Morc2a variants cause hydroxyl radical-mediated neuropathy and are rescued by restoring GHKL ATPase.

Mutations in the Microrchidia CW-type zinc finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high CNS transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss-of-function characteristics due to protein synthesis defects in Morc2a p.S87L were also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy.

Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase.

Many studies have been conducted to improve economically important livestock traits such as feed efficiency and muscle growth. Genome editing technologies represent a major advancement for both basic research and agronomic biotechnology development. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technical platform is a powerful tool used to engineer specific targeted loci. However, the potential occurrence of off-target effects, including the cleavage of unintended targets, limits the practical applications of Cas9-mediated genome editing. In this study, to minimize the off-target effects of this technology, we utilized D10A-Cas9 nickase to generate myostatin-knockout (MSTN KO) chickens via primordial germ cells. D10A-Cas9 nickase (Cas9n)-mediated MSTN KO chickens exhibited significantly larger skeletal muscles in the breast and leg. Degrees of skeletal muscle hypertrophy and hyperplasia induced by myostatin deletion differed by sex and muscle type. The abdominal fat deposition was dramatically lower in MSTN KO chickens than in wild-type chickens. Our results demonstrate that the D10A-Cas9 technical platform can facilitate precise and efficient targeted genome engineering and may broaden the range of applications for genome-edited chickens in practical industrialization and as animal models of human diseases.

Ten Cases of Taenia saginata Infection Confirmed by Analysis of the Internal Transcribed Spacer 1 rDNA Region in the Republic of Korea.

From October 2015 to August 2018, tapeworm proglottids were obtained from 10 patients who were residents of Daegu and Gyeongbuk provinces and had a history of raw beef consumption. Most of them had no overseas travel experience. The gravid proglottids obtained from the 10 cases had 15-20 lateral uterine branches. A part of internal transcribed spacer 1 (ITS1) DNA of the 10 cases, amplified by polymerase chain reaction (PCR) and digested with AleI restriction enzyme, produced the same band pattern of Taenia saginata, which differentiated from T. asiatica and T. solium. Sequences of ITS1 and cytochrome c oxidase subunit 1 (cox1) showed higher homology to T. saginata than to T. asiatica and T. solium. Collectively, these 10 cases were identified as T. saginata human infections. As taeniasis is one of the important parasitic diseases in humans, it is necessary to maintain hygienic conditions during livestock farming to avoid public health concerns.

Molecular and Biochemical Properties of a Cysteine Protease of Acanthamoeba castellanii.

Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their biochemical and functional properties are not clearly understood yet. In this study, we isolated a gene encoding cysteine protease of A. castellanii (AcCP) and its biochemical and functional properties were analyzed. Sequence analysis of AcCP suggests that this enzyme is a typical cathepsin L family cysteine protease, which shares similar structural characteristics with other cathepsin L-like enzymes. The recombinant AcCP showed enzymatic activity in acidic conditions with an optimum at pH 4.0. The recombinant enzyme effectively hydrolyzed human proteins including hemoglobin, albumin, immunoglobuins A and G, and fibronectin at acidic pH. AcCP mainly localized in lysosomal compartment and its expression was observed in both trophozoites and cysts. AcCP was also identified in cultured medium of A. castellanii. Considering to lysosomal localization, secretion or release by trophozoites and continuous expression in trophozoites and cysts, the enzyme could be a multifunctional enzyme that plays important biological functions for nutrition, development and pathogenicity of A. castellanii. These results also imply that AcCP can be a promising target for development of chemotherapeutic drug for Acanthamoeba infections.

A Case of Furuncular Myiasis Due to Cordylobia anthropophaga in a Korean Traveler Returning from Uganda.

A fly larva was recovered from a boil-like lesion on the left leg of a 33-year-old male on 21 November 2016. He has worked in an endemic area of myiasis, Uganda, for 8 months and returned to Korea on 11 November 2016. The larva was identified as Cordylobia anthropophaga by morphological features, including the body shape, size, anterior end, posterior spiracles, and pattern of spines on the body. Subsequent 28S rRNA gene sequencing showed 99.9% similarity (916/917 bp) with the partial 28S rRNA gene of C. anthropophaga. This is the first imported case of furuncular myiasis caused by C. anthropophaga in a Korean overseas traveler.

Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis.

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.

Prevalence of Trichomonas vaginalis in Women Visiting 2 Obstetrics and Gynecology Clinics in Daegu, South Korea.

This study explored epidemiological trends in trichomoniasis in Daegu, South Korea. Wet mount microscopy, PCR, and multiplex PCR were used to test for Trichomonas vaginalis in vaginal swab samples obtained from 621 women visiting 2 clinics in Daegu. Of the 621 women tested, microscopy detected T. vaginalis in 4 (0.6%) patients, PCR detected T. vaginalis in 19 (3.0%) patients, and multiplex PCR detected T. vaginalis in 12 (1.9%) patients. Testing via PCR demonstrated high sensitivity and high negative predictive value for T. vaginalis. Among the 19 women who tested positive for T. vaginalis according to PCR, 94.7% (18/19) reported vaginal signs and symptoms. Notably, more than 50% of T. vaginalis infections occurred in females younger than 30 years old, and 58% were unmarried. Multiplex PCR, which simultaneously detects pathogens from various sexually transmitted infections, revealed that 91.7% (11/12) of patients were infected with 2 or more pathogens. Mycoplasma hominis was the most prevalent co-infection pathogen with T. vaginalis, followed by Ureaplasma urealyticum and Chlamydia trachomatis. Our results indicate that PCR and multiplex PCR are the most sensitive tools for T. vaginalis diagnosis, rather than microscopy which has been routinely used to detect T. vaginalis infections in South Korea. Therefore, clinicians should take note of the high prevalence of T. vaginalis infections among adolescent and young women in order to prevent persistent infection and transmission of this disease.

Cysteine protease inhibitor (AcStefin) is required for complete cyst formation of Acanthamoeba.

The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protect Acanthamoeba from intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established in Acanthamoeba. In the present study, we identified and characterized Acanthamoeba cysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation of Acanthamoeba.

Two human cases of Diphyllobothrium nihonkaiense infection in Korea.

Diphyllobothrium latum and Diphyllobothrium nihonkaiense are the 2 reported main causes of human diphyllobothriasis in the Republic of Korea. However, the differentiation of these 2 species based on morphologic features alone is difficult. The authors used nucleotide sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene to diagnose Diphyllobothrium spp. Two patients visited the emergency room at Kyungpook National University Hospital on 3 April and 12 April 2013, respectively, with fragments of parasites found while defecating. The parasites were identified as Diphyllobothrium spp. based on morphologic characteristics, and subsequent cox1 gene sequencing showed 99.9% similarity (1,478/1,480 bp) with D. nihonkaiense. Our findings support the hypothesis that D. nihonkaiense is a dominant species in Korea.

Development of loop-mediated isothermal amplification targeting 18s ribosomal DNA for rapid detection of Azumiobodo hoyamushi (Kinetoplastea).

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.

Prevalence of Trichomonas vaginalis by PCR in men attending a primary care urology clinic in South Korea.

Trichomonas vaginalis, a causative agent of trichomoniasis, may trigger symptomatic or asymptomatic nongonococcal urethritis and chronic prostatitis in men. Despite the availability of highly sensitive diagnostic tests, such as nucleic acid amplification tests, including PCR, few prospective studies present data on male T. vaginalis infection in South Korea. In the present study, the prevalence of T. vaginalis and associated clinical conditions were evaluated in 201 male patients from a primary care urology clinic in South Korea. The prevalence of T. vaginalis infection in our cohort was 4% (8/201) by PCR. T. vaginalis infection was common in men older than 40 years (median age, 52 years). Among the 8 Trichomonas-positive patients, 87.5% (7/8) had prostatic diseases, such as prostatitis and benign prostatic hyperplasia, and 25.0% (2/8) and 12.5% (1/8) were coinfected with Chlamydia trachomatis and Mycoplasma genitalium, respectively. Our results suggest that T. vaginalis infection is not rare in men attending primary care urology clinics in South Korea, especially in those older than 40 years, in whom it may explain the presence of prostatic disease. The possibility of T. vaginalis infection should be routinely considered in older male patients with prostatic diseases in South Korea.

Assessing Individual Muscle Characteristics to Enhance Frozen-Thawed Meat Quality.

This study assessed previous research aimed at mitigating the adverse effects of freeze-thawing on meat quality. Specifically, it focuses on assessing the physicochemical alterations in meat resulting from freezing, freeze-thawing, or technologies to minimize these alterations. Recent studies have focused on conventional freeze-thaw technology applicable across various livestock species and muscle types. However, recent research has indicated the necessity for developing freeze-thaw technology considering the unique characteristics of individual muscles. In this review, we summarize previous studies that have compared alterations in the physicochemical properties of primary muscles owing to freezing or freeze-thawing. Despite the introduction of various technologies to significantly reduce the adverse effects on meat quality resulting from freeze-thawing, it is essential to consider the unique characteristics (proximate composition, pH, and muscle fiber characteristics) of individual muscles or cuts to develop enhanced the freeze-thaw processing technology.

Longitudinal and transversal intramuscular variation of muscle fiber and meat quality characteristics in bovine M. longissimus thoracis et lumborum.

This study evaluates longitudinal and transversal intramuscular variations in muscle fiber and meat quality characteristics in bovine M. longissimus thoracis et lumborum (LTL). The LTL muscles (n = 5) from the left side of the beef carcass were cut at intervertebral segment intervals (between 1st thoracic vertebra (TV) and 6th lumbar vertebra (LV)). The pennation angle demonstrated an increasing trend from the anterior to posterior regions regardless of the medial (M-zone) and lateral (L-zone) regions (P < 0.05). The M -zone had a higher pennation angle than the L-zone in the TV and 1st LV (P < 0.05). The cross-sectional area (CSA) of muscle fibers, excluding type I, was larger in the posterior region than the anterior region (P < 0.05). A larger CSA of type I/IIA, IIA, IIAX, and IIX was observed on the lateral side than on the medial side of the 13th TV (P < 0.05). Fiber types were more oxidative (types I and IIA) in the anterior region and more glycolytic (types IIA/IIX and IIX) in the posterior region. Fat content was higher in the anterior region than in the posterior region (P < 0.05). The lowest redness, yellowness, and Warner-Bratzler shear force values were observed in the middle of the muscle, whereas the lightness value was lower in the posterior region regardless of the transversal region (P < 0.05). Therefore, bovine LTL muscles exhibit unique morphological properties and contribute to understanding meat quality associated with morphological and muscle fiber characteristics in relation to their intramuscular variations.

Comparative review of muscle fiber characteristics between porcine skeletal muscles.

Meat derived from skeletal muscles of animals is a highly nutritious type of food, and different meat types differ in nutritional, sensory, and quality properties. This study was conducted to compare the results of previous studies on the muscle fiber characteristics of major porcine skeletal muscles to the end of providing basic data for understanding differences in physicochemical and nutritional properties between different porcine muscle types (or meat cuts). Specifically, the muscle fiber characteristics between 19 major porcine skeletal muscles were compared. The muscle fibers that constitute porcine skeletal muscle can be classified into several types based on their contractile and metabolic characteristics. In addition, the muscle fiber characteristics, including size, composition, and density, of each muscle type were investigated and a technology based on these muscle fiber characteristics for improving meat quality or preventing quality deterioration was briefly discussed. This comparative review revealed that differences in muscle fiber characteristics are primarily responsible for the differences in quality between pork cuts (muscle types) and also suggested that data on muscle fiber characteristics can be used to develop optimal meat storage and packaging technologies for each meat cut (or muscle type).

Comparison of Chemical Composition, Quality, and Muscle Fiber Characteristics between Cull Sows and Commercial Pigs: The Relationship between Pork Quality Based on Muscle Fiber Characteristics.

This study aims to compare the chemical composition, quality, and muscle fiber characteristics of cull sows and commercial pigs, investigating the effect of changes in muscle fiber characteristics on pork quality. The proximate composition, color, pH, water-holding capacity (drip loss and cooking loss), protein solubility, total collagen content, and muscle fiber characteristics of cull sows (n=20) and commercial pigs (n=20) pork were compared. No significant differences were found between cull sows and commercial pigs in terms of proximate composition, drip loss, protein solubility, or total collagen content of their meat (p<0.05). However, cull sow pork exhibited a red color and a higher pH (p<0.05). This appears to be the result of changes in muscle fiber number and area composition (p<0.05). Cull sow meat also displayed better water-holding capacity as evident in a smaller cooking loss (p<0.05), which may be related to an increase in muscle fiber cross-sectional area (p<0.05). In conclusion, muscle fiber composition influences the pork quality; cull sow pork retains more moisture when cooked, resulting in minimal physical loss during processing and can offer more processing suitability.

Effect of freezing raw meat on the physicochemical characteristics of beef jerky.

The effect of freezing raw meat on the quality characteristics of beef jerky was evaluated in the present study. Jerky was made using different types of raw beef (fresh, frozen, and frozen-thawed) with different curing times (6 h and 12 h). Frozen-thawed beef had a lower moisture content than fresh or frozen beef due to higher exudate loss (P < 0.05). Jerky made using frozen and frozen-thawed beef showed lower drying yield and higher shear force than jerky prepared using fresh beef (P < 0.05). Freezing raw beef decreased the fat content and increased the redness, yellowness, chroma, and hue values of jerky (P < 0.05). The microstructure of beef jerky was showed to increase the deformation and contraction of muscle fibers due to freezing. Longer curing times increased the moisture content of jerky made using frozen meat (P < 0.05). Jerky made using frozen or frozen-thawed meat was tough due to excessive fat and moisture loss.

Comparative Analysis of Volatile and Non-Volatile Metabolites Derived from Bacillus subtilis Strains Producing Different Levels of Biogenic Amines.

Biogenic amines (BAs), which are mainly generated by the microbial decarboxylation of amino acids, are important nitrogen compounds in fermented foods because of their toxicology. However, amino acids, the precursors of BAs, also play an important role in generating volatile and non-volatile metabolites, which are strongly associated with quality indicators for foods. Bacillus subtilis is one of dominant fermentative microorganism in various fermented foods and is well known as a BA-producing bacterium. In this study, B. subtilis strains which have different BAs-producing capacities, higher level of BAs production strain (BH) and lower level of BAs production strain (BL), were applied to compare the formations of volatile and non-volatile metabolite profiles according to cultivation times. In this study, histamine, putrescine, and spermidine were detected in all strains, however, 2-phenylethylamine was detected only in BH. Partial least squares discriminant analysis (PLS-DA) was applied to investigate the difference of metabolic profiles according to strains. In BH, some amino acids (phenylalanine, leucine, and threonine) and related volatile metabolites (3-methylbutanoic acid, pyrazines, styrene, and 1H-indole) were produced higher levels. On the other hand, BL produced significantly higher contents of metabolites associated with metabolism of fatty acids and nucleotides. It is necessary to consider the formation of metabolites in terms of quality as well as that of BAs during fermentation.

Comparison of Meat Quality Characteristics and Proteolysis Trends associated with Muscle Fiber Type Distribution between Duck Pectoralis Major and Iliotibialis Muscles.

This study was conducted to evaluate the proteolysis trends and change in meat quality during 10 days of cold storage in duck M. pectoralis major (PM) and M. iliotibialis (IL). Duck IL had a higher pH and greater degree of lightness but lower cooking loss than PM (p<0.05). During the 10-day cold storage, the pH value of PM declined significantly (p<0.05), while the meat quality traits of IL were not affected by cold storage (p>0.05). In PM, the redness increased from day 1 to day 5, while cooking loss was lower on day 10 compared to day 5 (p<0.05). There were no significant differences in the activities of cathepsin B and proteasome 20S during cold storage (p>0.05). The activity of calpains declined gradually during 10 days of storage (p<0.05), and the activity of calpains in PM was higher than that in IL (p<0.05). A total of 5,155 peptides were detected and derived from 34 proteins of duck PM muscle, whereas 4,222 peptides derived from 32 proteins were detected from duck IL muscle. Duck PM muscle was composed only of fast type of muscle fiber, whereas IL muscle was composed of both slow and fast types. The proteins responsible for glycolysis or myofibrillar proteins were closely related to changes in meat color or water-holding capacity during cold storage. These results suggest that changes in meat quality characteristics during cold storage are closely related to protein degradation, which is also related to the distribution of muscle fiber types.

Synthesis of cis-thiiranes as diastereoselective access to epoxide congeners via 4π-electrocyclization of thiocarbonyl ylides.

Organochalcogen heterocycles are ubiquitously present and widely utilized in various fields. Among them, oxirane has been extensively studied, and all of the stereoisomeric forms are readily available. In contrast, synthetic studies on thiirane were rarely reported, and thus the useful sulfur-congener of oxirane has been difficult to access in a stereodefined form. In this research, a general stereoselective synthesis of cis-thiiranes is accomplished by taking advantage of stereospecific electrocyclization of trans-thiocarbonyl ylides, which are generated in situ from readily available E,E-aldazine N-oxides upon treatment with Lawesson's reagent. This newly developed practical method provides a variety of cis-1,2-diarylthiiranes as essentially single diastereomers in high yields under mild reaction conditions. The intermediacy of trans-thiocarbonyl yilde is confirmed by mechanistic experiments, and the excellent stereocontrol is rationalized by DFT calculation.

Proteolysis and changes in meat quality of chicken pectoralis major and iliotibialis muscles in relation to muscle fiber type distribution.

The proteolysis trends and meat quality of the chicken pectoralis major (PM) and iliotibialis (IL) muscles stored at 4°C for 7 d were investigated. After 7 d of storage, the purge loss was higher (P < 0.05) in PM than in IL muscle. The difference in the composition of muscle fibers between PM (100% fast type) and IL (88.85% fast and 11.15% slow types) resulted in differences in proteolysis. Fructose-bisphosphate aldolase, troponin I, myosin heavy chain, and malate dehydrogenase exhibited the same tendencies, but pyruvate kinase, creatine kinase, L-lactate dehydrogenase, and triosephosphate isomerase exhibited different tendencies in the 2 muscles. The activity of cathepsin B was higher in PM than in IL during storage (P < 0.05). These results indicate that the proteolysis trend and changes in meat quality during cold storage are dependent on the different muscle fiber characteristics.