Development of non-ß-Lactam covalent allosteric inhibitors targeting PBP2a in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive bacterial pathogen, continues to pose a serious threat to the current public health system in our society. The high level of resistance to ß-lactam antibiotics in MRSA is attributed to the expression of penicillin-binding protein 2a (PBP2a), which catalyzes cell wall cross-linking. According to numerous research reports, the activity of the PBP2a protein is known to be regulated by an allosteric site distinct from the active site where cell wall cross-linking occurs. Here, we conducted a screening of 113 compounds containing a 1,3,4-oxadiazole core to design new covalent inhibitors targeting the allosteric site of PBP2a and establish their structural-activity relationship. The stereochemically selective synthesis of sulfonyl oxadiazole compounds identified in the initial screening resulted in a maximum eightfold enhancement in cell inhibition activity. The sulfonyl oxadiazole-based compounds formulated as PEG-based ointments, with low toxicity test results on human cells (CC 50 : >78µM), demonstrated potent antimicrobial effects not only in a mouse skin wound infection model but also against oxacillin-resistant clinical isolate MRSA (IC 50 ≈ 1µM), as evidenced by the results. Furthermore, additional studies utilizing LC-MS/MS and in-silico approaches clearly support the allosteric site covalent binding mechanism through the nucleophilic aromatic substitution (S N Ar) reaction, as well as its association with the closure of the major active site of PBP2a.

Carbapenem-induced ß-lactamase-isoform expression trends in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent bacterial threat to public health, with only a few treatment options and a >50% fatality rate. Although several resistance mechanisms are understood, the appearance of these mutations is generally considered stochastic. Recent reports have, however, begun to challenge this assumption. Here, we demonstrate that independent samples of Ab, exposed to different carbapenems with escalating concentrations, show concentration- and carbapenem-dependent trends in ß-lactamase-isoform expression. This result, based on the isoforms identified through label-free-quantification LC-MS/MS measurements of cell-free, gel-separated ß-lactamases, suggests that the appearance of antibiotic resistance may be somewhat non-stochastic. Specifically, several minor AmpC/ADC ß-lactamase-isoforms were found to exhibit both dose- and carbapenem-dependent expression, suggesting the possibility of non-stochastic mutations. Additionally, these also have high sequence similarity to major expressed isoforms, indicating a potential path over which resistance occurred in independent samples. Antibiotic resistance maybe somewhat antibiotic-directed by a hitherto unknown mechanism and further investigation may lead to new strategies for mitigating antibiotic resistance. Teaser: The emergence of antibiotic-resistant ß-lactamase proteins from mutations may exhibit patterns based on specific antibiotics.

Decoding Hepatic Portal Venous Gas: A Case Report.

Hepatic portal venous gas (HPVG) is an infrequent and life-threatening condition with high morbidity and mortality rates, which consists of the presence of gas in the portal vein and its branches. Improvements in imaging technologies have led to the diagnosis of HPVG in less severe circumstances, which, in turn, has only determined a small amelioration of the prognosis. We present a rare case of HPVG subsequent to paralytic ileus in a patient who attained long-term survival after the surgical treatment was performed. HPVG is considered to be associated with sepsis, parietal/mucosal damage, inflammation of the intraperitoneal organs, and meteorism, which may be found in a variety of pathologies. The severity of this pathology depends on the pre-existing conditions of the patients but also on how quickly a treatment plan is established and applied. As a correct and timely diagnosis is crucial for the increase of the survival rate in HPVG, greater attention shall be paid to the clinical manifestations and the differential diagnosis.

HLA Gene Polymorphisms in Romanian Patients with Chronic Lymphocytic Leukemia.

Materials and Methods: This study included 66 patients with CLL, diagnosed between 2020 and 2022, and 100 healthy controls. HLA class I and class II genes (HLA-A/B/C, HLA-DQA1/DQB1/DPA1/DPB1, and HLA-DRB1/3/4/5) were investigated using next-generation sequencing technology. Results: Several HLA alleles were strongly associated with CLL. The most important finding was that HLA-DRB1∗04:02:01 (p=0.001, OR = 1.05) and HLA-DRB3∗02:01:01 (p=0.009, OR = 1.03) have a predisposing role in CLL development. Moreover, we identified that HLA-A∗24:02:01 0.01 (p=0.01, OR = 0.38), HLA-DQA1∗05:05:01 (p=0.01, OR = 0.56), HLA-DQB1∗03:02:01 (p=0.03, OR = 0.40), and HLA-DRB4∗01:03:01 (p=0.03, OR = 0.54 alleles have protective roles. Correlations between HLA expression and gender showed that women had a higher expression of protective HLA alleles when compared to men. Conclusions: Our data are the first to indicate that in Romanian patients with CLL, the HLA-A∗24:02:01 and HLA-DQA1∗05:05:01 alleles have a protective role against CLL development, whereas HLA-DRB1∗04:02:01 and HLA-DRB3∗02:01:01alleles are positively associated with CLL.

Primary Retroperitoneal Hydatid Cyst: A Diagnostic and Treatment Conundrum.

Hydatid cysts are caused by accidental egg ingestion of the Echinococcus granulosus parasite. A 24-year-old female was admitted to our hospital for chronic left lumbar pain. Computed tomography (CT) and abdominal ultrasonography identified an 8/12 cm retroperitoneal cyst. The CT results coupled with enzyme-linked immunosorbent assay tests (positive IgG for Echinococcus granulosus) confirmed that the tumor was a hydatid cyst. Treatment consisted of preoperative chemotherapy with albendazole, intraoperative parasite inactivation, laparoscopic partial cystectomy, and drainage. The drain was removed after three days. Chemotherapy was maintained for two years after surgery. No relapse was observed at the six-month reevaluation. In this article, the diagnostic and therapeutic options and resources are discussed and compared with the published literature.

A High-Quality Blue Whale Genome, Segmental Duplications, and Historical Demography.

The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.

The effect of oral administration of zeolite on the energy metabolism and reproductive health of Romanian spotted breed in advanced gestation and post partum period.

The dairy cow experiences the most significant impact from negative energy balance during this period, which adversely affects reproductive health. Consequently, most pathologies affect dairy cows during this time frame. Thus, with the primary objective of reducing the incidence of these pathologies on dairy farms, we questioned whether supplemental zeolite administration in cattle feed would affect metabolism and reproductive health. Therefore, we proposed introducing an antepartum and postpartum supplementation of 400 g of zeolite in the basal diet. The control group received only the basal diet without zeolite supplementation. Monitoring the results stemmed from the consideration that reproductive health can only be present based on an unaltered energy metabolism. Hence, we deemed it necessary to analyze several metabolic markers in light of the expected outcomes concerning reproductive health. Cows treated with zeolite exhibited a calving to first service interval 12.78 days earlier than those in the control group. Moreover, the average number of services per conception used for future gestation was 0.44 lower in the zeolite-treated group compared to the control group (p<0.05). Additionally, the treatment group showed a lower presence of pathogens in the uterus and displayed a more favorable average uterine score. Observations following the completion of the research point towards an improvement in the health of transition dairy cows, opening a new path for dairy farms in terms of preventing postpartum pathologies. Indeed, the benefits from this study primarily impact the animals rather than directly influencing milk production. Therefore, further research is necessary in this regard.

Taxifolin as a Metallo-ß-Lactamase Inhibitor in Combination with Augmentin against Verona Imipenemase 2 Expressing Pseudomonas aeruginosa.

Among the various mechanisms that bacteria use to develop antibiotic resistance, the multiple expression of ß-lactamases is particularly problematic, threatening public health and increasing patient mortality rates. Even if a combination therapy-in which a ß-lactamase inhibitor is administered together with a ß-lactam antibiotic-has proven effective against serine-ß-lactamases, there are no currently approved metallo-ß-lactamase inhibitors. Herein, we demonstrate that quercetin and its analogs are promising starting points for the further development of safe and effective metallo-ß-lactamase inhibitors. Through a combined computational and in vitro approach, taxifolin was found to inhibit VIM-2 expressing P. aeruginosa cell proliferation at <4 µg/mL as part of a triple combination with amoxicillin and clavulanate. Furthermore, we tested this combination in mice with abrasive skin infections. Together, these results demonstrate that flavonol compounds, such as taxifolin, may be developed into effective metallo-ß-lactamase inhibitors.

GenArk: towards a million UCSC genome browsers.

Interactive graphical genome browsers are essential tools in genomics, but they do not contain all the recent genome assemblies. We create Genome Archive (GenArk) collection of UCSC Genome Browsers from NCBI assemblies. Built on our established track hub system, this enables fast visualization of annotations. Assemblies come with gene models, repeat masks, BLAT, and in silico PCR. Users can add annotations via track hubs and custom tracks. We can bulk-import third-party resources, demonstrated with TOGA and Ensembl gene models for hundreds of assemblies.Three thousand two hundred sixty-nine GenArk assemblies are listed at https://hgdownload.soe.ucsc.edu/hubs/ and can be searched for on the Genome Browser gateway page.

Electronic Structure and Transport Properties of Bi2Te3 and Bi2Se3 Single Crystals.

The electrical resistivity and the Hall effect of topological insulator Bi2Te3 and Bi2Se3 single crystals were studied in the temperature range from 4.2 to 300 K and in magnetic fields up to 10 T. Theoretical calculations of the electronic structure of these compounds were carried out in density functional approach, taking into account spin-orbit coupling and crystal structure data for temperatures of 5, 50 and 300 K. A clear correlation was found between the density of electronic states at the Fermi level and the current carrier concentration. In the case of Bi2Te3, the density of states at the Fermi level and the current carrier concentration increase with increasing temperature, from 0.296 states eV-1 cell-1 (5 K) to 0.307 states eV-1 cell-1 (300 K) and from 0.9 × 1019 cm-3 (5 K) to 2.6 × 1019 cm-3 (300 K), respectively. On the contrary, in the case of Bi2Se3, the density of states decreases with increasing temperature, from 0.201 states eV-1 cell-1 (5 K) to 0.198 states eV-1 cell-1 (300 K), and, as a consequence, the charge carrier concentration also decreases from 2.94 × 1019 cm-3 (5 K) to 2.81 × 1019 cm-3 (300 K).

Comparison Adsorption of Cd (II) onto Lignin and Polysaccharide-Based Polymers.

Given the predominantly negative impact of heavy metals on living organisms, the present study proposed to evaluate the adsorption performances under static conditions of Cd (II) from aqueous solutions on unmodified Sarkanda grass lignin compared to the adsorption performances of polysaccharide polymers chemically functionalized, obtained by synthesis and in their native state, but which, although effective, have a cost price that does not allow for large-scale expansion. To improve the retention of Cd (II) on this aromatic component of the biomass resulting from the processing of lignocellulosic materials, different experimental conditions (pH, concentration, dose and contact time) were followed. The Freundlich and Langmuir isotherms were used to describe the equilibrium conditions. Adsorption kinetics were assessed using the Lagergren I and Ho and McKay II kinetic models, furnishing informative insights into the process mechanism. Lignin adsorption capacity was also analyzed by performing biological tests on tomato seeds (Lypercosium esculentum), since heavy metals are known to be a stress factor for seeds by disturbing the osmotic equilibrium. Through the prism of the investigated parameters and under precisely established experimental conditions, unmodified Sarkanda grass lignin-an aromatic biopolymer-can be recommended as a promising adsorbent for the retention of Cd (II) from aqueous solutions, successfully replacing polysaccharide, especially cellulose-based polymers.

Characterization of a novel inhibitor for the New Delhi metallo-ß-lactamase-4: Implications for drug design and combating bacterial drug resistance.

The bacterial metallo-ß-lactamases (MBLs) catalyze the inactivation of ß-lactam antibiotics. Identifying novel pharmacophores remains crucial for the clinical development of additional MBL inhibitors. Previously, 1-hydroxypyridine-2(1H)-thione-6-carboxylic acid, hereafter referred to as 1,2-HPT-6-COOH, was reported as a low cytotoxic nanomolar ß-lactamase inhibitor of Verona-integron-encoded metallo-ß-lactamase 2, capable of rescuing ß-lactam antibiotic activity. In this study, we explore its exact mechanism of inhibition and the extent of its activity through structural characterization of its binding to New Delhi metallo-ß-lactamase 4 (NDM-4) and its inhibitory activity against both NDM-1 and NDM-4. Of all the structure-validated MBL inhibitors available, 1,2-HPT-6-COOH is the first discovered compound capable of forming an octahedral coordination sphere with Zn2 of the binuclear metal center. This unexpected mechanism of action provides important insight for the further optimization of 1,2-HPT-6-COOH and the identification of additional pharmacophores for MBL inhibition.

A novel strategy to characterize the pattern of ß-lactam antibiotic-induced drug resistance in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to ß-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in ß-lactamase expression that occurs in CRAb with different ß-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of ß-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC-MS-based quantitative proteomics. Thirteen proteins were identified and evaluated using a 1789 sequence database of Ab ß-lactamases from UniProt, the majority of which were Class C ß-lactamases (≥ 80%). Importantly, different antibiotics, even those of the same class (e.g. penicillin and amoxicillin), induced non-equivalent responses comprising various isoforms of Class C and D serine-ß-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on ß-lactamase expression.

Harnessing the Dual Antimicrobial Mechanism of Action with Fe(8-Hydroxyquinoline)3 to Develop a Topical Ointment for Mupirocin-Resistant MRSA Infections.

8-Hydroxyquinoline (8-hq) exhibits potent antimicrobial activity against Staphylococcus aureus (SA) bacteria with MIC = 16.0-32.0 µM owing to its ability to chelate metal ions such as Mn2+, Zn2+, and Cu2+ to disrupt metal homeostasis in bacterial cells. We demonstrate that Fe(8-hq)3, the 1:3 complex formed between Fe(III) and 8-hq, can readily transport Fe(III) across the bacterial cell membrane and deliver iron into the bacterial cell, thus, harnessing a dual antimicrobial mechanism of action that combines the bactericidal activity of iron with the metal chelating effect of 8-hq to kill bacteria. As a result, the antimicrobial potency of Fe(8-hq)3 is significantly enhanced in comparison with 8-hq. Resistance development by SA toward Fe(8-hq)3 is considerably delayed as compared with ciprofloxacin and 8-hq. Fe(8-hq)3 can also overcome the 8-hq and mupirocin resistance developed in the SA mutant and MRSA mutant bacteria, respectively. Fe(8-hq)3 can stimulate M1-like macrophage polarization of RAW 264.7 cells to kill the SA internalized in such macrophages. Fe(8-hq)3 exhibits a synergistic effect with both ciprofloxacin and imipenem, showing potential for combination therapies with topical and systemic antibiotics for more serious MRSA infections. The in vivo antimicrobial efficacy of a 2% Fe(8-hq)3 topical ointment is confirmed by the use of a murine model with skin wound infection by bioluminescent SA with a reduction of the bacterial burden by 99 ± 0.5%, indicating that this non-antibiotic iron complex has therapeutic potential for skin and soft tissue infections (SSTIs).

GenArk: Towards a million UCSC Genome Browsers.

Interactive graphical genome browsers are essential tools for biologists working with DNA sequences. Although tens of thousands of new genome assemblies have become available over the last decade, accessibility is limited by the work involved in manually creating browsers and curating annotations. The results can push the limits of data storage infrastructure. To facilitate managing this increasing number of genome assemblies, we created the Genome Archive (GenArk) collection of UCSC Genome Browsers from assemblies hosted at NCBI(1). Built on our established assembly hub system, this collection enables fast, on-demand visualization of chromosome regions without requiring a database server. Available annotations include gene models, some mapped through whole-genome alignments, repeat masks, GC content, and others. We also modified our popular BLAT(2) aligner and in-silico PCR to support a large number of genomes using limited RAM. Users can upload additional annotations themselves via track hubs(3) and custom tracks. We can import more annotations in bulk from third-party resources, demonstrated here with TOGA(4) gene models. 2,430 GenArk assemblies are listed at https://hgdownload.soe.ucsc.edu/hubs/ and can be found by searching on the main UCSC gateway page. We will continue to add human high-quality assemblies and for other organisms, we are looking forward to receiving requests from the research community for ever more browsers and whole-genome alignments via http://genome.ucsc.edu/assemblyRequest.html.

Integrating gene annotation with orthology inference at scale.

Annotating coding genes and inferring orthologs are two classical challenges in genomics and evolutionary biology that have traditionally been approached separately, limiting scalability. We present TOGA (Tool to infer Orthologs from Genome Alignments), a method that integrates structural gene annotation and orthology inference. TOGA implements a different paradigm to infer orthologous loci, improves ortholog detection and annotation of conserved genes compared with state-of-the-art methods, and handles even highly fragmented assemblies. TOGA scales to hundreds of genomes, which we demonstrate by applying it to 488 placental mammal and 501 bird assemblies, creating the largest comparative gene resources so far. Additionally, TOGA detects gene losses, enables selection screens, and automatically provides a superior measure of mammalian genome quality. TOGA is a powerful and scalable method to annotate and compare genes in the genomic era.

Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

A novel strategy to characterize the pattern of ß-lactam antibiotic-induced drug resistance in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to ß-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in ß-lactamase expression that occurs in CRAb with different ß-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of ß-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC-MS-based quantitative proteomics. Peptides were identified and evaluated using a 1789 sequence database of Ab ß-lactamases from UniProt. Importantly, we observed that different antibiotics, even those of the same class ( e.g. penicillin and amoxicillin), induce non-equivalent responses comprising various Class C and D serine-ß-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on ß-lactamase expression.

Emerging Perspectives on the Set of Conditions That Lead to the Emergence of Metabolic Syndrome.

Metabolic syndrome, as a medical condition, presents multifactorial complexity that is characterized by the resulting damage from genetic, environmental, and lifestyle factors (presence or absence of physical activity, food choices). Thus, metabolic syndrome qualifies unequivocally as a medical condition in which there are, simultaneously, several independent metabolic risk factors, namely, abdominal obesity, high triglyceride level, low HDL cholesterol level, arterial hypertension, and high glycemic level. Although age, sex, socio-economic status, and the precise definition of metabolic syndrome all influence the prevalence and risk of developing the condition, clinical and epidemiological studies clearly show that central obesity, as measured by an increased abdominal circumference, is the main risk factor. Thus, due to the growing global incidence of obesity, there has been an increase in the incidence of metabolic syndrome. Starting with obesity, all other metabolic risk factors are influenced: for example, as a result of insulin resistance with hyperglycemia, diabetes is linked to an increased risk of cardiovascular disease due to increased abdominal circumference. Through this review, we aimed to highlight the latest research studies and dietary nutritional interventions useful in the prevention of this disease but also implementation strategies for primary prevention among the healthy population.

Contradictory Phylogenetic Signals in the Laurasiatheria Anomaly Zone.

Relationships among laurasiatherian clades represent one of the most highly disputed topics in mammalian phylogeny. In this study, we attempt to disentangle laurasiatherian interordinal relationships using two independent genome-level approaches: (1) quantifying retrotransposon presence/absence patterns, and (2) comparisons of exon datasets at the levels of nucleotides and amino acids. The two approaches revealed contradictory phylogenetic signals, possibly due to a high level of ancestral incomplete lineage sorting. The positions of Eulipotyphla and Chiroptera as the first and second earliest divergences were consistent across the approaches. However, the phylogenetic relationships of Perissodactyla, Cetartiodactyla, and Ferae, were contradictory. While retrotransposon insertion analyses suggest a clade with Cetartiodactyla and Ferae, the exon dataset favoured Cetartiodactyla and Perissodactyla. Future analyses of hitherto unsampled laurasiatherian lineages and synergistic analyses of retrotransposon insertions, exon and conserved intron/intergenic sequences might unravel the conflicting patterns of relationships in this major mammalian clade.