The evidence to date: implications of l-ascorbic acid in the pathophysiology of aging.

L-Ascorbic acid, commonly known as vitamin C, has been used not only for disease prevention and in complementary and alternative medicine, but also for anti-aging purposes. However, the scientific evidence is not yet sufficient. Here, we review the physiological functions of vitamin C and its relationship with various pathological conditions, including our previous findings, and discuss the prospects of its application in healthy longevity. In summary, vitamin C levels are associated with lifespan in several animal models. Furthermore, clinical studies have shown that the blood vitamin C levels are lower in middle-aged and older adults than in younger adults. Lower blood vitamin C levels have also been observed in various pathological conditions such as chronic kidney disease and chronic obstructive pulmonary disease in the elderly. These observations suggest the implications of vitamin C in age-related pathological mechanisms owing to its physiological functions.

Inhibitory Effects of Alpha-Connexin Carboxyl-Terminal Peptide on Canine Mammary Epithelial Cells: A Study on Benign and Malignant Phenotypes.

Mammary cancer is highly prevalent in non-castrated female dogs. Cell-to-cell communication is an important mechanism to maintain homeostasis, and connexins are proteins that assemble to form the communicating gap junctions. In many cancers, communication capacity is reduced; several approaches are being tested in order to increase the communication capacity in cancer cells and, therefore, alter their viability. This study analyzed the effects of the alpha-connexin carboxyl-terminal peptide (αCT1) on canine mammary non-neoplastic and neoplastic epithelial cells. Seven canine epithelial mammary cell lines were used. Among these, one was a normal canine epithelial mammary cell line (LOEC-NMG), two canine mammary adenomas (LOEC-MAd1 and LOEC-MAd2), and four canine mammary adenocarcinomas (LOEC-MCA1, LOEC-MCA2, LOEC-MCA3 and CF41). The αCT1 corresponds to a short Cx43 C-terminal sequence linked to an internalization sequence called the antennapedia. After 24 h of incubation, the medium containing different αCT1 peptide concentrations was added to the cells, and only the culture medium was used for control. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to quantify cell viability before treatment and 48, 72, and 96 h after the treatment. Results showed that the normal mammary epithelial cell line (LOEC-NMG) was resistant to treatment with αCT1, which is consistent with a previous study on human mammary cell lines. One of the adenoma cell lines (LOEC-MAd2) was also resistant to treatment with αCT1, although the other (LOEC-MAd1) was susceptible to treatment, mostly at 72 h after treatment. Regarding the four canine adenocarcinoma cell lines, they differ regarding the susceptibility to the treatment with αCT1. Three cell lines, canine mixed adenocarcinoma (LOEC-MCA1), canine complex adenocarcinoma (LOEC-MCA2), and commercial canine mammary adenocarcinoma cell line CF41, were susceptible to treatment with αCT1, while one canine mammary adenocarcinoma cell line (LOEC-MCA3) was resistant to treatment. In most αCT1 treated cell lines, Cx43 was strongly detected in cell membranes by immunofluorescence. We propose that αCT1 restored the cell-to-cell communication capacity of neoplastic cells and induced inhibitory effects on cell viability.

Essential role of ROS - 8-Nitro-cGMP signaling in long-term memory of motor learning and cerebellar synaptic plasticity.

Although reactive oxygen species (ROS) are known to have harmful effects in organisms, recent studies have demonstrated expression of ROS synthases at various parts of the organisms and the controlled ROS generation, suggesting possible involvement of ROS signaling in physiological events of individuals. However, physiological roles of ROS in the CNS, including functional roles in higher brain functions or neuronal activity-dependent ROS production, remain to be elucidated. Here, we demonstrated involvement of ROS - 8-NO2-cGMP signaling in motor learning and synaptic plasticity in the cerebellum. In the presence of inhibitors of ROS signal or ROS synthases, cerebellar motor learning was impaired, and the stimulus inducing long-term depression (LTD), cellular basis for the motor learning, failed to induce LTD but induced long-term potentiation (LTP)-like change at cerebellar synapses. Furthermore, ROS was produced by LTD-inducing stimulus in enzyme-dependent manner, and excess administration of the antioxidant vitamin E impaired cerebellar motor learning, suggesting beneficial roles of endogenous ROS in the learning. As a downstream signal, involvement of 8-NO2-cGMP in motor learning and cerebellar LTD were also revealed. These findings indicate that ROS - 8-NO2-cGMP signal is activated by neuronal activity and is essential for cerebellum-dependent motor learning and synaptic plasticity, demonstrating involvement of the signal in physiological function of brain systems.

Resveratrol Upregulates Senescence Marker Protein 30 by Activating AMPK/Sirt1-Foxo1 Signals and Attenuating H2O2-Induced Damage in FAO Rat Liver Cells.

Resveratrol (RSV) is a polyphenol with numerous biological functions, including anti-inflammatory, antioxidant, and anti-aging activities. The novel senescence marker protein-30 (SMP30) indicates aging, and it suppresses hepatic oxidative stress. However, the effects of RSV on SMP30 expression regulation remain unclear. We observed that RSV positively regulates SMP30 expression in rat hepatoma-derived FAO cells. However, this was abolished by Compound C and EX-527 that specifically inhibit AMP-activated protein kinase (AMPK) and Silent Information Regulator T1 (Sirt1), respectively. We predicted binding sites for AMPK, forkhead box protein O1 (Foxo1), and Sirt1 downstream molecules as possible SMP30 promoters using the JASPAR and UniProtKB databases. We identified a Foxo1 binding site in the promoter region of SMP30. Inhibiting Foxo1 with AS1842527 also decreased the RSV-induced upregulation of SMP30 expression. Moreover, RSV suppressed the substantial downregulation of SMP30 expression caused by oxidative stress and hydrogen peroxide (H2O2) and released accumulated lactate dehydrogenase. These results demonstrate that, as a novel food factor, RSV-induced upregulation of SMP30 by activating AMPK/Sirt1-Foxo1 signaling and may attenuates H2O2-induced oxidative damage. The findings of this study offer new perspectives of the anti-ageing properties of RSV.

Effects of heated tobacco product aerosol extracts on DNA methylation and gene transcription in lung epithelial cells.

AIMS: Smoking causes DNA methylation (DNAm) alterations that lead to lung cancer development. Although the use of heated tobacco products (HTPs) has recently increased, their impact on health remains unclear. This study aimed to evaluate the effects of HTPs on DNAm and gene transcription in human lung epithelial cells in vitro. MAIN METHODS: Human lung adenocarcinoma (A549) cells with type II alveolar epithelial characteristics were treated with aerosol extracts of two HTPs or a smoke extract of combustible reference cigarette (RC). Global 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels were quantified using dot blot analysis. Furthermore, reduced representation of bisulfite sequencing, DNA microarray, and quantitative PCR analyses were performed to determine CpG methylation and gene transcription changes induced by HTP and RC. KEY FINDINGS: Global 5-mC and 5-hmC levels were decreased by the RC extract but not the HTP extracts. However, an HTP extract altered the CpG methylation pattern, and Gene Ontology enrichment analysis of the differentially methylated regions of the RC and HTP groups showed a similar pattern. The HTP extract affected gene expression, albeit to a lesser extent than the RC extract. In particular, the HTP extract markedly affected the mRNA expression and promoter methylation of cytochrome P450 family 1 subfamily A member 1 (CYP1A1), which is associated with carcinogenic risk. SIGNIFICANCE: The study results suggest that HTPs as well as conventional combustible cigarettes can alter CpG methylation and gene transcription in lung epithelial cells.

Piriform Fossa Injury during Calibration Tube Insertion in Laparoscopic Sleeve Gastrectomy.

Piriform fossa and/or esophageal injuries caused by calibration tubes are relatively rare and remain unelucidated. Herein, we report the case of a 36-year-old woman with morbid obesity, sleep apnea, and menstrual abnormalities who was scheduled to undergo laparoscopic sleeve gastrectomy (LSG). We inserted a 36-Fr Nelaton catheter made of natural rubber as a calibration tube during the surgery. However, excessive resistance was observed. We confirmed a submucosal layer detachment approximately 5 cm from the left piriform fossa to the esophagus using intraoperative endoscopy. Additionally, LSG was performed using an endoscope as the guiding calibration tube. We inserted a nasogastric tube under endoscopy with a guidewire before completing the surgery, hoping for a guiding effect on the saliva flow. After 17 months, the patient had successfully lost weight postoperatively without complaints of neck pain or discomfort during swallowing. Therefore, in cases where the damage is limited to the submucosal layer, as in this case, conservative therapy should be considered; this is similar to the concept of endoscopic submucosal dissection not requiring suture closure. This case highlights the risk of iatrogenic injuries to the piriform fossa and/or esophagus during LSG and the importance of careful calibration tube insertion to prevent them.

Treatments for Staple Line Leakage after Laparoscopic Sleeve Gastrectomy.

The number of laparoscopic sleeve gastrectomies (LSGs) performed in patients with obesity who are eligible for bariatric and metabolic surgery is currently much lower in Japan than in other countries. Considering the large number of potential patients with obesity and type 2 diabetes and the unique Japanese national health insurance system that guarantees fair healthcare delivery, there is room to increase the number of LSGs in Japan in the near future. However, strict health insurance regulations may limit access to mandatory devices needed to treat postoperative complications, such as staple line leakage, which can cause severe morbidity and even mortality. Therefore, understanding the pathogenesis and treatment options for this complication is crucial. This article examined the current situation in Japan and its impact on staple line leakage management, including the role of endoscopic treatment in reducing reoperation. The authors suggest increasing education and collaboration between healthcare professionals to optimize management and improve patient outcomes.

Resveratrol can induce differentiating phenotypes in canine oral mucosal melanoma cells.

This report described the differentiation induction of canine oral mucosal melanoma (OMM) cells by resveratrol. Exposure of canine OMM cells to resveratrol (maximum dose: 50 µM and treatment period: 72 hr) induced differentiating features like melanocytes, and enhanced chemosensitivity against cisplatin, but alone had no influence on cell viability. Additionally, resveratrol significantly enhanced mRNA expression of key melanoma differentiation markers such as microphthalmia-associated transcription factor (MITF). Of several inhibitors against mitogen-activated protein kinase subtypes, only the c-Jun N-terminal kinase (JNK) inhibitor, SP600125, induced melanocyte-like morphological change and enhanced MITF mRNA expression. Furthermore, resveratrol also suppressed JNK activation in OMM cells by approximately 33%. Overall, these findings suggest that resveratrol induces differentiation in canine OMM cells, due to the inhibition of JNK signaling.

Release of SMP30 in Extracellular Vesicles under Conditions of Ascorbic Acid Deficiency Is Involved with Acute Phase Response in ODS Rat.

Senescence marker protein-30 (SMP30) is a senescence marker molecule that exhibits lactonase activity in the ascorbic acid (AsA) biosynthesis pathway, except in primate mammals, including humans. Although numerous studies have shown that hepatic AsA deficiency causes acute-phase responses, details of the relationship between SMP30 expression and acute-phase responses in AsA-deficient conditions remain to be elucidated. Here, we investigated the effects of AsA deficiency on the relationship between SMP30 and acute liver injury in osteogenic disorder Shionogi (ODS) rats, which have a hereditary defect in AsA biosynthesis. Male-ODS rats (4 wk old) were pair-fed an AsA-free diet with distilled or 0.1% AsA-dissolved water for 14 d. Under AsA-deficient conditions, hepatic SMP30 protein level was decreased and liver injury markers, the serum aspartate aminotransferase/alanine transaminase ratio and cytokine-induced neutrophil chemoattractant-1 (CINC-1) concentration, were elevated. In contrast, SMP30 protein level in extracellular vesicles (EVs) was significantly increased in addition to the positive acute proteins haptoglobin and asialoglycoprotein receptor 1 (ASGPR1), hepatic-derived specific markers expression under AsA-deficient conditions. AsA deficiency also activated signal transducer and activator of transcription 3 (STAT3) which is linked to EVs release in the liver. These results suggest that the release of SMP30 in EVs by AsA deficiency is involved with acute-phase responses.

Signet-ring cell carcinoma of the appendix with ganglioneuromatosis: a case report.

BACKGROUND: Primary cancer of the appendix, especially signet-ring cell carcinoma, is an uncommon disease, and it is rarely suspected before surgery. Diffuse intestinal ganglioneuromatosis that is not associated with neurofibromatosis-1 or multiple endocrine neoplasia 2b is also rare. The most frequent symptoms caused by it are changes in bowel habits, abdominal pain, and occlusive episodes. CASE PRESENTATION: The patient was a 48-year-old woman who had a month-long history of chronic abdominal pain, fullness, constipation, and diarrhoea. Enhanced computed tomography showed a 100-mm irregular swelling in the appendix and thickening of the appendiceal wall with cystic dilatation. Based on a preoperative diagnosis of appendiceal cancer, the patient underwent laparoscopic ileocecal resection with D3 lymph node dissection. Pathological diagnosis revealed a signet-ring cell carcinoma of the appendix with ganglioneuromatosis. The patient completed four courses of capecitabine plus oxaliplatin (CAPEOX) as postoperative adjuvant chemotherapy, and 23-month postoperative outcome was noneventful without recurrence. CONCLUSION: We report a signet-ring cell carcinoma of the appendix that was detected early because of its presence with ganglioneuromatosis.

Influence of oestrogen on satellite cells and myonuclear domain size in skeletal muscles following resistance exercise.

BACKGROUND: Oestrogen deficiency reduces skeletal muscle mass and force generation in postmenopausal women. Muscle mass is maintained by satellite cells, which are regulated by oestrogen. Although oestrogen therapy enhances muscle hypertrophy induced by resistance training in postmenopausal women, the molecular mechanism is unclear. METHODS: Adult female rats (10 weeks old) were divided into six groups: sham sedentary (Sham-Sed), sham climbing training (Sham-CT), ovariectomy sedentary (OVX-Sed), ovariectomy climbing training (OVX-CT), ovariectomy plus oestrogen treatment sedentary (OVX+E-Sed), and ovariectomy plus oestrogen treatment climbing training (OVX+E-CT). At 8 weeks after ovariectomy, rats in the training group were trained (one session every 3 days for 8 weeks) to climb a ladder while bearing a load. Oestrogen treatment involved subcutaneous insertion of a 17ß-oestradiol pellet. After 8 weeks, the flexor hallucis longus muscle was collected and analysed. RESULTS: Following climbing training, the flexor hallucis longus muscle mass and muscle-to-body weight ratios were dramatically increased by training (main effect of training, P < 0.01); the OVX+E-CT group showed the highest values (main effect of group, P < 0.01). The cross-sectional area of all muscle fibre types was increased by training (main effect of training, P < 0.01). Particularly, the cross-sectional area of MHC IIa in the OVX+E-CT group was significantly larger than that in the Sham-CT and OVX-CT groups. Satellite cell numbers were increased in all training groups (main effect of training, P < 0.05), and the myonuclear number was increased by training (main effect of training, P < 0.01), but there was no main group effect. The myonuclear domain size of all muscle fibre types and MHC IIa was increased in all training groups (main effect of training, P < 0.01) and showed a main group effect (P < 0.01). The myonuclear domain sizes of all muscle fibre types and MHC IIa in the OVX+E-CT group were significantly larger than those in the Sham-CT and OVX-CT groups. The total RNA contents revealed main effects of training and the group (P < 0.01); the OVX+E-CT group showed the highest contents (main effect of group, P < 0.01). The mRNA and protein levels of rpS6 were increased in the OVX+E-Sed and CT groups (main effects of group, P < 0.05). Particularly, the 28S ribosomal RNA content in OVX+E-Sed group was significantly higher than that in the OVX-Sed group. CONCLUSIONS: Oestrogen enhanced the resistance training-induced increase in myonuclear domain size but did not affect satellite cells and ribosome biogenesis.

Changes in the Genotypic Characteristics of Community-Acquired Methicillin-Resistant Staphylococcus aureus Collected in 244 Medical Facilities in Japan between 2010 and 2018: a Nationwide Surveillance.

Although community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged worldwide, no nationwide CA-MRSA surveillance has been conducted in Japan to determine the changes in its molecular characteristics over time. We aimed to characterize the molecular epidemiology of Panton-Valentine leucocidin (PVL)-positive CA-MRSA strains collected from across Japan in the past decade. We isolated 1,770 MRSA strains from the skin and pus samples of outpatients of 244 medical facilities in 31 prefectures between 2010 and 2018 (2010, 2012, 2014, 2016, and 2018). Regions, hospitals, and periods in which strains were isolated and patient age group and sex were tabulated. Staphylococcal cassette chromosome mec (SCCmec) typing, detection of virulence factor genes, and antimicrobial susceptibility testing were performed. Whole-genome analysis was performed for the PVL-positive strains isolated in 2018. All strains harbored the mecA gene. Compared to that in 2010, the percentage of SCCmec type IV increased in 2018, with a corresponding increase in the proportion of PVL-positive strains (10% to 26%). Of the isolates obtained in 2018, clonal complex 8 (CC8) was dominant among PVL-positive strains. Core-genome single-nucleotide polymorphism analysis, using whole-genome sequencing, suggested that the CC8 PVL-positive strains spread throughout Japan over the last decade. Furthermore, a unique ST22 clone carrying both the PVL- and toxic shock syndrome toxin-1-encoding genes has emerged. We demonstrated that the molecular epidemiology of CA-MRSA in Japan differs from that in Europe and the United States; thus, it is crucial to monitor the trend of changes in CA-MRSA characteristics in Japan. IMPORTANCE Community-associated MRSA, which is a multidrug-resistant organism and can cause infections in otherwise-healthy individuals, has become a global problem. This paper describes a nationwide surveillance conducted in Japan to investigate changes in molecular epidemiological characteristics of CA-MRSA over the past decade and provides a detailed review of the characteristics of Panton-Valentine leucocidin (PVL)-positive strains isolated in 2018. Although CA-MRSA is rare in Japan to date, we found that the isolation of PVL-positive strains has been increasing over the past decade. In particular, the PVL-positive strains wherein CC8 was dominant exhibited high interstrain similarity, suggesting that a limited number of clones have spread over the past decade. Furthermore, a unique ST22 clone carrying both PVL-encoding and toxic shock syndrome toxin-1-encoding genes has emerged. This study shows that various changes can be observed when molecular epidemiological analysis, combined with next-generation sequencing, is conducted over a long period.

A Redox-Silent Analogue of Tocotrienol May Break the Homeostasis of Proteasomes in Human Malignant Mesothelioma Cells by Inhibiting STAT3 and NRF1.

6-O-Carboxypropyl-alpha-tocotrienol (α-T3E) is a multi-target redox-silent analogue of tocotrienol that exhibits cytotoxicity against many cancer cells, including malignant mesothelioma (MM) cells. α-T3E has several molecular targets to effectively induce cytotoxicity against MM cells; however, the mechanisms underlying this cytotoxicity remain unclear. In the present study, we demonstrated that the α-T3E-dependent disruption of the homeostasis of proteasomes strongly induced endoplasmic reticulum (ER) stress, which resulted in effective cytotoxicity against MM cells. The α-T3E-dependent disruption of the homeostasis of proteasomes depended on decreases in proteasome subunits via the inactivation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor erythroid 2 related factor-1 (NRF1), which inhibited protease activity, such as chymotrypsin-like activity, in proteasomes. The α-T3E-dependent inhibition of this activity also induced severe ER stress and ultimately resulted in effective cytotoxicity against MM cells with chemoresistance. The present results indicate that α-T3E acts as an effective anti-mesothelioma agent by disrupting the homeostasis of proteasomes through the simultaneous inactivation of STAT3 and NRF1.

Determination of tissue-specific interaction between vitamin C and vitamin E in vivo using senescence marker protein-30 knockout mice as a vitamin C synthesis deficiency model.

Vitamin E (α-tocopherol; VE) is known to be regenerated from VE radicals by vitamin C (L-ascorbic acid; VC) in vitro. However, their in vivo interaction in various tissues is still unclear. Therefore, we alternatively examined the in vivo interaction of VC and VE by measurement of their concentrations in various tissues of senescence marker protein-30 (SMP30) knockout (KO) mice as a VC synthesis deficiency model. Male SMP30-KO mice were divided into four groups (VC+/VE+, VC+/VE-, VC-/VE+ and VC-/VE-), fed diets with or without 500 mg/kg VE and given water with or without 1·5 g/l VC ad libitum. Then, VC and VE concentrations in the plasma and various tissues were determined. Further, gene expression levels of transporters associated with VC and VE, such as α-tocopherol transfer protein (α-TTP) and sodium-dependent vitamin C transporters (SVCTs), were examined. These results showed that the VE levels in the VC-depleted (VC-/VE+) group were significantly lower than those in the VC+/VE+ group in the liver and heart; the VC levels in the VE-depleted (VC+/VE-) group were significantly lower than those in the VC+/VE+ group in the kidneys. The α-TTP gene expression in the liver and kidneys was decreased by VC and/or VE depletion. Moreover, SVCT1 gene expression in the liver was decreased by both VC and VE depletion. In conclusion, these results indicate that VC spares VE mainly in the liver and heart and that VE spares VC in the kidneys of SMP30-KO mice. Thus, interaction between VC and VE is likely to be tissue specific.

Reduced Plasma Ascorbate and Increased Proportion of Dehydroascorbic Acid Levels in Patients Undergoing Hemodialysis.

Ascorbate functions as an electron donor and scavenges free radicals. Dehydroascorbic acid (DHA), the oxidized form of ascorbate, is generated as a result of these reactions. While low plasma ascorbate levels have been reported in hemodialysis patients worldwide, no studies have measured DHA because it is not generalized. In this study, we aimed to clarify whether plasma ascorbate levels are low in dialysis patients and whether plasma ascorbate levels fluctuate before and after dialysis. Moreover, we applied our previously established method to measure the plasma ascorbate and DHA levels in chronic kidney disease (CKD) stage G3-G5 non-hemodialysis-dependent patients, and pre- and post-dialysis plasma ascorbate and DHA levels in CKD stage G5D hemodialysis patients. The sample size was calculated using G-power software. The pre-dialysis plasma total ascorbate levels, including DHA, were significantly (56%) lower in hemodialysis patients than in non-hemodialysis-dependent CKD patients. After dialysis, there was a 40% reduction in the plasma total ascorbate levels. Hemodialysis increased the post-dialysis plasma proportions of DHA from 37% to 55%. The study results demonstrated lower plasma total ascorbate levels in hemodialysis patients compared with in non-hemodialysis-dependent CKD patients; these low levels in hemodialysis patients were further reduced by hemodialysis and increased DHA proportion.

Effects of Alpha-Connexin Carboxyl-Terminal Peptide (aCT1) and Bowman-Birk Protease Inhibitor (BBI) on Canine Oral Mucosal Melanoma (OMM) Cells.

Oral mucosal melanomas (OMM) are aggressive cancers in dogs, and are good models for human OMM. Gap junctions are composed of connexin units, which may have altered expression patterns and/or subcellular localization in cancer cells. Cell-to-cell communication by gap junctions is often impaired in cancer cells, including in melanomas. Meanwhile, the upregulated expression of the gap junction protein connexin 43 (Cx43) inhibits melanoma progression. The α-connexin carboxyl-terminal (aCT1) peptide reportedly maintains Cx43 expression and cell-cell communication in human mammary cells and increases the communication activity through gap junctions in functional assays, therefore causing decreased cell proliferation. The Bowman-Birk protease inhibitor (BBI), a component of soybeans, induces Cx43 expression in several tumor cells as a trypsin-chymotrypsin inhibition function, with antineoplastic effects. This study investigated the effect of aCT1 peptide and BBI treatment, alone or in combination, on TLM1 canine melanoma cell viability. Cell viability after treatment with aCT1, the reverse sequence peptide (R-pep), and/or BBI for 5 days was analyzed by PrestoBlue assay. Immunofluorescence was used to observe Cx43 localization and expression. aCT1 (200 µM) alone did not significantly decrease cell viability in TLM1 cells, whereas BBI (400 µg/ml) alone significantly decreased the TLM1 viability. Combined treatment with both aCT1 (200 µM) and BBI (400 µg/ml) significantly decreased cell viability in TLM1 cells. Cx43 expression, as identified by immunostainings in TLM1 cells, was increased in the cell membrane after the combination treatment with BBI and aCT1. This dual treatment can be combined to achieve the anticancer activity, possibly by increasing Cx 43 expression and affecting Cx43 migration to the cell membrane. In conclusion, a treatment strategy targeting Cx43 with BBI and aCT1 may possibly lead to new effective therapies for canine OMM.

Increased Incidence and Plasma-Biofilm Formation Ability of SCCmec Type IV Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated From Patients With Bacteremia.

In Japan, Staphylococcal cassette chromosome mec (SCCmec) type IV methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prominent cause of bacteremia, but the virulence of most of these strains is unclear. We aimed to investigate the relationship between the molecular characteristics and the ability to form biofilms in the presence of blood plasma (plasma-biofilms) of MRSA strains isolated from bloodstream infections. In this study, the molecular characteristics and biofilms of MRSA strains isolated from blood cultures between 2015 and 2017 were analyzed by PCR-based assays, crystal violet staining, and confocal reflection microscopy methods. Among the 90 MRSA isolates, the detection rate of SCCmec type II clones decreased from 60.7 to 20.6%. The SCCmec type IV clone replaced the SCCmec type II clone as the dominant clone, with a detection rate increasing from 32.1 to 73.5%. The plasma-biofilm formation ability of the SCCmec type IV clone was higher than the SCCmec type II clone and even higher in strains harboring the cna or arcA genes. Plasma-biofilms, mainly composed of proteins, were formed quickly and strongly. Our study demonstrated the increased plasma-biofilm formation ability of SCCmec type IV strains.

Severe pallid breath-holding spells treated with low-dose theophylline.

BACKGROUND: The medical treatment for severe pallid breath-holding spells accompanied with severe bradycardia or transient cardiac arrest is controversial. Although various medications have been reported to be effective, patients treated with pacemaker insertion are not always evaluated for pharmacological therapy beforehand. CASE REPORT: A 9-month-old boy developed pallid breath-holding spells. At 15 months of age, a Holter electrocardiogram revealed 12 s of asystole during a breath-holding spell. Treatment with low-dose theophylline sustained-release dry syrup (5.3 mg/kg/day) led to complete control of the spells. The peak concentration of theophylline was 4.4 µg/mL which was below the therapeutic range for bronchial asthma. When he turned 3 years and 5 months of age, theophylline treatment was discontinued without recurrence of pallid breath-holding spells. DISCUSSION: Theophylline is now infrequently used to treat pediatric bronchial asthma due to its limited effect coupled with its side effects, which include headache, digestive symptoms, and theophylline-associated convulsions. The effectiveness of theophylline as a treatment for pallid breath-holding spells has been reported in several reports. In our case, the theophylline dosage was approximately half the amount described in previous reports. CONCLUSIONS: In this case, low-dose theophylline was adequate in controlling the pallid breath-holding spells. Because theophylline-associated seizures are a major concern, we suggest an evaluation of low-dose theophylline for treating patients with severe pallid breath-holding spells without febrile convulsions or epilepsy before proceeding with permanent pacemaker insertion. Further development of preventive strategies for theophylline-associated seizures and characterization of patients who respond well to theophylline treatment is required.

Staphylococcal Cassette Chromosome mec (SCCmec) Analysis of MRSA.

Methicillin-susceptible S. aureus changes to methicillin-resistant S. aureus (MRSA) upon the acquisition of staphylococcal cassette chromosome mec (SCCmec), a genomic island that encodes methicillin resistance. SCCmec elements in S. aureus are classified into different types based on the combination of mec gene complexes and ccr gene complexes, which share variations, five classes in mec and eight in ccr. To date, at least 13 types of SCCmec elements have been identified and each SCCmec type has individual characteristics. It is known that hospital-associated MRSA strains carry SCCmec elements of types, I, II, and III, and the majority of community-acquired MRSA strains carry characteristic SCCmec elements, type IV SCCmec or type V SCCmec. We herein describe multiplex PCR methods to type SCCmec elements by identifying the mec gene complex class and ccr gene complex type.

AUTACs: Cargo-Specific Degraders Using Selective Autophagy.

Protein silencing represents an essential tool in biomedical research. Targeted protein degradation (TPD) strategies exemplified by PROTACs are rapidly emerging as modalities in drug discovery. However, the scope of current TPD techniques is limited because many intracellular materials are not substrates of proteasomal clearance. Here, we described a novel targeted-clearance strategy (autophagy-targeting chimera [AUTAC]) that contains a degradation tag (guanine derivatives) and a warhead to provide target specificity. As expected from the substrate scope of autophagy, AUTAC degraded fragmented mitochondria as well as proteins. Mitochondria-targeted AUTAC accelerated both the removal of dysfunctional fragmented mitochondria and the biogenesis of functionally normal mitochondria in patient-derived fibroblast cells. Cytoprotective effects against acute mitochondrial injuries were also seen. Canonical autophagy is viewed as a nonselective bulk decomposition system, and none of the available autophagy-inducing agents exhibit useful cargo selectivity. With its target specificity, AUTAC provides a new modality for research on autophagy-based drugs.