In silico functional elucidation of uncharacterized proteins of Chlamydia abortus strain LLG

Background: The Mycobacterium tuberculosis complex (MTBC) bacteria include the slowly growing, host-associated bacteria Mycobacterium tuberculosis, Mycobacterium Bovis, Mycobacterium microti, Mycobacterium africanum, Mycobacterium pinnipedii. Aim: Comparative Functional Genomics Studies for understanding the Hypothetical Proteins in Mycobacterium tuberculosis variant microti 12. Objective: A computational genomics study was performed to understand the 247 hypothetical protein genes. Functional annotation of virtual proteins was performed on different servers to maximize confidence level. Methods: Sequence Retrieval. The whole genome sequences for the Mycobacterium tuberculosis micro variant 12 were retrieved from the KEGG database ( http://www.genome.jp/kegg/) and were used for screening 247 hypothetical proteins (Fig. 1 ). Functional Annotation and Sub-cellular localization. The Mycobacterium tuberculosis micro variant 12 hypothetical proteins were screened and sorted out from the genome and were individually analyzed for the presence of conserved functional domains by using computational biology tools like CDD-BLAST ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) ;Pfam ( http://pfam.xfam.org/ncbiseq/398365647); The subcellular localization of hypothetical proteins was determined by CELLO2GO ( http://cello.life.nctu.edu.tw). These web tools can search the defined conserved domains in the sequences available in the online servers or databases and assist in the classification of proteins in the appropriate families. Protein Structure Prediction. The in-silico structure predictions of the hypothetical protein sequences showing functional properties were carried out by using the PS2 Protein Structure Prediction Server ( http://www.ps2.life.nctu.edu.tw/). The online server helps to generate the 3D structures of the hypothetical proteins. The server accepts the sequences in FASTA format as a query to generate resultant proteins 3D structures. The structure determination is completely based on the conserved template regions detected during functional annotations. Protein-protein interaction through String database: The interaction of each hypothetical protein analyzed for functional characteristics was subjected to a protein-protein interaction server for the prediction of a possible functional role in interaction amongst the available known proteins ( https://string-db.org/). This information can help us to further validated the functional role of such hypothetical proteins and their possible role in the Mycobacterium Tuberculosis micro variant. Protein secondary structure prediction through JPred4: The secondary structure prediction of all the hypothetical proteins was determined through JPred4 ( http://www.compbio.dundee.ac.uk/jpred4/index.html) and served to identify the available secondary structures in the unknown hypothetical protein sequences. These further help us to understand the available templates in the uncharacterized protein sequences for the prediction of novel functions associated with these proteins. The predictions were further characterized by the Phyre2 server for structural modeling and prediction of templates based on comparative analysis based on conserved domains. Protein modeling, prediction, and analysis through Phyre2. The hypothetical proteins which were identified to have functional properties were further characterized by the Phyre2 server ( http://www.sbg.bio.ic.ac.uk/phyre2) for structural modeling and prediction of templates based on comparative analysis based on conserved domains. Results: A computational genomics study was performed to understand the 247 hypothetical protein genes Functional annotation of virtual proteins, and was performed on different servers to maximize confidence level. The functional prediction was performed by CDD-Blast and Pfam. The gene sequences of proteins have probably been successfully functionally annotated, characterized, and their subcellular localization and 3-D structural predictions have been predicted computationally. Online automated bioinformatics tools such as CDD-Blast, Pfam, CELLO2GO and PS2-Server were used for the structural and functional characterization of screened hypothetical proteins. The structure, function, and subcellular localization of a hypothetical protein from Mycobacterium tuberculosis variant microti 12 have been obtained and presented (Fig. 2 ). Also, the three-dimensional structure generated after using the template with the highest score was displayed as the template ID in the structure column of the respective hypothetical protein. However, as systems biology denies hypothetical protein functions, the structures of such proteins can be tested through biological processes and experiments, making them suitable for understanding their role in the life cycle, pathogenesis, and drug development. We can further explore these predictive possibilities in pharmaceuticals, and other clinically relevant studies. This study by HP helped find structure-function relationships in Mycobacterium tuberculosis variant microti 12 using a variety of bioinformatics tools. The string database made predictions about protein-protein interactions and the template helped us predict a hypothetical protein structure and even helped us find its 3D protein structure. Protein profiling can be performed on structures retrieved from these servers. This is useful for proteomics studies, including protein-protein interactions, protein expression of specific hypothetical proteins, and post-translational modifications of protein-coding genes. Further understanding of these hypothetical proteins can help us to know more about the Mycobacterium tuberculosis complex (MTBC) and may assist in Drugs and inhibitors against different pathogens within this complex. Conclusion: The all-inclusive bioinformatic study has helped to functionally elucidate 247 hypothetical proteins, which have resulted and made it easier to understand many functional proteins available in the Mycobacterium tuberculosis micro variant 12. The subcellular localization of the 247 sorted hypothetical proteins was also carried & which further helped us understand the localization of identified enzymes or proteins. We have successfully characterized the 247 unknown proteins of hypothetical protein sequences from Mycobacterium tuberculosis micro variant 12 to validate their structure and functions of the gene products. These predicted functions and three-dimensional structures may lead to establishing their role in the life cycle of the bacterium. This computationally generated data can also be further used for developing new protocols for new vaccines against Mycobacterium tuberculosis micro variant 12 that are essential for preventing infection, diseases, and transmission. This complete result of Hypothetical Protein is needed for further studies of the whole genomic of the Mycobacterium Tuberculosis micro variant 12 for their function interpretation which further help in the understanding of its functions as well as structure. Moreover, this interpretation would help us to study the evolution of Mycobacterium Tuberculosis micro variant 12 which further helps in the process of discovering the drugs to inhibit the causes of diseases.

Suppression subtractive hybridization PCR isolation of cDNAs from a Caribbean soft coral

Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia. Currently, there are 18 different serotypes; the serotype 8 is the most widely distributed in the United States, Canada, United Kingdom, and southeastern Brazil. In this study, genomes of seven A. pleuropneumoniae serotype 8 clinical isolates were compared to the other genomes of twelve serotypes. The analyses of serotype 8 genomes resulted in a set of 2352 protein-coding sequences. Of these sequences, 76.6% are present in all serotypes, 18.5% are shared with some serotypes, and 4.9% were differential. This differential portion was characterized as a series of hypothetical and regulatory protein sequences: mobile element sequence. Synteny analysis demonstrated possible events of gene recombination and acquisition by horizontal gene transfer (HGT) in this species. A total of 30 sequences related to prophages were identified in the genomes. These sequences represented 0.3 to 3.5% of the genome of the strains analyzed, and 16 of them contained complete prophages. Similarity analysis between complete prophage sequences evidenced a possible HGT with species belonging to the family Pasteurellaceae. Thus, mobile genetic elements, such as prophages, are important components of the differential portion of the A. pleuropneumoniae genome and demonstrate a central role in the evolution of the species. This study represents the first study done to understand the genome of A. pleuropneumoniae serotype 8.

Disordered regions of a protein are segments that lack a fixed tertiary structure and are partially folded or unfolded. Proteins with such regions participate in important regulatory functions in the cell, including transcription, translation, cell cycle and numerous signal transduction events. Human proteome, which contains about 20% of sequences annotated as 'hypothetical proteins' and consisting of disordered regions, serve as an interesting dataset for investigating the roles of disordered regions in disease associations, drug target selections, biochemical validations, determination of novel folds, etc. An insilico attempt has been carried out here to investigate these disordered regions in hypothetical proteins from human genome to establish their relationships with diseases and as potential drug targets.

Monitored natural attenuation (MNA) is an in situ remediation technology that relies on naturally occurring and demonstrable processes in soil and groundwater which reduce the mass and concentration of the contaminants. Natural attenuation (NA) involves both aerobic and anaerobic degradation of the contaminants due to the fact that oxygen is used up near the core of the contaminant plume. The aerobic and anaerobic microbial processes can be assessed by microbial activity measurements and molecular biology methods in combination with chemical analyses. The sampling and knowledge on the site conditions are of major importance for the linkage of the results obtained to the conditions in situ. Rates obtained from activity measurements can, with certain limitations, be used in modeling of the fate of contaminants whereas most molecular methods mainly give qualitative information on the microbial community and gene abundances. However, molecular biology methods are fast and describe the in situ communities and avoid the biases inherent to activity assays requiring laboratory incubations.

Spinal infections (SI) are on the rise due to an aging population and the prevalence of more invasive procedures. This study aims to systematically review the microbiological spectrum of SI to enhance diagnostic accuracy and inform effective antibiotic treatment strategies. The last search was conducted on May 9th, 2024, from databases including EMBASE, PubMed, and Web of Science. The outcome variable is infection rate, and the detection method used should be blood culture, tissue culture, or molecular biology method. Two researchers independently extracted research data and evaluated its quality using the JBI Critical Appraisal Tools. Out of 14,639 identified records, 156 studies (encompassing 13,539 patients) were included. Staphylococcus aureus was identified as the most prevalent pathogen, with pooled infection rates of 17.6% (95%CI: 12.8–22.9%; I2=93%) in blood culture, 16.8% (95%CI: 14.0–19.8%; I2=96%) in tissue culture, and 12.0% (95%CI: 9.3–15.0%; I2=35%) in molecular methods. The bacterial spectrum also featured Staphylococcus epidermidis, Escherichia coli, and Mycobacterium tuberculosis (MTB). Molecular methods, particularly metagenomic next-generation sequencing (mNGS), demonstrated markedly superior sensitivity for MTB detection, with a pooled rate of 9.7% (95%CI: 4.6–16.3%; I2=90%) compared to 1.3% (95%CI: 0.6–2.1%; I2=86%) by tissue culture. The odds ratio for MTB detection with mNGS versus conventional culture was 4.24 (95%CI: 1.68–10.73). This review confirms that a core group of pathogens, including Staphylococcus aureus, Staphylococcus epidermidis, MTB, and Escherichia coli. Our findings underscore that tissue culture is fundamental for common pyogenic bacteria, while metagenomic next-generation sequencing is indispensable for detecting fastidious organisms like MTB. Trial registration: The protocol was registered with PROSPERO (No. CRD42023427429). Registered on May 28, 2023Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-28576-4.

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The haloarchaeal diversity of a salt mine, a natural cave in central Anatolia, was investigated using convential microbiological and molecular biology methods. Eight halophilic archaeal isolates selected based on their colony morphology and whole cell protein profiles were taxonomically classified on the basis of their morphological, physiological, biochemical properties, polar lipid and protein profiles and 16S rDNA sequences. From the 16S rDNA sequences comparisons it was established that the isolates CH2, CH3 and CHC resembled Halorubrum saccharovorum by 98.8%, 98.9% and 99.5%, respectively. There was a 99.7% similarity between the isolate CH11 and Halobacterium noricense and 99.2% between the isolate CHA1 and Haloarcula argentinensis. The isolate CH8K and CH8B revealed a similarity rate of 99.8% and 99.3% to Halococcus dombrowskii, respectively. It was concluded that the isolates named CH2, CH3 and CHC were clustered in the genus Halorubrum and that CHA1 and CH7 in the genus Haloarcula, CH8K and CH8B in the genus Halococcus and CH11 in the genus Halobacterium.

Detection of Toxoplasma gondii from amniotic fluid, a comparison of four different molecular biological methods

Relevance. Staphylococci are one of the most common pathogens in the etiological structure of nosocomial infections. Methicillinresistant staphylococci are resistant to the main groups of antibiotics and are one of bacteria that pose the greatest threat to human health according to the World Health Organization. The introduction of modern molecular biological methods in the monitoring of methicillin-resistant staphylococci is necessary for the rapid and accurate identification of microorganisms, monitoring the epidemiological situation and studying the need for anti-epidemic measures. Aims. Аnalysis of the results of monitoring methicillinresistant staphylococcus strains using molecular biological methods in a multidisciplinary hospital in Moscow for one year. Materials and methods. Single-center observational study with a one-year follow-up period (December 2016 to December 2017). The research included a molecular-biological analysis of biological material from 240 patients with signs of infection and washings samples (n=250) from the objects of the hospital environment. The whole genome sequencing was carried out for 24 samples, isolated from patients with different forms of staphylococcal infection and washings from a hospital environment. DNA detection of methicillin-resistant strains was performed using the «AmpliSens®MRSA-screen-titer-FL» reagent kit. Sequencing was performed on an Illumina HiSeq1500 instrument using the IlluminaHiSeq PE RapidClusterKit v2 and IlluminaHiSeqRapid SBS Kit v2. Results. DNA of methicillin-resistant staphylococci were detected in 6.3% of blood samples from patients with signs of infection and in 42.4% of washings from the objects of the hospital environment. The results of PCR of washing samples showed that DNA of methicillin-resistant coagulase-negative staphylococci were detected much more often than MRSA (p <0.001). DNA of methicillin-resistant staphylococci were detected more often in the intensive care and intensive care units compared with the hematology and surgical departments (p <0.001). The examine samples of Staphylococcus aureus belonged to 6 different sequence types (ST-5, ST-7, ST-8, ST-22, ST-30 and ST-5555) and 8 spatypes (t008, t021, t091, t1062, t12437, t1544, t223, t4573). As a result of monitoring, an isolate with a new allelic profile was found. Today it has been assigned the number ST5555. The predominant type of staphylococcal mec cassette was the type IV SCCmec cassette. Conclusion. Due to the widespread distribution of methicillin-resistant strains and the identification of epidemiologically significant genetic lines of staphylococci, it is necessary to conduct regular monitoring, take measures to limit the spread of such strains and introduce modern molecular biological methods for quick and accurate identification.

Intramuscular Immunization of Streptococcus pyogenes SF370 protein extract and identification of multiple virulence factors through proteomic profiling in RHD induced Balb/c mice

Biología molecular en el diagnóstico de la candidiasis profunda en el paciente crítico no neutropénico

Most fungal infections of the skin are caused by dermatophytes, both in Germany and globally. Tinea pedis is the most frequent fungal infection in Western industrial countries. Tinea pedis frequently leads to tinea unguium, while in the elderly, both may then spread causing tinea corporis. A variety of body sites may be affected, including tinea glutealis, tinea faciei and tinea capitis. The latter rarely occurs in adults, but is the most frequent fungal infection in childhood. Following antifungal treatment of tinea unguium and also tinea capitis a dermatophytid or hyperergic reaction to dermatophyte antigens may occur. Yeast infections affect the mucous membranes both of the gastro-intestinal system and the genital tract as candidiasis mostly due to Candida albicans. Cutaneous candidiasis affects predominantely the intertriginous regions such as groins and the inframammary area, but also the intertriginous space of fingers and toes. In contrast, pityriasis versicolor is a superficial epidermal fungal infection primarily on the the trunk. Mold infections are rare in dermatology; they play a role nearly exclusively in nondermatophyte-mold (NDM) onychomycosis. The diagnosis of dermatomycoses comprises the microscopic detection of fungi using the potassium hydroxide preparation or alternatively the fluorescence optical Blankophor preparation together with culture. The histological fungal detection with PAS staining possesses a high sensitivity, and it should play a more important role in particular for diagnosis of onychomycosis. Molecular biological methods, based on the amplification of fungal DNA with use of specific primers for the distinct causative agents are on the rise. With PCR, such as dermatophyte-PCR-ELISA, fungi can be detected directly in clinical material in a highly specific and sensitive manner without prior culture. Today, molecular methods, such as Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI TOF MS) as culture confirmation assay, complete the conventional mycological diagnostics.

Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.

Biofilms characteristic of aquifer conditions can be rapidly and efficiently collected using in situ microcosms or “bio‐traps” containing Bio‐Sep ® beads (25% Nomex and 75% powdered activated carbon [PAC]) (University of Tulsa, Tulsa, Oklahoma). Bio‐Sep beads can be “baited” with a variety of organic compounds by vapor‐phase adsorption onto the PAC component of the beads under reduced pressure. In the aquifer, the bait or the amendment does not substantially leach into the aquifer but is available to the bacteria as a carbon source within the bead. When the organic compound is labeled with 13 C, phospholipids may be extracted from bead biofilms postincubation and derived fatty acid methyl esters analyzed for 13 C. Incorporation of 13 C into biomass provides proof of current in situ degradation potential. We have now Bio‐Sep beads with 13 C‐labeled methyl‐ tert ‐butyl ether (MTBE) and tert ‐butyl alcohol (TBA). Deployment of these 13 C‐amended bio‐traps has been coupled with molecular biological methods and stable isotope probing to demonstrate the biodegradation of MTBE and TBA in an anaerobic gasoline plume in southern California. Nonamended bio‐traps were deployed at the same site as a preexperiment to determine if spatial variations in microbial community structure could be linked to concentrations of oxygenates or other electron donors. Analysis of biofilm phospholipids and DNA from nonamended bio‐traps demonstrated a clear relationship between various aspects of the subsurface microbial community structure and the concentration of TBA. There was no correlation of any phospholipid fatty acid or DNA attributes with the concentrations of MTBE; benzene, toluene, ethylbenzene, and xylenes (BTEX); or gasoline range total petroleum hydrocarbons (TPHg). Deployment of 13 C‐MTBE‐ or 13 C‐TBA‐amended bio‐traps in the plume clearly demonstrated the degradation of both MTBE and TBA under aquifer conditions through incorporation of 13 C into membrane phospholipids.