Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications.

Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localization of dynamic changes. Consequently, the HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase the data quality by providing an orthogonal mode of peptide ion separation in the gas phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multipass IM separations. Using one-pass and multipass cIM routines, we use the recently commercialized SELECT SERIES Cyclic IM spectrometer for HDX-MS analyses of four detergent solubilized IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multipass cIM data. Interestingly, use of one-pass and multipass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM that has the potential to enable the analysis of more complex systems with greater accuracy and speed.

The pathogenic role of the BRCA2 c.7847C>T (p.Ser2616Phe) variant in breast and ovarian cancer predisposition.

Substantial numbers of variants of unknown significance (VUSs) have been identified in BRCA1/2 through genetic testing, which poses a significant clinical challenge because the contribution of these VUSs to cancer predisposition has not yet been determined. Here, we report 10 Japanese patients from seven families with breast or ovarian cancer harboring the BRCA2 c.7847C>T (p.Ser2616Phe) variant that was interpreted as a VUS. This variant recurs only in families from Japan and has not been reported in the global general population databases. A Japanese patient with Fanconi anemia with compound heterozygous variants c.7847C>T (p.Ser2616Phe) and c.475+1G>A in BRCA2 was reported. In silico predictions and quantitative cosegregation analysis suggest a high probability of pathogenicity. The clinical features of the variant carriers were not specific to, but were consistent with, those of patients with hereditary breast and ovarian cancer. A validated functional assay, called the mixed-all-nominated-in-one-BRCA (MANO-B) method and the accurate BRCA companion diagnostic (ABCD) test, demonstrated the deleterious effects of the variant. Altogether, following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, this variant satisfied the "PS3," "PM2," "PM3," and "PP3" criteria. We thus conclude that the BRCA2 c.7847C>T (p.Ser2616Phe) variant is a "likely pathogenic" variant that is specifically observed in the Japanese population, leading to a breast and ovarian cancer predisposition.

[Acute Cardiac Tamponade Caused by Hemorrhage from Cardiac Hemangioma:Report of a Case].

A 68-year-old man with acute chest pain was transported into the emergency room in our hospital. He was in shock, with a systolic blood pressure of 63 mmHg and a heart rate of 115 beats per minute. Echocardiography revealed a cardiac tamponade, but no signs of acute aortic dissection were found by computed tomography. He immediately underwent pericardial fluid drainage and was transferred to the operation theatre. Pericardiotomy was performed and hemorrhage from the right atrium was found. Then, a mass of 35 mm in diameter was also detected by transesophageal echocardiography. Resection of the right atrial tumor was performed under cardiopulmonary bypass. A bovine pericardial patch was used for the repair of the defect in the right atrium. The pathological diagnosis was cardiac hemangioma with no malignant signs.

Qualitative investigation of the factors that generate ambivalent feelings in women who give birth after receiving negative results from non-invasive prenatal testing.

BACKGROUND: Women who receive negative results from non-invasive prenatal genetic testing (NIPT) may find that they later have mixed or ambivalent feelings, for example, feelings of accepting NIPT and regretting undergoing the test. This study aimed to investigate the factors generating ambivalent feelings among women who gave birth after having received negative results from NIPT. METHODS: A questionnaire was sent to women who received a negative NIPT result, and a contents analysis was conducted focusing on ambivalent expressions for those 1562 women who responded the questionnaire. The qualitative data gathered from the questionnaire were analyzed using the N-Vivo software package. RESULTS: Environmental factors, genetic counseling-related factors, and increased anticipatory anxiety, affected the feeling of ambivalence among pregnant women. Furthermore, pregnant women desired more information regarding the detailed prognosis for individuals with Down syndrome and living with them and/or termination, assuming the possibility that they were positive. CONCLUSIONS: Three major interrelated factors affected the feeling of ambivalence in women. Highlighting and discussing such factors during genetic counseling may resolve some of these ambivalences, thereby enhancing the quality of decisions made by pregnant women.

[Papillary Fibroelastoma on the Tricuspid Valve: Report of a Case].

68-year-old male was diagnosed as a cardiac tumor detected by echocardiography, incidentally. He was asymptomatic but the tumor was removed surgically for the diagnostic therapy. It was diagnosed as a papillary fibroelastoma (PFE) clinically and pathologically. He was discharged from the hospital without any complication. PFE on the tricuspid valve is uncommon benign tumor of the heart. Because it is at risk of embolic events, we think it should be operated even if it exists the right side of the heart.

[Aortic prosthetic valve endocarditis with aorto-left atrium fistula; report of a case].

An 83-year-old man had undergone aortic valve replacement (AVR)[CEP Magna 21 mm] and coronary aortic bypass grafting (CABG)[left internal thoracic artery (LITA)-left anterier descending artery( LAD)] 2 years ago in our hospital. He was admitted for fever of unknown origin and developed a stroke to another hospital. The echocardiography and computerized tomography showed an abscessaround the aortic prosthetic valve. Prosthetic valve endocarditis (PVE) was diagnosed, and he was transferred to our hospital for surgical treatment. Three days after admission, acute heart failure developed that led to an emergency operation. When the ascending aorta was dissected, an aorto-left atrium fistula and vegetation were recognized. Aortic valve replacement and patch plasty of the aorto-left atrium fistula were performed successfully. This case was diagnosed as PVE with aorto-left atrium fistula, which is quite a rare complication of PVE.

Complementary and alternative medicine use by pregnant women in Japan: a pilot survey.

BACKGROUND: Complementary and alternative medicine (CAM) are popular among women, and are used during their pregnancy in Japan. This study aimed to survey the prevalence of CAM use by healthy pregnant women as a health-care prescribed therapy or self-care and to investigate the factors associated with CAM use in Japan. METHODS: In this cross-sectional study, pregnant women after 34 weeks of gestation were asked to respond to a questionnaire at the clinic or online. The questionnaire comprised questions on the participants' characteristics and their use of CAM for therapy and self-care. Descriptive statistics were calculated in the analyses, and bivariate and multivariate logistic analyses were performed to evaluate the associations between factors and CAM use. RESULTS: A total of 394 women responded from three hospitals, two clinics, and two midwifery birth centers. CAM was received as treatment by practitioners during pregnancy by 75 women (19.0%). The following therapies were used: traditional Chinese medicine (7.9%), chiropractic (6.9%), moxibustion (6.4%), and acupuncture (5.3%). One or more types of therapy were used as self-care by 348 women (88.3%). Highly used CAM for self-care were: folic acid supplementation (75.4%), other supplements (51.5%), herbs (20.8%), and yoga (19.0%). Multiple logistic regression analyses revealed that the factors associated with CAM use as a therapy were midwifery birth centers for planned childbirth settings (adjusted odds ratio [AOR] 3.64, 95% confidence interval [CI] [1.69-7.83]) and pregnancy complications diagnosed (AOR 2.46, 95%CI [1.38-4.39]). The factors associated with CAM use for self-care were age 30-39 years (AOR 4.48, 95%CI [2.14-9.73]) and over 40 years (AOR 3.92, 95%CI [1.10-13.91]), junior college education or above (AOR 2.30, 95%CI [1.18-4.51]), and primiparas (AOR 3.82, 95%CI [1.86-7.86]). The most common source of information was the "Internet" (43.8%). CONCLUSIONS: Approximately 20% of Japanese pregnant women received CAM as therapy by practitioners, and the related factors were: tended to have baby at midwifery birth center and pregnancy complications. Almost 90% of respondents used CAM as self-care and the related factors were: older, had a higher educational level and tended to be primiparas. They used the Internet as their main source of information about CAM. Health care providers need to provide evidenced-based information on CAM and to help decision making to ensure safe and effective CAM utilization by pregnant women.

Dual-Uptake Mode of the Antibiotic Phazolicin Prevents Resistance Acquisition by Gram-Negative Bacteria.

Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer, Rhizobium sp. strain Pop5. Here, we show that the frequency of spontaneous PHZ-resistant mutants in Sinorhizobium meliloti is below the detection limit. We find that PHZ can enter S. meliloti cells through two distinct promiscuous peptide transporters, BacA and YejABEF, which belong to the SLiPT (SbmA-like peptide transporter) and ABC (ATP-binding cassette) transporter families, respectively. The dual-uptake mode explains the lack of observed resistance acquisition because the simultaneous inactivation of both transporters is necessary for resistance to PHZ. Since both BacA and YejABEF are essential for the development of functional symbiosis of S. meliloti with leguminous plants, the unlikely acquisition of PHZ resistance via the inactivation of these transporters is further disfavored. A whole-genome transposon sequencing screen did not reveal additional genes that can provide strong PHZ resistance when inactivated. However, it was found that the capsular polysaccharide KPS, the novel putative envelope polysaccharide PPP (PHZ-protecting polysaccharide), as well as the peptidoglycan layer jointly contribute to the sensitivity of S. meliloti to PHZ, most likely serving as barriers that reduce the amount of PHZ transported inside the cell. IMPORTANCE Many bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides act either by membrane disruption or by inhibiting essential intracellular processes. The Achilles' heel of the latter type of antimicrobials is their dependence on transporters to enter susceptible cells. Transporter inactivation results in resistance. Here, we show that a rhizobial ribosome-targeting peptide, phazolicin (PHZ), uses two different transporters, BacA and YejABEF, to enter the cells of a symbiotic bacterium, Sinorhizobium meliloti. This dual-entry mode dramatically reduces the probability of the appearance of PHZ-resistant mutants. Since these transporters are also crucial for S. meliloti symbiotic associations with host plants, their inactivation in natural settings is strongly disfavored, making PHZ an attractive lead for the development of biocontrol agents for agriculture.

CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

Clinical and genetic analysis of children with hearing loss and bilateral enlarged vestibular aqueducts.

OBJECTIVES: To evaluate the clinical and genetic features of children with hearing loss associated with one of the most common malformations of the inner ear: bilateral enlargement of vestibular aqueducts (EVA). METHODS: Clinical and genetic features were investigated in 28 children with hearing loss diagnosed with bilateral EVA by computed tomography from January 2008 to September 2019. RESULTS: Fourteen subjects had undergone newborn hearing screening (NHS). Nine subjects (64.3%) were referred in both ears, 4 subjects (28.6%) were referred in one ear, and one subject (7.1%) passed in both ears. Nineteen of 26 subjects (73.1%) who were followed for more than 3 years had hearing fluctuations, while 17 (65.4%) had hearing loss progression. Eleven of 28 subjects (39.2%) had vertigo attacks. Pathogenic variants were identified in two alleles of the SLC26A4 gene in 24 of 27 subjects (88.9%) by sequencing of all exons and flanking introns, leading to genetic diagnosis of Pendred syndrome/DFNB4. Our results indicate that genetic screening for specific SLC26A4 variants using a commercial clinical laboratory test in Japan would have achieved genetic diagnoses in 13 of the 27 subjects (54.2%). Although there was no statistically significance in the frequency of hearing fluctuation or progression depending on the presence or absence of the gene variant, mean hearing level was severe in subjects with two pathogenic variants in SLC26A4 gene. The most common variant detected in our subjects was p.His723Arg (13 alleles, 27.1%), followed by c. 919-2A > G (four alleles, 8.3%). Two novel variants were detected in this study: c.1544+1G > T and c.1614+5G > A. CONCLUSIONS: Our data suggest that some subjects may present with bilateral EVA that cannot be detected by NHS. We estimated that genetic diagnosis for SLC264 gene would not have been made in almost half subjects with the commercial genetic screening approach used in the present study in Japan. Although there were some limitations in this study, the subjects with pathogenic variants in two alleles of the SLC26A4 gene could have more severe hearing loss.

Arrhythmogenic right ventricular cardiomyopathy in a Japanese patient with a homozygous founder variant of DSG2 in the East Asian population.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary cardiomyopathy that results in fatal arrhythmias and heart failure. Herein, we report a Japanese patient with ARVC whose parents were blood relatives. Genetic testing identified a homozygous rare variant, c.1592T > G (p.Phe531Cys), of DSG2 that is presumed to be a founder variant among East Asians. Genetic counseling sessions with precise risk assessment and appropriate follow-up programs were provided to the patient and family members.

A novel pathogenic variant of the FH gene in a family with hereditary leiomyomatosis and renal cell carcinoma.

Hereditary leiomyomatosis and renal cell carcinoma caused by loss-of-function germline variants of the FH gene can develop into aggressive renal cell carcinoma (RCC). We report the case of a 27-year-old man who died of RCC. Genetic testing revealed a novel pathogenic variant of FH, NM_000143.3:c.1013_1014del (p.Ile338Serfs*3), that was also identified in healthy siblings. Identification of genetic causes in the proband helped us to provide relatives with precise genetic counseling and appropriate surveillance programs.

Identification of NRAS Downstream Genes with CRISPR Activation Screening.

Mutations in NRAS constitutively activate cell proliferation signaling in malignant neoplasms, such as leukemia and melanoma, and the clarification of comprehensive downstream genes of NRAS might lead to the control of cell-proliferative signals of NRAS-driven cancers. We previously established that NRAS expression and proliferative activity can be controlled with doxycycline and named as THP-1 B11. Using a CRISPR activation library on THP-1 B11 cells with the NRAS-off state, survival clones were harvested, and 21 candidate genes were identified. By inducting each candidate guide RNA with the CRISPR activation system, DOHH, HIST1H2AC, KRT32, and TAF6 showed higher cell-proliferative activity. The expression of DOHH, HIST1H2AC, and TAF6 was definitely upregulated with NRAS expression. Furthermore, MEK inhibitors resulted in the decreased expression of DOHH, HIST1H2AC, and TAF6 proteins in parental THP-1 cells. The knockdown of DOHH, HIST1H2AC, and TAF6 was found to reduce proliferation in THP-1 cells, indicating that they are involved in the downstream proliferation of NRAS. These molecules are expected to be new therapeutic targets for NRAS-mutant leukemia cells.

A Japanese boy with double diagnoses of 2p15p16.1 microdeletion syndrome and RP2-associated retinal disorder.

2p15p16.1 microdeletion syndrome is a recently recognized congenital disorder characterized by developmental delay and dysmorphic features. RP2-associated retinal disorder (RP2-RD) is an X-linked inherited retinal disease with a childhood onset caused by a loss-of-function variant in the RP2 gene. Here, we describe a 14-year-old boy with double diagnoses of 2p15p16.1 microdeletion syndrome and RP2-RD. The recurrence risk of each condition and the indication for potential therapeutic options for RP2-RD are discussed.

Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides.

Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo­electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.

[Aneurysm of the aortic cusp; report of a case].

We performed surgical therapy to the rare case of the perforation of an aneurysm of the aortic cusp which had caused the congestive heart failure. 71-year-old man having developed coughing was diagnosed as a severe aortic regurgitation and came to our hospital for surgery. The non coronary cusp was dilated and perforated. The aortic valves were removed and replaced with a bioprosthetic valve. The pathological examination of the valves showed healed infective endocarditis. The patient discharged home uneventfully.

Stanford Type A Acute Aortic Dissection with Intimal Intussusception.

In case of complete circumferential dissection of the ascending aorta, the dissected flap has the potential to fold backwards, causing several complications. We report two cases of Stanford type A acute aortic dissection (AAD) whose intimal flaps intussuscepted into the left ventricular outflow tract.Case 1: A 41-year-old man with AAD in whom transthoracic echocardiography (TTE) showed the dissected flap as folded back into the left ventricular outflow tract, causing severe aortic regurgitation (AR) with rapidly progressing acute pulmonary edema. Despite performing salvage surgery, the patient could not be rescued.Case 2: An 81-year-old man with annuloaortic ectasia developed Stanford type A AAD. TTE showed an extremely mobile intimal flap intussuscepting into the left ventricular outflow tract. However, AR was not severe as it was prevented by the flap itself. The patient was rescued by performance of the modified Bentall procedure.

Prophylactic management of postpartum haemorrhage in the third stage of labour: an overview of systematic reviews.

BACKGROUND: Postpartum haemorrhage is a direct cause of maternal death worldwide and usually occurs during the third stage of labour. Most women receive some type of prophylactic management, which may include pharmacological or non-pharmacological interventions. The objective of this study was to summarize systematic reviews that assessed the effects of postpartum haemorrhage prophylactic management during the third stage of labour. METHODS: We applied the guidelines for conducting an overview of reviews from the Cochrane Handbook for Systematic Reviews of Interventions. We searched MEDLINE, EMBASE, and the Cochrane Database of Systematic Reviews to identify all relevant systematic reviews of randomized controlled trials of prophylactic management of postpartum haemorrhage in the third stage of labour compared with no treatment, placebo, or another management technique. Two review authors independently extracted data and assessed methodological quality using a measurement tool to assess reviews and quality of evidence using the Grades of Recommendation, Assessment, Development, and Evaluation for primary outcomes, summarizing results narratively. RESULTS: We identified 29 systematic reviews: 18 Cochrane and 11 non-Cochrane. Cochrane systematic reviews were high quality, while the quality of non-Cochrane systematic reviews varied. The following techniques suggested effective, third-stage interventions to reduce the incidence of severe postpartum haemorrhage: active management of the third stage of labour compared to physiological management, active management compared to expectant management, administration of oxytocin compared to placebo, and use of tranexamic acid compared to placebo. The following third-stage management approaches reduced the need for blood transfusion: active management compared to physiological management, active management compared to expectant management, oral misoprostol compared to placebo, and tranexamic acid compared to placebo. CONCLUSIONS: No effective prophylactic management techniques were identified for maternal mortality. Most methods of effective prophylactic management of postpartum haemorrhage were supported by evidence; however, they were limited to low- or moderate-quality evidence, and high-quality studies are therefore needed. Outcome measures of the included systematic reviews varied. It is recommended that outcome measures in preventive postpartum haemorrhage intervention trials align with the World Health Organization guidelines. SYSTEMATIC REVIEW REGISTRATION: PROSPERO: CRD42016049220 .

Infected Abdominal Aortic Aneurysm Caused by Campylobacter Jejuni.

A 69-year-old man presented with abdominal pain. Enhanced computed tomography (ECT) showed abdominal aortic aneurysm (AAA) with vessel wall thickening. Follow-up ECT on day 3 of admission showed expansion of the AAA. Endovascular abdominal aortic repair (EVAR) was urgently performed. Since preoperative blood cultures revealed Campylobacter jejuni infection, the antibiotics imipenem/cilastatin were administered for five weeks, followed orally by Clarithromycin. The patient was discharged on postoperative day 45. There was no recurrence of the aneurysm at 9 months after EVAR. To the best of our knowledge, this is the first report of EVAR for infected AAA caused by Campylobacter jejuni.

[Type B intramural hematoma rupture].

A 70-year-old male, who had complained sudden chest pain and sweating, was admitted to our hospital and diagnosed as a type B intramural hematoma rupture. Emergency repair was performed with arch replacement and open-stent method. Post-operative recovery was satisfactory and the patient was discharged. A type B intramural hematoma rupture is relatively rare. Trans-esophagial echocardiography is quite useful to detect the aortic lesion and to determine the ideal depth of the stent-graft.