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.

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.

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.

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.

BMP11 regulates thermogenesis in white and brown adipocytes.

Bone morphogenetic protein-11 (BMP11), also known as growth differentiation factor-11 (GDF11), is implicated in skeletal development and joint morphogenesis in mammals. However, its functions in adipogenesis and energy homeostasis are mostly unknown. The present study investigates crucial roles of BMP11 in cultured 3T3-L1 white and HIB1B brown adipocytes, using Bmp11 gene depletion and pharmacological inhibition of BMP11. The silencing of Bmp11 markedly decreases the expression levels of brown-fat signature proteins and beige-specific genes in white adipocytes and significantly down-regulates the expression levels of brown fat-specific genes in brown adipocytes. The deficiency of Bmp11 reduces the expressions of lipolytic protein markers in white and brown adipocytes. Moreover, BMP11 induces browning of 3T3-L1 adipocytes via coordination of multiple signalling pathways, including mTORC1-COX2 and p38MAPK-PGC-1α as non-canonical pathways, as well as Smad1/5/8 as a canonical pathway. We believe this study is the first to provide evidence of the potential roles of BMP11 for improvement of lipid catabolism in both cultured white and brown adipocytes, as well as the effect on browning of white adipocytes. Taken together, these results demonstrate the therapeutic potential for the treatment of obesity.

A novel three-dimensionally printed model to assess biofilm removal by ultrasonically activated irrigation.

AIM: To apply an innovative three-dimensionally printed tooth model to investigate the efficacy of three ultrasonically activated irrigation (UAI) systems in removing multispecies biofilms from dentine samples. METHODOLOGY: Three-dimensionally printed teeth with a curved root canal were fabricated with a standardized slot in the apical third of the root to achieve precision fit of human root dentine specimens. Multispecies biofilms including Enterococcus faecalis, Streptococcus mitis and Campylobacter rectus were developed in the root canal for 21 days. The canals were allocated to be irrigated with 1% sodium hypochlorite (NaOCl) using a syringe and needle or ultrasonically activated NaOCl with a stainless-steel file (Irrisafe), a conventional nickel-titanium (Ni-Ti) file (CK) or a blue heat-treated Ni-Ti file (Endosonic Blue). Infected root canals irrigated with distilled water served as controls. Bacterial reduction was determined by colony-forming unit (CFU) counting (n = 20), whilst biofilms were analysed using confocal laser scanning microscopy (n = 7) and field emission scanning electron microscopy. For CFU counting, the independent two-sample t-test (Welch's t-test) was examined to compare overall bacterial reduction amongst groups. For CLSM analysis, the data were analysed using one-way analysis of variance (ANOVA), followed by the Scheffé post hoc test. The p-values <.05 were considered to indicate statistical significance. RESULTS: All groups in which NaOCl was ultrasonically activated had significantly lower CFU values than the syringe-and-needle irrigation and control groups (p < .05). Ultrasonic activation with the stainless-steel file and blue heat-treated Ni-Ti file significantly reduced the biofilm volume compared with other groups (p < .05). Overall, UAI with the blue heat-treated file resulted in the highest antibacterial and biofilm removal efficacy. CONCLUSIONS: UAI with different inserts had differential antibiofilm effects. The blue heat-treated Ni-Ti ultrasonic insert resulted in the greatest antibacterial and biofilm removal from dentine in this standardized root canal model.

A Membrane Filter-Assisted Mammalian Cell-Based Biosensor Enabling 3D Culture and Pathogen Detection.

We have developed a membrane filter-assisted cell-based biosensing platform by using a polyester membrane as a three-dimensional (3D) cell culture scaffold in which cells can be grown by physical attachment. The membrane was simply treated with ethanol to increase surficial hydrophobicity, inducing the stable settlement of cells via gravity. The 3D membrane scaffold was able to provide a relatively longer cell incubation time (up to 16 days) as compared to a common two-dimensional (2D) cell culture environment. For a practical application, we fabricated a cylindrical cartridge to support the scaffold membranes stacked inside the cartridge, enabling not only the maintenance of a certain volume of culture media but also the simple exchange of media in a flow-through manner. The cartridge-type cell-based analytical system was exemplified for pathogen detection by measuring the quantities of toll-like receptor 1 (TLR1) induced by applying a lysate of P. aeruginosa and live E. coli, respectively, providing a fast, convenient colorimetric TLR1 immunoassay. The color images of membranes were digitized to obtain the response signals. We expect the method to further be applied as an alternative tool to animal testing in various research areas such as cosmetic toxicity and drug efficiency.

Evaluation of Two Commercial Broth Microdilution Methods Using Different Interpretive Criteria for the Detection of Molecular Mechanisms of Acquired Azole and Echinocandin Resistance in Four Common Candida Species.

The abilities of the new Vitek 2 AST-YS08 (YS08) and Sensititre YeastOne (SYO) systems to detect the resistances of Candida isolates to azoles and echinocandins were evaluated. In total, 292 isolates, including 28 Candida albicans (6 Erg11 and 2 Fks mutants), 57 Candida parapsilosis (26 Erg11 mutants), 24 Candida tropicalis (10 Erg11 and 1 Fks mutants), and 183 Candida glabrata (39 Pdr1 and 13 Fks mutants) isolates, were tested. The categorical agreements (CAs) between the Clinical and Laboratory Standards Institute (CLSI) method and YS08 fluconazole MICs obtained using clinical breakpoints were 92.4% (C. albicans), 96.5% (C. parapsilosis), and 87.0% (C. tropicalis), and the CAs between the CLSI and SYO MICs were 92.3% (C. albicans), 77.2% (C. parapsilosis), 100% (C. tropicalis), and 98.9% (C. glabrata). For C. glabrata, the CAs with the CLSI micafungin MICs were 92.4% and 55.5% for the YS08 micafungin and caspofungin MICs, respectively; they were 100%, 95.6%, and 98.9% for the SYO micafungin, caspofungin, and anidulafungin MICs, respectively. YS08 does not provide fluconazole data for C. glabrata; the CA with the CLSI fluconazole MIC was 97.8% for the YS08 voriconazole MIC, using an epidemiological cutoff value (ECV) of 0.5 µg/ml. Increased CAs with the CLSI MIC were observed for the YS08 MIC using CLSI ECVs (for fluconazole and C. tropicalis, 100%; for micafungin and C. glabrata, 98.9%) and for the SYO MIC using method-specific ECVs (for fluconazole and C. parapsilosis, 91.2%; for caspofungin and C. glabrata, 98.9%). Therefore, the YS08 and SYO systems may have different abilities to detect mechanisms of azole and echinocandin resistance in four Candida species; the use of method-specific ECVs may improve the performance of both systems.

Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury.

BACKGROUND: Extracellular vesicle (EV)-mimetic nanovesicles (NVs) from embryonic stem cells have been observed to stimulate neurovascular regeneration in the streptozotocin-induced diabetic mouse. Pericytes play important roles in maintaining penile erection, yet no previous studies have explored the effects of pericyte-derived NVs (PC-NVs) in neurovascular regeneration in the context of erectile dysfunction. AIM: To investigate the potential effect of PC-NVs in neurovascular regeneration. METHODS: PC-NVs were isolated from mouse cavernous pericytes, and neurovascular regeneration was evaluated in an in vitro study. Twelve-week-old C57BL/6J mice were used to prepare cavernous nerve injury model. Erectile function evaluation, histologic examination of the penis, and Western blots were assessed 2 weeks after model creation and PC-NVs treatment. OUTCOMES: The main outcomes of this study are PC-NVs characterization, intracavernous pressure, neurovascular regeneration in the penis, and in vitro functional evaluation. RESULTS: The PC-NVs were extracted and characterized by cryotransmission electron microscopy and EV-positive (Alix, TSG101, CD81) and EV-negative (GM130) markers. In the in vivo studies, PC-NVs successfully improved erectile function in cavernous nerve injury mice (∼82% of control values). Immunofluorescence staining showed significant increases in pericytes, endothelial cell, and neuronal contents. In the in vitro studies, PC-NVs significantly increased mouse cavernous endothelial cells tube formation, Schwann cell migration, and dorsal root ganglion and major pelvic ganglion neurite sprouting. Finally, Western blot analysis revealed that PC-NVs upregulated cell survival signaling (Akt and eNOS) and induced the expression of neurotrophic factors (brain-derived neurotrophic factor, neurotrophin-3, and nerve growth factor). CLINICAL IMPLICATIONS: PC-NVs may be used as a strategy to treat erectile dysfunction after radical prostatectomy or in men with neurovascular diseases. STRENGTHS & LIMITATIONS: We evaluated the effect of PC-NVs in vitro and in a mouse nerve injury model, cavernous nerve injury. Additional studies are necessary to determine the detailed mechanisms of neurovascular improvement. Further study is needed to test whether PC-NVs are also effective when given weeks or months after nerve injury. CONCLUSION: PC-NVs significantly improved erectile function by enhancing neurovascular regeneration. Local treatment with PC-NVs may represent a promising therapeutic strategy for the treatment of neurovascular diseases. Yin GN, Park S-H, Ock J, et al. Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury. J Sex Med 2020;17:2118-2128.

Theobromine alleviates diet-induced obesity in mice via phosphodiesterase-4 inhibition.

PURPOSE: Modern science has given much attention to the treatment of obesity by activating brown adipose tissue (BAT) and browning of white adipose tissue (WAT). Recent studies have identified theobromine, a derivative of cocoa, as a potent natural component actively browning white fat cells. Here, we aimed to deduce the anti-obesity effect of theobromine involving phosphodiesterase (PDE) dependent-regulatory pathway in obese animal models. METHODS: For examining activity of theobromine, C57BL/6 mice were fed with high fat diet and treated with theobromine to determine the expression levels of protein markers by immunoblot analysis and gene targets by quantitative real-time PCR. Other methods used include histopathological studies, immunofluorescence and molecular docking approaches. RESULTS: Theobromine alleviated diet-induced obesity in mice by browning of iWAT and activating BAT. Further, theobromine actively interacted with PDE4D and inhibited its activity in adipose tissues and cells potentiating energy expenditure. Moreover, the regulatory action of theobromine via inhibition of PDE4D was mediated by ß3-AR signaling pathway. CONCLUSION: Altogether, the current results signifies critical role of theobromine in reducing obesity by regulation of lipid metabolism through inhibition of PDE4, indicating its potential as a major therapeutic medicinal compound.

Microsatellite Typing and Resistance Mechanism Analysis of Voriconazole-Resistant Aspergillus flavus Isolates in South Korean Hospitals.

A recent surveillance study in South Korea revealed that 14% (7/50) of Aspergillus flavus clinical isolates had a voriconazole minimum inhibitory concentration of ≥4 µg/ml. Of seven non-wild-type (non-WT) isolates, six ear isolates from four hospitals shared the same microsatellite genotype. None of the non-WT isolates showed cyp51 mutations associated with azole resistance. However, the mean expression levels of efflux pump (MDR2, atrF, and mfs1) and target (cyp51A) genes exhibited significant differences between non-WT and other isolates.

Candida auris Clinical Isolates from South Korea: Identification, Antifungal Susceptibility, and Genotyping.

Candida auris is an emerging worldwide fungal pathogen. Over the past 20 years, 61 patient isolates of C. auris (4 blood and 57 ear) have been obtained from 13 hospitals in Korea. Here, we reanalyzed those molecularly identified isolates using two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, including Biotyper and Vitek MS, followed by antifungal susceptibility testing, sequencing of the ERG11 gene, and genotyping. With a research-use-only (RUO) library, 83.6% and 93.4% of the isolates were correctly identified by Biotyper and Vitek MS, respectively. Using an in vitro diagnostic (IVD) library of Vitek MS, 96.7% of the isolates were correctly identified. Fluconazole-resistant isolates made up 62.3% of the isolates, while echinocandin- or multidrug-resistant isolates were not found. Excellent essential (within two dilutions, 96.7%) and categorical agreements (93.4%) between the Clinical and Laboratory Standards Institute (CLSI) and Vitek 2 (AST-YS07 card) methods were observed for fluconazole. Sequencing ERG11 for all 61 isolates revealed that only 3 fluconazole-resistant isolates showed the Erg11p amino acid substitution K143R. All 61 isolates showed identical multilocus sequence typing (MLST). Pulsed-field gel electrophoresis (PFGE) analyses revealed that both blood and ear isolates had the same or similar patterns. These results show that MALDI-TOF MS and Vitek 2 antifungal susceptibility systems can be reliable diagnostic tools for testing C. auris isolates from Korean hospitals. The Erg11p mutation was seldom found among Korean isolates of C. auris, and multidrug resistance was not found. Both MLST and PFGE analyses suggest that these isolates are genetically similar.

Inhibition of proNGF and p75NTR Pathway Restores Erectile Function Through Dual Angiogenic and Neurotrophic Effects in the Diabetic Mouse.

INTRODUCTION: Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in diabetic patients, which causes poor response to oral phosphodiesterase-5 inhibitors. Nerve growth factor precursor (proNGF) and its p75 neurotrophin receptor (p75NTR) have been known to be involved in microvascular complications and neurodegeneration. AIM: To examine the role of proNGF and its receptor p75NTR signaling pathway in diabetic ED, and to determine the effectiveness of proNGF neutralizing antibody (proNGF-Ab) in restoring erectile function in streptozotocin (STZ)-induced diabetic mice. METHODS: Diabetes mellitus was induced by intraperitoneal injection of STZ (50 mg/kg) into 8-week-old C57BL/6 male mice for 5 consecutive days. At 8 weeks after the induction of diabetes mellitus, the animals were distributed into 3 groups: controls and STZ-induced diabetic mice receiving 2 intracavernous injections of either saline (days -3 and 0; 20 µL) or proNGF-Ab (days -3 and 0; 20 µg in 20 µL of saline). We also examined the effect of proNGF-Ab or p75NTR small interfering RNA in primary cultured mouse cavernous endothelial cells, pericytes, and major pelvic ganglion. MAIN OUTCOME MEASURES: Erectile function was measured by electrical stimulation of the cavernous nerve at 2 weeks after treatment, and the penis was then harvested for histologic and biochemical studies. RESULTS: The cavernous expression of proNGF and p75NTR was upregulated under diabetic conditions. Intracavernous injection of proNGF-Ab successfully restored erectile function in diabetic mice, which reach 93-96% of control values. ProNGF-Ab significantly restored cavernous endothelial cell, pericyte, and neuronal cell content, and increased endothelial cell-to-cell junction proteins in the diabetic mice. Under the high-glucose condition, proNGF-Ab or p75NTR small interfering RNA promoted tube formation in mouse cavernous endothelial cells and pericytes, decreased apoptosis of endothelial cells and pericytes, and enhanced neurite sprouting from major pelvic ganglion. CLINICAL IMPLICATIONS: The ProNGF/p75NTR pathway will be a new therapeutic target for diabetic ED. STRENGTH & LIMITATIONS: This is the first study demonstrating the efficacy of the inhibition of proNGF/p75NTR pathway in diabetic ED. Further studies are needed to test whether a different dosing of proNGF-Ab would induce more durable erectile function recovery. CONCLUSION: Our findings suggest that inhibition of the proNGF/p75NTR signaling pathway is a promising therapeutic strategy for diabetic ED. Nguyen NM, Song K-M, Choi M-J, et al. Inhibition of proNGF and p75NTR Pathway Restores Erectile Function Through Dual Angiogenic and Neurotrophic Effects in the Diabetic Mouse. J Sex Med 2019;16:351-364.

Fluconazole-Resistant Candida parapsilosis Bloodstream Isolates with Y132F Mutation in ERG11 Gene, South Korea.

We recently observed the emergence of fluconazole-resistant Candida parapsilosis bloodstream isolates harboring a Y132F substitution in Erg11p in South Korea. These Y132F isolates had a higher propensity to cause clonal transmission than other fluconazole-resistant isolates and persisted within hospitals for several years, as revealed by microsatellite typing.

Antifungal susceptibilities to amphotericin B, triazoles and echinocandins of 77 clinical isolates of cryptic Aspergillus species in multicenter surveillance in Korea.

We investigated the in vitro antifungal susceptibilities of cryptic Aspergillus species from nine Korean hospitals. Based on the CLSI epidemiological cutoff values, resistance rates to amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin were as follows: A. awamori (34 isolates; all 0%), A. tubingensis (22; 0%, 4.5%, 0%, 0%, and 0%, respectively), A. sydowii (16; 0%, 6.3%, 0%, 0%, and 6.3%), A. lentulus (2; 50%, 0%, 100%, 50%, and 0%), and A. tamarii (2; all 0%). A. calidoustus (one isolate) showed resistance to multiple drugs. Thus, cryptic species identification can be mandatory for clinically important Aspergillus isolates, with their susceptibility data.

Three zinc-finger RNA-binding proteins in cabbage (Brassica rapa) play diverse roles in seed germination and plant growth under normal and abiotic stress conditions.

Despite the increasing understanding of the stress-responsive roles of zinc-finger RNA-binding proteins (RZs) in several plant species, such as Arabidopsis thaliana, wheat (Triticum aestivum) and rice (Oryza sativa), the functions of RZs in cabbage (Brassica rapa) have not yet been elucidated. In this study, the functional roles of the three RZ family members present in the cabbage genome, designated as BrRZ1, BrRZ2 and BrRZ3, were investigated in transgenic Arabidopsis under normal and environmental stress conditions. Subcellular localization analysis revealed that all BrRZ proteins were exclusively localized in the nucleus. The expression levels of each BrRZ were markedly increased by cold, drought or salt stress and by abscisic acid (ABA) treatment. Expression of BrRZ3 in Arabidopsis retarded seed germination and stem growth and reduced seed yield of Arabidopsis plants under normal growth conditions. Germination of BrRZ2- or BrRZ3-expressing Arabidopsis seeds was delayed compared with that of wild-type seeds under dehydration or salt stress conditions and cold stress conditions, respectively. Seedling growth of BrRZ3-expressing transgenic Arabidopsis plants was significantly inhibited under salt, dehydration or cold stress conditions. Notably, seedling growth of all three BrRZ-expressing transgenic Arabidopsis plants was inhibited upon ABA treatment. Importantly, all BrRZs possessed RNA chaperone activity. Taken together, these results indicate that the three cabbage BrRZs harboring RNA chaperone activity play diverse roles in seed germination and seedling growth of plants under abiotic stress conditions as well as in the presence of ABA.

Inhibition of Ninjurin 1 restores erectile function through dual angiogenic and neurotrophic effects in the diabetic mouse.

Penile erection is a neurovascular phenomenon, and erectile dysfunction (ED) is caused mainly by vascular risk factors or diseases, neurologic abnormalities, and hormonal disturbances. Men with diabetic ED often have severe endothelial dysfunction and peripheral nerve damage, which result in poor response to oral phosphodiesterase-5 inhibitors. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. Here, we demonstrate in streptozotocin-induced diabetic mice that inhibition of the Ninj1 pathway by administering Ninj1-neutralizing antibody (Ninj1-Ab) or by using Ninj1-knockout mice successfully restored erectile function through enhanced penile angiogenesis and neural regeneration. Angiopoietin-1 (Ang1) expression was down-regulated and angiopoietin-2 expression was up-regulated in the diabetic penis compared with that in controls, and these changes were reversed by treatment with Ninj1-Ab. Ninj1 blockade-mediated penile angiogenesis and neural regeneration as well as recovery of erectile function were abolished by inhibition of Ang1-Tie2 (tyrosine kinase with Ig and epidermal growth factor homology domain-2) signaling with soluble Tie2 antibody or Ang1 siRNA. The present results suggest that inhibition of the Ninj1 pathway will be a novel therapeutic strategy for treating ED.

Resistance Mechanisms and Clinical Features of Fluconazole-Nonsusceptible Candida tropicalis Isolates Compared with Fluconazole-Less-Susceptible Isolates.

We investigated the azole resistance mechanisms and clinical features of fluconazole-nonsusceptible (FNS) isolates of Candida tropicalis recovered from Korean surveillance cultures in comparison with fluconazole-less-susceptible (FLS) isolates. Thirty-five clinical isolates of C. tropicalis, comprising 9 FNS (fluconazole MIC, 4 to 64 µg/ml), 12 FLS (MIC, 1 to 2 µg/ml), and 14 control (MIC, 0.125 to 0.5 µg/ml) isolates, were assessed. CDR1, MDR1, and ERG11 expression was quantified, and the ERG11 and UPC2 genes were sequenced. Clinical features of 16 patients with FNS or FLS bloodstream isolates were analyzed. Both FNS and FLS isolates had >10-fold higher mean expression levels of CDR1, MDR1, and ERG11 genes than control isolates (P values of <0.02 for all). When FNS and FLS isolates were compared, FNS isolates had 3.4-fold higher mean ERG11 expression levels than FLS isolates (P = 0.004), but there were no differences in those of CDR1 or MDR1 Of all 35 isolates, 4 (2 FNS and 2 FLS) and 28 (8 FNS, 11 FLS, and 9 control) isolates exhibited amino acid substitutions in Erg11p and Upc2p, respectively. Both FNS and FLS bloodstream isolates were associated with azole therapeutic failure (3/4 versus 4/7) or uncleared fungemia (4/6 versus 4/10), but FNS isolates were identified more frequently from patients with previous azole exposure (6/6 versus 3/10; P = 0.011) and immunosuppression (6/6 versus 3/10; P = 0.011). These results reveal that the majority of FNS C. tropicalis isolates show overexpression of CDR1, MDR1, and ERG11 genes, and fungemia develops after azole exposure in patients with immunosuppression.

Effectiveness of Intracavernous Delivery of Recombinant Human Hepatocyte Growth Factor on Erectile Function in the Streptozotocin-Induced Diabetic Mouse.

INTRODUCTION: Diabetic erectile dysfunction is a disease mostly of vascular origin and men with diabetic erectile dysfunction respond poorly to oral phosphodiesterase-5 inhibitors. Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an essential role in the regulation of cell proliferation, survival, and angiogenesis. AIM: To determine the effectiveness of recombinant human (rh)-HGF in restoring erectile function in diabetic mice. METHODS: Four groups of mice were used: control non-diabetic mice and streptozotocin-induced diabetic mice receiving two successive intracavernous injections of phosphate buffered saline (days -3 and 0), a single intracavernous injection of rh-HGF (day 0), or two successive intracavernous injections of rh-HGF (days -3 and 0). We also examined the effect of rh-HGF in primary cultured mouse cavernous endothelial cells and in major pelvic ganglion culture in vitro, which was incubated under a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition. MAIN OUTCOME MEASURES: Two weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve and the penis was harvested for histologic studies. RESULTS: Repeated intracavernous injections of rh-HGF protein induced significant restoration of erectile function in diabetic mice (89-100% of control values), whereas a single intracavernous injection of rh-HGF protein elicited modest improvement. Rh-HGF significantly induced phosphorylation of its receptor c-Met, increased the content of endothelial cells and smooth muscle cells, and decreased the generation of reactive oxygen species (superoxide anion and peroxynitrite) and extravasation of oxidized low-density lipoprotein in diabetic mice. Under the high-glucose condition, rh-HGF protein also promoted tube formation in mouse cavernous endothelial cells and enhanced neurite sprouting in major pelvic ganglion culture in vitro. CONCLUSION: The dual angiogenic and neurotrophic effects of HGF could open a new avenue through which diabetic erectile dysfunction can be treated.