Cloning, Expression, Purification, and Characterization of Lactate Dehydrogenase from Plasmodium knowlesi: A Zoonotic Malaria Parasite.

Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by Plasmodium spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of P. knowlesi LDH (PkLDH) using a bacterial expression system for studying malaria caused by P. knowlesi. Recombinant pET-21a(+)-PkLDH plasmid was constructed by inserting the PkLDH gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Escherichia coli Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by P. knowlesi.

Photobiomodulation as a Potential Therapy for Erectile Function: A Preclinical Study in a Cavernous Nerve Injury Model.

PURPOSE: To identify the optimal photobiomodulation (PBM) parameters using molecular, histological, and erectile function analysis in cavernous nerve injury. MATERIALS AND METHODS: A cavernous nerve injury was induced in 8-week-old C57BL/6J male mice that were subsequently divided randomly into age-matched control groups. Erectile function tests, penile histology, and Western blotting were performed 2 weeks after surgery and PBM treatment. RESULTS: The PBM treatment was administered for five consecutive days with a light-emitted diode (LED) device that delivers 660 nm±3% RED light, and near infra-red 830 nm±2% promptly administered following nerve-crushing surgery and achieved a notable restoration of erectile function approximately 90% of the control values. Subsequent in-vitro and ex-vivo analyses revealed the regeneration of neurovascular connections in both the dorsal root ganglion and major pelvic ganglion, characterized by the sprouting of neurites. Furthermore, the expression levels of neurotrophic, survival, and angiogenic factors exhibited a substantial increase across all groups subjected to PBM treatment. CONCLUSIONS: The utilization of PBM employing LED with 660 nm, 830 nm, and combination of both these wavelengths, exhibited significant efficacy to restore erectile function in a murine model of cavernous nerve injury. Thus, the PBM emerges as a potent therapeutic modality with notable advantages such as efficacy, noninvasiveness, and non-pharmacological interventions for erectile dysfunction caused by nerve injury.

Characterization of Escherichia coli Strains for Novel Production of Plasmodium ovale Lactate Dehydrogenase.

Malaria is one of the most prevalent diseases worldwide with high incidence and mortality. Among the five species that can infect humans, Plasmodium ovale morphologically resembles Plasmodium vivax, resulting in misidentification and confusion in diagnosis, and is responsible for malarial disease relapse due to the formation of hypnozoites. P. ovale receives relatively less attention compared to other major parasites, such as P. falciparum and P. vivax, primarily due to its lower pathogenicity, mortality rates, and prevalence rates. To efficiently produce lactate dehydrogenase (LDH), a major target for diagnosing malaria, this study used three Escherichia coli strains, BL21(DE3), BL21(DE3)pLysS, and Rosetta(DE3), commonly used for recombinant protein production. These strains were characterized to select the optimal strain for P. ovale LDH (PoLDH) production. Gene cloning for recombinant PoLDH production and transformation of the three strains for protein expression were performed. The optimal PoLDH overexpression and washing buffer conditions in nickel-based affinity chromatography were established to ensure high-purity PoLDH. The yields of PoLDH expressed by the three strains were as follows: BL21(DE3), 7.6 mg/L; BL21(DE3)pLysS, 7.4 mg/L; and Rosetta(DE3), 9.5 mg/L. These findings are expected to be highly useful for PoLDH-specific diagnosis and development of antimalarial therapeutics.

BMP2 restores erectile dysfunction through neurovascular regeneration and fibrosis reduction in diabetic mice.

BACKGROUND: The odds of erectile dysfunction are three times more prevalent in diabetes. Severe peripheral vascular and neural damage in diabetic patients responds poorly to phosphodiesterase-5 (PDE5) inhibitors. However, bone morphogenetic protein 2 is known to be involved in angiogenesis. OBJECTIVES: To assess the efficacy of bone morphogenetic protein 2 in stimulating angiogenesis and augmenting nerve regeneration in a mouse model of diabetic-induced erectile dysfunction. MATERIALS AND METHODS: The induction of diabetes mellitus was performed by streptozotocin (50 mg/kg daily) administered intraperitoneally for 5 successive days to male C57BL/6 mice that were 8 weeks old. Eight weeks post-inductions, animals were allocated to one of five groups: a control group, a streptozotocin-induced diabetic mouse group receiving two intracavernous 20 µL phosphate-buffered saline injections, or one of three bone morphogenetic protein 2 groups administered two injections of bone morphogenetic protein 2 protein (1, 5, or 10 µg) diluted in 20 µL of phosphate-buffered saline within a 3-day interval between the first and second injections. The erectile functions were assessed 2 weeks after phosphate-buffered saline or bone morphogenetic protein 2 protein injections by recording the intracavernous pressure through cavernous nerve electrical stimulation. Angiogenic activities and nerve regenerating effects of bone morphogenetic protein 2 were determined in penile tissues, aorta, vena cava, the main pelvic ganglions, the dorsal roots, and from the primary cultured mouse cavernous endothelial cells. Moreover, fibrosis-related factor protein expressions were evaluated by western blotting. RESULTS: Erectile function recovery to 81% of the control value in diabetic mice was found with intracavernous bone morphogenetic protein 2 injection (5 µg/20 µL). Pericytes and endothelial cells were extensively restored. It was confirmed that angiogenesis was promoted in the corpus cavernosum of diabetic mice treated with bone morphogenetic protein 2 through increased ex vivo sprouting of aortic rings, vena cava and penile tissues, and migration and tube formation of mouse cavernous endothelial cells. Bone morphogenetic protein 2 protein enhanced cell proliferation and reduced apoptosis in mouse cavernous endothelial cells and penile tissues, and promoted neurite outgrowth in major pelvic ganglia and dorsal root ganglia under high-glucose conditions. Furthermore, bone morphogenetic protein 2 suppressed fibrosis by reducing mouse cavernous endothelial cell fibronectin, collagen 1, and collagen 4 levels under high-glucose conditions. CONCLUSION: Bone morphogenetic protein 2 modulates neurovascular regeneration and inhibits fibrosis to revive the mouse erection function in diabetic conditions. Our findings propose that the bone morphogenetic protein 2 protein represents a novel and promising approach to treating diabetes-related erectile dysfunction.

Virulence Traits and Azole Resistance in Korean Candida auris Isolates.

We analyzed the virulence traits and azole resistance mechanisms of 104 Candida auris isolates collected from 13 Korean hospitals from 1996 to 2022. Of these 104 isolates, 96 (5 blood and 91 ear isolates) belonged to clade II, and 8 (6 blood and 2 other isolates) belonged to clade I. Fluconazole resistance (minimum inhibitory concentration ≥32 mg/L) was observed in 68.8% of clade II and 25.0% of clade I isolates. All 104 isolates were susceptible to amphotericin B and three echinocandins. In 2022, six clade I isolates indicated the first nosocomial C. auris cluster in Korea. Clade II C. auris isolates exhibited reduced thermotolerance at 42 °C, with diminished in vitro competitive growth and lower virulence in the Galleria mellonella model compared to non-clade II isolates. Of the 66 fluconazole-resistant clade II isolates, several amino acid substitutions were identified: Erg11p in 14 (21.2%), Tac1Ap in 2 (3.0%), Tac1Bp in 62 (93.9%), and Tac1Bp F214S in 33 (50.0%). Although there were a limited number of non-clade II isolates studied, our results suggest that clade II C. auris isolates from Korean hospitals might display lower virulence traits than non-clade II isolates, and their primary fluconazole resistance mechanism is linked to Tac1Bp mutations.

Antibody production and characterization of the nucleoprotein of sever fever with thrombocytopenia syndrome virus (SFTSV) for effective diagnosis of SFTSV.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by the Dabie bandavirus, [or SFTS virus (SFTSV)] that has become increasingly widespread since it was first reported in 2009. The SFTSV comprises three essential single-stranded RNA gene segments, with the S segment encoding the nucleocapsid (N) protein. Since the N protein is the most abundant and stable viral protein, it is a useful diagnostic marker of infection. Various SFTSV N-protein-based detection methods have been developed. However, given the limited research on antibodies of an SFTSV N-protein, here we report the characterization of the antibodies against SFTSV N protein especially their mapping results which is essential for more efficient and optimized detection of SFTSV. METHODS: To generate SFTSV-N-protein-specific monoclonal antibodies, recombinant full-length SFTSV N protein was expressed in E. coli, and the purified N protein was immunized to mice. The binding epitope positions of the antibodies generated were identified through binding-domain mapping. An antibody pair test using a lateral flow immunoassay (LFIA) was performed to identify effective diagnostic combinations of paired antibodies. RESULTS: Nine monoclonal antibodies specific for the SFTSV N protein were generated. Antibodies #3(B4E2) and #5(B4D9) were specific for sequential epitopes, while the remainder were specific for conformational epitopes. Antibody #4(C2G1) showed the highest affinity for the SFTSV N protein. The binding domain mapping results indicated the binding regions of the antibodies were divided into three groups. The antibody pair test demonstrated that #3(B4E2)/#4(C2G1) and #4(C2G1)/#5(B4D9) were effective antibody pairs for SFTSV diagnosis. CONCLUSIONS: Effective virus detection requires at least two strong antibodies recognizing separate epitope binding sites of the virus antigen. Here, we generated SFTSV-N-protein-specific monoclonal antibodies and subsequently performed epitope mapping and an antibody pair test to enhance the diagnostic efficiency and accuracy of SFTSV. Confirmation of epitope mappings and their combination immune response to the N protein provide valuable information for effective detection of SFTSV as well as can respond actively to detect a variant SFTSV.

PATHWAYS ASSOCIATED WITH POSITIVE SEPSIS SURVIVAL OUTCOMES IN AFRICAN AMERICAN/BLACK AND NON-HISPANIC WHITE PATIENTS WITH URINARY TRACT INFECTION.

ABSTRACT: Urinary tract infections (UTIs) are a common cause of sepsis worldwide. Annually, more than 60,000 US deaths can be attributed to sepsis secondary to UTIs, and African American/Black adults have higher incidence and case-fatality rates than non-Hispanic White adults. Molecular-level factors that may help partially explain differences in sepsis survival outcomes between African American/Black and Non-Hispanic White adults are not clear. In this study, patient samples (N = 166) from the Protocolized Care for Early Septic Shock cohort were analyzed using discovery-based plasma proteomics. Patients had sepsis secondary to UTIs and were stratified according to self-identified racial background and sepsis survival outcomes. Proteomics results suggest patient heterogeneity across mechanisms driving survival from sepsis secondary to UTIs. Differentially expressed proteins (n = 122, false discovery rate-adjusted P < 0.05) in Non-Hispanic White sepsis survivors were primarily in immune system pathways, while differentially expressed proteins (n = 47, false discovery rate-adjusted P < 0.05) in African American/Black patients were mostly in metabolic pathways. However, in all patients, regardless of racial background, there were 16 differentially expressed proteins in sepsis survivors involved in translation initiation and shutdown pathways. These pathways are potential targets for prognostic intervention. Overall, this study provides information about molecular factors that may help explain disparities in sepsis survival outcomes among African American/Black and Non-Hispanic White patients with primary UTIs.

Molecular and Clinical Features of Fluconazole Non-susceptible Candida albicans Bloodstream Isolates Recovered in Korean Multicenter Surveillance Studies.

Acquired fluconazole resistance (FR) in bloodstream infection (BSI) isolates of Candida albicans is rare. We investigated the FR mechanisms and clinical features of 14 fluconazole non-susceptible (FNS; FR and fluconazole-susceptible dose-dependent) BSI isolates of C. albicans recovered from Korean multicenter surveillance studies during 2006-2021. Mutations causing amino acid substitutions (AASs) in the drug-target gene ERG11 and the FR-associated transcription factor genes TAC1, MRR1, and UPC2 of the 14 FNS isolates were compared with those of 12 fluconazole-susceptible isolates. Of the 14 FNS isolates, eight and seven had Erg11p (K143R, F145L, or G464S) and Tac1p (T225A, R673L, A736T, or A736V) AASs, respectively, which were previously described in FR isolates. Novel Erg11p, Tac1p, and Mrr1p AASs were observed in two, four, and one FNS isolates, respectively. Combined Erg11p and Tac1p AASs were observed in seven FNS isolates. None of the FR-associated Upc2p AASs were detected. Of the 14 patients, only one had previous azole exposure, and the 30-day mortality rate was 57.1% (8/14). Our data show that Erg11p and Tac1p AASs are likely to contribute to FR in C. albicans BSI isolates in Korea and that most FNS C. albicans BSIs develop without azole exposure.

Whole-Genome Sequence Analysis of Candida glabrata Isolates from a Patient with Persistent Fungemia and Determination of the Molecular Mechanisms of Multidrug Resistance.

Whole-genome sequencing (WGS) was used to determine the molecular mechanisms of multidrug resistance for 10 serial Candida glabrata bloodstream isolates obtained from a neutropenic patient during 82 days of amphotericin B (AMB) or echinocandin therapy. For WGS, a library was prepared and sequenced using a Nextera DNA Flex Kit (Illumina) and the MiseqDx (Illumina) instrument. All isolates harbored the same Msh2p substitution, V239L, associated with multilocus sequence type 7 and a Pdr1p substitution, L825P, that caused azole resistance. Of six isolates with increased AMB MICs (≥2 mg/L), three harboring the Erg6p A158fs mutation had AMB MICs ≥ 8 mg/L, and three harboring the Erg6p R314K, Erg3p G236D, or Erg3p F226fs mutation had AMB MICs of 2-3 mg/L. Four isolates harboring the Erg6p A158fs or R314K mutation had fluconazole MICs of 4-8 mg/L while the remaining six had fluconazole MICs ≥ 256 mg/L. Two isolates with micafungin MICs > 8 mg/L harbored Fks2p (I661_L662insF) and Fks1p (C499fs) mutations, while six isolates with micafungin MICs of 0.25-2 mg/L harbored an Fks2p K1357E substitution. Using WGS, we detected novel mechanisms of AMB and echinocandin resistance; we explored mechanisms that may explain the complex relationship between AMB and azole resistance.

Establishing Quality Control Procedures for Large-Scale Plasma Proteomics Analyses.

Proteomics research has been transformed due to high-throughput liquid chromatography (LC-MS/MS) tandem mass spectrometry instruments combined with highly sophisticated automated sample preparation and multiplexing workflows. However, scaling proteomics experiments to large sample cohorts (hundreds to thousands) requires thoughtful quality control (QC) protocols. Robust QC protocols can help with reproducibility, quantitative accuracy, and provide opportunities for more decisive troubleshooting. Our laboratory conducted a plasma proteomics study of a cohort of N = 335 patient samples using tandem mass tag (TMTpro) 16-plex batches. Over the course of a 10-month data acquisition period for this cohort we collected 271 pooled QC LC-MS/MS result files obtained from MS/MS analysis of a patient-derived pooled plasma sample, representative of the entire cohort population. This sample was tagged with TMTzero or TMTpro reagents and used to inform the daily performance of the LC-MS/MS instruments and to allow within and across sample batch normalization. Analytical variability of a number of instrumental and data analysis metrics including protein and peptide identifications, peptide spectral matches (PSMs), number of obtained MS/MS spectra, average peptide abundance, percent of peptides with a Δ m/z between ±0.003 Da, percent of MS/MS spectra obtained at the maximum injection time, and the retention time of selected tracking peptides were evaluated to help inform the design of a robust LC-MS/MS QC workflow for use in future cohort studies. This study also led to general tips for using selected metrics to inform real-time troubleshooting of LC-MS/MS performance issues with daily QC checks.

Detection and Characterization of Two Phenotypes of Candida parapsilosis in South Korea: Clinical Features and Microbiological Findings.

We newly detected two (sinking and floating) phenotypes of Candida parapsilosis among bloodstream infection (BSI) isolates from Korean hospitals and assessed their microbiological and clinical characteristics. During the performance of a Clinical and Laboratory Standards Institute (CLSI) broth microdilution antifungal susceptibility testing, the sinking phenotype had a characteristic smaller button-like appearance because all yeast cells sank to the bottoms of the CLSI U-shaped round-bottom wells, whereas the floating phenotype comprised dispersed cells. Phenotypic analysis, antifungal susceptibility testing, ERG11 sequencing, microsatellite genotyping, and clinical analysis were performed on C. parapsilosis isolates from 197 patients with BSI at a university hospital during 2006 to 2018. The sinking phenotype was detected in 86.7% (65/75) of the fluconazole-nonsusceptible (FNS) isolates, 92.9% (65/70) of the isolates harboring the Y132F ERG11 gene substitution, and 49.7% (98/197) of all isolates. Clonality was more frequently observed for the Y132F-sinking isolates (84.6% [55/65]) than for all other isolates (26.5% [35/132]; P < 0.0001). Annual incidence of Y132F-sinking isolates increased 4.5-fold after 2014, and two dominant genotypes, persistently recovered for 6 and 10 years, accounted for 69.2% of all Y132F-sinking isolates. Azole breakthrough fungemia (odds ratio [OR], 6.540), admission to the intensive care unit (OR, 5.044), and urinary catheter placement (OR, 6.918) were independent risk factors for BSIs with Y132F-sinking isolates. The Y132F-sinking isolates exhibited fewer pseudohyphae, a higher chitin content, and lower virulence in the Galleria mellonella model than the floating isolates. These long-term results illustrate the increasing BSIs caused by clonal transmission of the Y132F-sinking isolates of C. parapsilosis. IMPORTANCE We believe that this is the first study describe the microbiological and molecular characteristics of bloodstream isolates of C. parapsilosis in Korea exhibiting two phenotypes (sinking and floating). An important aspect of our findings is that the sinking phenotype was observed predominantly in isolates harboring a Y132F substitution in the ERG11 gene (92.9%), fluconazole-nonsusceptible (FNS) isolates (86.7%), and clonal BSI isolates (74.4%) of C. parapsilosis. Although the increase in the prevalence of FNS C. parapsilosis isolates has been a major threat in developing countries, in which the vast majority of candidemia cases are treated with fluconazole, our long-term results show increasing numbers of BSIs caused by clonal transmission of Y132F-sinking isolates of C. parapsilosis in the period with an increased echinocandin use for candidemia treatment in Korea, which suggests that C. parapsilosis isolates with the sinking phenotype continue to be a nosocomial threat in the era of echinocandin therapy.

A combination of BR101801 and venetoclax enhances antitumor effect in DLBCL cells via c-Myc/Bcl-2/Mcl-1 triple targeting.

Double hit diffuse large B-cell lymphoma (DLBCL) with rearrangement and overexpression of both c-Myc and Bcl-2 responds poorly to standard R-CHOP therapy. In a recent phase I study, Venetoclax (ABT-199) targeting Bcl-2 also exhibited disappointing response rates in patients with relapsed/refractory DLBCL, suggesting that targeting only Bcl-2 is not sufficient for achieving successful efficacy due to the concurrent oncogenic function of c-Myc expression and drug resistance following an increase in Mcl-1. Therefore, co-targeting c-Myc and Mcl-1 could be a key combinatorial strategy to enhance the efficacy of Venetoclax. In this study, BR101801 a novel drug for DLBCL, effectively inhibited DLBCL cell growth/proliferation, induced cell cycle arrest, and markedly inhibited G0/G1 arrest. The apoptotic effect of BR101801 was also observed by increased Cytochrome C, cleaved PARP, and Annexin V-positive cell populations. This anti-cancer effect of BR101801 was confirmed in animal models, where it effectively inhibited tumor growth by reducing the expression of both c-Myc and Mcl-1. Furthermore, BR101801 exhibited a significant synergistic antitumor effect even in late xenograft models when combined with Venetoclax. Our data strongly suggest that c-Myc/Bcl-2/Mcl-1 triple targeting through a combination of BR101801 and Venetoclax could be a potential clinical option for double-hit DLBCL.

Whole blood transcript and protein abundance of the vascular endothelial growth factor family relate to cognitive performance.

The vascular endothelial growth factor (VEGF) family of genes has been implicated in the clinical development of Alzheimer's Disease (AD). A previous study identified associations between gene expression of VEGF family members in the prefrontal cortex and cognitive performance and AD pathology. This study explored if those associations were also observed in the blood. Consistent with previous observations in brain tissue, higher blood gene expression of placental growth factor (PGF) was associated with a faster rate of memory decline (p=0.04). Higher protein abundance of FMS-related receptor tyrosine kinase 4 (FLT4) in blood was associated with biomarker levels indicative of lower amyloid and tau pathology, opposite the direction observed in brain. Also, higher gene expression of VEGFB in blood was associated with better baseline memory (p=0.008). Notably, we observed that higher gene expression of VEGFB in blood was associated with lower expression of VEGFB in the brain (r=-0.19, p=0.02). Together, these results suggest that the VEGFB, FLT4, and PGF alterations in the AD brain may be detectable in the blood compartment.

Evolution of Fluconazole Resistance Mechanisms and Clonal Types of Candida parapsilosis Isolates from a Tertiary Care Hospital in South Korea.

We investigated the evolution of fluconazole resistance mechanisms and clonal types of Candida parapsilosis isolates from a tertiary care hospital in South Korea. A total of 45 clinical isolates, including 42 collected between 2017 and 2021 and 3 collected between 2012 and 2013, were subjected to antifungal susceptibility testing, sequencing of fluconazole resistance genes (ERG11, CDR1, TAC1, and MRR1), and microsatellite typing. Twenty-two isolates carried Y132F (n = 21; fluconazole MIC = 2 to >256 mg/L) or Y132F+R398I (n = 1; fluconazole MIC = 64 mg/L) in ERG11 and four isolates harbored N1132D in CDR1 (fluconazole MIC = 16 to 64 mg/L). All 21 Y132F isolates exhibited similar microsatellite profiles and formed a distinct group in the dendrogram. All four N1132D isolates displayed identical microsatellite profiles. Fluconazole MIC values of the Y132F isolates varied depending on their MRR1 mutation status (number of isolates, year of isolation, and MIC): K177N (n = 8, 2012 to 2020, 2 to 8 mg/L); K177N + heterozygous G982R (n = 1, 2017, 64 mg/L); K177N + heterozygous S614P (n = 2, 2019 to 2020, 16 mg/L); and K177N + homozygous S614P (n = 10, 2020 to 2021, 64 to > 256 mg/L). Our study revealed that Y132F in ERG11 and N1132D in CDR1 were the major mechanisms of fluconazole resistance in C. parapsilosis isolates. Furthermore, our results suggested that the clonal evolution of Y132F isolates persisting and spreading in hospital settings for several years occurred with the acquisition of heterozygous or homozygous MRR1 mutations associated with a gradual increase in fluconazole resistance.

Functional and Immunofluorescence Evaluations of Vascular and Neural Integrities in Urinary Bladder of Streptozotocin-Induced Diabetic Mice.

PURPOSE: To assess functional and structural changes in vascular and neural structures associated with diabetic bladder dysfunction (DBD) in the bladders of streptozotocin (STZ)-induced diabetic mice. METHODS: Eight-week-old C57BL/6 mice were injected with STZ at 50 mg/kg daily for 5 consecutive days. Catheters were inserted 12 weeks later, and 5 days after catheter placement bladder functions were assessed by conscious cystometry. Neurovascular and extracellular matrix marker changes in harvested urinary bladders were investigated by immunofluorescent staining. Body weights and fasting and postprandial blood glucose levels were measured 12 weeks after STZ injection. RESULTS: STZ-induced diabetic mice had significantly lower body weights and significantly higher blood glucose levels. Assessment of bladder function in STZ-induced diabetic mice revealed a nearly 3-fold increase in bladder capacity and intercontractile interval compared to controls. However, basal pressure, maximal bladder pressure, and threshold pressure were not significantly different. Morphological and structural analysis showed that STZ-induced diabetic mice had significantly reduced microvascular density in lamina propria (33% of the nondiabetic control values), and severely decreased nerve contents in the detrusor region (42% of the nondiabetic control values). CONCLUSION: STZ-induced diabetic mice exhibit functional and structural derangements in urinary bladder. The present study provides a foundation and describes a useful means of evaluating the efficacies of therapeutic targets and exploring the detailed mechanism of DBD.

Nationwide Surveillance of Antifungal Resistance of Candida Bloodstream Isolates in South Korean Hospitals: Two Year Report from Kor-GLASS.

We incorporated nationwide Candida antifungal surveillance into the Korea Global Antimicrobial Resistance Surveillance System (Kor-GLASS) for bacterial pathogens. We prospectively collected and analyzed complete non-duplicate blood isolates and information from nine sentinel hospitals during 2020−2021, based on GLASS early implementation protocol for the inclusion of Candida species. Candida species ranked fourth among 10,758 target blood pathogens and second among 4050 hospital-origin blood pathogens. Among 766 Candida blood isolates, 87.6% were of hospital origin, and 41.3% occurred in intensive care unit patients. Adults > 60 years of age accounted for 75.7% of cases. Based on species-specific clinical breakpoints, non-susceptibility to fluconazole, voriconazole, caspofungin, micafungin, and anidulafungin was found in 21.1% (154/729), 4.0% (24/596), 0.1% (1/741), 0.0% (0/741), and 0.1% (1/741) of the isolates, respectively. Fluconazole resistance was determined in 0% (0/348), 2.2% (3/135, 1 Erg11 mutant), 5.3% (7/133, 6 Pdr1 mutants), and 5.6% (6/108, 4 Erg11 and 1 Cdr1 mutants) of C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis isolates, respectively. An echinocandin-resistant C. glabrata isolate harbored an F659Y mutation in Fks2p. The inclusion of Candida species in the Kor-GLASS system generated well-curated surveillance data and may encourage global Candida surveillance efforts using a harmonized GLASS system.

Fungal Endophthalmitis Outbreak after Cataract Surgery, South Korea, 2020.

In November 2020, an unusual increase in fungal endophthalmitis cases after cataract surgery was reported to the Korea Disease Control and Prevention Agency, South Korea. We initiated an outbreak investigation to identify the cause. We identified 156 cases nationwide, 62 confirmed and 94 probable. Most case-patients were exposed during surgery to ocular viscoelastic devices (OVDs) from the same manufacturer (company A). We isolated Fusarium spp. from 50 confirmed cases. Molecular identification of 39 fungal isolates from clinical samples and 13 isolates from OVDs confirmed F. oxysporum caused the infections. The risk ratio for fungal endophthalmitis from company A's OVDs was 86.0 (95% CI 27.4-256.9), much higher than risk from other manufacturers' products. We determined this fungal endophthalmitis outbreak was caused by a contaminated lot of OVDs and recommended discontinued use of this product. Early recognition of outbreaks and joint responses from related government agencies can reduce risk for fungal endophthalmitis.

Advancements in automation for plasma proteomics sample preparation.

Automation is necessary to increase sample processing throughput for large-scale clinical analyses. Replacement of manual pipettes with robotic liquid handler systems is especially helpful in processing blood-based samples, such as plasma and serum. These samples are very heterogenous, and protein expression can vary greatly from sample-to-sample, even for healthy controls. Detection of true biological changes requires that variation from sample preparation steps and downstream analytical detection methods, such as mass spectrometry, remains low. In this mini-review, we discuss plasma proteomics protocols and the benefits of automation towards enabling detection of low abundant proteins and providing low sample error and increased sample throughput. This discussion includes considerations for automation of major sample depletion and/or enrichment strategies for plasma toward mass spectrometry detection.

Latrophilin-2 is a novel receptor of LRG1 that rescues vascular and neurological abnormalities and restores diabetic erectile function.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by inappropriate hyperglycemia, which causes endothelial dysfunction and peripheral neuropathy, ultimately leading to multiple complications. One prevalent complication is diabetic erectile dysfunction (ED), which is more severe and more resistant to treatment than nondiabetic ED. The serum glycoprotein leucine-rich ɑ-2-glycoprotein 1 (LRG1) is a modulator of TGF-ß-mediated angiogenesis and has been proposed as a biomarker for a variety of diseases, including DM. Here, we found that the adhesion GPCR latrophilin-2 (LPHN2) is a TGF-ß-independent receptor of LRG1. By interacting with LPHN2, LRG1 promotes both angiogenic and neurotrophic processes in mouse tissue explants under hyperglycemic conditions. Preclinical studies in a diabetic ED mouse model showed that LRG1 administration into the penile tissue, which exhibits significantly increased LPHN2 expression, fully restores erectile function by rescuing vascular and neurological abnormalities. Further investigations revealed that PI3K, AKT, and NF-κB p65 constitute the key intracellular signaling pathway of the LRG1/LPHN2 axis, providing important mechanistic insights into LRG1-mediated angiogenesis and nerve regeneration in DM. Our findings suggest that LRG1 can be a potential new therapeutic option for treating aberrant peripheral blood vessels and neuropathy associated with diabetic complications, such as diabetic ED.