Genetic Diversity, Virulence, and Antibiotic Resistance Determinants of Campylobacter jejuni Isolates in Romania.

The emergence of antibiotic-resistant Campylobacter jejuni, a leading cause of gastroenteritis worldwide, presents a significant public health challenge requiring vigilant surveillance and disease control. This study aimed to characterize C. jejuni strains isolated in Romania from 2017 to 2020, focusing on genetic diversity, virulence, and antibiotic resistance determinants. The isolates underwent phenotypical testing, PCR, and antibiotic resistance assessment using the Kirby-Bauer disc diffusion method for ciprofloxacin, erythromycin, and tetracycline. Genetic analysis identified resistance and virulence genes, point mutations, and performed sequence typing (7-gene MLST) to determine genetic relatedness. Results indicated substitutions at position 86 in the amino acid sequence or position 257 in the nucleotide sequence of the gyrA gene in 47 fluoroquinolone-resistant isolates. Additionally, mutations in the rRNA 23S gene at positions 2074 and 2075, associated with macrolide resistance, were found in 12 of the 66 isolates. Allelic profiles generated 38 sequence types (STs), including three new STs not present in the reference database. The sequence data analysis revealed a genetically diverse C. jejuni population with a weak clonal structure. This study provides crucial insights into the genetic diversity and antibiotic resistance of C. jejuni strains in Romania, highlighting the need for ongoing surveillance and control measures.

The Molecular Detection of Germline Mutations in the BRCA1 and BRCA2 Genes Associated with Breast and Ovarian Cancer in a Romanian Cohort of 616 Patients.

The objective of this study was to identify and classify the spectrum of mutations found in the BRCA1 and BRCA2 genes associated with breast and ovarian cancer in female patients in Romania. Germline BRCA1 and BRCA2 mutations were investigated in a cohort of 616 female patients using NGS and/or MLPA methods followed by software-based data analysis and classification according to international guidelines. Out of the 616 female patients included in this study, we found that 482 patients (78.2%) did not have any mutation present in the two genes investigated; 69 patients (11.2%) had a BRCA1 mutation, 34 (5.5%) had a BRCA2 mutation, and 31 (5%) presented different type of mutations with uncertain clinical significance, moderate risk or a large mutation in the BRCA1 gene. Our investigation indicates the most common mutations in the BRCA1 and BRCA2 genes, associated with breast and ovarian cancer in the Romanian population. Our results also bring more data in support of the frequency of the c.5266 mutation in the BRCA1 gene, acknowledged in the literature as a founder mutation in Eastern Europe. We consider that the results of our study will provide necessary data regarding BRCA1 and BRCA2 mutations that would help to create a genetic database for the Romanian population.

Recent Advancements in Research on DNA Methylation and Testicular Germ Cell Tumors: Unveiling the Intricate Relationship.

Testicular germ cell tumors (TGCTs) are the most common type of testicular cancer, with a particularly high incidence in the 15-45-year age category. Although highly treatable, resistance to therapy sometimes occurs, with devastating consequences for the patients. Additionally, the young age at diagnosis and the treatment itself pose a great threat to patients' fertility. Despite extensive research concerning genetic and environmental risk factors, little is known about TGCT etiology. However, epigenetics has recently come into the spotlight as a major factor in TGCT initiation, progression, and even resistance to treatment. As such, recent studies have been focusing on epigenetic mechanisms, which have revealed their potential in the development of novel, non-invasive biomarkers. As the most studied epigenetic mechanism, DNA methylation was the first revelation in this particular field, and it continues to be a main target of investigations as research into its association with TGCT has contributed to a better understanding of this type of cancer and constantly reveals novel aspects that can be exploited through clinical applications. In addition to biomarker development, DNA methylation holds potential for developing novel treatments based on DNA methyltransferase inhibitors (DNMTis) and may even be of interest for fertility management in cancer survivors. This manuscript is structured as a literature review, which comprehensively explores the pivotal role of DNA methylation in the pathogenesis, progression, and treatment resistance of TGCTs.

Antibacterial Activity of PVA Hydrogels Embedding Oxide Nanostructures Sensitized by Noble Metals and Ruthenium Dye.

Nanostructured oxides (SiO2, TiO2) were synthesized using the sol-gel method and modified with noble metal nanoparticles (Pt, Au) and ruthenium dye to enhance light harvesting and promote the photogeneration of reactive oxygen species, namely singlet oxygen (1O2) and hydroxyl radical (•OH). The resulting nanostructures were embedded in a transparent polyvinyl alcohol (PVA) hydrogel. Morphological and structural characterization of the bare and modified oxides was performed using scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), UV-Vis spectroscopy, and X-ray photoelectron spectroscopy (XPS). Additionally, electrokinetic potential measurements were conducted. Crystallinity data and elemental analysis of the investigated systems were obtained through X-ray diffraction and X-ray fluorescence analyses, while the chemical state of the elements was determined using XPS. The engineered materials, both as simple powders and embedded in the hydrogel, were evaluated for their ability to generate reactive oxygen species (ROS) under visible and simulated solar light irradiation to establish a correlation with their antibacterial activity against Staphylococcus aureus. The generation of singlet oxygen (1O2) by the samples under visible light exposure can be of significant importance for their potential use in biomedical applications.

The Landscape of the DNA Transposons in the Genome of the Horezu_LaPeri Strain of Drosophila melanogaster.

Natural transposons (NTs) represent mobile DNA sequences found in both prokaryotic and eukaryotic genomes. Drosophila melanogaster (the fruit fly) is a eukaryotic model organism with NTs standing for about 20% of its genome and has contributed significantly to the understanding of various aspects of transposon biology. Our study describes an accurate approach designed to map class II transposons (DNA transposons) in the genome of the Horezu_LaPeri fruit fly strain, consecutive to Oxford Nanopore Technology sequencing. A whole genome bioinformatics analysis was conducted using Genome ARTIST_v2, LoRTE and RepeatMasker tools to identify DNA transposons insertions. Then, a gene ontology enrichment analysis was performed in order to evaluate the potential adaptive role of some DNA transposons insertions. Herein, we describe DNA transposon insertions specific for the Horezu_LaPeri genome and a predictive functional analysis of some insertional alleles. The PCR validation of P-element insertions specific for this fruit fly strain, along with a putative consensus sequence for the KP element, is also reported. Overall, the genome of the Horezu_LaPeri strain contains several insertions of DNA transposons associated with genes known to be involved in adaptive processes. For some of these genes, insertional alleles obtained via mobilization of the artificial transposons were previously reported. This is a very alluring aspect, as it suggests that insertional mutagenesis experiments conducting adaptive predictions for laboratory strains may be confirmed by mirroring insertions which are expected to be found at least in some natural fruit fly strains.

H3 histone methylation landscape in male urogenital cancers: from molecular mechanisms to epigenetic biomarkers and therapeutic targets.

During the last decades, male urogenital cancers (including prostate, renal, bladder and testicular cancers) have become one of the most frequently encountered malignancies affecting all ages. While their great variety has promoted the development of various diagnosis, treatment and monitoring strategies, some aspects such as the common involvement of epigenetic mechanisms are still not elucidated. Epigenetic processes have come into the spotlight in the past years as important players in the initiation and progression of tumors, leading to a plethora of studies highlighting their potential as biomarkers for diagnosis, staging, prognosis, and even as therapeutic targets. Thus, fostering research on the various epigenetic mechanisms and their roles in cancer remains a priority for the scientific community. This review focuses on one of the main epigenetic mechanisms, namely, the methylation of the histone H3 at various sites and its involvement in male urogenital cancers. This histone modification presents a great interest due to its modulatory effect on gene expression, leading either to activation (e.g., H3K4me3, H3K36me3) or repression (e.g., H3K27me3, H3K9me3). In the last few years, growing evidence has demonstrated the aberrant expression of enzymes that methylate/demethylate histone H3 in cancer and inflammatory diseases, that might contribute to the initiation and progression of such disorders. We highlight how these particular epigenetic modifications are emerging as potential diagnostic and prognostic biomarkers or targets for the treatment of urogenital cancers.

Easy method for six-fragment Golden Gate Assembly of modular vectors.

Efficient cloning techniques are a requirement for synthetic biology. This study provides a simplified cloning method based on Golden Gate Assembly that can be used for rapid vector construction. The building of multiple expression vectors with customizable modules is achieved in a timely manner with minimal hands-on time by removing unnecessary steps in the workflow. The authors constructed a total of 21 mammalian episomal expression vectors and conducted a fluorescence expression comparison for different regulatory region combinations post-transfection in HEK293T and HEPG2 cells. Screening revealed that using the EF-1α promoter in combination with the bovine growth hormone polyadenylation sequence seemed to perform best in the types of cells tested compared with other variants.

ONT-Based Alternative Assemblies Impact on the Annotations of Unique versus Repetitive Features in the Genome of a Romanian Strain of Drosophila melanogaster.

To date, different strategies of whole-genome sequencing (WGS) have been developed in order to understand the genome structure and functions. However, the analysis of genomic sequences obtained from natural populations is challenging and the biological interpretation of sequencing data remains the main issue. The MinION device developed by Oxford Nanopore Technologies (ONT) is able to generate long reads with minimal costs and time requirements. These valuable assets qualify it as a suitable method for performing WGS, especially in small laboratories. The long reads resulted using this sequencing approach can cover large structural variants and repetitive sequences commonly present in the genomes of eukaryotes. Using MinION, we performed two WGS assessments of a Romanian local strain of Drosophila melanogaster, referred to as Horezu_LaPeri (Horezu). In total, 1,317,857 reads with a size of 8.9 gigabytes (Gb) were generated. Canu and Flye de novo assembly tools were employed to obtain four distinct assemblies with both unfiltered and filtered reads, achieving maximum reference genome coverages of 94.8% (Canu) and 91.4% (Flye). In order to test the quality of these assemblies, we performed a two-step evaluation. Firstly, we considered the BUSCO scores and inquired for a supplemental set of genes using BLAST. Subsequently, we appraised the total content of natural transposons (NTs) relative to the reference genome (ISO1 strain) and mapped the mdg1 retroelement as a resolution assayer. Our results reveal that filtered data provide only slightly enhanced results when considering genes identification, but the use of unfiltered data had a consistent positive impact on the global evaluation of the NTs content. Our comparative studies also revealed differences between Flye and Canu assemblies regarding the annotation of unique versus repetitive genomic features. In our hands, Flye proved to be moderately better for gene identification, while Canu clearly outperformed Flye for NTs analysis. Data concerning the NTs content were compared to those obtained with ONT for the D. melanogaster ISO1 strain, revealing that our strategy conducted to better results. Additionally, the parameters of our ONT reads and assemblies are similar to those reported for ONT experiments performed on various model organisms, revealing that our assembly data are appropriate for a proficient annotation of the Horezu genome.

Revealing the Phenotypic and Genomic Background for PHA Production from Rapeseed-Biodiesel Crude Glycerol Using Photobacterium ganghwense C2.2.

Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain's metabolic capabilities is necessary in choosing economically feasible production conditions. This study aimed to create an in-depth view of the utilization of Photobacterium ganghwense C2.2 for PHA production by linking a wide array of characterization methods: metabolic pathway annotation from the strain's complete genome, high-throughput phenotypic tests, and biomass analyses through plate-based assays and flask and bioreactor cultivations. We confirmed, in PHA production conditions, urea catabolization, fatty acid degradation and synthesis, and high pH variation and osmotic stress tolerance. With urea as a nitrogen source, pure and rapeseed-biodiesel crude glycerol were analyzed comparatively as carbon sources for fermentation at 20 °C. Flask cultivations yielded 2.2 g/L and 2 g/L PHA at 120 h, respectively, with molecular weights of 428,629 g/mol and 81,515 g/mol. Bioreactor batch cultivation doubled biomass accumulation (10 g/L and 13.2 g/L) in 48 h, with a PHA productivity of 0.133 g/(L·h) and 0.05 g/(L·h). Thus, phenotypic and genomic analyses determined the successful use of Photobacterium ganghwense C2.2 for PHA production using urea and crude glycerol and 20 g/L NaCl, without pH adjustment, providing the basis for a viable fermentation process.

Bioactive Coatings Based on Nanostructured TiO2 Modified with Noble Metal Nanoparticles and Lysozyme for Ti Dental Implants.

This work presents the synthesis of nanostructured TiO2 modified with noble metal nanoparticles (Au, Ag) and lysozyme and coated on titanium foil. Moreover, the specific structural and functional properties of the resulting inorganic and hybrid materials were explored. The purpose of this study was to identify the key parameters for developing engineered coatings on titanium foil appropriate for efficient dental implants with intrinsic antibacterial activity. TiO2 nanoparticles obtained using the sol-gel method were deposited on Ti foil and modified with Au/Ag nanoparticles. Morphological and structural investigations (scanning electron and atomic force microscopies, X-ray diffraction, photoluminescence, and UV-Vis spectroscopies) were carried out for the characterization of the resulting inorganic coatings. In order to modify their antibacterial activity, which is essential for safe dental implants, the following aspects were investigated: (a) singlet oxygen (1O2) generation by inorganic coatings exposed to visible light irradiation; (b) the antibacterial behavior emphasized by titania-based coatings deposited on titanium foil (TiO2/Ti foil; Au-TiO2/Ti foil, Ag-TiO2/Ti foil); (c) the lysozyme bioactivity on the microbial substrate (Micrococcus lysodeicticus) after its adsorption on inorganic surfaces (Lys/TiO2/Ti foil; Lys/Au-TiO2/Ti foil, Lys/Ag-TiO2/Ti foil); (d) the enzymatic activity of the above-mentioned hybrids materials for the hydrolysis reaction of a synthetic organic substrate usually used for monitoring the lysozyme biocatalytic activity, namely, 4-Methylumbelliferyl ß-D-N,N',N″-triacetylchitotrioside [4-MU-ß- (GlcNAc)3]. This was evaluated by identifying the presence of a fluorescent reaction product, 7-hydroxy-4-metyl coumarin (4-methylumbelliferone).

Phylogenetic analysis of torque teno virus in Romania: possible evidence of distinct geographical distribution.

Torque teno virus (TTV) is highly prevalent, but little is known about its circulation in humans. Here, we investigated the geographical distribution and phylogeny of TTV in Romania. A fragment of TTV untranslated region B was sequenced in samples from volunteers across the country. Additional sequences from dialyzed patients were also included in the study. Phylogenetic analysis showed that more than 80% of Romanian sequences clustered with isolates assigned to the species Torque teno virus 1 and Torque teno virus 3 (former genogroup 1), and this analysis discriminated between isolates from the North-East and West regions. Further studies assessing the pathogenic potential of TTV isolates should employ analysis based on genomic regions with phylogenetic resolution below the species level.

Quality of Life in Patients with Chronic Psychotic Disorders: A Practical Model for Interventions in Romanian Mental Health Centers.

Background and Objectives: Current psychiatric care is increasingly focusing on patients' quality of life (QoL). Research is still trying to determine the main factors which influence QoL. The present study aims to assess the QoL of patients with chronic psychotic-spectrum disorders, as well as its relation to symptomatology, functionality, adaptive behavior, and perceived level of recovery. Materials and Methods: The study included a sample of 78 patients with chronic psychosis. Symptomatology and illness severity were assessed with the Positive and Negative Syndrome Scale (PANSS) and the Clinical Global Impression Scale-Severity (CGI-S) scales, respectively. The Global Assessment of Functioning Scale (GAFS) and the Adaptive Behavior Assessment System II (ABAS-II)-Adult Form were used for the assessment of patients' functionality, and the Quality-of-Life Inventory (QOLI) scale was applied for the evaluation of QoL. Results: According to the CGI-Severity scale, 20.5% of the patients were borderline mentally ill, and 24.4% were mildly ill. The highest number of patients (34.6%) were moderately ill, while 14.1% and 2.6% were markedly ill and severely ill, respectively. Among the moderately ill patients, more than half (63%) were patients with schizophrenia, 18.5% were patients with delusional disorder, and 18.5% were patients with schizoaffective disorder. Most of the patients (43.6%) presented moderate functioning deficiency, while 38.5% of the patients presented severe deficiency, according to the GAFS score. When assessed with the ABAS, we observed that almost half of the patients (44.9%) showed an average functioning across skill areas in the conceptual, social, and practical domains. A percent of 67.9% of the patients presented an average QoL, while 15.4% and 12.8% showed a very low and low QoL. QoL was not influenced by the patients' symptomatology, gender, and education level. Having children, family support, better social and conceptual skills, and a higher perceived level of recovery was correlated with an increased QoL, hierarchical multiple regression R2 = 0.379, F(9, 68) = 2.616, and p = 0.012. Conclusions: Psychiatric interventions in psychosis should focus not only on symptoms' control, but also on improving social and family support, as well as adaptive skills to increase the patients' QoL.

Epigenetic Regulation of Angiogenesis in Development and Tumors Progression: Potential Implications for Cancer Treatment.

Angiogenesis is a multi-stage process of new blood vessel development from pre-existing vessels toward an angiogenic stimulus. The process is essential for tissue maintenance and homeostasis during embryonic development and adult life as well as tumor growth. Under normal conditions, angiogenesis is involved in physiological processes, such as wound healing, cyclic regeneration of the endometrium, placental development and repairing certain cardiac damage, in pathological conditions, it is frequently associated with cancer development and metastasis. The control mechanisms of angiogenesis in carcinogenesis are tightly regulated at the genetic and epigenetic level. While genetic alterations are the critical part of gene silencing in cancer cells, epigenetic dysregulation can lead to repression of tumor suppressor genes or oncogene activation, becoming an important event in early development and the late stages of tumor development, as well. The global alteration of the epigenetic spectrum, which includes DNA methylation, histone modification, chromatin remodeling, microRNAs, and other chromatin components, is considered one of the hallmarks of cancer, and the efforts are concentrated on the discovery of molecular epigenetic markers that identify cancerous precursor lesions or early stage cancer. This review aims to highlight recent findings on the genetic and epigenetic changes that can occur in physiological and pathological angiogenesis and analyze current knowledge on how deregulation of epigenetic modifiers contributes to tumorigenesis and tumor maintenance. Also, we will evaluate the clinical relevance of epigenetic markers of angiogenesis and the potential use of "epi-drugs" in modulating the responsiveness of cancer cells to anticancer therapy through chemotherapy, radiotherapy, immunotherapy and hormone therapy as anti-angiogenic strategies in cancer.

Burnout Toll on Empathy Would Mediate the Missing Professional Support in the COVID-19 Outbreak.

PURPOSE: The year 2020 was marked by the COVID-19 pandemic, massively disruptive at the general population level and for healthcare systems. We aimed to evaluate the psychological distress associated with work-related experiences among medical professionals and supporting staff during the pandemic outbreak. PATIENTS AND METHODS: A cross-sectional survey was conducted between April and May 2020, employing a self-administered on-line questionnaire that included the collection of socio-demographic and professional status information, Hamilton Anxiety Rating Scale, nine-item Patient Health Questionnaire (PHQ-9), Maslach Burnout Inventory - General Survey, Connor-Davidson Resilience Scale, and a subscale of the International Personality Item Pool (IPIP) for empathy. A total of 364 professionals of the county-coordinated area responded. Descriptive statistics summarized the findings and a mediation model was analyzed, applying the causal step strategy. The specific direct and causal mediation effects were estimated with the bias-corrected and accelerated bootstrap sampling method. RESULTS: Anxiety, burnout, stress, resilience, and empathy proved to be significantly associated with both the professional category (ie, consultant, specialty doctor, trainee doctor, senior nurse, trainee nurse or other) and the perceived professional support (the nonparametric multivariate permutation test resulted in p=0.048 and p<0.001, respectively). When controlling for the marital and professional status, the female gender had an OR=2.26, 95% CI (1.21; 4.22) toward a high level of empathy compared to males. The causal mediation effect of the perceived lack of professional support on empathy through burnout-depersonalization was highly significant (p<0.001) with an average of 0.0599, 95% CI (0.0238; 0.10), while the direct effect was non-significant (p=0.536) with an average of 0.0295, 95% CI (-0.0774; 0.15). CONCLUSION: We highlighted the impact of the lack of professional support on healthcare workers' empathy through burnout-depersonalization in a context of a high workload, time pressure, job stress beyond previous training, and inherently limited organizational support, which are potentially modifiable factors in the mid-term.

Complete genome sequence of Photobacterium ganghwense C2.2: A new polyhydroxyalkanoate production candidate.

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that can be manufactured sustainably and represent a promising green alternative to petrochemical-based plastics. Here, we describe the complete genome of a new marine PHA-producing bacterium-Photobacterium ganghwense (strain C2.2), which we have isolated from the Black Sea seashore. This new isolate is psychrotolerant and accumulates PHA when glycerol is provided as the main carbon source. Transmission electron microscopy, specific staining with Nile Red visualized via epifluorescence microscopy and gas chromatography analysis confirmed the accumulation of PHA. This is the only PHA-producing Photobacterium for which we now have a complete genome sequence, allowing us to investigate the pathways for PHA production and other secondary metabolite synthesis pathways. The de novo assembly genome, obtained using open-source tools, comprises two chromosomes (3.5, 2 Mbp) and a megaplasmid (202 kbp). We identify the entire PHA synthesis gene cluster that encodes a class I PHA synthase, a phasin, a 3-ketothiolase, and an acetoacetyl-CoA reductase. No conventional PHA depolymerase was identified in strain C2.2, but a putative lipase with extracellular amorphous PHA depolymerase activity was annotated, suggesting that C2.2 is unable to degrade intracellular PHA. A complete pathway for the conversion of glycerol to acetyl-CoA was annotated, in accordance with its ability to convert glycerol to PHA. Several secondary metabolite biosynthetic gene clusters and a low number of genes involved in antibiotic resistance and virulence were also identified, indicating the strain's suitability for biotechnological applications.

Complete genome sequence of Tsukamurella sp. MH1: A wide-chain length alkane-degrading actinomycete.

Tsukamurella sp. strain MH1, capable to use a wide range of n-alkanes as the only carbon source, was isolated from petroleum-contaminated soil (Pitești, Romania) and its complete genome was sequenced. The 4,922,396 bp genome contains only one circular chromosome with a G + C content of 71.12%, much higher than the type strains of this genus (68.4%). Based on the 16S rRNA genes sequence similarity, strain MH1 was taxonomically identified as Tsukamurella carboxydivorans. Genome analyses revealed that strain MH1 is harboring only one gene encoding for the alkB-like hydroxylase, arranged in a complete alkane monooxygenase operon. This is the first complete genome of the specie T. carboxydivorans, which will provide insights into the potential of Tsukamurella sp. MH1 and related strains for bioremediation of petroleum hydrocarbons-contaminated sites and into the environmental role of these bacteria.

Newly isolated alkalophilic Advenella species bioaugmented in activated sludge for high p-cresol removal.

In this work, an alkalophilic bacterium (LVX-4) capable of using p-cresol as sole source of carbon and energy was screened and isolated from soil polluted by used oil. Phylogenetic (16S rRNA) and phenotypic characterization using Biolog GN microplates and API 20NE strips indicated that LVX-4 strain is a new Advenella species. It showed both the capability to degrade of p-cresol at high concentrations (750 mg/L) and to use p-cresol for growth in a pH from 7 to 10, although the optimum pH was 9. Moreover bioaugmentation of activated sludge with this strain lead to the complete removal of p-cresol in less than 100 h. This is the first study that shows the potential of Advenella sp. to be bioaugmented in activated sludge system for p-cresol biodegradation.

Phenotypic profiles of virulence in different Candida species isolated from vulvovaginal infections.

In order to establish an infection, pathogenic microorganisms have to colonize, survive, multiply, evade the immune system and spread to other tissues [1, 2, 3]. Although some Candida species are normally commensal in humans, in the last decades the frequency and the severity of nosocomial diseases due to Candida strains have increased dramatically [4]. The aim of the present study was to characterize some Candida strains isolated from vulvovaginal infections by determining the virulence and pathogenicity profile. The assessment of the in vitro expression of virulence cell wall associated factors (hyphal formation, adherence to HeLa cell line, biofilm development), soluble secreted enzymes (aspartyl protease, lipase, phospholipase, DN-ase) and Fe3+ accumulation was achieved by phenotypic methods on 13 yeast strains belonging to five Candida (C.) species (C. albicans, C. krusei, C. parapsilopsis, C. catenulata and C. kefyr). Candida sp. strains isolated from vulvovaginal infections showed species/ strain specific virulence profile.

[The concept of bacterial species]. / Conceptul de specie bacteriana.

Prokaryotes are the most abundant living organisms and also most diverse from genetically and metabolically point of view, being responsible for the majority of biogeochemical processes playing the most important role in life cycle on the planet. Considering this, there is a general agreement among taxonomists, that there is a very small number of bacterial species recognized and described today, mostly because of controversial issues concerning bacterial species concept. One of the most accepted approaches, even today, is the polyphasic taxonomy because it is based on diverse information, obtained from classic taxonomy but also from molecular level. The development of new molecular techniques, especially sequencing rRNA genes conducted to an improved concept, that we intended to evaluate in this review, and even more, to reconstruction of group specific phylogenetic tree.

Investigation of the utility of selective methyl protonation for determination of membrane protein structures.

Polytopic alpha-helical membrane proteins present one of the final frontiers for protein structural biology, with significant challenges causing severe under-representation in the protein structure databank. However, with the advent of hardware and methodology geared to the study of large molecular weight complexes, solution NMR is being increasingly considered as a tool for structural studies of these types of membrane proteins. One method that has the potential to facilitate these studies utilizes uniformly deuterated samples with protons reintroduced at one or two methyl groups of leucine, valine and isoleucine. In this work we demonstrate that in spite of the increased proportion of these amino acids in membrane proteins, the quality of structures that can be obtained from this strategy is similar to that obtained for all alpha-helical water soluble proteins. This is partly attributed to the observation that NOEs between residues within the transmembrane helix did not have an impact on structure quality. Instead the most important factors controlling structure accuracy were the strength of dihedral angle restraints imposed and the number of unique inter-helical pairs of residues constrained by NOEs. Overall these results suggest that the most accurate structures will arise from accurate identification of helical segments and utilization of inter-helical distance restraints from various sources to maximize the distribution of long-range restraints.