPase FtsZ (a key component of the bacterial and euryarchaeal cell-division machinery), and the tRNA-specific pseudouridine synthase, previously considered universal. A. pernix is represented in 48 COGs that do not contain any euryarchaeal members. Many of these proteins are TCA cycle and electron transport chain enzymes, reflecting the aerobic lifestyle of A. pernix. CONCLUSIONS: Special-purpose databases organized on the basis of phylogenetic analysis and carefully curated with respect to known and predicted protein functions provide for a significant improvement in genome annotation. A differential genome display approach helps in a systematic investigation of common and distinct features of gene repertoires and in some cases reveals unexpected connections that may be indicative of functional similarities between phylogenetically distant organisms and of lateral gene exchange.

The COG database: a tool for genome-scale analysis of protein functions and evolution.

Rational classification of proteins encoded in sequenced genomes is critical for making the genome sequences maximally useful for functional and evolutionary studies. The database of Clusters of Orthologous Groups of proteins (COGs) is an attempt on a phylogenetic classification of the proteins encoded in 21 complete genomes of bacteria, archaea and eukaryotes (http://www. ncbi.nlm. nih.gov/COG). The COGs were constructed by applying the criterion of consistency of genome-specific best hits to the results of an exhaustive comparison of all protein sequences from these genomes. The database comprises 2091 COGs that include 56-83% of the gene products from each of the complete bacterial and archaeal genomes and approximately 35% of those from the yeast Saccharomyces cerevisiae genome. The COG database is accompanied by the COGNITOR program that is used to fit new proteins into the COGs and can be applied to functional and phylogenetic annotation of newly sequenced genomes.

Absence of an unusual "densely methylated island" at the hamster dhfr ori-beta.

An unusual "densely methylated island" (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has been reported at mammalian origins of DNA replication. This report had far-reaching implications in understanding of DNA methylation and DNA replication. For example, since this DMI appeared in about 90% of proliferating cells, but not in stationary cells, it may regulate origin activation. In an effort to confirm and extend these observations, the DMI at the well characterized ori-beta locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was checked for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cells and in cell populations enriched in the G1, S, or G2 + M phases of their cell division cycle. DNA was subjected to 1) cleavage by methylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate modification of 5-methylcytosines (mC) followed by piperidine cleavage. The permanganate reaction is a novel method for direct detection of mC residues that complements the more commonly used hydrazine method. These methods were capable of detecting mC in 2% of the cells. At the region of the proposed DMI, only one mC at a CpG site was detected. However, the ori-beta DMI was not detected in any of these cell populations using any of these methods.

Ease of DNA unwinding is a conserved property of yeast replication origins.

Autonomously replicating sequence (ARS) elements function as plasmid replication origins. Our studies of the H4 ARS and ARS307 have established the requirement for a DNA unwinding element (DUE), a broad easily-unwound sequence 3' to the essential consensus that likely facilitates opening of the origin. In this report, we examine the intrinsic ease of unwinding a variety of ARS elements using (1) a single-strand-specific nuclease to probe for DNA unwinding in a negatively-supercoiled plasmid, and (2) a computer program that calculates DNA helical stability from the nucleotide sequence. ARS elements that are associated with replication origins on chromosome III are nuclease hypersensitive, and the helical stability minima correctly predict the location and hierarchy of the hypersensitive sites. All well-studied ARS elements in which the essential consensus sequence has been identified by mutational analysis contain a 100-bp region of low helical stability immediately 3' to the consensus, as do ARS elements created by mutation within the prokaryotic M13 vector. The level of helical stability is, in all cases, below that of ARS307 derivatives inactivated by mutations in the DUE. Our findings indicate that the ease of DNA unwinding at the broad region directly 3' to the ARS consensus is a conserved property of yeast replication origins.

DNA helical stability accounts for mutational defects in a yeast replication origin.

Earlier studies on the H4 autonomously replicating sequence (ARS) identified a DNA unwinding element (DUE), a required sequence that is hypersensitive to single-strand-specific nucleases and serves to facilitate origin unwinding. Here we demonstrate that a DUE can be identified in the C2G1 ARS, a chromosomal replication origin, by using a computer program that calculates DNA helical stability from the base sequence. The helical stability minima correctly predict the location and hierarchy of the nuclease-hypersensitive sites in a C2G1 ARS plasmid. Nucleotide-level mapping shows that the nuclease-hypersensitive site at the ARS spans a 100-base-pair sequence in the required 3'-flanking region. Mutations that stabilize the DNA helix in the broad 3'-flanking region reduce or abolish ARS-mediated plasmid replication, indicating that helical instability is required for origin function. The level of helical instability is quantitatively related to the replication efficiency of the ARS mutants. Multiple copies of either a consensus-related sequence present in the C2G1 ARS or the consensus sequence itself in synthetic ARS elements contribute to DNA helical instability. Our findings indicate that a DUE is a conserved component of the C2G1 ARS and is a major determinant of replication origin activity.

Stable DNA unwinding, not "breathing," accounts for single-strand-specific nuclease hypersensitivity of specific A+T-rich sequences.

A long A+T-rich sequence in supercoiled pBR322 DNA is hypersensitive to single-strand-specific nucleases at 37 degrees C but not at reduced temperature. The basis for the nuclease hypersensitivity is stable DNA unwinding as revealed by (i) the same temperature dependence for hypersensitivity and for stable unwinding of plasmid topoisomers after two-dimensional gel electrophoresis, (ii) preferential nuclease digestion of stably unwound topoisomers, and (iii) quantitative nicking of stably unwound topoisomers in the A+T-rich region. Nuclease hypersensitivity of A+T-rich sequences is hierarchical, and either deletion of the primary site or a sufficient increase in the free energy of supercoiling leads to enhanced nicking at an alternative A+T-rich site. The hierarchy of nuclease hypersensitivity reflects a hierarchy in the free energy required for unwinding naturally occurring sequences in supercoiled DNA. This finding, along with the known hypersensitivity of replication origins and transcriptional regulatory regions, has important implications for using single-strand-specific nucleases in DNA structure-function studies.