Analysis of virulence profiles in clinical isolates of Klebsiella pneumoniae from renal abscesses: clinical significance of hypervirulent isolates.

Introduction: Klebsiella pneumoniae can cause a wide range of infections. Hypervirulent K. pneumoniae (hvKp), particularly associated with the K1 and K2 capsular types, is an increasingly significant microorganism with the potential to cause invasive infections, including renal abscesses. Despite the rising prevalence of hvKp infections, information on renal abscesses caused by K. pneumoniae is limited, and the clinical significance of hvKp associated with specific virulence genes remains elusive. Methods: This study performed at a 1200-bed tertiary hospital sought to identify the clinical and microbiological characteristics of renal abscesses caused by K. pneumoniae, focusing on various virulence genes, including capsular serotypes and multilocus sequence typing (MLST). Results: Over an 8-year period, 64 patients with suspected renal abscesses were reviewed. Ten patients diagnosed with K. pneumoniae-related renal abscesses were ultimately enrolled in the study. Among the isolates from the 10 patients, capsular serotype K2 was predominant (40.0%), followed by K1 (30.0%). The most common sequence type by MLST was 23 (40.0%). In particular, six patients (60.0%) harbored specific genes indicative of hvKp: iucA, peg-344, rmpA, and rmpA2. Conclusions: Our findings highlight the importance of hvKp as a pathogen in renal abscesses. Although the nature of hvKp is relatively unknown, it is widely recognized as a highly virulent pathogen that can infect relatively healthy individuals of various ages and simultaneously cause infections at multiple anatomical sites. Therefore, when treating patients with K. pneumoniae-related renal abscesses, caution is necessary when considering the characteristics of hvKp, such as potential bacteremia, multi-organ abscess formation, and metastatic spread.

Development of an automated amplicon-based next-generation sequencing pipeline for rapid detection of bacteria and fungi directly from clinical specimens.

The timely identification of microbial pathogens is essential to guide targeted antimicrobial therapy and ultimately, successful treatment of an infection. However, the yield of standard microbiology testing (SMT) is directly related to the duration of antecedent antimicrobial therapy as SMT culture methods are dependent on the recovery of viable organisms, the fastidious nature of certain pathogens, and other pre-analytic factors. In the last decade, metagenomic next-generation sequencing (mNGS) has been successfully utilized as a diagnostic tool for various applications within the clinical laboratory. However, mNGS is resource, time, and labor-intensive-requiring extensive laborious preliminary benchwork, followed by complex bioinformatic analysis. We aimed to address these shortcomings by developing a largely Automated targeted Metagenomic next-generation sequencing (tmNGS) PipeLine for rapId inFectIous disEase Diagnosis (AMPLIFIED) to detect bacteria and fungi directly from clinical specimens. Therefore, AMPLIFIED may serve as an adjunctive approach to complement SMT. This tmNGS pipeline requires less than 1 hour of hands-on time before sequencing and less than 2 hours of total processing time, including bioinformatic analysis. We performed tmNGS on 50 clinical specimens with concomitant cultures to assess feasibility and performance in the hospital laboratory. Of the 50 specimens, 34 (68%) were from true clinical infections. Specimens from cases of true infection were more often tmNGS positive compared to those from the non-infected group (82.4% vs 43.8%, respectively, P = 0.0087). Overall, the clinical sensitivity of AMPLIFIED was 54.6% with 85.7% specificity, equating to 70.6% and 75% negative and positive predictive values, respectively. AMPLIFIED represents a rapid supplementary approach to SMT; the typical time from specimen receipt to identification of potential pathogens by AMPLIFIED is roughly 24 hours which is markedly faster than the days, weeks, and months required to recover bacterial, fungal, and mycobacterial pathogens by culture, respectively. IMPORTANCE: To our knowledge, this represents the first application of an automated sequencing and bioinformatics pipeline in an exclusively pediatric population. Next-generation sequencing is time-consuming, labor-intensive, and requires experienced personnel; perhaps contributing to hesitancy among clinical laboratories to adopt such a test. Here, we report a strong case for use by removing these barriers through near-total automation of our sequencing pipeline.

Smallholders' vulnerability in the maize market: An analysis of marketing channels to improve the role of cooperatives in Benin.

Smallholder households in developing countries often face challenges related to market access. Despite being proposed as a potential solution, cooperatives in the north of Benin have reportedly failed to effectively address this issue. The alternatives channels used by smallholder famers and the factors that influence their choices remain unclear. This paper adopts a mixed approach, combining qualitative analysis with quantitative methods, particularly the multivariate probit model, to investigate the distribution channels of maize used by farmers in the Kandi district of Benin. The study aims to identify the current distribution channels of maize in Kandi and analyze factors that affect, smallholder producers choice of marketing channel. Initially, the study identified four primary channels for marketing maize-namely, collectors, wholesalers, brokers, and cooperatives-and producers typically commonly engage in multiple channels simultaneously. Subsequently, It becomes evident that factors such as age, cooperative membership, distance to market, reliance on informal credit, average maize bag prices, and the timing of sales significantly influence producers' selection of marketing channels. Interestingly, despite cooperatives offering comparatively higher prices, the majority of farmers opt for collectors due to the informal credit they provide and their practice of purchasing large volumes of maize bags at pre-agreed prices. In conclusion, enhancing joint-selling capabilities of cooperatives and establishing a credit provision business tailored to maize producers are identified as crucial steps. These measures aim to alleviate producers' financial strain, diminish their dependence on collectors for credit, and bolster their bargaining power in the market.

A taxonomic study of four rare pteromalid genera: Amblyharma Huang & Tong, Fusta Xiao & Ye, Nazgulia Hedqvist and Platecrizotes Ferrière from the Eastern Palaearctic (Chalcidoidea, Pteromalidae, Pachyneurinae).

The four morphologically similar genera Amblyharma Huang & Tong, 1993, Fusta Xiao & Ye, 2015, Nazgulia Hedqvist, 1973 and Platecrizotes Ferrière, 1934 from the Eastern Palaearctic are reviewed. Redescriptions of genera and all available types of Eastern Palaearctic species are provided. An identification key to genera is given. A new species from South Korea, Platecrizotesjediisp. nov. is described and illustrated.

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.

Doxorubicin covalently conjugated heparin displays anti-cancer activity as a self-assembled nanoparticle with a low-anticoagulant effect.

Heparin is a glycosaminoglycans (GAGs) member and well-known FDA-approved anticoagulant that has been widely used in the clinic for 100 years. It has also been evaluated in various fields for further clinical applications, such as in anti-cancer or anti-inflammatory therapy beyond its anticoagulant effect. Here, we sought to utilize heparin molecules as drug carriers by directly conjugating the anticancer drug doxorubicin to the carboxyl group of unfractionated heparin. Given the molecular action of doxorubicin in intercalating DNA, it is expected to be less effective when structurally combined with other molecules. However, by utilizing doxorubicin molecules to produce reactive oxygen species (ROS), we found that the heparin-doxorubicin conjugates have significant cytotoxic ability to kill CT26 tumor cells with low anticoagulant activity. Several doxorubicin molecules were bound to heparin to provide sufficient cytotoxic capability and self-assembly ability due to their amphiphilic properties. The self-assembled formation of these nanoparticles was demonstrated through DLS, SEM and TEM. The cytotoxic ROS-generating doxorubicin-conjugated heparins could inhibit tumor growth and metastasis in CT26-bearing Balb/c animal models. Our results demonstrate that this cytotoxic doxorubicin-based heparin conjugate can significantly inhibit tumor growth and metastasis, thus showing promise as a potential new anti-cancer therapeutic.

Review of the Palaearctic species of Miscogasteriella Girault, 1915 (Chalcidoidea, Pteromalidae).

Palaearctic species of the genus Miscogasteriella Girault, 1915 are reviewed. Miscogasteriellaolgaesp. nov. from South Korea and M.vladimirisp. nov. from Japan are described. Type material of M.nigricans (Masi) and M.sulcata (Kamijo) is redescribed and illustrated. Miscogasteriellanigricans is recorded from the Palaearctic region for the first time. An identification key to females of all Palaearctic species of Miscogasteriella is given.

Accuracy Validation of the Elecsys HBsAg II Quant Assay and Its Utility in Resolving Equivocal Qualitative HBsAg Results.

Background and Objectives: There are reports of false qualitative HBsAg results, because of various causes, such as samples with low HBsAg concentrations that may produce false positives. The main aims of this study were to validate the analytical accuracy and to assess the utility of the Elecsys assay compared to that of the qualitative HbsAg assay as a screening test in resolving equivocal qualitative HbsAg results. Materials and Methods: The limit of blank (LoB), the limit of detection (LoD), the limit of quantification (LoQ), and linearity were estimated to validate the analytical accuracy of the Elecsys HBsAg II Quant assay. A total of 449 serum samples showing initial equivocal results (1-50 index) were evaluated by Elecsys HBsAg II Quant and ADVIA Centaur HBsAg II assays. Results: The LoQ of the assay was determined to be 0.050 IU/mL, as provided by the manufacturer. The Kappa agreement between the two assays was almost perfect, at 0.9669, despite seven discordant results. With a specificity of 100% at new cut-off index value ≥5.42, about 78 samples (17%, 78/449) with index value ≥5.42 were interpreted as positives without further duplicate tests, however the remaining 371 samples with index value <5.42 need to be confirmed with additional HBV marker assays. Conclusions: We confirm that the Elecsys HBsAg II Quant assay is accurate and sensitive for HBV infection and recommend it as an alternative confirmatory HBsAg assay for resolving equivocal qualitative HBsAg results.

Clinical Evaluation of BioFire COVID-19 Test, BioFire Respiratory Panel 2.1, and Cepheid Xpert Xpress SARS-CoV-2 Assays for Sample-to-Answer Detection of SARS-CoV-2.

Background: Due to the extreme infectivity of SARS-CoV-2, sample-to-answer SARS-CoV-2 reverse transcription (RT) polymerase chain reaction (PCR) assays are urgently needed in order to facilitate infectious disease surveillance and control. The purpose of this study was to evaluate three sample-to-answer SARS-CoV-2 RT-PCR assays­BioFire COVID-19 Test, BioFire RP 2.1, and Cepheid Xpert Xpress SARS-CoV-2­using clinical samples. Methods: A total of 77 leftover nasopharyngeal swab (NP) swabs (36 positives and 41 negatives) confirmed by reference SARS-CoV-2 RT real-time (q) PCR assay were collected. The clinical sample concordance, as specified by their respective emergency use authorizations (EUAs), in comparison to the reference SARS-CoV-2 RT-qPCR assay, was assessed. Results: The results showed that all three sample-to-answer SARS-CoV-2 RT-PCR assays provided perfectly concordant results consistent with the reference SARS-CoV-2 RT-qPCR assay. The BioFire COVID-19 Test exhibited the best turnaround time (TAT) compared to the other assays, regardless of the test results, using one-way analysis of variance followed by Scheffe's post hoc test (p < 0.001). The Xpert Xpress SARS-CoV-2 showed a shorter average TAT (mean ± standard deviation, 49.9 ± 3.1 min) in the positive samples compared to that (55.7 ± 2.5 min) of the negative samples. Conclusions: Our evaluation demonstrates that the BioFire COVID-19 Test, BioFire RP 2.1, and Cepheid Xpert Xpress SARS-CoV-2 assays compare favorably to the reference SARS-CoV-2 RT-qPCR assay, along with a 100% concordance in assay results for clinical samples and an acceptable analytical performance at their guaranteed limits of detection. The addition of a widely used simultaneous sample-to-answer SARS-CoV-2 RT-PCR assay will contribute to the number of medical laboratories able to test for COVID-19.

FPGA Implementation of the Chirp-Scaling Algorithm for Real-Time Synthetic Aperture Radar Imaging.

Synthetic aperture radar (SAR), which can generate images of regions or objects, is an important research area of radar. The chirp scaling algorithm (CSA) is a representative SAR imaging algorithm. The CSA has a simple structure comprising phase compensation and fast Fourier transform (FFT) operations by replacing interpolation for range cell migration correction (RCMC) with phase compensation. However, real-time processing still requires many computations and a long execution time. Therefore, it is necessary to develop a hardware accelerator to improve the speed of algorithm processing. In addition, the demand for a small SAR system that can be mounted on a small aircraft or drone and that satisfies the constraints of area and power consumption is increasing. In this study, we proposed a CSA-based SAR processor that supports FFT and phase compensation operations and presents field-programmable gate array (FPGA)-based implementation results. We also proposed a modified CSA flow that simplifies the traditional CSA flow by changing the order in which the transpose operation occurs. Therefore, the proposed CSA-based SAR processor was designed to be suitable for modified CSA flow. We designed the multiplier for FFT to be shared for phase compensation, thereby achieving area efficiency and simplifying the data flow. The proposed CSA-based SAR processor was implemented on a Xilinx UltraScale+ MPSoC FPGA device and designed using Verilog-HDL. After comparing the execution times of the proposed SAR processor and the ARM cortex-A53 microprocessor, we observed a 136.2-fold increase in speed for the 4096 × 4096-pixel image.

The current state of laboratory mycology in Asia/Pacific: A survey from the European Confederation of Medical Mycology (ECMM) and International Society for Human and Animal Mycology (ISHAM).

INTRODUCTION: Invasive fungal infections (IFIs) in Asia/Pacific are a particular threat to patients with malignancies, uncontrolled diabetes mellitus or undiagnosed/untreated human immunodeficiency virus infection and acquired immunodeficiency syndrome (HIV/AIDS). Adequate and early access to diagnostic tools and antifungals is essential for IFI clinical management and patient survival. METHODS: Details on institution profile, self-perception on IFI, and access to microscopy, culture, serology, antigen detection, molecular testing, and therapeutic drug monitoring for IFI were collected in a survey. RESULTS: As of June 2022, 235 centres from 40 countries/territories in Asia/Pacific answered the questionnaire. More than half the centres were from six countries: India (25%), China (17%), Thailand (5%), Indonesia, Iran, and Japan (4% each). Candida spp. (93%) and Aspergillus spp. (75%) were considered the most relevant pathogens. Most institutions had access to microscopy (98%) or culture-based approaches (97%). Furthermore, 79% of centres had access to antigen detection, 66% to molecular assays, and 63% to antibody tests. Access to antifungals varied between countries/territories. At least one triazole was available in 93% of the reporting sites (voriconazole [89%] was the most common mould-active azole), whereas 80% had at least one amphotericin B formulation, and 72% had at least one echinocandin. CONCLUSION: According to the replies provided, the resources available for IFI diagnosis and management vary among Asia/Pacific countries/territories. Economical or geographical factors may play a key role in the incidence and clinical handling of this disease burden. Regional cooperation may be a good strategy to overcome shortcomings.

Guidelines for the Laboratory Diagnosis of Monkeypox in Korea.

While the coronavirus disease 2019 pandemic is ongoing, monkeypox has been rapidly spreading in non-endemic countries since May 2022. Accurate and rapid laboratory tests are essential for identifying and controlling monkeypox. Korean Society for Laboratory Medicine and the Korea Disease Prevention and Control Agency have proposed guidelines for diagnosing monkeypox in clinical laboratories in Korea. These guidelines cover the type of tests, selection of specimens, collection of specimens, diagnostic methods, interpretation of test results, and biosafety. Molecular tests are recommended as confirmatory tests. Skin lesion specimens are recommended for testing in the symptomatic stage, and the collection of both blood and oropharyngeal swabs is recommended in the presymptomatic or prodromal stage.

Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae Causing Invasive Pneumococcal Disease in Korea Between 2017 and 2019 After Introduction of the 13-Valent Pneumococcal Conjugate Vaccine.

Background: Streptococcus pneumoniae is a serious pathogen causing various infections in humans. We evaluated the serotype distribution and antimicrobial resistance of S. pneumoniae causing invasive pneumococcal disease (IPD) after introduction of pneumococcal conjugate vaccine (PCV)13 in Korea and investigated the epidemiological characteristics of multidrug-resistant (MDR) isolates. Methods: S. pneumoniae isolates causing IPD were collected from 16 hospitals in Korea between 2017 and 2019. Serotyping was performed using modified sequential multiplex PCR and the Quellung reaction. Antimicrobial susceptibility tests were performed using the broth microdilution method. Multilocus sequence typing was performed on MDR isolates for epidemiological investigations. Results: Among the 411 S. pneumoniae isolates analyzed, the most prevalent serotype was 3 (12.2%), followed by 10A (9.5%), 34 (7.3%), 19A (6.8%), 23A (6.3%), 22F (6.1%), 35B (5.8%), 11A (5.1%), and others (40.9%). The coverage rates of PCV7, PCV10, PCV13, and pneumococcal polysaccharide vaccine (PPSV)23 were 7.8%, 7.8%, 28.7%, and 59.4%, respectively. Resistance rates to penicillin, ceftriaxone, erythromycin, and levofloxacin were 13.1%, 9.2%, 80.3%, and 4.1%, respectively. MDR isolates accounted for 23.4% of all isolates. Serotypes 23A, 11A, 19A, and 15B accounted for the highest proportions of total isolates at 18.8%, 16.7%, 14.6%, and 8.3%, respectively. Sequence type (ST)166 (43.8%) and ST320 (12.5%) were common among MDR isolates. Conclusions: Non-PCV13 serotypes are increasing among invasive S. pneumoniae strains causing IPD. Differences in antimicrobial resistance were found according to the specific serotype. Continuous monitoring of serotypes and antimicrobial resistance is necessary for the appropriate management of S. pneumoniae infections.

Multiple ligation-Assisted recombinase polymerase amplification for highly sensitive and selective colorimetric detection of SARS-CoV-2.

In this paper we present a new method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), targeting a specific region "N gene." Under isothermal reaction conditions, we integrated ligation (Lig; high selectivity) and recombinase polymerase amplification (RPA; high sensitivity) processes, obtaining a robust method of detection. For point-of-care testing, we incorporated our laboratory-produced pyrophosphate ion (PPi)-sensing probe (PK-probe) for colorimetric analysis of the reaction. The total detection system was efficient and effective at diagnosing this RNA virus-mediated disease rapidly (30 min). In a full-genome SARS-CoV-2 study, our PK-probe/Lig-RPA system functioned with a limit of detection of 1160 copies/ml, with a single-mismatch level of selectively, and it was highly selective even in the presence of bacterial genomes commonly found in the human mouth and nose. This robust, straightforward, selective, efficient, and ultrasensitive colorimetric detection method, with potential for point-of-care analysis, should also be effective in detecting a diverse range of other RNA-based diseases.

Case report: Compound heterozygosity in PKLR gene with a large exon deletion and a novel rare p.Gly536Asp variant as a cause of severe pyruvate kinase deficiency.

Red cell pyruvate kinase (PK) deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia and the most frequent enzyme abnormality of the glycolytic pathway. To the best of our knowledge, this is the first Korean PK deficiency study that analyzes copy number variation (CNV) using next-generation sequencing (NGS). A 7-year-old girl with jaundice was admitted for evaluation of a persistent hemolytic anemia. The proband appeared chronically ill, showing a yellowish skin color, icteric sclera, hepatomegaly, and splenomegaly on physical examination. Sequence variants and CNV generated from NGS data were estimated to determine if there was a potential genetic cause. As a result, compound heterozygosity in the PKLR gene for a large exon deletion between exon 3 and exon 9 accompanied with a novel rare p.Gly536Asp variant located on exon 10 was identified as a cause of severe PK deficiency in the proband. The PK activity of the proband had been measured at the time of day 1, 21, and 28 after receiving transfusion to indirectly assume the effect of the transfused blood, and the results were 100.9%, 73.0%, and 48.5%, compared with average of normal controls, respectively. Our report emphasizes the need to perform complete CNV analysis of NGS data and gene dosage assays such as multiplex ligation-dependent probe amplification to evaluate large deletions or duplications/insertions of the PKLR gene in patients with suspected PK deficiency.

Invasive Hypervirulent Klebsiella pneumoniae Syndrome Originating from an Anorectal Abscess as Opposed to a Pyogenic Liver Abscess.

An immunocompetent 49-year-old man presented with swelling and pain in the lower region of his left leg that had lasted for 4 weeks. The diagnosis was severe pyomyositis and osteomyelitis in the lower left leg caused by hypervirulent Klebsiella pneumoniae (hvKP) along with multiple metastatic infections in the kidneys, lungs, and brain originating from an anorectal abscess. A virulence-gene analysis revealed that the isolated K. pneumoniae harbored rmpA, entB, ybtS, kfu, iutA, mrkD, and allS-virulence genes and belonged to the K1 capsular serotype. After repeated abscess drainage procedures, intravenous ceftriaxone was administered for more than 10 weeks, and the patient's infection was controlled. We focused on the clinical features of hvKP originating from an anorectal abscess without a pyogenic liver abscess. We suggest that hvKP be considered a causative pathogen of pyomyositis and osteomyelitis resulting in multiple metastatic infections in an immunocompetent patient, and more information on the unexpected multiple metastatic infections should be obtained from a virulence analysis of K. pneumoniae.

Pyogenic spondylitis caused by Klebsiella pneumoniae: should the possibility of hypervirulent Klebsiella pneumoniae be considered?

BACKGROUND: Klebsiella pneumoniae is rare but the second most common causative agent among gram-negative bacteria that cause pyogenic spondylitis. However, there are no available studies on the serotype, virulence factors, and clinical characteristics associated with K. pneumoniae-caused pyogenic spondylitis. Accordingly, we investigated the clinical characteristics of pyogenic spondylitis, K1 and K2 serotypes, and virulence factors of K. pneumoniae. METHODS: We reviewed the microbiological reports of specimens collected between January 2014 and December 2019 as well as the medical records of patients with pyogenic spondylitis caused by K. pneumoniae. We also evaluated K1 and K2 serotypes and the virulent genes rmpA, iutA, mrkD, ybtS, entB, and kfu. Strains that possessed rmpA and iutA were defined as hypervirulent K. pneumoniae. RESULTS: Six patients with pyogenic spondylitis caused by K. pneumoniae were enrolled in the study. The capsular serotypes K1 and K2 were present in 66.7% (4/6) of cases, and the hypervirulent strains were present in 88.3% (5/6) of cases. All patients had community-acquired infections, and all strains isolated were susceptible to antimicrobial agents. Intravenous antibiotic treatment continued for 2-7 weeks, and no patient underwent decompressive operation or surgical debridement. There was no recurrence. One patient died from pneumonia with a septic lung. CONCLUSION: Hypervirulent K. pneumoniae is a rare but possible causative agent associated with pyogenic spondylitis.

Phthalate plasticizer decreases the prion-like protein doppel essential for structural integrity and function of spermatozoa.

Di-n-butyl phthalate (DBP), a well-known endocrine disruptor, causes male reproductive dysfunction. To understand the underlying mechanisms, we performed histological, endocrinological, and biochemical analyses and assessed the expression of genes involved in spermatogenesis and sperm function according to OECD test guideline 407. Following 28 days of administration of the lowest observed adverse effect level dose of DBP to mice, no significant changes in body weight, testis and epididymis weights and histology, serum testosterone level, or testicular daily sperm production were found. Nonetheless, the motility of the epididymal sperm of the DBP group was significantly decreased together with an increase in the incidence of bent tails and abnormal heads. In the testes of the DBP group, lipid peroxidation (LPO) level was significantly increased and testicular Bcl-2 mRNA level was significantly decreased together with an increase in the Bax/Bcl-2 mRNA ratio. In the testes of the DBP group, levels of Prnd mRNA and protein and Pou4f1 mRNA, an activator of the Prnd promotor, were significantly decreased. Of note, prion-like protein doppel (PRND) was significantly decreased together with decreased PRND immunoreactivity in the head, midpiece, and tail of sperm. In the testes of the DBP group, levels of Sox9, Sgp1, and Sgp2 mRNA, which are functional Sertoli cell markers, were significantly decreased. Level of Amh mRNA, a Sertoli cell immaturity marker, was significantly increased together with that of Inha mRNA, suggesting deregulation of the brain-gonadal axis. Together, our findings suggest that DBP at present dosage may potentiate LPO generation and Sertoli cell immaturity via downregulation of Sox9 and disruption of the Pou4f1-Prnd gene network in post-meiotic germ cells without visible changes in spermatogenesis or testosterone level. This may result in structural and functional abnormalities in spermatozoa. Additionally, our findings suggest that assessment of the male reproductive toxicity of phthalate ester plasticizers based on conventional OECD test guidelines should be reconsidered.

Gene Panel Sequencing Identifies a Novel RYR1 p.Ser2300Pro Variant as Candidate for Malignant Hyperthermia with Multi-Minicore Myopathy.

Malignant hyperthermia (MH), a rare autosomal dominant pharmacogenetic disorder of skeletal muscle calcium regulation, is triggered by sevoflurane in susceptible individuals. We report a Korean having MH with multi-minicore myopathy functionally supported by RYR1-mediated intracellular Ca2+ release testing in B lymphocytes. A 14-year-old boy was admitted for the evaluation of progressive torticollis accompanied by cervicothoracic scoliosis. During the preoperative drape of the patient for the release of the sternocleidomastoid muscle under general anesthesia, his wrist and ankle were observed to have severe flexion contracture. The body temperature was 37.1 °C. To treat MH, the patient was administered a bolus of dantrolene intravenously (1.5 mg/kg) and sodium bicarbonate. After a few minutes, muscle rigidity, tachycardia, and EtCO2 all resolved. Next-generation panel sequencing for hereditary myopathy identified a novel RYR1 heterozygous missense variant (NM_000540.2: c.6898T > C; p.Ser2300Pro), which mapped to the MH2 domain of the protein, a hot spot for MH mutations. Ex vivo RYR1-mediated intracellular Ca2+ release testing in B lymphocytes showed hypersensitive Ca2+ responses to isoflurane and caffeine, resulting in an abnormal Ca2+ release only in the proband, not in his family members. Our findings expand the clinical and pathological spectra of information associated with MH with multi-minicore myopathy.