Assessment of Genetic Stability in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Using Droplet Digital PCR.

Unintended genetic modifications that occur during the differentiation and proliferation of human induced pluripotent stem cells (hiPSCs) can lead to tumorigenicity. This is a crucial concern in the development of stem cell-based therapies to ensure the safety and efficacy of the final product. Moreover, conventional genetic stability testing methods are limited by low sensitivity, which is an issue that remains unsolved. In this study, we assessed the genetic stability of hiPSCs and hiPSC-derived cardiomyocytes using various testing methods, including karyotyping, CytoScanHD chip analysis, whole-exome sequencing, and targeted sequencing. Two specific genetic mutations in KMT2C and BCOR were selected from the 17 gene variants identified by whole-exome and targeted sequencing methods, which were validated using droplet digital PCR. The applicability of this approach to stem cell-based therapeutic products was further demonstrated with associated validation according to the International Council for Harmonisation (ICH) guidelines, including specificity, precision, robustness, and limit of detection. Our droplet digital PCR results showed high sensitivity and accuracy for quantitatively detecting gene mutations, whereas conventional qPCR could not avoid false positives. In conclusion, droplet digital PCR is a highly sensitive and precise method for assessing the expression of mutations with tumorigenic potential for the development of stem cell-based therapeutics.

Telangiectasia-ectodermal dysplasia-brachydactyly-cardiac anomaly syndrome is caused by de novo mutations in protein kinase D1.

BACKGROUND: We describe two unrelated patients who display similar clinical features including telangiectasia, ectodermal dysplasia, brachydactyly and congenital heart disease. METHODS: We performed trio whole exome sequencing and functional analysis using in vitro kinase assays with recombinant proteins. RESULTS: We identified two different de novo mutations in protein kinase D1 (PRKD1, NM_002742.2): c.1774G>C, p.(Gly592Arg) and c.1808G>A, p.(Arg603His), one in each patient. PRKD1 (PKD1, HGNC:9407) encodes a kinase that is a member of the protein kinase D (PKD) family of serine/threonine protein kinases involved in diverse cellular processes such as cell differentiation and proliferation and cell migration as well as vesicle transport and angiogenesis. Functional analysis using in vitro kinase assays with recombinant proteins showed that the mutation c.1808G>A, p.(Arg603His) represents a gain-of-function mutation encoding an enzyme with a constitutive, lipid-independent catalytic activity. The mutation c.1774G>C, p.(Gly592Arg) in contrast shows a defect in substrate phosphorylation representing a loss-of-function mutation. CONCLUSION: The present cases represent a syndrome, which associates symptoms from several different organ systems: skin, teeth, bones and heart, caused by heterozygous de novo mutations in PRKD1 and expands the clinical spectrum of PRKD1 mutations, which have hitherto been linked to syndromic congenital heart disease and limb abnormalities.

Longitudinal analysis of symptom-based clustering in patients with primary Sjogren's syndrome: a prospective cohort study with a 5-year follow-up period.

BACKGROUND: Sjogren's syndrome (SS) is a heterogenous disease with various phenotypes. We aimed to provide a relevant subclassification based on symptom-based clustering for patients with primary (p) SS. METHODS: Data from patients in a prospective pSS cohort in Korea were analysed. Latent class analysis (LCA) was performed using patient reported outcomes, including pain, fatigue, dryness, and anxiety/depression. Clinical and laboratory differences between the classes were analysed. Latent transition analysis (LTA) was applied to the longitudinal data (annually for up to 5 years) to assess temporal stability of the classifications. RESULTS: LCA identified three classes among 341 patients with pSS (i.e., 'high symptom burden', 'dryness dominant', 'low symptom burden'). Each group had distinct laboratory and clinical phenotypes. LTA revealed that class membership remained stable over time. Baseline class predicted future salivary gland function and damage accrual represented by a Sjogren's syndrome disease damage index. CONCLUSION: Symptom-based clustering of heterogenous patients with primary Sjogren's syndrome provided a relevant classification supported by temporal stability over time and distinct phenotypes between the classes. This clustering strategy may provide more homogenous groups of pSS patients for novel treatment development and predict future phenotypic evolvement.

Valproic acid protects intestinal organoids against radiation via NOTCH signaling.

Radiotherapy is a leading treatment for various types of cancer. However, exposure to high-dose ionizing radiation causes acute gastrointestinal injury and gastrointestinal syndrome. This has significant implications for human health, and therefore, radioprotection is a major area of research. Radiation induces the loss of intestinal stem cells; hence, the protection of stem cells expressing LGR5 (a marker of intestinal epithelial stem cells) is a key strategy for the prevention of radiation-induced injury. In this study, we identified valproic acid (VPA) as a potent radioprotector using an intestinal organoid culture system. VPA treatment increased the number of LGR5+ stem cells and organoid regeneration after irradiation. N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT, an inhibitor of NOTCH signaling) blocked the radioprotective effects of VPA, indicating that NOTCH signaling is a likely mechanism underlying the observed effects of VPA. In addition, VPA acted as a radiosensitizer via the inhibition of histone deacetylase (HDAC) in a colorectal cancer organoid. These results demonstrate that VPA exerts strong protective effects on LGR5+ stem cells via NOTCH signaling and that the inhibition of NOTCH signaling reduces these protective effects, providing a basis for the improved management of radiation injury.

Preemptive intravenous iron therapy versus autologous whole blood therapy for early postoperative hemoglobin level in patients undergoing bimaxillary orthognathic surgery: a prospective randomized noninferiority trial.

BACKGROUND: Previous studies have reported the efficacy and safety of intravenous (IV) iron therapy during the perioperative period as an alternative and adjunct to allogeneic blood transfusion. Preemptive IV iron therapy provides noninferior hemoglobin levels on postoperative day (POD) 1 compared to autologous whole blood therapy (AWBT) in healthy patients who had undergone bimaxillary orthognathic surgery. METHODS: This was a prospective, patient-randomized, noninferiority trial. After excluding 2 patients, 64 patients were divided into two groups: the IV iron therapy group (patients received IV iron infusion 4 weeks before surgery; n = 32) and the AWBT group (2 units of autologous whole blood were collected 4 and 2 weeks before surgery; n = 32). The primary outcome was hemoglobin level on POD 1 and the prespecified noninferiority limit was - 1 g/dL. RESULTS: Baseline data were comparable, including hemoglobin and iron levels, between the two groups. Immediately before surgery, the levels of hemoglobin, iron, and ferritin were higher in the IV iron group than in the AWBT group. The mean treatment difference (iron group-whole blood group) in hemoglobin level on POD 1 between the two groups was 0.09 (95% CI = - 0.83 to 1.0). As the lower limit of the 95% CI (- 0.83) was higher than the prespecified noninferiority margin (δ = - 1), noninferiority was established. On POD 2, the hemoglobin level became lower in the iron group, which eventually led to greater requirement of allogeneic blood transfusion compared to the whole blood group. However, the iron group did not require allogeneic blood transfusion during or early after surgery, and the whole blood group showed continuously higher incidence of overt iron deficiency compared to the iron group. CONCLUSION: As collection of autologous whole blood caused overt iron loss and anemia before surgery and intraoperative transfusion of whole blood was not able to prevent the occurrence of persistent iron deficiency after surgery, IV iron therapy was found to have potential benefits for iron homeostasis and subsequent erythropoiesis in healthy patients early after bimaxillary orthognathic surgery. TRIAL REGISTRATION: Clinical Research Information Service, Republic of Korea, approval number: KCT0003680 on March 27, 2019. https://cris.nih.go.kr/cris/search/search_result_st01_kren.jsp?seq=15769&sLeft=2<ype=my&rtype=my .

Effects of birth months on rheumatic diseases in South Korea: a nationwide case-control study.

OBJECTIVES: Birth month/season impacts the development of certain diseases. However, the effect of birth month/season on the development of rheumatic diseases has not been thoroughly investigated. Thus, the objective of this study was to determine whether birth month/season might affect the development of rheumatic diseases. METHODS: Birth month patterns of patients with various rheumatic diseases were compared with those of the general population. The dataset included 17,247,458 individuals from the health insurance review and assessment service database of Korea. RESULTS: Among 24 rheumatic diseases, the development of Crohn's disease, ulcerative colitis (UC), rheumatoid arthritis, Sjögren's syndrome, polymyalgia rheumatica, ankylosing spondylitis (AS), gout, and fibromyalgia (FM) was significantly associated with birth month/season. UC and AS were more prevalent in individuals born in February/winter. On the contrary, those who were born in June or July/summer were at a higher risk of gout and FM. CONCLUSIONS: Seasonal variations in infectious agents, sun exposure, and food ingestion during gestation or early infancy seem to explain the association between birth month/season and development of rheumatic diseases.

Neurobehavioral recovery in patients who emerged from prolonged disorder of consciousness: a retrospective study.

BACKGROUND: We investigated the clinical course of patients with prolonged disorders of consciousness (PDoC), predictors of emergence from PDoC (EDoC), and the temporal dynamics of six neurobehavior domains based on the JFK Coma Recovery Scale-Revised (CRS-R) during the recovery. METHODS: A total of 50 traumatic and non-traumatic patients with PDoC were enrolled between October 2014 and February 2017. A retrospective analysis of the clinical findings and neurobehavioral signs was conducted using standardized methodology such as CRS-R. The findings were used to investigate the incidence and predictors of EDoC and determine the cumulative pattern of neurobehavioral recovery at 6 months, 1 year, and 2 years post-injury. RESULTS: The results showed that 46% of the subjects emerged from PDoC after 200 median days (64-1197 days) of injury onset. The significant predictors of EDoC included minimally conscious state (MCS) (vs. vegetative state), higher auditory, communication, arousal, total CRS-R scores, shorter lag time post-injury, and the absence of intra-axial lesions. In terms of cumulative recovery of motor and communication signs in patients who emerged from PDoC, 39 and 32% showed EDoC at 6 months post-injury, and 88 and 93% exhibited EDoC at 2 years post-injury, respectively. CONCLUSIONS: Nearly half of the patients with PDoC recovered consciousness during inpatient rehabilitation. MCS, shorter lag time, the absence of intra-axial lesions, higher auditory, communication, arousal, and total CRS-R scores were important predictors for EDoC. Motor scores in the early stage of recovery and communication scores after prolonged intervals contributed to the higher levels of cumulative EDoC.

Self- and informant-reported cognitive functioning and awareness in subjective cognitive decline, mild cognitive impairment, and very mild Alzheimer disease.

OBJECTIVES: The present study examined self-reports and informant reports of cognitive function and discrepancies between the two reporting methods in healthy controls (HC), subjective cognitive decline (SCD), mild cognitive impairment (MCI), and very mild Alzheimer disease (AD) using three questionnaires. METHODS: The study included a total of 300 individuals (mean age: 74.4 ± 5.7 y), including 130 HC, 70 SCD, 51 MCI, and 49 very mild AD patients. Self-ratings and informant ratings of cognitive function were assessed using the Korean Dementia Screening Questionnaire-Cognition (KDSQ-C), AD8, and Subjective Memory Complaints Questionnaire (SMCQ). Awareness of cognitive functioning was measured on the basis of the discrepancy scores between self-reports and informant reports. RESULTS: Group comparisons on questionnaire scores adjusting for age, education, and depressive symptoms showed that self-reports were lowest in HC than other groups, with no differences between SCD and MCI groups. Informant reports were lower in SCD than in MCI, while discrepancy scores were higher in SCD than in MCI (P < .001 for KDSQ-C and SMCQ; P = .076 for AD8). There were no differences in self-reports, informant reports, and discrepancy scores between MCI and AD groups. CONCLUSIONS: These results support the usefulness of informant-reported cognitive functioning to classify MCI among elderly with subjective cognitive complaints. In addition, discrepancies between self-reports and informant reports demonstrate that overestimation and underestimation of cognitive function may serve as a clinical indicator of SCD and MCI across the cognitive continuum, respectively.

Delayed remnant kidney function recovery is less observed in living donors who receive an analgesic, intrathecal morphine block in laparoscopic nephrectomy for kidney transplantation: a propensity score-matched analysis.

BACKGROUND: This study analyzed remnant kidney function recovery in living donors after laparoscopic nephrectomy to establish a risk stratification model for delayed recovery and further investigated clinically modifiable factors. PATIENTS AND METHODS: This retrospective study included 366 adult living donors who underwent elective donation surgery between January 2017 and November 2019 at our hospital. ITMB was included as an analgesic component in the living donor strategy for early postoperative pain relief from November 2018 to November 2019 (n = 116). Kidney function was quantified based on the estimated glomerular filtration rate (eGFR), and delayed functional recovery of remnant kidney was defined as eGFR < 60 mL/min/1.73 m2 on postoperative day (POD) 1 (n = 240). RESULTS: Multivariable analyses revealed that lower risk for development of eGFR < 60 mL/min/1.73 m2 on POD 1 was associated with ITMB, female sex, younger age, and higher amount of hourly fluid infusion (area under the receiver operating characteristic curve = 0.783; 95% confidence interval = 0.734-0.832; p < 0.001). Propensity score (PS)-matching analyses showed that prevalence rates of eGFR < 60 mL/min/1.73 m2 on PODs 1 and 7 were higher in the non-ITMB group than in the ITMB group. ITMB adjusted for PS was significantly associated with lower risk for development of eGFR < 60 mL/min/1.73 m2 on POD 1 in PS-matched living donors. No living donors exhibited severe remnant kidney dysfunction and/or required renal replacement therapy at POD 7. CONCLUSIONS: We found an association between the analgesic impact of ITMB and better functional recovery of remnant kidney in living kidney donors. In addition, we propose a stratification model that predicts delayed functional recovery of remnant kidney in living donors: male sex, older age, non-ITMB, and lower hourly fluid infusion rate.

Intraoperative changes in whole-blood viscosity in patients undergoing robot-assisted laparoscopic prostatectomy in the steep Trendelenburg position with pneumoperitoneum: a prospective nonrandomized observational cohort study.

BACKGROUND: The aim of this study was to investigate the effect of the steep Trendelenburg position (STP) with pneumoperitoneum on whole-blood viscosity (WBV) in patients undergoing robot-assisted laparoscopic prostatectomy (RALP). The study also analyzed the associations of clinical patient-specific and time-dependent variables with WBV and recorded postoperative outcomes. METHODS: Fifty-eight adult male patients (ASA physical status of I or II) undergoing elective RALP were prospectively analyzed in this study. WBV was intraoperatively measured three times: at the beginning of surgery in the supine position without pneumoperitoneum; after 30 min in the STP with pneumoperitoneum; and at the end of surgery in the supine position without pneumoperitoneum. The WBV at a high shear rate (300 s- 1) was recorded as systolic blood viscosity (SBV) and that at a low shear rate (5 s- 1) was recorded as diastolic blood viscosity (DBV). Systolic blood hyperviscosity was defined as > 13.0 cP at 300 s- 1 and diastolic blood hyperviscosity was defined as > 4.1 cP at 5 s- 1. RESULTS: The WBV and incidences of systolic and diastolic blood hyperviscosity significantly increased from the supine position without pneumoperitoneum to the STP with pneumoperitoneum. When RALP was performed in the STP with pneumoperitoneum, 12 patients (27.3%) who had normal SBV at the beginning of surgery and 11 patients (26.8%) who had normal DBV at the beginning of surgery developed new systolic and diastolic blood hyperviscosity, respectively. The degree of increase in WBV after positioning with the STP and pneumoperitoneum was higher in the patients with hyperviscosity than in those without hyperviscosity at the beginning of surgery. Higher preoperative body mass index (BMI) and hematocrit level were associated with the development of both systolic and diastolic blood hyperviscosity in the STP with pneumoperitoneum. All patients were postoperatively discharged without fatal complications. CONCLUSIONS: Changes in surgical position may influence WBV, and higher preoperative BMI and hematocrit level are independent factors associated with the risk of hyperviscosity during RALP in the STP with pneumoperitoneum. TRIAL REGISTRATION: Clinical Research Information Service, Republic of Korea, approval number: KCT0003295 on October 25, 2018.

Spoligotype Variation of Mycobacterium tuberculosis Strains Prevailing in Korea.

Tuberculosis (TB) is an ongoing global health problem, including in South Korea. To manage TB efficiently, it is necessary to understand the epidemiology, transmission route, and characteristics of prevailing Mycobacterium tuberculosis strains. In this study, we investigated microevolutions over time in the spoligotype patterns of M. tuberculosis isolated from TB patients in Korea. We collected 1,055 clinical M. tuberculosis isolates from 16 provinces in Korea from 1994 to 2006 and analyzed them by spoligotyping. We observed 26 subfamilies, including two large predominant families: a Beijing family (72.7%) and the T family (19.1%). Specifically, the abundance of spoligotype SIT269 from the Beijing-like subfamily significantly increased in the 2000s relative to the 1990s in Korea. This study provides an overview of the M. tuberculosis genotype trends over time in Korea. These data also indicate that we should consider the influence of the newly growing SIT269 subtype identified in the Beijing family.

A Comparative Evaluation of the KDSQ-C, AD8, and SMCQ as a Cognitive Screening Test to Be Used in National Medical Check-ups in Korea.

BACKGROUND: Korea has a periodic general health check-up program that uses the Korean Dementia Screening Questionnaire-Cognition (KDSQ-C) as a cognitive dysfunction screening tool. The Alzheimer Disease 8 (AD8) and Subjective Memory Complaints Questionnaire (SMCQ) are also used in clinical practice. We compared the diagnostic ability of these screening questionnaires for cognitive impairment when completed by participants and their caregivers. Hence, we aimed to evaluate whether the SMCQ or AD8 is superior to the KDSQ-C and can be used as its replacement. METHODS: A total of 420 participants over 65 years and their informants were recruited from 11 hospitals for this study. The patients were grouped into normal cognition, mild cognitive impairment, and dementia subgroups. The KDSQ-C, AD8, and SMCQ were completed separately by participants and their informants. RESULTS: A receiver operating characteristic analysis of questionnaire scores completed by participants showed that the areas under the curve (AUCs) for the KDSQ-C, AD8, and SMCQ for diagnosing dementia were 0.75, 0.8, and 0.73, respectively. Regarding informant-completed questionnaires, the AD8 (AUC of 0.93), KDSQ-C (AUC of 0.92), and SMCQ (AUC of 0.92) showed good discriminability for dementia, with no differences in discriminability between the questionnaires. CONCLUSION: When an informant-report is possible, we recommend that the KDSQ-C continues to be used in national medical check-ups as its discriminability for dementia is not different from that of the AD8 or SMCQ. Moreover, consistent data collection using the same questionnaire is important. When an informant is not available, either the KDSQ-C or AD8 may be used. However, in the cases of patient-reports, discriminability is lower than that for informant-completed questionnaires.

PTK7 regulates radioresistance through nuclear factor-kappa B in esophageal squamous cell carcinoma.

Tumor radioresistance is a major reason for decreased efficiency of cancer radiation therapy. Although a number of factors involved in radioresistance have been identified, the molecular mechanisms underlying radioresistance of esophageal squamous cell carcinoma (ESCC) have not been elucidated. In this study, we investigated the role of oncogenic protein tyrosine kinase 7 (PTK7) in the resistance of ESCC to radiation therapy. ESCC cell lines with high PTK7 expression were more refractive to radiation than those with low PTK7 levels. In radioresistant ESCC cells, PTK7 knockdown by specific siRNAs decreased the survival of irradiated cells and increased radiation-induced apoptosis, while in radiosensitive ESCC cells, PTK7 overexpression promoted cell survival and inhibited radiation-induced apoptosis. We hypothesized that PTK7 could regulate the activation of transcription factor NF-kB known for its role in cancer radioresistance. Our results indicated that the inhibition of PTK7 suppressed nuclear translocation of NF-kB subunit p65 induced by radiation, suggesting relevance of PTK7 expression with NF-kB activation in radioresistant ESCC. Furthermore, the levels of inhibitor of apoptosis proteins (IAPs), XIAP, and survivin, encoded by NF-kB-regulated genes, were induced in irradiated radioresistant cells but not in radiosensitive cells, while PTK7 knockdown downregulated IAP expression. Our findings revealed a novel mechanism underlying radioresistance in ESCC, which is associated with PTK7 and NF-kB-dependent apoptosis. These results suggest that the manipulation of PTK7 expression can be instrumental in enhancing ESCC response to radiotherapy. This study demonstrates that PTK7 plays a significant role in ESCC radioresistance via the NF-kB pathway.

Epigenetic regulation of the novel tumor suppressor cysteine dioxygenase 1 in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC), the most common subtype of esophageal cancer in East Asian countries, is still associated with a poor prognosis because of the high frequency of lymph node metastasis and invasion. In our previous study, we identified a novel methylation gene, cysteine dioxygenase 1 (CDO1) that is involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. Decreased expression of CDO1 was observed in ESCC cell lines and tumors derived from patient tissues, and CDO1 silencing could be reversed by treatment with 5-aza-2'-deoxycytidine in six ESCC cell lines. Forced expression of CDO1 in three different ESCC cell lines, TE-4, TE-6, and TE-14, significantly decreased tumor cell growth, cell migration, invasion, and the ability of colony formation. Although CDO1 expression was not found to significantly correlate with survival in ESCC patients, our results suggest that methylation-regulated CDO1 may represent a functional tumor suppressor and a potentially valuable diagnostic biomarker for ESCC.

Type 5 adenylyl cyclase increases oxidative stress by transcriptional regulation of manganese superoxide dismutase via the SIRT1/FoxO3a pathway.

BACKGROUND: For reasons that remain unclear, whether type 5 adenylyl cyclase (AC5), 1 of 2 major AC isoforms in heart, is protective or deleterious in response to cardiac stress is controversial. To reconcile this controversy we examined the cardiomyopathy induced by chronic isoproterenol in AC5 transgenic (Tg) mice and the signaling mechanisms involved. METHODS AND RESULTS: Chronic isoproterenol increased oxidative stress and induced more severe cardiomyopathy in AC5 Tg, as left ventricular ejection fraction fell 1.9-fold more than wild type, along with greater left ventricular dilation and increased fibrosis, apoptosis, and hypertrophy. Oxidative stress induced by chronic isoproterenol, detected by 8-OhDG was 15% greater, P=0.007, in AC5 Tg hearts, whereas protein expression of manganese superoxide dismutase (MnSOD) was reduced by 38%, indicating that the susceptibility of AC5 Tg to cardiomyopathy may be attributable to decreased MnSOD expression. Consistent with this, susceptibility of the AC5 Tg to cardiomyopathy was suppressed by overexpression of MnSOD, whereas protection afforded by the AC5 knockout (KO) was lost in AC5 KO×MnSOD heterozyous KO mice. Elevation of MnSOD was eliminated by both sirtuin and MEK inhibitors, suggesting both the SIRT1/FoxO3a and MEK/ERK pathway are involved in MnSOD regulation by AC5. CONCLUSIONS: Overexpression of AC5 exacerbates the cardiomyopathy induced by chronic catecholamine stress by altering regulation of SIRT1/FoxO3a, MEK/ERK, and MnSOD, resulting in oxidative stress intolerance, thereby shedding light on new approaches for treatment of heart failure.

Characterization of gene expression and activated signaling pathways in solid-pseudopapillary neoplasm of pancreas.

Solid-pseudopapillary neoplasm is an uncommon pancreatic tumor with distinct clinicopathologic features. Solid-pseudopapillary neoplasms are characterized by mutations in exon 3 of CTNNB1. However, little is known about the gene and microRNA expression profiles of solid-pseudopapillary neoplasms. Thus, we sought to characterize solid-pseudopapillary neoplasm-specific gene expression and identify the signaling pathways activated in these tumors. Comparisons of gene expression in solid-pseudopapillary neoplasm to pancreatic ductal carcinomas, neuroendocrine tumors, and non-neoplastic pancreatic tissues identified solid-pseudopapillary neoplasm-specific mRNA and microRNA profiles. By analyzing 1686 (1119 upregulated and 567 downregulated) genes differentially expressed in solid-pseudopapillary neoplasm, we found that the Wnt/ß-catenin, Hedgehog, and androgen receptor signaling pathways, as well as genes involved in epithelial mesenchymal transition, are activated in solid-pseudopapillary neoplasms. We validated these results experimentally by assessing the expression of ß-catenin, WIF-1, GLI2, androgen receptor, and epithelial-mesenchymal transition-related markers with western blotting and immunohistochemistry. Our analysis also revealed 17 microRNAs, especially the miR-200 family and miR-192/215, closely associated with the upregulated genes associated with the three pathways activated in solid-pseudopapillary neoplasm and epithelial mesenchymal transition. Our results provide insight into the molecular mechanisms underlying solid-pseudopapillary neoplasm tumorigenesis and its characteristic less epithelial cell differentiation than the other common pancreatic tumors.

The effectiveness of a suicide prevention program in primary care clinics supported by community public health resources: A difference-in-differences analysis.

The importance of appropriate and intensive follow-up management for individuals identified with suicide risk through screening is highlighted. The Link between Primary Care Clinic and Public Health Resources Intervention(LinkPC-PH), a suicide prevention program in primary care clinics supported by community public health resources, was implemented at the district level in 2017. The purpose of the present study is to evaluate the effectiveness of the LinkPC-PH intervention by comparing suicide rates before(2014-2016) and after(2017-2019) implementation of the intervention using a difference-in-differences design. The LinkPC-PH comprises several dimensions of intervention including screening, risk assessment of suicidality, and referral in primary care clinics and crisis contact within 24 hours, case management, and safety planning led by public health professionals. After adjustment for district-level confounders, an intervention-implemented district had 2.87 fewer suicide deaths per 100,000 people in a population sample at post-intervention than would have been expected from the same trend in suicide rates as non-implemented intervention districts. In other words, the suicide rate in the intervention area decreased by 25% following the intervention. These results empirically substantiate suicide prevention programs in primary care clinics by community public health resources for reduced suicide rates to support effective community-based suicide prevention interventions.

Adenylyl cyclase type 5 in cardiac disease, metabolism, and aging.

G protein-coupled receptor/adenylyl cyclase (AC)/cAMP signaling is crucial for all cellular responses to physiological and pathophysiological stimuli. There are nine isoforms of membrane-bound AC, with type 5 being one of the two major isoforms in the heart. Since the role of AC in the heart in regulating cAMP and acute changes in inotropic and chronotropic state are well known, this review will address our current understanding of the distinct regulatory role of the AC5 isoform in response to chronic stress. Transgenic overexpression of AC5 in cardiomyocytes of the heart (AC5-Tg) improves baseline cardiac function but impairs the ability of the heart to withstand stress. For example, chronic catecholamine stimulation induces cardiomyopathy, which is more severe in AC5-Tg mice, mediated through the AC5/sirtuin 1/forkhead box O3a pathway. Conversely, disrupting AC5, i.e., AC5 knockout, protects the heart from chronic catecholamine cardiomyopathy as well as the cardiomyopathies resulting from chronic pressure overload or aging. Moreover, AC5 knockout results in a 30% increase in a healthy life span, resembling the most widely studied model of longevity, i.e., calorie restriction. These two models of longevity share similar gene regulation in the heart, muscle, liver, and brain in that they are both protected against diabetes, obesity, and diabetic and aging cardiomyopathy. A pharmacological inhibitor of AC5 also provides protection against cardiac stress, diabetes, and obesity. Thus AC5 inhibition has novel, potential therapeutic applicability to several diseases not only in the heart but also in aging, diabetes, and obesity.

Novel mechanisms for caspase inhibition protecting cardiac function with chronic pressure overload.

Myocyte apoptosis is considered a major mechanism in the pathogenesis of heart failure. Accordingly, manipulations that inhibit apoptosis are assumed to preserve cardiac function by maintaining myocyte numbers. We tested this assumption by examining the effects of caspase inhibition (CI) on cardiac structure and function in C57BL/6 mouse with pressure overload model induced by transverse aortic constriction (TAC). CI preserved left ventricular (LV) function following TAC compared with the vehicle. TAC increased apoptosis in non-myocytes more than in myocytes and these increases were blunted more in non-myocytes by CI. Total myocyte number, however, did not differ significantly among control and TAC groups and there was no correlation between myocyte number and apoptosis, but there was a strong correlation between myocyte number and an index of myocyte proliferation, Ki67-positive myocytes. Despite comparable pressure gradients, LV hypertrophy was less in the CI group, likely attributable to decreased wall stress. Since changes in myocyte numbers did not account for protection from TAC, several other CI-mediated mechanisms were identified including: (a) lessening of TAC-induced fibrosis, (b) augmentation of isolated myocyte contractility, and (c) increased angiogenesis and Ki67-positive myocytes, which were due almost entirely to the non-myocyte apoptosis, but not myocyte apoptosis, with CI. CI maintained LV function following TAC not by protecting against myocyte loss, but rather by augmenting myocyte contractile function, myocyte proliferation, and angiogenesis resulting in reduced LV wall stress, hypertrophy, and fibrosis.

Rapid and sensitive LC-MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study.

A rapid, simple and fully validated LC-MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one-step liquid-liquid extraction with methyl-tert-butyl-ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C18 column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)-methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal-to-noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1-2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra- and inter-day precision and accuracy, recovery, matrix effect and stability. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace™) to five healthy Korean male volunteers under fed conditions.