Matrix-assisted Laser Desorption/ionization Time-of-flight Research Articles

Actinomyces funkei bacteraemia and infected pulmonary cavities in an intravenous drug user: a case report

BackgroundActinomyces spp. are most commonly found in human commensal flora; however, they have also been shown to cause suppurative infections. We present a case of a rare Actinomyces funkei bacteraemia from an infected deep vein thrombosis in a patient who went on to develop pulmonary cavities secondary to septic emboli. Infected thrombi and septic emboli have been associated with other Actinomyces spp. in the literature, often posing a diagnostic challenge and, in some cases, causing drastic clinical deterioration in patients. The literature regarding Actinomyces funkei is scarce and to our knowledge there are no reports of a relationship between this Actinomyces subspecies and infected thrombi or septic emboli.Case presentationThe patient was a 39-year-old known intravenous drug user who presented with a groin injecting site sinus and systemic symptoms. The bacteria was first observed by gram staining of a blood culture sample after 48 h of incubation and the species was identified using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) as Actinomyces funkei. Sputum cytology/histology with cell block revealed a branching gram-positive species suspicious of slow growing bacteria or fungus. CT imaging of his lower limb and chest revealed an extensive DVT with inflammatory changes and pulmonary cavities respectively. The patient was treated with Ceftriaxone before being discharged with a 6-month course of Linezolid. He made a good recovery with reduction in size of the cavitating lung lesions on follow-up imaging.ConclusionsThis case report presents a difficult-to-diagnose bacterial infection in an intravenous drug user, complicated by bacteraemia and secondary septic emboli. Relatively little is known about Actinomyces funkei, and therefore this report aims to increase clinician awareness of diagnosis, management, and complications.

Detecting M-Protein via Mass Spectrometry and Affinity Beads: Enrichment With Mixed Kappa-Lambda Beads Enables Prompt Application in Clinical Laboratories.

Detecting monoclonal protein (M-protein), a hallmark of plasma cell disorders, traditionally relies on methods such as protein electrophoresis, immune-electrophoresis, and immunofixation electrophoresis (IFE). Mass spectrometry (MS)-based methods, such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-quadrupole time-of-flight (ESI-qTOF) MS, have emerged as sensitive methods. We explored the M-protein-detection efficacies of different MS techniques. To isolate immunoglobulin and light chain proteins, six types of beads (IgG, IgA, IgM, kappa, lambda, and mixed kappa and lambda) were used to prepare samples along with CaptureSelect nanobody affinity beads (NBs). After purification, both MALDI-TOF MS and liquid chromatography coupled with Synapt G2 ESI-qTOF high-resolution MS analysis were performed. We purified 25 normal and 25 abnormal IFE samples using NBs and MALDI-TOF MS (NB-MALDI-TOF). Abnormal samples showed monoclonal peaks, whereas normal samples showed polyclonal peaks. The IgG and mixed kappa and lambda beads showed monoclonal peaks following the use of daratumumab (an IgG/kappa type of monoclonal antibody) with both MALDI-TOF and ESI-qTOF MS analysis. The limits of detection for MALDI-TOF MS and ESI-qTOF MS were established as 0.1 g/dL and 0.025 g/dL, respectively. NB-MALDI-TOF and IFE exhibited comparable sensitivity and specificity (92% and 92%, respectively). NBs for M-protein detection, particularly with mixed kappa-lambda beads, identified monoclonal peaks with both MALDI-TOF and ESI-qTOF analyses. Qualitative analysis using MALDI-TOF yielded results comparable with that of IFE. NB-MALDI-TOF might be used as an alternative method to replace conventional tests (such as IFE) to detect M-protein with high sensitivity.

Candida auris: Understanding the dynamics of C. auris infection versus colonization.

Candida auris is a pathogen of growing public health concern worldwide. However, risk factors contributing to C. auris infection in patients colonized with C. auris remain unclear. Understanding these risk factors is crucial to prevent colonization-to-infection transition and devise effective preventive strategies. This study aimed to investigate risk factors associated with C. auris infection compared to colonization. The study included 97 patients who acquired laboratory-confirmed C. auris in either matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry or VITEK 2 system from October 2019 to June 2023. Baseline demographics and known risk factors associated with C. auris infection were collected from electronic medical records. The infection group had C. auris from a sterile site or non-sterile site with evidence of infection. The colonization group was followed up for a median of 30 days for any signs of infection. Associations between relevant variables and C. auris infection were assessed using multivariable logistic regression. The infection group (n=31) was more likely to be bedbound, with longer hospital stays and more arterial catheters. Chronic kidney disease (odds ratio [OR] 45.070), carriage of multidrug-resistant organisms (OR 64.612), and vasopressor use for > 20 days (OR 68.994) were associated with C. auris infection, after adjusting for sex, age, and prior colonization with C. auris. Chronic kidney disease, carriage of multidrug-resistant organisms, and prolonged vasopressor use emerged as significant risk factors for C. auris infection compared to colonization. They could be used to predict C. auris infection early in patients colonized with C. auris.

Artificial Photosynthesis: Visible Light-Activated TiOCl<sub>2</sub>/2-Phenyl Indole Complexes for Atmospheric CO<sub>2</sub> and H<sub>2</sub>O Capture and Long-Chain Organic Products Generation

We present a novel self-organized chemical "living" system that mimics natural photosynthesis by capturing CO2 and H2O from the atmosphere at ambient conditions. This system autonomously reduces CO2 with H2O protons to produce long-chain aliphatic oxygenated products. Starting with 2-phenyl indole (PI) complexes with TiCl4, the hydrolyzed complexes react with CO2 to form organotitanium carbonates. The 2:1 PI/TiCl4 complex was found to be the most effective. Key findings include: Photocatalytic Reduction: Visible light reduces TiIV to TiIII and TiII, forming peroxotitanium complexes and releasing OH radicals. CO2 Reduction: CO2 is reduced to CO, H2CO, and CH3OH, which couple to form HOCH2CH2OH. Organic Compound Synthesis: The system generates oxygenated organic compounds with carbon chains from C2 to C16. Ligand Exchange: The PI ligand exchanges with donor molecules, forming adducts involved in the photocatalytic process. PI Oligomerization: Contributes to the formation of PI oligomers. These processes were monitored using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectra, proton and Carbon-13 nuclear magnetic resonance (13C NMR), and infrared (IR) spectra, identifying over two dozen intermediates and products. This system offers a prototype for new applications with potential improvements using other metals and ligands.

Bactofencin YH, a novel bacteriocin with high inhibitory activity against clinical Streptococcus species.

Strain Lactiplantibacillus plantarum D1 with bacteriocin producing ability was found in the intestine of Gambusia affinis. The bacteriocin was found to have high inhibitory activity against multiple Streptococcus species and several other Gram-positive and Gram-negative bacteria. Bacteriocin was purified from culture supernatant by ion-exchange chromatography, Sep-Pak C18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectral analysis determined that purified bacteriocin has a molecular mass of 2,731 Da. A partial N-terminal sequence KRKKHKXQIYNNGM was obtained from the Edman analysis. The N-terminal sequence was employed to search against a translation of the draft genome of strain D1. The translated full amino acid sequence of the mature peptide is as follows: NH2- KRKKHKCQIYNNGMPTGQYRWC, which has a molecular weight of 2738 Da. A BLAST search revealed that this bacteriocin was most similar to bactofencin A but differed from it with three amino acid residues. No identical peptide or protein has been previously reported, and this peptide, termed bactofencin YH, was therefore considered to be a new bacteriocin produced by Lactiplantibacillus plantarum D1.

Analysis of serum proteomic in cats with polycystic kidney disease-1 gene mutation

Feline autosomal dominant polycystic kidney disease (PKD) is common in Persian and Persian-cross cats. This study aims to investigate proteins that can be potential biomarkers for early disease diagnosis in cats with PKD1 heterozygous gene mutations and compare them with chronic kidney disease cats and normal wild-type cats. Thirty-three client-owned cats of variable breeds (ten PKD1 gene mutation cats, twelve wild-type cats with normal blood profiles, and eleven wild-type cats with chronic renal disease) were enrolled in this study. This study used serum-based proteomic profiling analysis in cats. Abdominal ultrasounds were examined in all cats. Proteomic analysis was conducted by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). One hundred and fifty-nine proteins were significantly differentially expressed between each group. The proteins identified in this study are known to regulate the apoptosis pathway. The apoptosis regulator Bcl-2 (BCL2) was overexpressed in the chronic kidney disease (CKD) group, while apoptotic peptidase activating factor 1 (APAF1) and BCL2-associated X apoptosis regulator (BAX) were only expressed in the PKD group. Ingenuity pathway analysis revealed that proteins uniquely expressed in the PKD group were linked to the Wnt signaling pathway and MAPK pathway. Asparagine synthetase domain-containing and secreted frizzled-related proteins were interesting proteins that should be studied further for the possibility of a candidate protein for disease detection. The proteomic profiles identified in this study could be used as potential novel biomarkers for the early detection of PKD in cats.

Proteome identification of common immunological proteins of two nematode parasites.

Although helminth parasites have different life cycles, their hosts share similar immune responses involving Th2 cell-type. Here, we extracted proteins from the larvae of Anisakis simplex complex and Trichinella spiralis to identify common and specific antigens (or allergens) associated with the Th2 immune response. We performed two-dimensional electrophoresis analysis and Matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) experiments. We found 13 potentially immunogenic proteins, which included 5 spots specific to T. spiralis and 8 common to T. spiralis and A. simplex, by tandem mass spectrometry. These molecules were identified structurally as actin, tropomyosin, col cuticle N domain-containing protein, and heat shock proteins. We also identified molecules related to parasite-host immune modulation and interactions. Our results may contribute to reveal potential roles of immunological proteins in parasite-derived immune modulation.

Starch phosphorylation—A needle in a haystack

Phosphoesterification is the only naturally occurring covalent starch modification identified to date, and it has a major impact on overall starch metabolism. The incorporation of phosphate groups mediated by dikinases [α-glucan, water dikinase (GWD), EC 2.7.9.4; phosphoglucan, water dikinase (PWD), EC 2.7.9.5] massively alters the starch granule properties; however, previous studies did not determine whether the starch-related dikinases bind the phosphate to the glucosyl units within the amylopectin molecules in a specific pattern or randomly. In order to answer this challenging question, a number of approaches were initially pursued until a protocol could be established that enabled a massive step forward in the in vitro analysis of phosphorylated glucan chains obtained from starch. For this purpose, phosphorylation by GWD was investigated, including the final state of phosphorylation i.e., the state of substrate saturation when GWD lacks further free hydroxyl groups at OH-C6 for the catalysis of monophosphate esters. Since the separated phosphorylated glucan chains were required for the analysis, isoamylase digestion was performed to cleave the α-1,6-glycosidic bonds and to allow for the removal of the huge number of existing neutral chains by means of anion exchange chromatography. Via Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) MS and MALDI-MS/MS, the phosphorylated α-glucan chains were analysed, and the position of the phosphate group within the chain in relation to the reducing end was determined. Here, we demonstrate a protocol that enables the analysis of phosphorylated oligosaccharides, even in small quantities.

About alcohol-initiated polymerization of glycolide and separate crystallization of cyclic and linear polyglycolides

Alcohol-initiated polymerizations of glycolide (GL) catalyzed by tin(II) 2-ethylhexanoate (SnOct2) were carried out in bulk with variation of GA/In ratio, temperature and time. Due to a rather strong competition of cyclization polyglycolide (PGA) free of cycles were never obtained. When the cyclic catalysts 2,2-dibutal-2-stanna −1,3-dithiolane (DSTL) or 2-stanna 1,3-dioxo-4,5,6,7 bibenzepane (SnBiph) were used in combination with 1,4-butanediol the influence of cyclization was even stronger. Furthermore, the degrees of polymerization were higher than the GA/alcohol ratio due to rapid polycondensation in the solid state. At 160 °C or below, the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra indicated separate crystallization of low molar mass cyclic and linear PGAs from the same reaction mixture (also observed for poly(l-lactide)s).

Innovative approaches to microbial identification enhancing accuracy and speed in infectious disease diagnostics

The rapid and accurate identification of microbial pathogens is crucial for effective management of infectious diseases. Traditional methods, though reliable, are often time-consuming. Recent advancements in molecular diagnostics, mass spectrometry, and next-generation sequencing (NGS) have revolutionized the field by enhancing both the speed and accuracy of microbial identification. Polymerase chain reaction (PCR) and its derivatives, such as real-time PCR, enable rapid amplification of genetic material, significantly reducing diagnostic times. Techniques like nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP) further extend these capabilities to resource-limited settings by operating at constant temperatures. Mass spectrometry, particularly matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), provides rapid and accurate microbial identification by analyzing protein profiles. This method has high throughput, is cost-effective, and offers the ability to detect antimicrobial resistance, significantly impacting clinical decision-making and patient management. NGS technologies offer comprehensive pathogen detection by sequencing entire microbial genomes. This approach is invaluable in diagnosing complex infections, tracking outbreaks, and identifying antibiotic resistance genes, though its high cost and complexity limit widespread use. The integration of these innovative technologies into clinical practice enhances the ability to diagnose and manage infectious diseases effectively. Molecular diagnostics, mass spectrometry, and NGS each bring unique strengths, from rapid and precise pathogen detection to comprehensive genetic analysis and resistance profiling. These advancements contribute to improved patient outcomes and more effective public health strategies. The continued development and implementation of these technologies are essential to address the evolving challenges in microbial diagnostics, paving the way for more precise and personalized approaches in infectious disease management.

Imaging Mass Spectrometry and Genome Mining Reveal Antimicrobial Peptides of Novel Pediococcus acidilactici CCFM18.

The mechanism of metabolites produced by lactic acid bacteria in mediating microbial interactions has been difficult to ascertain. This study comparatively evaluated the antimicrobial effect of the novel bacterium Pediococcus acidilactici CCFM18 and explored the global chemical view of its interactions with indicator bacteria. P. acidilactici CCFM18 had sufficiently strong antimicrobial activity to effectively inhibit the growth of the indicator bacteria and enhance their intracellular reactive oxygen species (ROS) level. The emerging technique of matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) imaging mass spectrometry indicated that P. acidilactici CCFM18 increased the production of pediocin PA-1 and the penocin A profile during its interaction with the indicator bacteria, thus differing from P. acidilactici CCFM28 (a commonly used laboratory strain). Strikingly, the production of coagulin A was triggered only by signaling molecules made by the competing strain L. thermophilus, suggesting an idiosyncratic response from P. acidilactici CCFM18. Bioinformatic mining of the P. acidilactici CCFM18 draft genome sequence revealed gene loci that code for the complex secondary metabolites analyzed via MSI. Taken together, these results illustrate that chemical interactions between P. acidilactici CCFM18 and indicator bacteria exhibit high complexity and specificity and can drive P. acidilactici CCFM18 to produce different secondary metabolites.

Investigation of coagulation and proteomics profiles in symptomatic feline hypertrophic cardiomyopathy and healthy control cats

BackgroundHypertrophic cardiomyopathy (HCM) is a crucial heart disease in cats. The clinical manifestations of HCM comprise pulmonary edema, dyspnea, syncope, arterial thromboembolism (ATE), and sudden cardiac death. D-dimer and prothrombin time (PT) are powerful biomarkers used to assess coagulation function. Dysregulation in these two biomarkers may be associated with HCM in cats. This study aims to assess D-dimer levels, PT, and proteomic profiling in healthy cats in comparison to cats with symptomatic HCM.ResultsTwenty-nine client-owned cats with HCM were enrolled, including 15 healthy control and 14 symptomatic HCM cats. The D-dimer concentration and PT were examined. Proteomic analysis was conducted by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In symptomatic cats, D-dimer levels were statistically significantly higher (mean ± SEM: 372.19 ng/ml ± 58.28) than in healthy cats (mean ± SEM: 208.54 ng/ml ± 10.92) with P-value of less than 0.01, while PT was statistically significantly lower in symptomatic cats (mean ± SEM: 9.8 s ± 0.15) compared to healthy cats (mean ± SEM: 11.08 s ± 0.23) with P-value of less than 0.0001. The proteomics analysis revealed upregulation of integrin subunit alpha M (ITGAM), elongin B (ELOB), and fibrillin 2 (FBN2) and downregulation of zinc finger protein 316 (ZNF316) and ectonucleoside triphosphate diphosphohydrolase 8 (ENTPD8) in symptomatic HCM cats. In addition, protein-drug interaction analysis identified the Ras signaling pathway and PI3K-Akt signaling pathway.ConclusionsCats with symptomatic HCM have higher D-dimer and lower PT than healthy cats. Proteomic profiles may be used as potential biomarkers for the detection and management of HCM in cats. The use of D-dimer as a biomarker for HCM detection and the use of proteomic profiling for a better understanding of disease mechanisms remain to be further studied in cats.

Resistance to Antifungals in Non-albicans Candida Species Isolates in the Southeast Region

Background: Antimicrobial resistance is a growing problem in Candida spp., leading to treatment challenges and increased morbidity and mortality. The World Health Organization (WHO) fungal priority pathogens list classifies C. glabrata, C. tropicalis, and C. parapsilosis as high priority and leading causes of candidemia with high fluconazole resistance. In the US, these organisms are the most frequently isolated non-albicans Candida species. In 2016, the Antibiotic Resistance Laboratory Network (ARLN) was created to monitor resistance threats, including in Candida spp. This study describes the proportion of resistance in C. glabrata, C. parapsilosis, and C. tropicalis isolates sent to the Southeast ARLN from 2017 to 2023. Methods: This study evaluated C. glabrata, C. parapsilosis, and C. tropicalis submitted to the Southeast ARLN from Alabama, Florida, Georgia, Louisiana, Mississippi, and Tennessee from February 2017- September 2023. Species identification was confirmed by Bruker Biotyper matrix assisted laser desorption-ionization time of flight (MALDI-TOF). Antifungal susceptibility testing (AFST) was performed using TREK frozen broth microdilution panels. Minimum inhibitory concentration values from the clinical instrument were used to determine susceptibility based on Clinical and Laboratory Standards Institute (CLSI) standard interpretations from the 2020 CLSI M60 guidelines. Data were extracted from the laboratory information management system. Analyses were conducted using SAS v9.4. Results: AFST testing was performed on 660 C. glabrata, 500 C. parapsilosis, and 233 C. tropicalis isolates from within the Southeast region. The predominant specimen sources by species were blood 25.30% C. glabrata; other/not specified 27.80% C. parapsilosis; and lower respiratory 36.91% C. tropicalis. Resistance to fluconazole is as follows: C. glabrata, 12.88%; C. parapsilosis, 3.41%; C. tropicalis, 36.64%. Resistance to voriconazole is as follows: C. parapsilosis, 1.00%; C. tropicalis 30.04%. Resistance to at least one echinocandin (Anidulafungin, Capsofungin, Micafungin) is as follows: C. glabrata, 1.67%; C. parapsilosis, 0.60%; C. tropicalis, 0.43%. Overall, there was a decreasing trend in resistance to fluconazole, and voriconazole in all three species between 2017 and 2023. Conclusions: Antifungal resistance in non-albicans Candida species represents an emerging public health threat, however, within the Southeast region, ARLN data has shown a decreasing trend of azole resistance. This may be due in part to changes in reporting requirements and submission criteria from within the region. Nevertheless, C. tropicalis showed high resistance to azoles within the Southeast region. These Candida species should be monitored to inform clinical decision making and identify resistance patterns in other US regions due to their increase in resistance worldwide.

Utilizing Direct MALDI-TOF MS from Positive Blood Culture for Diagnosing Anaerobic Bacteremia

Anaerobic bacteraemia is a significant cause of morbidity and mortality among hospitalized patients worldwide. Early identification of these microorganisms from blood culture is pivotal in diagnosing and can improve treatment and outcomes. We present a case of anaerobic bacteraemia in a patient with diabetic foot ulcer, which was detected rapidly by the Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) from a positive blood culture bottle in a laboratory setting with minimal facilities for anaerobic culture methods.

Sulfatide imaging identifies tumor cells in colorectal cancer peritoneal metastases

Even with systemic chemotherapy, cytoreductive surgery (CRS), and hyperthermic intraperitoneal chemotherapy (HIPEC), peritoneal metastases (PM) remain a common site of disease progression for colorectal cancer (CRC) and are frequently associated with a poor prognosis. The mass spectrometry (MS) method known as Matrix-Assisted Laser Desorption/Ionization – Time of Flight (MALDI-TOF) is frequently used in medicine to identify structural compounds and biomarkers. It has been demonstrated that lipids are crucial in mediating the aggressive growth of tumors. In order to investigate the lipid profiles, particularly with regard to histological distribution, we used MALDI-TOF MS (MALDI-MS) and MALDI-TOF imaging MS (MALDI-IMS) on patient-derived tumor organoids (PDOs) and PM clinical samples. According to the MALDI-IMS research shown here, the predominant lipid signature of PDOs in PM tissues, glycosphingolipid (GSL) sulfates or sulfatides, or STs, is unique to the areas containing tumor cells and absent from the surrounding stromal compartments. Bioactive lipids are derived from arachidonic acid (AA), and AA-containing phosphatidylinositol (PI), or PI (18:0–20:4), is shown to be highly expressed in the stromal components. On the other hand, the tumor components contained a higher abundance of PI species with shorter and more saturated acyl chains (C34 and C36 carbons). The cellular subversion of PI and ST species may alter in ways that promote the growth, aggressiveness, and metastasis of tumor cells. Together, these findings suggest that the GSL/ST metabolic programming of PM may contain novel therapeutic targets to impede or halt PM progression.

Physicochemical and Biological Properties of Vancomycin-Containing Antibacterial Polysaccharide Gels for Biocomposite Bone Implant Impregnation.

Polymeric drugs containing up to 60% by weight of the antibiotic vancomycin were synthesized based on dextran carriers activated with epichlorohydrin. Vancomycin was covalently bound, involving the primary amino group of the molecule through the hydroxypropyl radical to the C6 position of the anhydroglucose units of the dextran main chain. Covalent binding is necessary to prevent spontaneous release of the antibiotic from the gel, thereby reducing the risk of bacterial multiresistance. Antibacterial depot gels were obtained from those polymers, containing up to 17.5% by weight of polysaccharide with a cross-linking density of q = 3-5 nodes per macromolecule for the deposition of another type of drugs not covalently bound to the polymer gel. They were used to coat the surface of the internal pores of biocomposite bone implants based on bovine cancellous bone used in orthopedics. The chemical structure of the polymer was studied using 13C NMR spectroscopy and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The stiffness of the gels was evaluated by the values of the accumulation modulus G' = 170-270 kPa and the loss modulus G″ = 3.7-4.2 kPa determined on a rheometer. Their values are close to those typical for materials used to replace soft tissue in plastic surgery. The minimum inhibitory concentration of the gels against Staphylococcus aureus P209 depends on the antibiotic content in the polymer. It equals 2.5 mg/L for vancomycin we used and 100 mg/L for a polymer containing 50% by weight of covalently bound antibiotic. The cytotoxic concentration measured with cell culture HEK 293T exceeds 1200 mg/L in 24 h exposure. The release dynamics of drugs not covalently bound to dextran from the depot gel were studied using fluorescein as a model. The release time is independent of the gel density and lasts up to 6 days for a 2 mm thick layer. Both the gel and the bone implants impregnated with it maintained consistently high antibacterial activity throughout the experiment, up to its completion after 168 h, with the local concentration of the released antibiotic at the site of bacterial attack exceeding the therapeutic level by 200 times.

Janus and Amphiphilic MoS2 2D Sheets for Surface-Directed Orientational Assemblies toward Ex Vivo Dual Substrate Release.

The two-dimensional (2-D) Janus and amphiphilic molybdenum disulfide (MoS2) nanosheet with opposite optical activities on each side (amphichiral) is synthesized by modifying sandwich-like bulk MoS2 with tannic acid and cholesterol through biphasic emulsion method. This new type of amphichiral Janus MoS2 nanosheet consists of a hydrophilic and positive optical activity tannic acid side as well as a hydrophobic and negative optical activity cholesterol side thereby characterized by circular dichroism. Surface-directed orientational differentiation assemblies are performed for the as-synthesized 2D material and are characterized by contact angle, infrared spectroscopy, X-ray photoelectron, and circular dichroism spectroscopies. The amphiphilic nature of the materials is demonstrated by the pre-organization of the nanosheets on either hydrophobic or hydrophilic surfaces, providing unprecedented properties of circular dichroism signal enhancement and wettability. Selective detachment of the surface organic groups (cholesterol and tannic acid fragments) is realized by matrix-assisted laser desorption/ionisation - time-of-flight(MALDI-TOF) mass spectrometry, and the dual substrate release in tissue is detected by ex vivo mass spectrometry imaging.

Diagnosis of Septic Body Cavity Effusion in Dogs and Cats: Cytology vs. Bacterial Culture

The elective test for the determination of the effusions etiopathogenesis is represented by physico-chemical analysis and cytology. Nevertheless, the bacterial culture and antibiotic sensitivity tests are crucial for setting therapy and for the outcome. This study compared cytology with microbiology in the etiologic diagnosis of exudative body cavity effusions in dogs and cats collected from October 2018 to October 2022. All samples underwent aerobic and anaerobic culture and cytology examination. Bacterial identifications were confirmed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, whereas cytological samples were blindly evaluated either in May Grunwald-Giemsa (MGG) or Gram-stained samples by two board-certified clinical pathologists. A moderate agreement (κ = 0.454) between cytology and bacterial culture was revealed. The sensitivity of the cytological evaluation in our study ranged from 38.5% to 67.9%, and the specificity ranged from 88.9% to 100%, depending on the type of the effusion, so cytology may not be representative of the etiopathogenesis, whereas bacterial culture can misidentify or fail to isolate the correct pathogen for difficult in vitro growing due to the presence of inhibitory substances or contamination. Cytology and bacterial culture results for exudative body cavity effusions in dogs and cats can be misleading if conducted separately, so these two tests should be performed together to increase diagnostic accuracy.

Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects. Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken. Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP. A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation. KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

A Comprehensive Review of Innovative Paradigms in Microbial Detection and Antimicrobial Resistance: Beyond Traditional Cultural Methods.

Microbial detection and antimicrobial resistance (AMR) surveillance are critical components of public health efforts to combat infectious diseases and preserve the efficacy of antimicrobial agents. While foundational in microbial identification, traditional cultural methods are often laborious, time-consuming, and limited in their ability to detect AMR markers. In response to these challenges, innovative paradigms have emerged, leveraging advances in molecular biology, genomics, proteomics, nanotechnology, and bioinformatics. This comprehensive review provides an overview of innovative approaches beyond traditional cultural methods for microbial detection and AMR surveillance. Molecular-based techniques such as polymerase chain reaction (PCR) and next-generation sequencing (NGS) offer enhanced sensitivity and specificity, enabling the rapid identification of microbial pathogens and AMR determinants. Mass spectrometry-based methods provide rapid and accurate detection of microbial biomarkers, including matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and biosensor technologies. Nanotechnology approaches, such as nanoparticle-based assays and nanopore sequencing, offer novel platforms for sensitive and label-free detection of pathogens and AMR markers. Embracing these innovative paradigms holds immense promise for improving disease diagnosis, antibiotic stewardship, and AMR containment efforts. However, challenges such as cost, standardization, and integration with existing healthcare systems must be addressed to realize the full potential of these technologies. By fostering interdisciplinary collaboration and innovation, we can strengthen our ability to detect, monitor, and combat AMR, safeguarding public health for generations.