Integrated longitudinal multiomics study identifies immune programs associated with acute COVID-19 severity and mortality.

BACKGROUNDPatients hospitalized for COVID-19 exhibit diverse clinical outcomes, with outcomes for some individuals diverging over time even though their initial disease severity appears similar to that of other patients. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity.METHODSWe performed deep immunophenotyping and conducted longitudinal multiomics modeling, integrating 10 assays for 1,152 Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study participants and identifying several immune cascades that were significant drivers of differential clinical outcomes.RESULTSIncreasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, formation of neutrophil extracellular traps, and T cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma Igs and B cells and dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to failure of viral clearance in patients with fatal illness.CONCLUSIONOur longitudinal multiomics profiling study revealed temporal coordination across diverse omics that potentially explain the disease progression, providing insights that can inform the targeted development of therapies for patients hospitalized with COVID-19, especially those who are critically ill.TRIAL REGISTRATIONClinicalTrials.gov NCT04378777.FUNDINGNIH (5R01AI135803-03, 5U19AI118608-04, 5U19AI128910-04, 4U19AI090023-11, 4U19AI118610-06, R01AI145835-01A1S1, 5U19AI062629-17, 5U19AI057229-17, 5U19AI125357-05, 5U19AI128913-03, 3U19AI077439-13, 5U54AI142766-03, 5R01AI104870-07, 3U19AI089992-09, 3U19AI128913-03, and 5T32DA018926-18); NIAID, NIH (3U19AI1289130, U19AI128913-04S1, and R01AI122220); and National Science Foundation (DMS2310836).

A multi-omics systems vaccinology resource to develop and test computational models of immunity.

Systems vaccinology studies have identified factors affecting individual vaccine responses, but comparing these findings is challenging due to varying study designs. To address this lack of reproducibility, we established a community resource for comparing Bordetella pertussis booster responses and to host annual contests for predicting patients' vaccination outcomes. We report here on our experiences with the "dry-run" prediction contest. We found that, among 20+ models adopted from the literature, the most successful model predicting vaccination outcome was based on age alone. This confirms our concerns about the reproducibility of conclusions between different vaccinology studies. Further, we found that, for newly trained models, handling of baseline information on the target variables was crucial. Overall, multiple co-inertia analysis gave the best results of the tested modeling approaches. Our goal is to engage community in these prediction challenges by making data and models available and opening a public contest in August 2024.

Targeted Isolation of Antibiofilm Compounds from Halophytic Endophyte Bacillus velezensis 7NPB-3B Using LC-HR-MS-Based Metabolomics.

The discovery of new natural products has become more challenging because of the re-isolation of compounds and the lack of new sources. Microbes dwelling in extreme conditions of high salinity and temperature are huge prospects for interesting natural metabolites. In this study, the endophytic bacteria Bacillus velezensis 7NPB-3B isolated from the halophyte Salicornia brachiata was screened for its biofilm inhibition against methicillin-resistant Staphylococcus aureus (MRSA). The fractionation of the crude extract was guided by bioassay and LC-HRMS-based metabolomics using multivariate analysis. The 37 fractions obtained by high-throughput chromatography were dereplicated using an in-house MS-Excel macro coupled with the Dictionary of Natural Products database. Successive bioactivity-guided separation yielded one novel compound (1), a diketopiperazine (m/z 469.258 [M - H]-) with an attached saturated decanoic acid chain, and four known compounds (2-5). The compounds were identified based on 1D- and 2D-NMR and mass spectrometry. Compounds 1 and 5 exhibited excellent biofilm inhibition properties of >90% against the MRSA pathogen at minimum inhibition concentrations of 25 and 35 µg/mL, respectively. The investigation resulted in the isolation of a novel diketopiperazine from a bacterial endophyte of an untapped plant using an omics approach.

Integrated longitudinal multi-omics study identifies immune programs associated with COVID-19 severity and mortality in 1152 hospitalized participants.

Hospitalized COVID-19 patients exhibit diverse clinical outcomes, with some individuals diverging over time even though their initial disease severity appears similar. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity. In this study, we carried out deep immunophenotyping and conducted longitudinal multi-omics modeling integrating ten distinct assays on a total of 1,152 IMPACC participants and identified several immune cascades that were significant drivers of differential clinical outcomes. Increasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, NETosis, and T-cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma immunoglobulins and B cells, as well as dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to the failure of viral clearance in patients with fatal illness. Our longitudinal multi-omics profiling study revealed novel temporal coordination across diverse omics that potentially explain disease progression, providing insights that inform the targeted development of therapies for hospitalized COVID-19 patients, especially those critically ill.

A systems vaccinology resource to develop and test computational models of immunity.

Computational models that predict an individual's response to a vaccine offer the potential for mechanistic insights and personalized vaccination strategies. These models are increasingly derived from systems vaccinology studies that generate immune profiles from human cohorts pre- and post-vaccination. Most of these studies involve relatively small cohorts and profile the response to a single vaccine. The ability to assess the performance of the resulting models would be improved by comparing their performance on independent datasets, as has been done with great success in other areas of biology such as protein structure predictions. To transfer this approach to system vaccinology studies, we established a prototype platform that focuses on the evaluation of Computational Models of Immunity to Pertussis Booster vaccinations (CMI-PB). A community resource, CMI-PB generates experimental data for the explicit purpose of model evaluation, which is performed through a series of annual data releases and associated contests. We here report on our experience with the first such 'dry run' for a contest where the goal was to predict individual immune responses based on pre-vaccination multi-omic profiles. Over 30 models adopted from the literature were tested, but only one was predictive, and was based on age alone. The performance of new models built using CMI-PB training data was much better, but varied significantly based on the choice of pre-vaccination features used and the model building strategy. This suggests that previously published models developed for other vaccines do not generalize well to Pertussis Booster vaccination. Overall, these results reinforced the need for comparative analysis across models and datasets that CMI-PB aims to achieve. We are seeking wider community engagement for our first public prediction contest, which will open in early 2024.

Bacillinaphthin A: A New Naphthohydroquinone from the Endophyte Bacillus subtilis NPROOT3.

The present study explores the endophyte associated with the halophyte Salicornia brachiata for uncovering new biologically important compounds. Thus, HPLC-PDA guided chemical investigation of the ethyl acetate extract of the Bacillus subtilis NPROOT3 led to the isolation of a new compound named bacillinaphthin A (1) along with previously known rubinaphthin A (2). The structure of 2 was determined by a comparison of HR-ESI-MS, 1 H and 13 C nuclear magnetic resonances (NMR) with those of reported data, whereas the structure of new compound 1 was elucidated by interpretation of 1D- and 2D-NMR and MS data. Bacillinaphthin (1) and rubinaphthin (2) feature 1,4-dihydroxy-2-naphthoic acid derivatives which have been isolated herein for the first time from the genus Bacillus. Bacillinaphthin (1) is a new congener of 2 with an additional succinic acid side chain attached to the sugar moiety. Production of succinoglycan compounds was reported to regulate symbiosis, hence the isolation of 1 exhibits an example of chemical ecology between the halophyte and its endophyte. In silico tools were used to assess the bioactive potential of both isolated molecules.

Dactylides A-C, three new bioactive 22-membered macrolides produced by Dactylosporangium aurantiacum.

Three new 22-membered polyol macrolides, dactylides A-C (1-3), were isolated from Dactylosporangium aurantiacum ATCC 23491 employing repeated chromatographic separations, and their structures were established based on detailed analysis of NMR and MS data. The relative configurations at the stereocenters were established via vicinal 1H-1H coupling constants, NOE correlations, and by application of Kishi's universal NMR database. In order to get insights into the biosynthetic pathway of 1-3, the genome sequence of the producer strain D. aurantiacum was obtained and the putative biosynthetic gene cluster encoding their biosynthesis was identified through bioinformatic analysis using antiSMASH. Compounds 1-3 showed significant in-vitro antimycobacterial and cytotoxic activity.

Bacillibactin class siderophores produced by the endophyte Bacillus subtilis NPROOT3 as antimycobacterial agents.

The bacterial endophytes isolated from the halophyte Salicornia brachiata were explored for the antimicrobial potential to discover novel microbial inhibitors that combat multidrug resistance. Upon investigation, ethyl acetate extract of the endophyte Bacillus subtilis NPROOT3 displayed significant potency against Mycobacterium smegmatis MTCC6 as well as Mycobacterium tuberculosis H37Rv strain. Further investigation of ethyl acetate crude extract by repeated chromatographic separations followed by characterization using UV, HR-ESI-MS, MALDI-MS, MALDI-MS/MS, CD, and NMR spectroscopy yielded a series of five known siderophores, namely, SVK21 (1), bacillibactin C (2), bacillibactin B (3), tribenglthin A (4), and bacillibactin (5). A total of two out of five compounds, 4 (MIC 38.66 µM) and 5 (MIC 22.15 µM) exhibited significant inhibition against the strain M. smegmatis MTCC6 comparable with positive control rifampicin (MIC 12.15 µM). None of these five bacillibactin molecules are previously reported to exhibit bioactivity against Mycobacterium sp. Herein for the first time, all the compounds were screened for their antibacterial activities against a panel of bacterial pathogens of humans. Furthermore, the probable mechanism of action of bacillibactin compounds for their antimycobacterial activity is also discussed. The findings of this study open up a new chemotype for inhibition of the Mycobacterium sp. and other multidrug-resistant pathogens.

Selective Partition of Lipopeptides from Fermentation Broth: A Green and Sustainable Approach.

Lipopeptide (LP) biosurfactants from microbes have the potential to gradually replace chemical synthetic surfactants and fit the contemporary green and sustainable industrial production concept. However, their active participation is comparatively low in the global market pertaining to their low yield in microbial broth and costly downstream processes arising due to tedious isolation and purification methods. Herein, an efficient extraction method is developed that utilizes an aqueous biphasic system (ABS) comprising ionic liquids and polypropylene glycol 400 (PPG) to selectively extract a mixture of cyclic lipopeptides, namely, surfactin and fengycin from the culture broth of Bacillus amyloliquefaciens 5NPA-1, isolated from the halophyte Salicornia brachiata Roxb. Out of four different ABSs, the ABS composed of 2-hydroxyethyl ammonium formate and PPG displayed a maximum extraction efficiency of 82.30%. PPG-rich phase containing lipopeptides exhibited excellent antimicrobial and mosquito larvicidal properties with no toxic effect on plants. The developed method is simple, novel and accelerates the application of cyclic lipopeptides produced by the microbial source.

Nucleotide-based genetic networks: Methods and applications.

Genomic variations have been acclaimed as among the key players in understanding the biological mechanisms behind migration, evolution, and adaptation to extreme conditions. Due to stochastic evolutionary forces, the frequency of polymorphisms is affected by changes in the frequency of nearby polymorphisms in the same DNA sample, making them connected in terms of evolution. This article presents all the ingredients to understand the cumulative effects and complex behaviors of genetic variations in the human mitochondrial genome by analyzing co-occurrence networks of nucleotides, and shows key results obtained from such analyses. The article emphasizes recent investigations of these co-occurrence networks, describing the role of interactions between nucleotides in fundamental processes of human migration and viral evolution. The corresponding co-mutation-based genetic networks revealed genetic signatures of human adaptation in extreme environments. This article provides the methods of constructing such networks in detail, along with their graph-theoretical properties, and applications of the genomic networks in understanding the role of nucleotide co-evolution in evolution of the whole genome.

Indazole-based microtubule-targeting agents as potential candidates for anticancer drugs discovery.

Tremendous research is focused on developing novel drug candidates targeting microtubules to inhibit their function in several cellular processes, including cell division. In this regard, several indazole derivatives were sought to target the colchicine binding site on the ß-tubulin, a crucial protein required to form microtubules, to develop microtubule targeting agents. Even though there are several reviews on the indazole-based compounds, none of them focused on using indazole scaffold to develop microtubule targeting agents. Therefore, this review aims to present the advances in research on compounds containing indazole scaffolds as microtubule targeting agents based on the articles published in the last two decades. Among the articles reviewed, we found that compounds 6 and 7 showed the lowest IC50 values of 0.6 âˆ¼ 0.9 nM in the cell line studies, making them the strongest indazole derivatives that target microtubules. The compounds 30, 31, 37 (IC50 = ∼ 1 nM) and compounds 8, 38 (IC50 = ∼ 2 nM) have proved to be potent microtubule inhibitors. The compounds 18, 31, 44, 45 also showed strong anticancer activity (IC50 = ∼ 8 nM). It is important to notice that except for compounds 9, 12, 13, 15, and SRF, the top activity compounds including 6, 7, 8, 10, 11, 30, 31, 37, 44, and 45 contain 3,4,5­trimethoxyphenyl substitution similar to that of colchicine. Therefore, it appears that the 3,4,5­trimethoxyphenyl substituent on the indazole scaffold is crucial for targeting CBS.

Synthesis and evaluation of 2-aryl-1H-benzo[d]imidazole derivatives as potential microtubule targeting agents.

Microtubule targeting agents (MTAs) are the potential drug candidates for anticancer drug discovery. Disrupting the microtubule formation or inhibiting the de-polymerization process by a synthetic molecule can lead to an excellent anticancer drug candidate. Here, we present the 2,5-substituted-1H-benzo[d]imidazole derivatives as potential colchicine, nocodazole binding site targeting agents. About 20 benzimidazole derivatives were synthesized with 82.0%-94.0% yield using mild reaction conditions. The synthesized compounds showed moderate to excellent anticancer activity established in three cell lines, including Hela cells, A549 cells, MRC-5 cells. The compounds B15, B16, B19, and B20 are the potential candidates with the IC50 values <15 µM in the three different cell lines. In MTT assay, compounds B15, B16, B19, and B20 showed excellent antiproliferation activity indicated by IC50 values in the range of 5.3 ± 0.21 to 18.1 ± 0.32 µM using HeLa and A549 cell lines. The predicted absorption, distribution, metabolism and excretion (ADME) properties and drug-likeness properties of B15, B16, B19, and B20 indicate that these compounds can be used as lead compounds for further study to develop excellent MTAs.

Development of a Novel Benzimidazole-Based Probe and Portable Fluorimeter for the Detection of Cysteine in Human Urine.

The measurement of cysteine in human urine and live cells is crucial for evaluating biological metabolism, monitoring and maintaining the immune system, preventing tissue/DNA damage caused by free radicals, preventing autoimmune diseases, and diagnosing disorders such as cystinuria and cancer. A method that uses a fluorescence turn-on probe and a portable fluorescence spectrometer device are crucial for highly sensitive, simple, rapid, and inexpensive cysteine detection. Herein, we present the synthesis and application of a benzimidazole-based fluorescent probe (ABIA) along with the design and development of a portable fluorescence spectrometer device (CysDDev) for detecting cysteine in simulated human urine. ABIA showed excellent selectivity and sensitivity in detecting cysteine over homocysteine, glutathione, and other amino acids with the response time of 1 min and demonstrated a detection limit of 16.3 nM using the developed CysDDev. Further, ABIA also demonstrated its utility in detecting intracellular cysteine, making it an excellent probe for bio-imaging assay.

Microbial melanin: Recent advances in biosynthesis, extraction, characterization, and applications.

Melanin is a common name for a group of biopolymers with the dominance of potential applications in medical sciences, cosmeceutical, bioremediation, and bioelectronic applications. The broad distribution of these pigments suggests their role to combat abiotic and biotic stresses in diverse life forms. Biosynthesis of melanin in fungi and bacteria occurs by oxidative polymerization of phenolic compounds predominantly by two pathways, 1,8-dihydroxynaphthalene [DHN] or 3,4-dihydroxyphenylalanine [DOPA], resulting in different kinds of melanin, i.e., eumelanin, pheomelanin, allomelanin, pyomelanin, and neuromelanin. The enzymes responsible for melanin synthesis belong mainly to tyrosinase, laccase, and polyketide synthase families. Studies have shown that manipulating culture parameters, combined with recombinant technology, can increase melanin yield for large-scale production. Despite significant efforts, its low solubility has limited the development of extraction procedures, and heterogeneous structural complexity has impaired structural elucidation, restricting effective exploitation of their biotechnological potential. Innumerable studies have been performed on melanin pigments from different taxa of life in order to advance the knowledge about melanin pigments for their efficient utilization in diverse applications. These studies prompted an urgent need for a comprehensive review on melanin pigments isolated from microorganisms, so that such review encompassing biosynthesis, bioproduction, characterization, and potential applications would help researchers from diverse background to understand the importance of microbial melanins and to utilize the information from the review for planning studies on melanin. With this aim in mind, the present report compares conventional and modern ideas for environment-friendly extraction procedures for melanin. Furthermore, the characteristic parameters to differentiate between eumelanin and pheomelanin are also mentioned, followed by their biotechnological applications forming the basis of industrial utilization. There lies a massive scope of work to circumvent the bottlenecks in their isolation and structural elucidation methodologies.

Biodiversity and antimicrobial potential of bacterial endophytes from halophyte Salicornia brachiata.

Extreme natural habitats like halophytes, marsh land, and marine environment are suitable arena for chemical ecology between plants and microbes having environmental impact. Endophytes are an ecofriendly option for the promotion of plant growth and to serve as sustainable resource of novel bioactive natural products. The present study, focusing on biodiversity of bacterial endophytes from Salicornia brachiata, led to isolation of around 336 bacterial endophytes. Phylogenetic analysis of 63 endophytes revealed 13 genera with 27 different species, belonging to 3 major groups: Firmicutes, Proteobacteria, and Actinobacteria. 30% endophytic isolates belonging to various genera demonstrated broad-spectrum antibacterial and antifungal activities against a panel of human, plant, and aquatic infectious agents. An endophytic isolate Bacillus amyloliquefaciens 5NPA-1, exhibited strong in-vitro antibacterial activity against human pathogen Staphylococcus aureus and phytopathogen Xanthomonas campestris. Investigation through LC-MS/MS-based molecular networking and bioactivity-guided purification led to the identification of three bioactive compounds belonging to lipopeptide class based on 1H-, 13C-NMR and MS analysis. To our knowledge, this is the first report studying bacterial endophytic biodiversity of Salicornia brachiata and the isolation of bioactive compounds from its endophyte. Overall, the present study provides insights into the diversity of endophytes associated with the plants from the extreme environment as a rich source of metabolites with remarkable agricultural applications and therapeutic properties.

Impact of modular mitochondrial epistatic interactions on the evolution of human subpopulations.

Investigation of human mitochondrial (mt) genome variation has been shown to provide insights to the human history and natural selection. By analyzing 24,167 human mt-genome samples, collected for five continents, we have developed a co-mutation network model to investigate characteristic human evolutionary patterns. The analysis highlighted richer co-mutating regions of the mt-genome, suggesting the presence of epistasis. Specifically, a large portion of COX genes was found to co-mutate in Asian and American populations, whereas, in African, European, and Oceanic populations, there was greater co-mutation bias in hypervariable regions. Interestingly, this study demonstrated hierarchical modularity as a crucial agent for these co-mutation networks. More profoundly, our ancestry-based co-mutation module analyses showed that mutations cluster preferentially in known mitochondrial haplogroups. Contemporary human mt-genome nucleotides most closely resembled the ancestral state, and very few of them were found to be ancestral-variants. Overall, these results demonstrated that subpopulation-based biases may favor mitochondrial gene specific epistasis.

Analysis of human mitochondrial genome co-occurrence networks of Asian population at varying altitudes.

Networks have been established as an extremely powerful framework to understand and predict the behavior of many large-scale complex systems. We studied network motifs, the basic structural elements of networks, to describe the possible role of co-occurrence of genomic variations behind high altitude adaptation in the Asian human population. Mitochondrial DNA (mtDNA) variations have been acclaimed as one of the key players in understanding the biological mechanisms behind adaptation to extreme conditions. To explore the cumulative effects of variations in the mitochondrial genome with the variation in the altitude, we investigated human mt-DNA sequences from the NCBI database at different altitudes under the co-occurrence motifs framework. Analysis of the co-occurrence motifs using similarity clustering revealed a clear distinction between lower and higher altitude regions. In addition, the previously known high altitude markers 3394 and 7697 (which are definitive sites of haplogroup M9a1a1c1b) were found to co-occur within their own gene complexes indicating the impact of intra-genic constraint on co-evolution of nucleotides. Furthermore, an ancestral 'RSRS50' variant 10,398 was found to co-occur only at higher altitudes supporting the fact that a separate route of colonization at these altitudes might have taken place. Overall, our analysis revealed the presence of co-occurrence interactions specific to high altitude at a whole mitochondrial genome level. This study, combined with the classical haplogroups analysis is useful in understanding the role of co-occurrence of mitochondrial variations in high altitude adaptation.

Macrocycles and Supramolecules as Antioxidants: Excellent Scaffolds for Development of Potential Therapeutic Agents.

Oxidative stress due to the high levels of reactive oxygen species (ROS) that damage biomolecules (lipids, proteins, DNA) results in acute inflammation. However, without proper intervention, acute inflammation progresses to chronic inflammation and then to several chronic diseases, including cancer, myocardial infarction, cardiovascular diseases, chronic inflammation, atherosclerosis, and more. There has been extensive research on the antioxidants of natural origin. However, there are myriad possibilities for the development of synthetic antioxidants for pharmacological applications. There is an increasing interest in the identification of novel synthetic antioxidants for the modulation of biochemical processes related to ROS. In this regard, derivatives of supramolecules, such as calix[n]arene, resorcinarene, calixtyrosol, calixpyrrole, cucurbit[n]uril, porphyrin etc. are gaining attention for their abilities to scavenge the free radicals. Supramolecular chemistry offers excellent scaffolds for the development of novel antioxidants that can be used to modulate free radical reactions and to improve the disorders related to oxidative stress. This review focuses on the interdisciplinary approach for the design and development of novel synthetic antioxidants based on supramolecular scaffolds, with potentially protective effects against oxidative stress.

Natural Melanin Produced by the Endophytic Bacillus subtilis 4NP-BL Associated with the Halophyte Salicornia brachiata.

Natural melanin with many interesting properties has potential applications in cosmetics, drug delivery, semiconductors, etc. However, conventional production methods are not efficient, resulting in its high cost (350-650 USD g-1), which has been a bottleneck for its efficient commercial utilization. To explore a faster extraction method with a higher yield, a melanin-producing endophytic bacterium was isolated from the halophyte Salicornia brachiata and further identified as Bacillus subtilis 4NP-BL by phylogenetic analysis of 16S rRNA gene sequences. The maximum melanin yield of up to 1.5 g dry wt L-1 of production media was obtained through central composite design (CCD). The isolated melanin belonged to the eumelanin class with an irregular structure on the basis of elemental analysis, UV-vis, Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), electron paramagnetic resonance (EPR), and NMR studies. Furthermore, purified melanin displayed antioxidant activity and antimicrobial activity against pathogens Xanthomonas campestris and Alteromonas macleodii. Thus, this study further suggests a probable role of endophytes that produce melanin in aiding host plant protection from environmental stress and other pathogens.

Codon based co-occurrence network motifs in human mitochondria.

The nucleotide polymorphism in the human mitochondrial genome (mtDNA) tolled by codon position bias plays an indispensable role in human population dispersion and expansion. Herein, genome-wide nucleotide co-occurrence networks were constructed using data comprised of five different geographical regions and around 3000 samples for each region. We developed a powerful network model to describe complex mitochondrial evolutionary patterns among codon and non-codon positions. We found evidence that the evolution of human mitochondria DNA is dominated by adaptive forces, particularly mutation and selection, which was supported by many previous studies. The diversity observed in the mtDNA was compared with mutations, co-occurring mutations, network motifs considering codon positions as causing agent. This comparison showed that long-range nucleotide co-occurrences have a large effect on genomic diversity. Most notably, codon motifs apparently underpinned the preferences among codon positions for co-evolution which is probably highly biased during the origin of the genetic code. Our analysis also showed that variable nucleotide positions of different human sub-populations implemented the independent mtDNA evolution to its geographical dispensation. Ergo, this study has provided both a network framework and a codon glance to investigate co-occurring genomic variations that are critical in underlying complex mitochondrial evolution.