Spectrometric Characterization of Clinical and Environmental Isolates of Aspergillus Series Versicolores.

Aspergillus series Versicolores are molds distributed among 17 species, commonly found in our environment, and responsible for infections. Since 2022, a new taxonomy has grouped them into 4 major lineages: A. versicolor, A. subversicolor, A. sydowii, and A. creber. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) could be a faster and more cost-effective alternative to molecular techniques for identifying them by developing a local database. To evaluate this technique, 30 isolates from Aspergillus series Versicolores were used. A total of 59 main spectra profiles (MSPs) were created in the local database. This protocol enabled accurate identification of 100% of the extracted isolates, of which 97% (29/30) were correctly identified with a log score ≥ 2.00. Some MSPs recorded as Aspergillus versicolor in the supplier's database could lead to false identifications as they did not match with the correct lineages. Although the local database is still limited in the number and diversity of species of Aspergillus series Versicolores, it is sufficiently effective for correct lineage identification according to the latest taxonomic revision, and better than the MALDI-TOF MS supplier's database. This technology could improve the speed and accuracy of routine fungal identification for these species.

γ-enolase (ENO2) is methylated at the Nτ position of His-190 among enolase isozymes.

Protein methylation is mainly observed in lysine, arginine and histidine residues. Histidine methylation occurs at one of two different nitrogen atoms of the imidazole ring, producing Nτ-methylhistidine and Nπ-methylhistidine, and it has recently attracted attention with the identification of SETD3, METTL18 and METTL9 as catalytic enzymes in mammals. Although accumulating evidence had suggested the presence of more than 100 proteins containing methylated histidine residues in cells, much less information has been known regarding histidine-methylated proteins than lysine- and arginine-methylated ones, because no method has been developed to identify substrates for histidine methylation. Here, we established a method to screen novel target proteins for histidine methylation, using biochemical protein fractionation combined with the quantification of methylhistidine by LC-MS/MS. Interestingly, the differential distribution pattern of Nτ-methylated proteins was found between the brain and skeletal muscle, and identified γ-enolase where the His-190 at the Nτ position is methylated in mouse brain. Finally, in silico structural prediction and biochemical analysis showed that the His-190 in γ-enolase is involved in the intermolecular homodimeric formation and enzymatic activity. In the present study, we provide a new methodology to find histidine-methylated proteins in vivo and suggest an insight into the importance of histidine methylation.

Conventional methods and future trends in antimicrobial susceptibility testing.

Antimicrobial susceptibility testing is an essential task for selecting appropriate antimicrobial agents to treat infectious diseases. Constant evolution has been observed in methods used in the diagnostic microbiology laboratories. Disc diffusion or broth microdilution are classical and conventional phenotypic methods with long turnaround time and labour-intensive but still widely practiced as gold-standard. Scientists are striving to develop innovative, novel and faster methods of antimicrobial susceptibility testing to be applicable for routine microbiological laboratory practice and research. To meet the requirements, there is an increasing trend towards automation, genotypic and micro/nano technology-based innovations. Automation in detection systems and integration of computers for online data analysis and data sharing are giant leaps towards versatile nature of automated methods currently in use. Genotypic methods detect a specific genetic marker associated with resistant phenotypes using molecular amplification techniques and genome sequencing. Microfluidics and microdroplets are recent addition in the continuous advancement of methods that show great promises with regards to safety and speed and have the prospect to identify and monitor resistance mechanisms. Although genotypic and microfluidics methods have many exciting features, however, their applications into routine clinical laboratory practice warrant extensive validation. The main impetus behind the evolution of methods in antimicrobial susceptibility testing is to shorten the overall turnaround time in obtaining the results and to enhance the ease of sample processing. This comprehensive narrative review summarises major conventional phenotypic methods and automated systems currently in use, and highlights principles of some of the emerging genotypic and micro/nanotechnology-based methods in antimicrobial susceptibility testing.

Cryptic aspergillosis: a rare entity and a diagnostic challenge.

Introduction: Cryptic aspergillosis, caused by cryptic species of Aspergillus, is increasingly reported in humans and causes significant morbidity and mortality in immunocompromised individuals. The main aim of this study was to describe the occurrence of this entity at a large tertiary care centre and analyse the challenges in identifying them in a routine diagnostic laboratory. Methods: This was a retrospective case review of all patients diagnosed with cryptic Aspergillus species from April 2019 to February 2020. The isolates were identified using conventional microbiological techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF MS), 28S rRNA and internal transcribed spacer (ITS) sequencing. Results: The species identified were Aspergillus tamarii, Aspergillus lentulus and Aspergillus sydowii. Identification by MALDI- TOF MS and sequencing was concordant for all except A. sydowii, with MALDI- TOF MS misidentifying it as Aspergillus thermomutans. All isolates showed low minimum inhibitory concentrations (MICs) for the panel of antifungal drugs. Conclusion: Aspergillosis caused by cryptic Aspergillus species presents a diagnostic challenge. This study confirms the importance of molecular methods for accurate identification.

First report of disseminated Mycobacterium abscessus in an immunocompetent adult patient in Oman.

Mycobacterium abscessus is a ubiquitous rapid-growing mycobacterium that belongs to a diverse group of non-tuberculous mycobacteria. It can cause frequent and serious infections in patients with or without risk factors. This article reports a case of disseminated M. abscessus in an immunocompetent Omani male with native joint septic arthritis.

Biochemistry and toxicology of proteins and peptides purified from the venom of Vipera berus berus.

The isolation and characterization of individual snake venom components is important for a deeper understanding of the pathophysiology of envenomation and for improving the therapeutic procedures of patients. It also opens possibilities for the discovery of novel toxins that might be useful as tools for understanding cellular and molecular processes. The variable venom composition, toxicological and immunological properties of the common vipers (Vipera berus berus) have been reviewed. The combination of venom gland transcriptomics, bottom-up and top-down proteomics enabled comparison of common viper venom proteomes from multiple individuals. V. b. berus venom contains proteins and peptides belonging to 10-15 toxin families: snake venom metalloproteinase, phospholipases A2 (PLA2), snake venom serine proteinase, aspartic protease, L-amino acid oxidase (LAAO), hyaluronidase, 5'-nucleotidase, glutaminyl-peptide cyclotransferase, disintegrin, C-type lectin (snaclec), nerve growth factor, Kunitz type serine protease inhibitor, snake venom vascular endothelial growth factor, cysteine-rich secretory protein, bradykinin potentiating peptide, natriuretic peptides. PLA2 and LAAO from V. b. berus venom produce more pronounced cytotoxic effects in cancer cells than normal cells, via induction of apoptosis, cell cycle arrest and suppression of proliferation. Proteomic data of V. b. berus venoms from different parts of Russia and Slovakian Republic have been compared with analogous data for Vipera nikolskii venom. Proteomic studies demonstrated quantitative differences in the composition of V. b. berus venom from different geographical regions. Differences in the venom composition of V. berus were mainly driven by the age, sex, habitat and diet of the snakes. The venom variability of V. berus results in a loss of antivenom efficacy against snakebites. The effectiveness of antibodies is discussed. This review presents an overview with a special focus on different toxins that have been isolated and characterized from the venoms of V. b. berus. Their main biochemical properties and toxic actions are described.

Capture and identification of bacteria from fish muscle based on immunomagnetic beads and MALDI-TOF MS.

In the present study, E. coli was taken as a model bacterium, anti-E. coli functionalized magnetic beads were constructed and used to capture E. coli from aqueous extracts of fish sarcoplasmic protein (FSP) and fish muscle protein of sablefish. The excellency of the reproducibility of the present protocol was demonstrated by capturing E. coli from sablefish FSP extracts. The presence of 10 CFU/mL E. coli is still detectable. A microbial safety test on the surface of fish muscle was successfully performed. The bacterial identification accuracy from samples with different matrices was found to be excellent with RSD = 3%. High specific detection of target bacteria in complex biological samples was testified by spiking Staphylococcus aureus and Klebsiella pneumoniae in samples as interference. Ten biomarker ions were discovered for E. coli's recognition. It is promising to apply the present protocol in bacterial analysis in muscle food samples to ensure their safety.

COVID-19-associated brain abscess caused by Trichosporon dohaense: A case report and review of literature.

We report the first case of Coronavirus Disease 2019 (COVID-19)-associated brain abscess caused by a rare Trichosporon species, T. dohaense. The patient was a known diabetic and had received systemic corticosteroids for the treatment of COVID-19. He underwent craniotomy and evacuation of abscess. The pus aspirate grew a basidiomycetous yeast, morphologically resembling Trichosporon species. The isolate was initially misidentified by VITEK® MS due to lack of mass spectral database of T. dohaense. Accurate identification was achieved by internal transcribed spacer-directed panfungal polymerase chain reaction. The patient had a favorable outcome following surgical intervention and antifungal therapy.

The first case of Janibacter hoylei bacteremia in an adult.

The Janibacter species are Gram positive, coryneform bacteria that belong to the Actinobacteria phylum and have been linked to bacteremia in immunocompromised children. We present the first documented adult case of Janibacter hoylei bacteremia. The patient was a 52-year-old woman with a history of recurrent Clostridioides difficile infection, sinus tachycardia and high-risk AML who had been admitted one month prior to presentation for matched unrelated donor hematopoietic stem cell transplant with reduced intensity fludarabine-melphalan. Thirty days post-transplant, the infectious disease team was consulted because blood cultures grew Janibacter hoylei, from one of two blood cultures It took nine days to identify the species. She was treated with linezolid and imipenem. Janibacter are rarely implicated in human pathology, and therein, usually identified in the context of malignancy and relative immunosuppression. J. hoylei was only previously reported from the bloodstream of a previously healthy 8-week-old infant without underlying medical conditions. Antimicrobial susceptibility testing is challenging as only in vitro susceptibility testing of Janibacter terrae has been reported. Given these challenges, it is our hope to illustrate the clinical approach to diagnosis as well as subsequent recommendations for treatment in a particularly challenging case of bacteremia in an AML patient.

MALDI-ToF protein profiling as a potential rapid diagnostic platform for COVID-19.

More than a year after the COVID-19 pandemic was declared, the need still exists for accurate, rapid, inexpensive and non-invasive diagnostic methods that yield high specificity and sensitivity towards the current and newly emerging SARS-CoV-2 strains. Compared to the nasopharyngeal swabs, several studies have established saliva as a more amenable specimen type for early detection of SARS-CoV-2. Considering the limitations and high demand for COVID-19 testing, we employed MALDI-ToF mass spectrometry in the analysis of 60 gargle samples from human donors and compared the resultant spectra against COVID-19 status. Several standards, including isolated human serum immunoglobulins, and controls, such as pre-COVID-19 saliva and heat inactivated SARS-CoV-2 virus, were simultaneously analyzed to provide a relative view of the saliva and viral proteome as they would appear in this workflow. Five potential biomarker peaks were established that demonstrated high concordance with COVID-19 positive individuals. Overall, the agreement of these results with RT-qPCR testing on nasopharyngeal swabs was ≥90% for the studied cohort, which consisted of young and largely asymptomatic student athletes. From a clinical standpoint, the results from this pilot study suggest that MALDI-ToF could be used to develop a relatively rapid and inexpensive COVID-19 assay.

Duration of carriage of multidrug-resistant bacteria in dogs and cats in veterinary care and co-carriage with their owners.

BACKGROUND: The emergence and spread of multidrug-resistant organisms (MDROs) represent a threat to human and animal health. OBJECTIVES: To assess duration of carriage of MDROs in dogs and cats presented to veterinary clinics/hospitals in Switzerland. To estimate prevalence, duration of and risk factors for MDRO carriage in their owners and the occurrence of co-carriage in owner-pet pairs. METHODS: Prospective, longitudinal, observational study. Nasal swabs and fecal samples were collected from 50 owners of dogs and cats presented to 3 large veterinary hospitals, 1 medium-sized clinic and 1 practice. If pet or owner tested positive for a MDRO, follow-up samples were collected for up to 8 months. Methicillin-resistant (MR) Staphylococcus aureus, MR S. pseudintermedius, MR coagulase-negative staphylococci (MRCoNS), MR Macrococcus spp., cephalosporinase- and carbapenemase-producing (CP) Enterobacterales were isolated and further characterized by MALDI-TOF MS, microdilution, ß-lactam resistance gene detection, REP/ERIC-PCR, multilocus sequence typing or whole-genome sequencing. Risk factors for MDRO carriage in owners were explored based on questionnaire-derived data. RESULTS: Five out of 50 owners carried 3rd generation cephalosporin-resistant Enterobacterales (3GC-R-Ent.), and 5/50 MRCoNS. In 3 dogs and 4 cats carriage of 3GC-R-Ent. persisted for up to 136 days after discharge (median 99 days, IQR 83 days, range 36-136 days), in two cats isolates were carbapenem-resistant. Owner-pet co-carriage was not observed. No specific risk factors for MDRO carriage in owners were identified. CONCLUSIONS: After discharge from veterinary care, dogs and cats may carry 3GC-R-Ent. for prolonged time periods. Carriage of MDROs was common in owners, but pet-owner co-carriage of the same MDRO was not observed.

Endogenous modulators of neurotrophin signaling: Landscape of the transient ATP-NGF interactions.

The Nerve Growth Factor (NGF) neurotrophin acts in the maintenance and growth of neuronal populations. Despite the detailed knowledge of NGF's role in neuron physiology, the structural and mechanistic determinants of NGF bioactivity modulated by essential endogenous ligands are still lacking. We present the results of an integrated structural and advanced computational approach to characterize the extracellular ATP-NGF interaction. We mapped by NMR the interacting surface and ATP orientation on NGF and revealed the functional role of this interaction in the binding to TrkA and p75NTR receptors by SPR. The role of divalent ions was explored in conjunction with ATP. Our results pinpoint ATP as a likely transient molecular modulator of NGF signaling, in health and disease states.

N-glycome and N-glycoproteome of a hematophagous parasitic nematode Haemonchus.

N-glycosylation is a physiologically vital post-translational modification of proteins in eukaryotic organisms. Initial work on Haemonchus contortus - a blood-sucking nematode of ruminants with a broad geographical distribution - has shown that this parasite harbors N-glycans with exclusive chitobiose modifications. Besides, several immunogenic proteins (e.g., amino- and metallo-peptidases) are known to be N-glycosylated in adult worms. However, an informative atlas of N-glycosylation in H. contortus is not yet available. Herein, we report 291 N-glycosylated proteins with a total of 425 modification sites in the parasite. Among them, many peptidase families (e.g., peptidase C1 and M1) including potential vaccine targets were enriched. Notably, the glycan-rich conjugates are distributed primarily in the intestine and gonads of adult worms, and consequently hidden from the host's immune system. Collectively, these data provide a comprehensive atlas of N-glycosylation in a prevalent parasitic nematode while underlining its significance for infection, immunity and prevention.

A case of pulmonary Mycobacterium heckeshornense infection in a healthy Japanese man.

A 72-year-old man, healthy, smoker, with long-standing cough, was referred to our hospital and his chest X-ray (CXR) revealed a cavity lesion in the right upper lobe. Direct sputum smears, but not culture in solid medium, were positive for acid-fast bacilli (AFB) without tuberculosis DNA. The preliminary diagnosis was of a non-tuberculosis infection that progressed slowly, and the CXR showed the condition to worsen daily. Four years later, a commercialized mycobacteria growth indicator tube system was used to culture the colonies of AFB successfully in liquid medium, and the species Mycobacterium heckeshornense was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The patient responded well to triple therapy with rifampicin, ethambutol, and clarithromycin, the sputum cultures remained negative and the roentgenogram showed minor improvement over the following 6 months.

Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE.

Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab')2, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications.

In vitro interactions of histones and α-crystallin.

The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone-α-crystallin binding with a Kd of 4 × 10-7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone-α-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone-α-crystallin mixtures showed a decrease in the oligomeric molecular weight of α-crystallin, indicating that histones alter the oligomerization of α-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of α-crystallin, indicating that the interaction between α-crystallin and histones in the lens is functionally important.

Protective effect of aqueous extract of Phyllanthus fraternus against bromobenzene induced changes on cytosolic glutathione S-transferase isozymes in rat liver.

The aim of this study was to investigate beneficial effect of aqueous extract of Phyllanthus fraternus (AEPF) on bromobenzene (BB) induced changes on cytosolic glutathione S-transferase (GST) isozymes in rat liver. Administration of BB significantly decreased the activity of GST, however, prior administration of AEPF prevented the BB induced decrease in GST activity. Further the cytosolic GSTs were purified from 3 groups of animals (control, BB and AEPF+BB administered) and resolved into three protein bands on SDS-PAGE. Densitometric analysis showed a significant decrease in BB group compared to control. Further, 2D PAGE analysis resolved these proteins into 8 bands which were identified as five isozymes of alpha, two of Mu and one of theta by MALDI-TOF MS and also observed decreased levels of isozymes in BB group. However, on prior administration of AEPF significantly prevented the BB induced decrease in GSTs and restored to normal levels.

Novel angiotensin-converting enzyme inhibitory peptides from caseins and whey proteins of goat milk.

Angiotensin-converting enzyme (ACE) plays a central role in blood pressure regulation by producing the vasoconstrictor angiotensin II. The inhibition of ACE with natural inhibitors, as alternatives to avoid the side effect of synthetic drugs, is a major target in the prevention of hypertension. In this study, we examined the separated caseins and whey proteins of goat milk for the presence of ACE inhibitory peptides. Digestion of isolated whey proteins and caseins of goat milk by gastric pepsin generated soluble hydrolysates exhibiting significant inhibition of ACE compared to weak inhibition by undigested proteins. The hydrolysates were fractionated by size exclusion chromatography, Sephacryl S-100 column, into four fractions (F1-F4). The late-eluting fraction (F4) of either whey or caseins exhibited greater ACE inhibition. Peptides in both F4 fractions, isolated by RP-HPLC, exhibited variable ACE inhibitory activities with the hydrophobic peptide peaks being the most potent ACE inhibitors. MALDI-TOF MS/MS resulted in identification of three potent ACE inhibitory peptides: PEQSLACQCL from ß-lactoglobulin (residues 113-122), QSLVYPFTGPI from ß-casein (residues 56-66), and ARHPHPHLSFM from κ-casein (residues 96-106). The peptides from whey and caseins exert significant ACE inhibitory activities comparable to that of captopril, an antihypertensive drug, exhibiting IC50 values of 4.45 µM and 4.27 µM, respectively. The results introduce, for the first time, new potent ACE-inhibitory peptides that can be released by gastric pepsin of goat milk whey and caseins and thus may pave the way for their candidacy as anti-hypertensive bioactive peptides and prevention of associated disorders.

2D DIGE proteomic analysis highlights delayed postnatal repression of α-fetoprotein expression in homocystinuria model mice.

Cystathionine ß-synthase-deficient (Cbs (-/-)) mice, an animal model for homocystinuria, exhibit hepatic steatosis and juvenile semilethality via as yet unknown mechanisms. The plasma protein profile of Cbs (-/-) mice was investigated by proteomic analysis using two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. We found hyperaccumulation of α-fetoprotein (AFP) and downregulation of most other plasma proteins. AFP was highly expressed in fetal liver, but its expression declined dramatically via transcriptional repression after birth in both wild-type and Cbs (-/-) mice. However, the repression was delayed in Cbs (-/-) mice, causing high postnatal AFP levels, which may relate to transcriptional repression of most plasma proteins originating from liver and the observed hepatic dysfunction.

FLJ25439, a novel cytokinesis-associated protein, induces tetraploidization and maintains chromosomal stability via enhancing expression of endoplasmic reticulum stress chaperones.

Investigation of the mechanisms leading to aneuploidy and polyploidy is critical to cancer research. Previous studies have provided strong evidence of the importance of tetraploidization as an early step in tumorigenesis. In cancer cells, tetraploid cells may contribute to abnormal mitotic progression, which may be associated with cytokinesis failure. Tetraploidy leads to genomic instability due to centrosome and chromosome over-replication. Until now, the mechanism by which cells maintain tetraploid status has been unknown. Here, we identified a novel D box-containing protein, FLJ25439, which displays a dynamic expression profile during mitosis/cytokinesis with the midbody as the most prominent associated structure. To understand the function of FLJ25439, we established stable cell lines overexpressing FLJ25439. FLJ25439-overexpression cells grew slower and displayed a tetraploid DNA content in comparison with diploid parental cells. They also showed aberrant mitosis and dysregulated expression of p53, pRb and p21, suggesting a defect in cell cycle progression. To explore the molecular mechanisms responsible for FLJ25439-induced tetraploidization, we conducted a comparative analysis of the global protein expression patterns of wild type and overexpressors using proteomics and bioinformatics approaches. Protein category profiling indicated that FLJ25439 is involved in pathways related to anti-apoptosis, protein folding, the cell cycle, and cytoskeleton regulation. Specifically, genotoxic-stress- and ER stress-related chaperone proteins greatly contributed to the FLJ25439 overexpression phenotypes. The results of this study pave the way to our further understanding of the role of this novel cytokinesis-related protein in protecting cells from environmental stress and tetraploid formation.