Cooperative Carbene Photocatalysis for ß-Amino Ester Synthesis.

ß-Amino acids are useful building blocks of bioactive molecules, including peptidomimetics and pharmaceutical compounds. The current limited accessibility to ß2,2-type amino acids which bear an α-quaternary center has limited their use in chemical synthesis and biological investigations. Disclosed herein is the development of a new N-heterocyclic carbene/photocatalyzed aminocarboxylation of olefins, affording ß2,2-amino esters with high regioselectivity. The generation of nitrogen-centered radicals derived from simple imides via a sequence of deprotonation and single-electron oxidation allows for the subsequent addition to geminal-disubstituted olefins regioselectively. The intermediate tertiary radicals then cross-couple with a stabilized azolium-based radical generated in situ to efficiently construct the quaternary centers. Mechanistic studies, including Stern-Volmer fluorescence quenching experiments, support the proposed catalytic cycle.

Chondroprotective effects of CDK4/6 inhibition via enhanced ubiquitin-dependent degradation of JUN in synovial fibroblasts.

OBJECTIVE: Targeting synovial fibroblasts (SF) using a cyclin-dependent kinase (CDK) 4/6 inhibitor (CDKI) could be a potent therapy for RA via inhibition of proliferation and MMP-3 production. This study was designed to elucidate the mechanism of chondroprotective effects on SFs by CDK 4/6 inhibition. METHODS: CDK4/6 activity was inhibited using CDKI treatment or enhanced by adenoviral gene transduction. Chondroprotective effects were evaluated using a collagen-induced arthritis model (CIA). Gene and protein expression were evaluated with quantitative PCR, ELISA and Western blotting. The binding of nuclear extracts to DNA was assessed with an electrophoresis mobility shift assay. RNA-Seq was performed to identify gene sets affected by CDKI treatment. RESULTS: CDKI attenuated cartilage destruction and MMP-3 production in CIA. In RASFs, CDKI impaired the binding of AP-1 components to DNA and inhibited the production of MMP-1 and MMP-3, which contain the AP-1 binding sequence in their promoter. CDK4/6 protected JUN from proteasome-dependent degradation by inhibiting ubiquitination. The RNA-Seq analysis identified CDKI-sensitive inflammatory genes, which were associated with the pathway of RA-associated genes, cytokine-cytokine receptor interaction and IL-17 signalling. Notably, the AP-1 motif was enriched in these genes. CONCLUSION: The mechanism of chondroprotective effects by CDK4/6 inhibition was achieved by the attenuation of AP-1 transcriptional activity via the impaired stability of JUN. Because the pharmacologic inhibition of CDK4/6 has been established as tolerable in cancer treatment, it could also be beneficial in patients with RA due to its chondroprotective and anti-inflammatory effects.

Bifidobacterium breve during infancy attenuates mobility in low birthweight rats.

BACKGROUND: Children with low birthweight (LBW) have a higher risk for developing attention-deficit/hyperactivity disorder, for which no prophylactic measure exists. The gut microbiota in infants with LBW is different from that in infants with normal birthweight and is associated with attention-deficit/hyperactivity disorder. Oral supplementation with Bifidobacterium has several health benefits, such as suppressing inflammation. METHODS: We examined the effect of gavage supplementation with Bifidobacterium breve M-16V from postnatal days 1-21 in a rat model of intrauterine hypoperfusion. RESULTS: The open-field test at 5 weeks of age (equivalent to human pubertal age) showed that rats in the LBW-vehicle group were marginally hyperactive compared with rats in the sham group, while rats in the LBW-B.breve group were significantly hypoactive compared with rats in the LBW-vehicle group. The gut microbiota in the LBW-vehicle group exhibited a profile significantly different from that in the sham group, whereas the gut microbiota in the LBW-B.breve group did not exhibit a significant difference from that in the sham group. Anatomical/histological evaluation at 6 weeks of age demonstrated that the brain weight and the cerebral areas on coronal sections were reduced in the LBW groups compared with the sham group. Probiotic supplementation did not ameliorate these morphological brain anomalies in LBW animals. The percentage of Iba-1+ cells in the brain was not different among the LBW-B.breve, LBW-vehicle, and sham groups. CONCLUSION: Bifidobacterium breve supplementation during early life is suggested to have the potential to help children with LBW attenuate hypermobility in adolescence.

Eight novel susceptibility loci and putative causal variants in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is the most common allergic disease in the world. While genetic components play critical roles in its pathophysiology, a large proportion of its genetic background is still unexplored. OBJECTIVES: This study sought to illuminate the genetic associations with AD using genome-wide association study (GWAS) and its downstream analyses. METHODS: This study conducted a GWAS for AD comprising 2,639 cases and 115,648 controls in the Japanese population, followed by a trans-ethnic meta-analysis with UK Biobank data and downstream analyses including partitioning heritability analysis by linkage disequilibrium score regression. RESULTS: This study identified 17 significant susceptibility loci, among which 4 loci-AFF1, ITGB8, EHMT1, and EGR2-were novel in the Japanese GWAS. The trans-ethnic meta-analysis revealed 4 additional novel loci, namely-ZBTB38,LOC105755953/LOC101928272, TRAF3, andIQGAP1. This study found a missense variant (R243W) with a deleterious functional effect in NLRP10 and a variant altering expression of CCDC80 via enhancer expression as highly likely causal variants. These 2 regions were Asian-specific, and these population-specific associations could be explained by the frequency of causal variants. The gene-based test showed SMAD4 as an additional novel significant locus. Downstream analyses revealed substantial overlap of GWAS significant signals in enhancers of skin cells and immune cells, especially CD4 T cells. A highly shared polygenic architecture of AD between Europeans and Asians was also found. CONCLUSIONS: This study identified Japanese-specific loci and novel significant loci shared by different populations. Two putative causal variants were illuminated in Japanese-specific loci. Trans-ethnic analyses revealed strong heritability enrichment in immune-related pathways, and relevant cell types shared among populations.

Chichibabin/isoChichibabin pyridinium synthesis of ma'edamines C and D.

Ma'edamines C and D were isolated from an Okinawan marine sponge and exhibited a unique tetrasubstituted pyridinium skeleton. The proposed biosynthetic pathway is similar to that of desmosine and isodesmosine, which are elastin-crosslinking amino acids. In this study, first total synthesis of ma'edamines C and D was achieved via Pr(OTf)3-promoted Chichibabin/isoChichibabin pyridinium synthesis starting from the corresponding aldehydes and amine.

IsoChichibabin desmosine-13C3,15N1 synthesis and quantitative LC-MS/MS analysis of desmosine and isodesmosine in human skin.

Desmosine and isodesmosine are crosslinking amino acids of elastin, which is an essential component of the dermal extracellular matrix protein. Quantitative analysis of crosslinker desmosines in human skin dermis has not been fully achieved due to the insoluble nature of elastin protein. In the present study, chemical synthesis of isotopically labeled desmosine, desmosine-13C3,15N1, was carried out via isoChichibabin pyridinium synthesis starting from corresponding isotopically labeled amino acids. Isotope-dilution LC-MS/MS analysis of desmosine and isodesmosine utilizing synthetic desmosine-13C3,15N1 enabled the quantitative analysis of desmosines in human skin for the first time. Thus, ca. 1.43 µg of desmosines was detected from analysis of 1 mg of dry human skin.

Safety of vancomycin in patients with moderate and severe renal dysfunction.

AIM: The new guidelines in Japan do not recommend a vancomycin (VCM) loading dose for patients with an estimated glomerular filtration rate (eGFR) 30 < and ≤ 80 mL×min-1×1.73m-2 (moderate renal dysfunction) or administration to those with the eGFR < 30 mL×min-1×1.73m-2 (severe renal dysfunction). We investigated the safety and efficiency of VCM in patients with moderate and severe renal dysfunction based on the new guidelines. MATERIALS AND METHODS: The study involved patients admitted to our hospital between April 2014 and March 2018 with an eGFR < 80 mL×min-1×1.73m-2 and treated with VCM. VCM trough concentration and pre- and post-administration renal function were investigated retrospectively. The primary endpoints were the proportion of patients who achieved an effective trough concentration of 10 - 20 µg/mL and rate of acute kidney injury (AKI). RESULTS: We included 64 patients (32 moderate, 32 severe). The mean VCM trough concentration achieved for the first time was 9.3 and 11.6 µg/mL in the moderate and severe renal dysfunction groups, respectively (p = 0.91). The effective trough concentration endpoint was achieved by 50% and 43% of the patients in the severe and moderate renal dysfunction groups, respectively, and no significant difference was found in the AKI rate. The serum creatinine change was significantly different between the groups - the moderate group showed a slight deterioration and the severe renal dysfunction group an improvement. CONCLUSION: It may be necessary to increase the dose for these patients with severe renal dysfunction while implementing a VCM loading dose and monitoring trough concentrations and adverse effects.

Dermatomyositis associated with prostate adenocarcinoma with neuroendocrine differentiation.

BACKGROUND: Although it is known that malignancies can be associated with dermatomyositis, there are few reports on dermatomyositis associated with prostate cancer with neuroendocrine differentiation. CASE PRESENTATION: A 63-year-old man visited our hospital due to pollakiuria. High levels of PSA and NSE were observed, and prostate biopsy revealed an adenocarcinoma with neuroendocrine differentiation. Multiple metastases to the lymph nodes, bones, and liver were identified, and androgen deprivation therapy (ADT) was started immediately. Following 2 weeks of treatment, erythema on the skin, and muscle weakness with severe dysphagia appeared. The patient was diagnosed with dermatomyositis, and high-dose glucocorticoid therapy was initiated. ADT and subsequent chemotherapy with etoposide and cisplatin (EP) were performed for prostate cancer, which resulted in decreased PSA and NSE and reduction of all metastases. After the initiation of EP therapy, dermatomyositis improved, and the patient regained oral intake function. Although EP therapy was replaced by docetaxel, abiraterone, and enzalutamide because of adverse events, no cancer progression was consistently observed. Dermatomyositis worsened temporarily during the administration of abiraterone, but it improved upon switching from abiraterone to enzalutamide and dose escalation of glucocorticoid. CONCLUSIONS: We successfully treated a rare case of dermatomyositis associated with prostate adenocarcinoma with neuroendocrine differentiation.

Preparation of isodesmosine-KLH conjugate for ELISA system.

Chronic obstructive pulmonary disease (COPD) is a degenerative condition with limited diagnostic detection efficiency. Currently with no available cure, COPD is associated with irreversible elastic tissue degradation in lungs, which results in release of unusual amino acids, isodesmosine and desmosine. These biomarkers are potential key elements in enzyme-linked immunosorbent assay (ELISA), an analytical method, which can detect certain compounds including antigens and proteins in easy and affordable manner. In order to target a biomarker with ELISA, it is necessary to prepare its specific antibody, which can be achieved by immunization of host organism with appropriate antigen containing the biomarker. Although preparation of these types of conjugates has been published, desmosine and isodesmosine used by researchers are obtained from natural sources such as animal tissues. Here, we report the first synthetic preparation of isodesmosine and keyhole limpet hemocyanin (KLH) conjugate from commercially available chiral amino acids and carrier protein. Formation of the core pyridinium of isodesmosine was achieved through key reaction-Chichibabin pyridinium synthesis-to deliver a 1,2,3,5-tetrasubstituted pyridinium amino acid selectively. Further modifications involving KLH and maleimide linker provided the target conjugate, which could potentially invoke an immune response to produce anti-isodesmosine antibody for the ELISA system.

A clinical review of ovarian tumors in children and adolescents.

PURPOSE: To determine the features which predict torsion and the pre-operative indicators of malignancy in cases of ovarian torsion in ovarian tumors (OTs) in children. METHODS: The medical records of 35 pediatric patients who underwent surgery for OT, except for neonate cases, from 1997 to 2018 at our institution were reviewed retrospectively. RESULTS: The pathological diagnosis was mature teratoma in 17, immature teratoma in 9, yolk sac tumor in 3, and others in 6. The preoperative diagnosis, which was made based on the imaging findings and the serum tumor marker values, matched with the pathological diagnosis in 29/35 (83%). Ovarian torsion occurred in 14/35 (40%). All but one case that presented with torsion had intermittent abdominal pain as the primary symptom. The preoperative white blood cell count was significantly higher in cases where ovary preservation was impossible than where it was possible (p = 0.01) among the cases presenting with torsion. CONCLUSION: Preoperative imaging findings and the serum tumor marker values enabled us to make an accurate preoperative diagnosis. Patients with intermittent abdominal primary symptoms were more likely to have ovarian torsion than those without such symptoms, and leukocytosis may indicate irreversible ischemic changes in the affected ovary.

Evaluation of antitumor effects of folate-conjugated methyl-ß-cyclodextrin in melanoma.

Melanoma is a life-threatening disorder and its incidence is increasing gradually. Despite the numerous treatment approaches, conventional systemic chemotherapy has not reduced the mortality rate among melanoma patients, probably due to the induction of toxicity to normal tissues. Recently, we have developed folate-conjugated methyl-ß-cyclodextrin (FA-M-ß-CyD) and clarified its potential as a new antitumor agent involved in autophagic cell death. However, it remains uncertain whether FA-M-ß-CyD exerts anticancer effects against melanomas. Therefore, in this study, we investigated the effects of FA-M-ß-CyD on the folate receptor-α (FR-α)-expressing melanoma cell-selective cytotoxic effect. FA-M-ß-CyD showed cytotoxic effects in Ihara cells, a human melanoma cell line expressing FR-α. In sharp contrast to methyl-ß-cyclodextrin, FA-M-ß-CyD entered Ihara cells [FR-α(+)] through FR-α-mediated endocytosis. Additionally, FA-M-ß-CyD elicited the formation of autophagosomes in Ihara cells. Notably, FA-M-ß-CyD suppressed melanoma growth in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double deficient mice bearing Ihara cells. Therefore, these results suggest that FA-M-ß-CyD could be utilized as a potent anticancer agent for melanoma chemotherapy by regulating autophagy.

Career development expectations and challenges of midwives in Urban Tanzania: a preliminary study.

BACKGROUND: Approaches to addressing the shortage of midwives are a great need especially in Sub-Saharan Africa including Tanzania. The midwifery shortage in Tanzania consists of two major causes; the first is the shortage of pre-service nursing training and the second is the low rate of retention as it is difficult to sustain midwives' career motivations. Lack of opportunities for career development, is one of the most related problems to keep midwives motivated. Continuing education as an approach to career development can heighten midwives' motivation and cultivate more skilled midwives who can educate other midwives or students and who could raise the status of midwives. Effective continuing education is ongoing, interactive, contextually relevant and based on needs assessment, however there is very limited research that describes Tanzanian midwives perspective of expectations for career development; hence this research is significant for revealing important and meaningful professional desires of midwives in Tanzania. METHODS: This was a preliminary qualitative study, using snowball sampling to recruit 16 midwives in Tanzania. The researchers used a semi-structured interview including probing questions with both a focus group and several individuals. The data were collected from July to December 2013 and coded into categories and sub-categories. RESULTS: There were 14 midwives in the focus group interview and two midwives in the individual interviews. Through data analysis, four major categories (with subcategories) emerged: (1) motivation for learning (to achieve the MDGs, and to raise reproductive health), (2) knowledge is power (to provide good practice based on knowledge, to be a role model, knowledge gives higher position and courage, and knowledge enables one to approach to the government), (3) there is no end to learning (hunger for learning, and ripple effect). CONCLUSIONS: From findings, four major categories plainly showed midwives' desire for learning, however they experienced a number of barriers to access further education. Continuing education is one of the most important and effective ways to cultivate and retain midwives. In order to respond to the midwives expectations and challenges to overcome the barriers inherent in providing more continuing education, it will be necessary to increase accessible opportunities for career development in Tanzania.

Extensive-Disease Small-Cell Lung Cancer With Severe Immune-Related Adverse Events Due to Atezolizumab Maintaining a Complete Response for Two Years: A Case Report.

A 75-year-old male with diabetes mellitus was referred to our hospital with an abnormal shadow on chest radiography, based on which he was diagnosed with extensive-disease small-cell lung cancer (ED-SCLC; cT2bN2M1a). The first-line therapy comprised atezolizumab, carboplatin, and etoposide. After four cycles, the patient achieved complete response (CR), and maintenance therapy was initiated with atezolizumab. However, even though CR was maintained, maintenance therapy was discontinued after 16 cycles due to persistent grade 2 anorexia and fatigue. Simultaneously, the HbA1c decreased to 5.5%, and antidiabetic therapy was discontinued. Six months after the last dose of atezolizumab, the patient visited the emergency room because of anorexia, dry mouth, and fatigue. Laboratory findings were as follows: blood glucose was 668 mg/dL, glycated hemoglobin (HbA1c) was 8.8%, urine ketone was 2+, sodium (Na) was 127 mmol/L, potassium (K) was 6.5 mmol/L, creatinine (Cre) was 1.43 mg/dL, and arterial pH was 7.29. Based on these findings, his presentation was consistent with fulminant type 1 diabetes mellitus (T1DM) complicated by diabetic ketoacidosis (DKA). Regular continuous insulin and saline administration was initiated in the intensive care unit, and acidosis and electrolyte abnormalities were corrected. His C-peptide was <0.03 ng/mL. His insulin secretory capacity was considered to be depleted, and he required continuous subcutaneous insulin injections. Glutamic acid decarboxylase and insulin autoantibodies were absent. The complete response persisted without further therapy until two years since the event.

An atlas of transcribed enhancers across helper T cell diversity for decoding human diseases.

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5' single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4+ T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type-specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type-resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.

GWAS for systemic sclerosis identifies six novel susceptibility loci including one in the Fcγ receptor region.

Here we report the largest Asian genome-wide association study (GWAS) for systemic sclerosis performed to date, based on data from Japanese subjects and comprising of 1428 cases and 112,599 controls. The lead SNP is in the FCGR/FCRL region, which shows a penetrating association in the Asian population, while a complete linkage disequilibrium SNP, rs10917688, is found in a cis-regulatory element for IRF8. IRF8 is also a significant locus in European GWAS for systemic sclerosis, but rs10917688 only shows an association in the presence of the risk allele of IRF8 in the Japanese population. Further analysis shows that rs10917688 is marked with H3K4me1 in primary B cells. A meta-analysis with a European GWAS detects 30 additional significant loci. Polygenic risk scores constructed with the effect sizes of the meta-analysis suggest the potential portability of genetic associations beyond populations. Prioritizing the top 5% of SNPs of IRF8 binding sites in B cells improves the fitting of the polygenic risk scores, underscoring the roles of B cells and IRF8 in the development of systemic sclerosis. The results also suggest that systemic sclerosis shares a common genetic architecture across populations.

Various Organ Damages in Rats with Fetal Growth Restriction and Their Slight Attenuation by Bifidobacterium breve Supplementation.

Children with fetal growth restriction (FGR) and its resultant low birthweight (LBW) are at a higher risk of developing various health problems later in life, including renal diseases, metabolic syndrome, and sarcopenia. The mechanism through which LBW caused by intrauterine hypoperfusion leads to these health problems has not been properly investigated. Oral supplementation with probiotics is expected to reduce these risks in children. In the present study, rat pups born with FGR-LBW after mild intrauterine hypoperfusion were supplemented with either Bifidobacterium breve (B. breve) or a vehicle from postnatal day 1 (P1) to P21. Splanchnic organs and skeletal muscles were evaluated at six weeks of age. Compared with the sham group, the LBW-vehicle group presented significant changes as follows: overgrowth from infancy to childhood; lighter weight of the liver, kidneys, and gastrocnemius and plantaris muscles; reduced height of villi in the ileum; and increased depth of crypts in the jejunum. Some of these changes were milder in the LBW-B.breve group. In conclusion, this rat model could be useful for investigating the mechanisms of how FGR-LBW leads to future health problems and for developing interventions for these problems. Supplementation with B. breve in early life may modestly attenuate these problems.

A comprehensive microbial analysis of pediatric patients with acute appendicitis.

BACKGROUND: Pathogenesis of pediatric acute appendicitis (AA) is yet to be elucidated. Therefore, we performed a comprehensive microbial analysis of saliva, feces, and appendiceal lumen of AA patients using 16S ribosomal RNA (rRNA) gene amplicon sequencing to elucidate the pathogenesis of pediatric AA. METHODS: This study included 33 AA patients and 17 healthy controls (HCs) aged <15 y. Among the AA patients, 18 had simple appendicitis, and 15 had complicated appendicitis. Salivary and fecal samples were obtained from both groups. The contents of the appendiceal lumen were collected from the AA group. All samples were analyzed using 16S rRNA gene amplicon sequencing. RESULTS: The relative abundance of Fusobacterium was significantly higher in the saliva of AA patients as compared to that in HCs (P = 0.011). Bacteroides, Escherichia, Fusobacterium, Coprobacillus, and Flavonifractor were significantly increased in the feces of AA patients, as compared to that in HCs (P = 0.020, 0.010, 0.029, 0.031, and 0.002, respectively). In the appendiceal lumen, Bacteroides, Parvimonas, Fusobacterium, and Alloprevotella were the top bacterial genera with an average relative abundance >5% (16.0%, 9.1%, 7.9%, and 6.0%, respectively). CONCLUSIONS: The relative abundance of Fusobacterium was high in the appendiceal lumen of pediatric AA patients. Moreover, the relative abundance of Fusobacterium was significantly higher in the saliva and feces of pediatric AA patients than in those of healthy children. These results suggest that ectopic colonization of oral Fusobacterium in the appendix might play an important role in the pathogenesis of pediatric AA.

Interlaboratory comparison of electron paramagnetic resonance tooth enamel dosimetry with investigations of the dose responses of the standard samples.

A total of seven Japanese laboratories participated in an intercomparison study to estimate the dose given to tooth enamel samples, using the electron spin resonance method. Each of four of the participating laboratories prepared a set of tooth enamel samples, using the electron spin resonance method. Four of the participating laboratories each prepared a set of tooth enamel samples, consisting of seven standard aliquots irradiated from 100 to 2000 mGy and three samples with an 'unknown' dose between 140 and 960 mGy, were intended to eliminate bias from sample preparation. Although not all seven laboratories measured all four sets of samples, the major finding was that systematic biases in estimating doses may be caused by differences in laboratory measurements rather than by the enamel extracting procedures. When doses were averaged by measurements made by multiple laboratories, the averaged values were close to the actual values. Scattering in the intercepts in the standard dose response would be a serious problem in actual dosimetry where no background sample is available.