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Abstract High-throughput sequencing technologies have increasingly led to discovery of disease-causing genetic variants, primarily in postnatal multi-cell DNA samples. However, applying these technologies to preimplantation genetic testing (PGT) in nuclear or mitochondrial DNA from single or few-cells biopsied from in vitro fertilised (IVF) embryos is challenging. PGT aims to select IVF embryos without genetic abnormalities. Although genotyping-by-sequencing (GBS)-based haplotyping methods enabled PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A), they are labour intensive, only partially cover the genome and are troublesome for difficult loci and consanguineous couples. Here, we devise a simple, scalable and universal whole genome sequencing haplarithmisis-based approach enabling all forms of PGT in a single assay. In a comparison to state-of-the-art GBS-based PGT for nuclear DNA, shallow sequencing-based PGT, and PCR-based PGT for mitochondrial DNA, our approach alleviates technical limitations by decreasing whole genome amplification artifacts by 68.4%, increasing breadth of coverage by at least 4-fold, and reducing wet-lab turn-around-time by ~2.5-fold. Importantly, this method enables trio-based PGT-A for aneuploidy origin, an approach we coin PGT-AO, detects translocation breakpoints, and nuclear and mitochondrial single nucleotide variants and indels in base-resolution.

Yun Han,1,* Jianping Zhu,1,* Jieqiong Liu,2 Ying Zheng,2 Gang Liang,1 Yi Yang,1 Lingyan Yu,3 Zhenwei Yu,1 Gang Han1 1Research Center for Clinical Pharmacy, Sir Run Run Shaw Hospital, School of Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People’s Republic of China; 2The 903rd Hospital of PLA Joint Logistic Support Force, Hangzhou, People’s Republic of China; 3Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhenwei Yu; Gang Han, Email yzw_srrsh@zju.edu.cn; 3199022@zju.edu.cnIntroduction: Recent studies suggested the potential benefits of extended infusion times to optimize the treatment efficacy of ceftazidime/avibactam, which indicated that the current pharmacokinetic/pharmacodynamic (PK/PD) target may not be sufficient, especially for severe infections. The purpose of this study is to assess the adequacy of dosing strategies and infusion durations of ceftazidime/avibactam when applying higher PK/PD targets.Methods: This study utilized published PK parameters to conduct Monte Carlo simulations. Different dosages including the recommended regimen based on renal function were simulated and evaluated by the probability of target attainment (PTA) and cumulative fraction of response (CFR). Different PK/PD targets were set for ceftazidime and avibactam. MIC distributions from various sources were used to calculate the CFR.Results: Multiple PK/PD targets have been set in this study, All recommended dosage could easily achieve the target of 50%fT ≥ MIC (ceftazidime) and 50%fT ≥ CT=1.0 mg/L (avibactam). However, for severe infection patients with normal renal function and augmented renal clearance at the recommended dosage (2000 mg/500 mg, every 8 hours), the infusion duration needs to be extended to 3 hours and 4 hours to achieve the targets of 100%fT ≥ MIC and 100%fT ≥ CT=1.0 mg/L. Only continuous infusion at higher dosages achieved 100%fT ≥ 4×MIC and 100%fT ≥ CT=4.0 mg/L targets to all currently recommended regimens. According to the varying MIC distributions, higher concentrations are needed for Pseudomonas aeruginosa, with the attainment rates vary across different regions.Conclusion: The current recommended dosing regimen of ceftazidime/avibactam is insufficient for severe infection patients, and continuous infusion is suggested.Keywords: ceftazidime, avibactam, pharmacokinetic/pharmacodynamic, probability of target attainment

Xiaodong Zhu,1 Ya Huang,2 Jia Ding,1 Jianguo Liu,1 Changmeng Cui,1 Guangkui Han1 1Department of Neurosurgery, Affiliated Hospital of Jining Medical University, Jining, 272000, People’s Republic of China; 2College of Traditional Chinese Medicine, Shandong Polytechnic College, Jining, 272000, People’s Republic of ChinaCorrespondence: Guangkui Han, Department of Neurosurgery, Affiliated Hospital of Jining Medical University, 89 Guhuai Road, Jining, Shandong, 272000, People’s Republic of China, Tel +8618266808351, Email hanguangkui1997@163.comPurpose: SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of irinotecan, has been extensively studied in drug delivery systems. However, its impact on neural metabolism remains unclear. This study aims to investigate the toxic effects of SN-38 on mouse brain metabolism.Methods: Male mice were divided into an SN-38 group and a control group. The SN-38 group received SN-38 (20 mg/kg/day) via intraperitoneal injection, while the control group was given an equal volume of a blank solvent mixture (DMSO and saline, ratio 1:9). Gas chromatography-mass spectrometry (GC-MS) was employed to analyze differential metabolites in the cortical and hippocampal regions of the SN-38-treated mice.Results: SN-38 induced metabolic disturbances in the central nervous system. Eighteen differential metabolites were identified in the hippocampus and twenty-four in the cortex, with six common to both regions. KEGG pathway enrichment analysis revealed statistically significant alterations in six metabolic pathways in the hippocampus and ten in the cortex (P< 0.05).Conclusion: This study is the first to demonstrate the neurotoxicity of SN-38 in male mice through metabolomics. Differential metabolites in the hippocampal and cortical regions were closely linked to purine metabolism, pyrimidine metabolism, amino acid metabolism, and glyceride metabolism, indicating disruptions in the blood-brain barrier, energy metabolism, and central signaling pathways.Keywords: SN-38 (7-ethyl-10-hydroxycamptothecin), biomarker, gas chromatography mass spectrometry, metabolomics, toxicity mechanism

Summary: Mechanisms by which Porphyromonas gingivalis (P. gingivalis) infection enhances oral tumor growth or resistance to cell death remain elusive. Here, we determined that P. gingivalis infection mediates therapeutic resistance via inhibiting lethal mitophagy in cancer cells and tumors. Mechanistically, P. gingivalis targets the LC3B-ceramide complex by associating with LC3B via bacterial major fimbriae (FimA) protein, preventing ceramide-dependent mitophagy in response to various therapeutic agents. Moreover, ceramide-mediated mitophagy is induced by Annexin A2 (ANXA2)-ceramide association involving the E142 residue of ANXA2. Inhibition of ANXA2-ceramide-LC3B complex formation by wild-type P. gingivalis prevented ceramide-dependent mitophagy. Moreover, a FimA-deletion mutant P. gingivalis variant had no inhibitory effects on ceramide-dependent mitophagy. Further, 16S rRNA sequencing of oral tumors indicated that P. gingivalis infection altered the microbiome of the tumor macroenvironment in response to ceramide analog treatment in mice. Thus, these data provide a mechanism describing the pro-survival roles of P. gingivalis in oral tumors.

Abstract Background To diagnose the full spectrum of hereditary and congenital diseases, genetic laboratories use many different workflows, ranging from karyotyping to exome sequencing. A single generic high-throughput workflow would greatly increase efficiency. We assessed whether genome sequencing (GS) can replace these existing workflows aimed at germline genetic diagnosis for rare disease. Methods We performed short-read GS (NovaSeq™6000; 150 bp paired-end reads, 37 × mean coverage) on 1000 cases with 1271 known clinically relevant variants, identified across different workflows, representative of our tertiary diagnostic centers. Variants were categorized into small variants (single nucleotide variants and indels

Xiaolei Ma,1 Tianying Li,2 Gangwen Han1 1Department of Dermatology, Peking University International Hospital, Beijing, People’s Republic of China; 2Department of Pathology, Peking University International Hospital, Beijing, People’s Republic of ChinaCorrespondence: Xiaolei Ma, Peking University International Hospital, Life Park Road No. 1 Life Science Park of Zhong Guancun, Chang Ping District, Beijing, People’s Republic of China, Email superma.xiaolei@163.comAbstract: Atopic dermatitis and psoriasis are both common chronic inflammatory skin conditions that can significantly affect the quality of life for individuals affected by them. With the growing use of biologic agents, there have been remarkable clinical advancements in the treatment of these diseases. Interestingly, during biologic therapy for either condition, a phenomenon has emerged where treatment can paradoxically induce a transition to the phenotype of the other condition.We present two cases of immune drift phenomena caused by biologic agents for treating psoriasis and atopic dermatitis.The first one is a case of psoriasis lesion that developed in an old patient with AD who was treated with dupilumab for two months. The second one is a case of eczematoid lesion that developed in an adult patient with ankylosing spondylitis who was treated with Secukinumab for 1 year.Keywords: immune drift phenomena, secukinumab, dupilumab, psoriasis and atopic dermatitis

Yan-Hua Sha,1 Song-Ling Cao,1 Lu Zhang,2 Li-Sha Lai,2 Pei-Feng Ke,1 Ke-Wei Yu,1 Xiu-Zhu Fang,2 Ren-Tang Deng,2 Ze-Min Wan,1 Xiao-Bin Wu,1 Guang Han,1 Yu-Bang Jie,2 Lan-Lan Song,2 Xian-Zhang Huang,1 Wen-Jin Fu2 1Department of Laboratory Medicine, The Second Clinical College of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China; 2Clinical Laboratory, Houjie Hospital of Guangdong Medical University, Dongguan, Guangdong, People’s Republic of ChinaCorrespondence: Xian-Zhang Huang, Department of Laboratory Medicine, The Second Clinical College of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China, Email huangxz020@163.com; Wen-Jin Fu, Clinical Laboratory, Houjie Hospital of Guangdong Medical University, Dongguan, Guangdong, People’s Republic of China, Email 332689892@qq.comIntroduction: Circular RNA (circRNAs) are a type of non-coding RNA (ncRNAs) with a wealth of functions. Recently, circRNAs have been identified as important regulators of diabetic kidney disease (DKD), owing to their stability and enrichment in exosomes. However, the role of circRNAs in exosomes of tubular epithelial cells in DKD development has not been fully elucidated.Methods: In our study, microarray technology was used to analyze circRNA expression in cell supernatant exosomes isolated from HK-2 cells with or without high glucose (HG) treatment. The small interfering RNAs (siRNA) and plasmid overexpression were used to validate functions of differentially expressed circRNAs.Results: We found that exosome concentration was higher in HG-stimulated HK-2 cells than in controls. A total of 235 circRNAs were significantly increased and 458 circRNAs were significantly decreased in the exosomes of the HG group. In parallel with the microarray data, the qPCR results showed that the expression of circ_0009885, circ_0043753, and circ_0011760 increased, and the expression of circ_0032872, circ_0004716, and circ_0009445 decreased in the HG group. Rescue experiments showed that the effects of high glucose on regulation of CCL2, IL6, fibronetin, n cadherin, e cadherin and epcam expression can be reversed by inhibiting or overexpressing these circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses indicated that circRNA parental genes are associated with glucose metabolism, lipid metabolism, and inflammatory processes, which are important in DKD development. Further analysis of circRNA/miRNA interactions indicated that 152 differentially expressed circRNAs with fold change (FC) ≥ 1.5 could be paired with 43 differentially expressed miRNAs, which are associated with diabetes or DKD.Discussion: Our results indicate that exosomal circRNAs may be promising diagnostic and therapeutic biomarkers, and may play a critical role in the progression of DKD.Keywords: exosome, cicrRNA, diabetic nephropathy, high glucose, microarray

Batudeligen, Zhiqiang Han, Hongmei Chen, Narisu, Yanhua Xu, Anda, Gegentaoli Han Institute of Clinical Pharmacology of Traditional Mongolian Medicine, Affiliated Hospital of Inner Mongolia Minzu University, Tongliao City, Inner Mongolia, People’s Republic of ChinaCorrespondence: Batudeligen, Institute of Clinical Pharmacology of Traditional Mongolian Medicine, Affiliated Hospital of Inner Mongolia Minzu University, 1742 East Huolinhe Dajie, Tongliao City, Inner Mongolia, 028007, People’s Republic of China, Tel +86- 475- 8215505, Email Batudeligen123@163.comBackground: Traditional Chinese medicine (TCM) with single or compound materials is an effective cure for liver fibrosis. Hepatic stellate cells (HSCs) play a key role in liver fibrosis pathology and have become a novel drug target for this condition.Methods: CCK-8 assay was used to determine the cytotoxicity of four components, SYPA, HSYPA, Apigenin, and Luteolin, from Deduhonghua-7 powder on HSC-T6 cells. Transforming Growth Factor β 1 (TGFβ 1)-induced fibrotic cell model and CCI4-induced fibrotic rat model were constructed, the expression of fibrosis-related genes, the pathological changes and serum biochemical markers were evaluated. Proteomic analysis was performed to determine the mechanism by which luteolin attenuated liver fibrosis, which were further confirmed by Western blot.Results: Luteolin attenuates liver fibrosis in HSC-T6 cells and luteolin decreases the liver fibrosis index level in vivo. A total of 5000 differentially expressed proteins (DEPs) were obtained using proteomic analysis. KEGG analysis found that DEPs were concentrated in various metabolic pathways, including DNA replication and repair and lysosomal signaling. GO analysis showed that molecular functions included the activity and binding of various enzymes, related cellular components included the extracellular space, lysosomal lumen, mitochondrial matrix, and nucleus, and biological processes included collagen organization and biosynthesis and the positive regulation of cell migration. Western blot results showed that CCR1, CD59, and NAGA were downregulated in TGFβ 1 treatment, while upregulated both in Lut2 and Lut10 treatment. Meanwhile, eight proteins, ITIH3, MKI67, KIF23, DNMT1, P4HA3, CCDC80, APOB, FBLN2, that were upregulated in TGFβ 1 treatment, while downregulated both in Lut2 and Lut10 treatment.Conclusion: Luteolin was shown to have a strong protective effect on liver fibrosis. CCR1, CD59, and NAGA may promote liver fibrosis while ITIH3, MKI67, KIF23, DNMT1, P4HA3, CCDC80, APOB, and FBLN2 may facilitate protection against fibrosis.Keywords: luteolin, deduhonghua-7 powder, liver fibrosis, proteomic analysis, TGFβ 1

Introduction: Rapid exome sequencing (rES) has become the first-choice genetic test for critically ill patients, mostly neonates, young infants, or fetuses in prenatal care, in time-sensitive situations and when it is expected that the genetic test result may guide clinical decision making. The implementation of rES has revolutionized medicine by enabling timely identification of genetic causes for various rare diseases. The utilization of rES has increasingly been recognized as an essential diagnostic tool for the identification of complex and undiagnosed genetic disorders.Methods: We conducted a retrospective evaluation of our experiences with rES performed on 575 critically ill patients from various age groups (prenatal to adulthood), over a four-year period (2016–2019). These patients presented with a wide spectrum of rare diseases, including but not limited to neurological disorders, severe combined immune deficiency, and cancer.Results: During the study period, there was a significant increase in rES referrals, with a rise from a total of two referrals in Q1-2016 to 10 referrals per week in Q4-2019. The median turnaround time (TAT) decreased from 17 to 11 days in the period 2016–2019, with an overall median TAT of 11 days (IQR 8–15 days). The overall diagnostic yield for this cohort was 30.4%, and did not significantly differ between the different age groups (e.g. adults 22.2% vs children 31.0%; p-value 0.35). However, variability in yield was observed between clinical entities: craniofacial anomalies yielded 58.3%, while for three clinical entities (severe combined immune deficiency, aneurysm, and hypogonadotropic hypogonadism) no diagnoses were obtained.Discussion: Importantly, whereas clinical significance is often only attributed to a conclusive diagnosis, we also observed impact on clinical decision-making for individuals in whom no genetic diagnosis was established. Hence, our experience shows that rES has an important role for patients of all ages and across the broad spectrum of rare diseases to impact clinical outcomes.

Abstract Background Long-read sequencing (LRS) techniques have been very successful in identifying structural variants (SVs). However, the high error rate of LRS made the detection of small variants (substitutions and short indels

Qin Qian,&ast; Yu-Zhen Wang,&ast; Lian-Di Kan,&ast; Jie Chen, Chen Wang, Gang Han, Liu-Cheng Li, Wei-Jian Lou Department of Pharmacy, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, 310016, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Liu-Cheng Li; Wei-Jian Lou, Department of Pharmacy, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, 310016, People’s Republic of China, Tel/Fax +86-571-8600-6803, Email 3415116@zju.edu.cn; 13588708537@163.comBackground: Pediatric hypertension become an early marker of cardiovascular diseases, but their antihypertensive drug use patterns are rarely known.Purpose: To investigate the epidemiological characteristics of pediatric hypertension and the use of antihypertensive drugs in the real world in China.Methods: In this study, the demographic, diagnosis, and medication prescription data including the antihypertensive drug types and comorbidities, were analyzed. The antihypertensive drugs use were evaluated according to the Chinese guidelines for hypertension.Results: 1301 prescriptions (number of visits) containing 1880 antihypertensive medical orders were collected. The average number of antihypertensive drugs per prescription was 1.45 (± 0.75). The patients aged 16 to 18 (70.18%) accounted for the highest proportion. Kidney diseases (33.28%) were the most common comorbidities. Calcium channel blocker (CCB), angiotensin II receptor blocker (ARB), and β receptor blocker (BB) were the most used antihypertensive drugs. The most frequent monotherapy was CCB, while that of two and three drugs combination were ARB+CCB and ARB+BB+CCB, respectively. Metoprolol (11.44%), nifedipine (10.64%), amlodipine (10.59%), valsartan (6.12%) were the most commonly used antihypertensive drugs. The utilization rate of fixed compound preparations was 7.34%. However, the percentage of recommended antihypertensive drugs was just 14.20%, while the recommended drug combination was 84.93% according to the guidelines.Conclusion: For the first time ever we reported the antihypertensive prescription analysis in children in a large area of China. Our data provided new insights into the epidemiological characteristics and drug use in hypertensive children. We found that the guidelines for medication management in hypertensive children were not routinely followed. The wide use of antihypertensive drugs in children and those with weak clinical evidence raised concerns regarding its rational use. The findings could lead to more effective management of hypertension in children.Keywords: hypertensive children, antihypertensive drugs, drug combination, prescription

Jingjing Yan,1 Jing Tian,1,2 Hong Yang,1 Gangfei Han,2 Yanling Liu,1 Hangzhi He,1 Qinghua Han,2 Yanbo Zhang3,4 1Department of Health Statistics, School of Public Health, Shanxi Medical University, Taiyuan, People’s Republic of China; 2Department of Cardiology, The First Hospital of Shanxi Medical University, Taiyuan, People’s Republic of China; 3Shanxi Provincial Key Laboratory of Major Diseases Risk Assessment, Shanxi Medical University, Taiyuan, People’s Republic of China; 4Shanxi University of Chinese Medicine, Taiyuan, People’s Republic of ChinaCorrespondence: Yanbo Zhang, Shanxi Provincial Key Laboratory of Major Diseases Risk Assessment, Shanxi Medical University, No. 56 South Xinjian Road, Taiyuan, Shanxi Province, 030001, People’s Republic of China, Tel +86 3543985051, Email sxmuzyb@126.com Qinghua Han, Department of Cardiology, The First Hospital of Shanxi Medical University, No. 85 South Jiefang Road, Taiyuan, Shanxi Province, 030001, People’s Republic of China, Tel +86 3100113031, Fax +86 3514867146, Email syhqh@sohu.comBackground: Chronic heart failure (CHF) affects more than 3.8 million people worldwide. There is a paucity of studies focusing on psychosocial issues in CHF patients. This study aimed to investigate the association of social support, mental adjustment and death to exploring whether mental adjustment could mediate the relationship.Methods: From May 2017 to June 2021, we conducted a multicenter clinical study to collect 1552 patients data. The Patient Report Outcome (PRO) scale were disseminated to collect information in the physical, psychological, social and therapeutic domains of patients. Marginal structural model was used to investigate the association of social support and CHF death, and the role of mental adjustment in their mediation.Results: The direct effect of social support accounted for 44.76% of the total effect. High social support (≥ 14 points) reduced mortality by 46.3% (RR=0.537, P=0.027), medium social support (11– 14 points) reduced mortality by 45.3% (RR=0.547, P=0.042). Anxiety (effect percentage: 24.63%) and appetite-sleep quality (effect percentage: 30.61%) played a mediating role between social support and death in CHF patients. In women, aged > 75 years, divorced or widowed patients were most prone to anxiety due to inadequate support (RR=0.519, P=0.019; RR=0.403, P=0.002; RR=0.413, P=0.041). Family care and socioeconomic assistance significantly reduced the risk of death (RR=0.689, P=0.040; RR=0.584, P=0.012).Conclusion: Social support can reduce patient mortality, especially family care and social economic assistance. The elderly, female, divorced or widowed patients are more likely to cause mental illness due to inadequate social support.Keywords: chronic heart failure, social support, marginal structural model, mediation analysis

Jing Tian,1,2 Fengqin Ding,3 Ruoya Wang,3 Gangfei Han,1 Jingjing Yan,3 Na Yuan,3 Yutao Du,3 Qinghua Han,1 Yanbo Zhang2– 4 1Department of Cardiology, the 1st Hospital of Shanxi Medical University, Taiyuan, People’s Republic of China; 2Shanxi Provincial Key Laboratory of Major Diseases Risk Assessment, Taiyuan, People’s Republic of China; 3Department of Health Statistics, School of Public Health, Shanxi Medical University, Taiyuan, People’s Republic of China; 4Shanxi University of Chinese Medicine, Jinzhong, People’s Republic of ChinaCorrespondence: Yanbo Zhang, Department of Health Statistics, School of Public Health, Shanxi Medical University, Taiyuan, People’s Republic of China, Tel +86 3327518812, Email sxmuzyb@126.com Correspondence: Qinghua Han, Department of Cardiology, the 1st Hospital of Shanxi Medical University, Taiyuan, People’s Republic of China, Tel +86 3100113031, Email syhqh@sohu.comPurpose: This study aimed to identify subgroups of chronic heart failure (CHF) patients with distinct trajectories of quality of life (QOL) and to identify baseline characteristics associated with the trajectories.Patients and methods: Two-year, prospective, cohort study including 315 patients with CHF was conducted from July 2017. Information on QOL assessed by CHF-patient-reported outcomes measure (CHF-PROM) was collected at baseline, 6, 12, 18, and 24 months. Demographic and clinical variables were recorded at baseline. Growth mixture model was used to identify distinct trajectories of CHF-PROM and its physical, psychological, social, and therapeutic domains. Single factor analysis was employed to assess the factors associated with development of CHF-PROM over time.Results: Two classes of overall score of CHF-PROM were identified: poorer (14.0%) and better (86.0%). Poorer class tended to be aged, have low diastolic blood pressure, have concomitant atrial fibrillation, diabetes, chronic obstructive pulmonary disease, cancers, and central nervous system diseases, and used nitrates. Three classes of physical scores were identified: unstable-poorer (5.2%), stable-poorer (29.4%) and better (65.4%). Age, NYHA grade, chronic obstructive pulmonary disease, combined with cancers and central nervous system diseases were related to the grouping. Poorer (8.6%) and better (91.4%) classes of psychological scores were identified. Poorer class tended to be female and had concomitant atrial fibrillation. Degenerate class (34.6%) and meliorate class (65.4%) of therapeutic scores were identified. Degenerate class tended to have concomitant chronic obstructive pulmonary disease and use less angiotensin converting enzyme inhibitors.Conclusion: We identified different classes with distinct trajectories of QOL that may help proper evaluate QOL and further improve its status for patients CHF.Keywords: patient-reported outcome, chronic heart failure, growth mixture model, dynamic trajectory

Summary: Background: Haematopoietic clones caused by somatic mutations with ≥2% variant allele frequency (VAF) increase with age and are linked to risk of haematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones (VAF

Abstract Background Approximately two third of patients with a rare genetic disease remain undiagnosed after exome sequencing (ES). As part of our post-test counseling procedures, patients without a conclusive diagnosis are advised to recontact their referring clinician to discuss new diagnostic opportunities in due time. We performed a systematic study of genetically undiagnosed patients 5 years after their initial negative ES report to determine the efficiency of diverse reanalysis strategies. Methods We revisited a cohort of 150 pediatric neurology patients originally enrolled at Radboud University Medical Center, of whom 103 initially remained genetically undiagnosed. We monitored uptake of physician-initiated routine clinical and/or genetic re-evaluation (ad hoc re-evaluation) and performed systematic reanalysis, including ES-based resequencing, of all genetically undiagnosed patients (systematic re-evaluation). Results Ad hoc re-evaluation was initiated for 45 of 103 patients and yielded 18 diagnoses (including 1 non-genetic). Subsequent systematic re-evaluation identified another 14 diagnoses, increasing the diagnostic yield in our cohort from 31% (47/150) to 53% (79/150). New genetic diagnoses were established by reclassification of previously identified variants (10%, 3/31), reanalysis with enhanced bioinformatic pipelines (19%, 6/31), improved coverage after resequencing (29%, 9/31), and new disease-gene associations (42%, 13/31). Crucially, our systematic study also showed that 11 of the 14 further conclusive genetic diagnoses were made in patients without a genetic diagnosis that did not recontact their referring clinician. Conclusions We find that upon re-evaluation of undiagnosed patients, both reanalysis of existing ES data as well as resequencing strategies are needed to identify additional genetic diagnoses. Importantly, not all patients are routinely re-evaluated in clinical care, prolonging their diagnostic trajectory, unless systematic reanalysis is facilitated. We have translated our observations into considerations for systematic and ad hoc reanalysis in routine genetic care.

Abstract Aims Heart failure (HF) represents a clinical syndrome resulting from different aetiologies and degrees of heart diseases. Among these, a key role is played by primary heart muscle disease (cardiomyopathies), which are the combination of multifactorial environmental insults in the presence or absence of a known genetic predisposition. The aim of the Maastricht Cardiomyopathy registry (mCMP‐registry; NCT04976348) is to improve (early) diagnosis, risk stratification, and management of cardiomyopathy phenotypes beyond the limits of left ventricular ejection fraction (LVEF). Methods and results The mCMP‐registry is an investigator‐initiated prospective registry including patient characteristics, diagnostic measurements performed as part of routine clinical care, treatment information, sequential biobanking, quality of life and economic impact assessment, and regular follow‐up. All subjects aged ≥16 years referred to the cardiology department of the Maastricht University Medical Center (MUMC+) for HF‐like symptoms or cardiac screening for cardiomyopathies are eligible for inclusion, irrespective of phenotype or underlying causes. Informed consented subjects will be followed up for 15 years. Two central approaches will be used to answer the research questions related to the aims of this registry: (i) a data‐driven approach to predict clinical outcome and response to therapy and to identify clusters of patients who share underlying pathophysiological processes; and (ii) a hypothesis‐driven approach in which clinical parameters are tested for their (incremental) diagnostic, prognostic, or therapeutic value. The study allows other centres to easily join this initiative, which will further boost research within this field. Conclusions The broad inclusion criteria, systematic routine clinical care data‐collection, extensive study‐related data‐collection, sequential biobanking, and multi‐disciplinary approach gives the mCMP‐registry a unique opportunity to improve diagnosis, risk stratification, and management of HF and (early) cardiomyopathy phenotypes beyond the LVEF limits.

Wenhao Shen,1,&ast; Gaohua Han,1,&ast; Lei Yu,1 Song Yang,1 Xiangyi Li,2 Wei Zhang,3 Pei Pei4 1Department of Oncology, Hospital Affiliated 5 to Nantong University (Taizhou People’s Hospital), Taizhou, Jiangsu, People’s Republic of China; 2Department of Endocrinology, Hospital Affiliated 5 to Nantong University (Taizhou People’s Hospital), Taizhou, Jiangsu, People’s Republic of China; 3Department of Infectious Disease, Hospital Affiliated 5 to Nantong University (Taizhou People’s Hospital), Taizhou, Jiangsu, People’s Republic of China; 4School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Wei Zhang; Pei Pei, Email zw562652291@126.com; ppei1204@outlook.comIntroduction: Photodynamic therapy (PDT) as a new technique for theranostics is to kill tumor cells by activating photosensitizer and interacting with oxygen (O2) to produce reactive oxygen species (ROS). However, the hypoxic tumor microenvironment (TME) may constrain the efficacy of PDT. Moreover, the lack of O2 in TME also up-regulates the expression of HIF-1α and promotes tumor metastasis, which is also a leading cause of death for terminal cancer patients.Methods: Prussian blue (PBs) was firstly synthesized by hydrothermal method, which was then etched by hydrochloric acid to obtained hollow Prussian blue nanoparticles (HPBs). Afterwards, Au-Pt nanozymes were in situ growing on the HPBs by reduction method to prepare Au-Pt@HPBs (APHPBs). Owing to the hollow structure of APHPBs, photosensitizer Ce6 can be easily and efficiently loaded into it to obtain Ce6-Au-Pt@HPBs (Ce6-APHPBs). After ce6-APHPBS regulation, photoacoustic imaging and hypoxic fluorescence imaging were then used to evaluate changes in hypoxic TME in vivo. Finally, under the assistant of Ce6-APHPBs, we evaluated the inhibitory effect of enhanced PDT on primary and metastatic tumors.Results: We first designed and synthesized Ce6 loaded hollow prussian blue nanoparticles with Au-Pt nanozymes grown in situ on it. Both in vitro and in vivo experiments show that the prepared Ce6-APHPBs have good biosafety and could effectively degrade the overexpressed H2O2 in TME to generate O2, further relieve the hypoxic TME and thus enhance the effect of PDT. At the same time, the increasing O2 content could also reduce the expression of HIF-1α at the tumor site, which could reduce lung metastasis.Conclusion: Ce6-APHPBs designed by us could not only efficiently enhance PDT but also regulate TME to reduce tumor metastasis and prolong survival of mice, which provide a novel idea and strategy for clinical PDT and metastatic tumor.Keywords: Au-Pt nanozymes, prussian blue, enhanced PDT, TME, HIF-1α, tumor metastasis

Summary: WDR5 is a broadly studied, highly conserved key protein involved in a wide array of biological functions. Among these functions, WDR5 is a part of several protein complexes that affect gene regulation via post-translational modification of histones. We collected data from 11 unrelated individuals with six different rare de novo germline missense variants in WDR5; one identical variant was found in five individuals and another variant in two individuals. All individuals had neurodevelopmental disorders including speech/language delays (n = 11), intellectual disability (n = 9), epilepsy (n = 7), and autism spectrum disorder (n = 4). Additional phenotypic features included abnormal growth parameters (n = 7), heart anomalies (n = 2), and hearing loss (n = 2). Three-dimensional protein structures indicate that all the residues affected by these variants are located at the surface of one side of the WDR5 protein. It is predicted that five out of the six amino acid substitutions disrupt interactions of WDR5 with RbBP5 and/or KMT2A/C, as part of the COMPASS (complex proteins associated with Set1) family complexes. Our experimental approaches in Drosophila melanogaster and human cell lines show normal protein expression, localization, and protein-protein interactions for all tested variants. These results, together with the clustering of variants in a specific region of WDR5 and the absence of truncating variants so far, suggest that dominant-negative or gain-of-function mechanisms might be at play. All in all, we define a neurodevelopmental disorder associated with missense variants in WDR5 and a broad range of features. This finding highlights the important role of genes encoding COMPASS family proteins in neurodevelopmental disorders.

Guangchao Han, Min Li, Junfeng Du, Yang Chen, Chen Xu Department of Respiratory and Critical Care Medicine, Cangzhou Central Hospital, Cangzhou, 061000, Hebei, People’s Republic of ChinaCorrespondence: Guangchao HanDepartment of Respiratory and Critical Care Medicine, Cangzhou Central Hospital, No. 16 Xinhua West Road, Cangzhou, 061000, Hebei, People’s Republic of ChinaTel +86-15833371555Email guangchao1020@126.comIntroduction: Chronic obstructive pulmonary disease (COPD) is a progressive condition related to abnormal inflammatory responses. As an inflammatory driver, nucleotide-binding oligomerizing domain-1 (NOD1) is highly expressed in pulmonary inflammatory cells; however, the roles of NOD1 in COPD are unknown.Methods: A COPD mouse model was established by lipopolysaccharides tracheal instillation plus cigarette smoke (CS) exposure. NOD1 activation was induced by C12-iE-DAP (iE) treatment in both control and COPD mice. Inflammatory infiltration, pulmonary histological damage and gene expression were measured to evaluate the lung function of treated mice.Results: The results showed that NOD1 was up-regulated in COPD mice, which significantly exaggerated CS-induced impairment of lung function, demonstrated by increased airway resistance, functional residual capacity and pulmonary damages. Mechanistically, NOD1 activation strongly activated the TLR4/NF-κB signaling pathway and then increased inflammatory responses and promoted the secretion of inflammatory cytokines.Discussion: This study demonstrates that NOD1 is an important risk factor in the progression of COPD; therefore, targeting NOD1 in lung tissues is a potential strategy for COPD treatment.Keywords: COPD, pulmonary damage, inflammation, NOD1, TLR4/NF-κB signaling

Background: Despite (neo) adjuvant chemotherapy with cisplatin, doxorubicin and methotrexate, some patients with primary osteosarcoma progress during first-line systemic treatment and have a poor prognosis. In this study, we investigated whether patients with early disease progression (EDP), are characterized by a distinctive pharmacogenetic profile.Methods and Findings: Germline DNA from 287 Dutch high-grade osteosarcoma patients was genotyped using the DMET Plus array (containing 1,936 genetic markers in 231 drug metabolism and transporter genes). Associations between genetic variants and EDP were assessed using logistic regression models and associated variants (p

Abstract Monoamine oxidase A (MAO‐A) deficiency is a rare inborn error of metabolism with impaired degradation of biogenic amines including 5‐hydroxytryptamine (5‐HT), resulting in borderline intellectual disability and behavioral abnormalities. Genetic variants in MAOA need functional confirmation to enable a definite diagnosis. To this end, we developed an inexpensive, simple and nonradioactive MAO‐A activity assay based on the conversion of 5‐HT into 5‐hydroxyindoleacetic acid (5‐HIAA). Fibroblast cell lysates were incubated with 5‐HT and aldehyde dehydrogenase to allow 5‐HIAA production. 5‐HIAA was quantified using high‐performance liquid chromatography with fluorimetric detection. We optimized reaction mixture components, pH, and substrate concentration and tested linearity and specificity of the assay. We verified the functional validity of the enzyme assay using fibroblasts of controls, female mutation carriers and MAO‐A deficient patients. This included a newly described patient with a novel MAOA variant (c.1336G>A, p.(Glu446Lys)), who represents the fifth MAO‐A deficiency family so far. The optimized enzyme assay showed good linearity and specificity. Application to clinical samples showed a 100% differentiation of affected patients (with negligible MAO‐A enzyme activity) and controls or mutation carriers. In conclusion, the described MAO‐A activity assay is easy to implement and can readily be used to test the pathogenicity of variants in the MAOA gene in a clinical setting. Especially in this era of whole‐exome (and whole‐genome) sequencing, this functional assay fulfills a clinical need for functional confirmation of a suspected diagnosis of MAO‐A deficiency.

Song Yang, 1, 2,* Gaohua Han, 1, 2,* Quan Chen, 2, 3 Lei Yu, 1, 2 Peng Wang, 1, 2 Qi Zhang, 1, 2 Jiang Dong, 1, 2 Wei Zhang, 2, 4 Junxing Huang 1, 2 1Department of Oncology, Taizhou People’s Hospital, Taizhou, Jiangsu, People’s Republic of China; 2Medical School of Nantong University, Nantong, Jiangsu, People’s Republic of China; 3Department of Thoracic Surgery, Taizhou People’s Hospital, Taizhou, Jiangsu, People’s Republic of China; 4Department of Infectious Disease, Taizhou People’s Hospital, Taizhou, Jiangsu, People’s Republic of China*These authors contributed equally to this workCorrespondence: Wei ZhangDepartment of Infectious Disease, Taizhou People’s Hospital, No. 399, South Hailing Road, Taizhou, 225300 Jiangsu, People’s Republic of ChinaTel +86-523-86606739Email zw562652291@126.comJunxing HuangDepartment of Oncology, Taizhou People’s Hospital, No. 399, South Hailing Road, Taizhou, 225300 Jiangsu, People’s Republic of ChinaTel +86-523-86606739Email hjxtz@sina.cnBackground: Radiotherapy occupies an essential position as one of the most significant approaches for the clinical treatment of cancer. However, we cannot overcome the shortcoming of X-rays which is the high value of the oxygen enhancement ratio (OER). Radiosensitizers with the ability to enhance the radiosensitivity of tumor cells provide an alternative to changing X-rays to protons and heavy ion radiotherapy.Materials and Methods: We prepared the Au-Pt nanoparticles (Au-Pt NPs) using a one-step method. The characteristics of the Au-Pt NPs were determined using TEM, HAADF-STEM, elemental mapping images, and DLS. The enhanced radiotherapy was demonstrated in vitro using MTT assays, colony formation assays, fluorescence imaging, and flow cytometric analyses of the apoptosis. The biodistribution of the Au-Pt NPs was analyzed using ICP-OES, and thermal images. The enhanced radiotherapy was demonstrated in vitro using immunofluorescence images, tumor volume and weigh, and hematoxylin & eosin (H&E) staining.Results: Polyethylene glycol (PEG) functionalized nanoparticles composed of the metallic elements Au and Pt were designed to increase synergistic radiosensitivity. The mechanism demonstrated that heavy metal NPs possess a high X-ray photon capture cross-section and Compton scattering effect which increased DNA damage. Furthermore, the Au-Pt NPs exhibited enzyme-mimicking activities by catalyzing the decomposition of endogenous H 2O 2 to O 2 in the solid tumor microenvironment (TME).Conclusion: Our work provides a systematically administered radiosensitizer that can selectively reside in a tumor via the EPR effect and enhances the efficiency of treating cancer with radiotherapy.Keywords: radiosensitizer, nano-enzyme, TME, radiotherapy

Laterality defects are defined by the perturbed left–right arrangement of organs in the body, occurring in a syndromal or isolated fashion. In humans, primary ciliary dyskinesia (PCD) is a frequent underlying condition of defective left–right patterning, where ciliary motility defects also result in reduced airway clearance, frequent respiratory infections, and infertility. Non-motile cilia dysfunction and dysfunction of non-ciliary genes can also result in disturbances of the left–right body axis. Despite long-lasting genetic research, identification of gene mutations responsible for left–right patterning has remained surprisingly low. Here, we used whole-exome sequencing with Copy Number Variation (CNV) analysis to delineate the underlying molecular cause in 35 mainly consanguineous families with laterality defects. We identified causative gene variants in 14 families with a majority of mutations detected in genes previously associated with PCD, including two small homozygous CNVs. None of the patients were previously clinically diagnosed with PCD, underlining the importance of genetic diagnostics for PCD diagnosis and adequate clinical management. Identified variants in non-PCD-associated genes included variants in PKD1L1 and PIFO, suggesting that dysfunction of these genes results in laterality defects in humans. Furthermore, we detected candidate variants in GJA1 and ACVR2B possibly associated with situs inversus. The low mutation detection rate of this study, in line with other previously published studies, points toward the possibility of non-coding genetic variants, putative genetic mosaicism, epigenetic, or environmental effects promoting laterality defects.

AGO2 binds to miRNAs to repress expression of cognate target mRNAs. Here the authors report that heterozygous AGO2 mutations result in defects in neurological development and impair RNA interference.

Song Yang,1,* Peng Wang,1,* Songhua Wang,1 Aihua Cong,1 Qi Zhang,1 Wenhao Shen,1 Xiangyi Li,2 Wei Zhang,3 Gaohua Han1 1Department of Oncology, Taizhou People’s Hospital, Taizhou, Jiangsu 225300, People’s Republic of China; 2Department of Endocrinology, Taizhou People’s Hospital, Taizhou, Jiangsu 225300, People’s Republic of China; 3Department of Infectious Diseases, Taizhou People’s Hospital, Taizhou 225300, Jiangsu, People’s Republic of China*These authors contributed equally to this workCorrespondence: Wei Zhang; Gaohua Han Email tgc6nl@163.com; y0ztiin@163.comBackground: Cisplatin (CDDP) is extensively used for esophageal adenocarcinoma (EAC) chemotherapy, while cisplatin resistance is getting worse. microRNA-181a-5p (miR-181a-5p) has been reported to play an important role in various human cancers. However, the effect and underlying mechanism of miR-181a-5p in cisplatin resistance of EAC remain unclear.Methods: Cisplatin-resistant EAC cells OE19/CDDP and parental sensitive OE19 cells were applied for experiments in vitro. The expressions of miR-181a-5p and CBLB were measured by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. The cisplatin resistance of cells was expressed by cell viability, IC50 and apoptosis rate by using CCK-8 assay or flow cytometry. The interaction between miR-181a-5p and CBLB was evaluated by luciferase reporter assay and RIP assay. In vivo experiments were conducted via the murine xenograft model.Results: miR-181a-5p was highly expressed while CBLB was lowly expressed in OE19 cell lines compared with OE19/CDDP cells. In cisplatin-resistant OE19/CDDP cells, miR-181a-5p up-regulation or CBLB knockdown inhibited cell viability and inducted apoptosis. In cisplatin-sensitive OE19 cells, miR-181a-5p inhibition or CBLB overexpression promoted cell viability and suppressed apoptosis. CBLB was confirmed to be a target of miR-181a-5p, and rescue assay showed CBLB overexpression reversed the suppression of OE19/CDDP cell viability induced by miR-181a-5p up-regulation, and its down-regulation attenuated miR-181a-5p-inhibition-mediated enhancement of OE19 cell viability. In addition, miR-181a-5p up-regulation enhanced the cytotoxicity of cisplatin in EAC in vivo.Conclusion: miR-181a-5p enhanced the sensitivity of cells to cisplatin in EAC by targeting CBLB, indicating a promising sensitizer of cisplatin therapy in clinical esophageal cancer.Keywords: EAC, cisplatin resistance, miR-181a-5p, CBLB

Neurodevelopmental disorders (NDDs) are a heterogeneous group of diseases for which the genetic basis is still unknown in more than half of the cases. Here, the authors report a NDD associated with disruptive variants in the TANC2 gene and show that rols, the TANC2 homolog in flies, is required for synapse growth and function.

The HTML and PDF versions of this Article were updated after publication to remove images of one individual from Figure 1.

Yong-Da Liu,1 Zhi-Bin Wang,1 Guang Han,1 Li Jin,2 Ping Zhao1 1Department of Anesthesiology and Pain Management, Shengjing Hospital of China Medical University, Shenyang 110004, China; 2Department of Orthopaedic Surgery, University of Virginia, Charlottesville, VA 22908, USA Background: Our previous study suggested that HBO treatment attenuated neuropathic pain by inhibiting mTOR to induce autophagy in SNL neuropathic pain model. The aim of this study was to evaluate the role of AKT/TSC2/mTOR pathway in SNL and autophagy and determine whether HBO treatment could relieve neuropathic pain via modulating AKT/TSC2/mTOR pathway.Materials and methods: Rats were randomly divided into sham, SNL, SNL + HBO treatment, SNL + vehicle, and SNL + AKT inhibitor groups. Neuropathic pain was induced following SNL procedure. Rats in the SNL + HBO group received HBO treatment for 7 consecutive days beginning on postoperative day 1. The SNL + vehicle group received 10 µL of 3% dimethyl sulfoxide in saline. SNL + AKT inhibitor group received 10 µL AKT inhibitor IV intrathecally. Mechanical withdrawal threshold tests were performed to evaluate mechanical hypersensitivity. AKT, p-AKT, TSC2, mTOR, p-mTOR, and LC3-II protein expressions were examined by Western blot analysis.Results: HBO reversed AKT/TSC2/mTOR upregulation induced by SNL and attenuated neuropathic pain. Intrathecal injection of AKT inhibitor IV decreased the activity of AKT/TSC2/mTOR pathway and increased LC3-II expression accompanied by analgesic effect in SNL rats.Conclusion: Taken together, our findings demonstrated AKT/TSC2/mTOR pathway was activated in SNL-induced neuropathic pain, and HBO treatment attenuated neuropathic pain via neutralizing AKT/TSC2/mTOR pathway activation. Keywords: neuropathic pain, autophagy, mTOR, TSC2, AKT, hyperbaric oxygen

The Asian Pacific American Medical Student Association (APAMSA) is a national student organization that advocates for the health of the Asian American Pacific Islanders. In May 2020, our APAMSA chapter at Oakland University William Beaumont (OUWB) School of Medicine located in Michigan, USA hosted a virtual mental health campaign titled, “Socially Distant but Emotionally Connected: 6ft Closer During Quarantine.” We reached out to medical students and faculty within the OUWB community to share their experiences during the initial phases of quarantine. Our goal was to create a space for everyone at OUWB to engage in meaningful conversations about mental health and support each other during the pandemic. The responses we received varied across numerous topics, including xenophobia, loneliness, and lack of motivation. Participants also followed up with words of encouragement for their peers and guidance on how to cope with social isolation. Our virtual campaign was very feasible and successful under the constraints of social distancing, and we urge other medical schools to implement their own mental health awareness initiatives to destigmatize the topic in their communities. General steps on how to start your own campaign include: collaborating with interest groups, deciding on social media platforms, and sharing with your community.

Background: The VATER/VACTERL association (VACTERL) is defined as the non-random occurrence of the following congenital anomalies: Vertebral, Anal, Cardiac, Tracheal-Esophageal, Renal, and Limb anomalies. As no unequivocal candidate gene has been identified yet, patients are diagnosed phenotypically. The aims of this study were to identify patients with monogenic disorders using a genetics-first approach, and to study whether variants in candidate genes are involved in the etiology of VACTERL or the individual features of VACTERL: Anorectal malformation (ARM) or esophageal atresia with or without trachea-esophageal fistula (EA/TEF).Methods: Using molecular inversion probes, a candidate gene panel of 56 genes was sequenced in three patient groups: VACTERL (n = 211), ARM (n = 204), and EA/TEF (n = 95). Loss-of-function (LoF) and additional likely pathogenic missense variants, were prioritized and validated using Sanger sequencing. Validated variants were tested for segregation and patients were clinically re-evaluated.Results: In 7 out of the 510 patients (1.4%), pathogenic or likely pathogenic variants were identified in SALL1, SALL4, and MID1, genes that are associated with Townes-Brocks, Duane-radial-ray, and Opitz-G/BBB syndrome. These syndromes always include ARM or EA/TEF, in combination with at least two other VACTERL features. We did not identify LoF variants in the remaining candidate genes.Conclusions: None of the other candidate genes were identified as novel unequivocal disease genes for VACTERL. However, a genetics-first approach allowed refinement of the clinical diagnosis in seven patients, in whom an alternative molecular-based diagnosis was found with important implications for the counseling of the families.

Abstract Background A causal genetic mutation is found in 40% of families with dilated cardiomyopathy (DCM), leaving a large percentage of families genetically unsolved. This prevents adequate counseling and clear recommendations in these families. We aim to identify novel genes or modifiers associated with DCM. Methods We performed computational ranking of human genes based on coexpression with a predefined set of genes known to be associated with DCM, which allowed us to prioritize gene candidates for their likelihood of being involved in DCM. Top candidates will be checked for variants in the available whole‐exome sequencing data of 142 DCM patients. RNA was isolated from cardiac biopsies to investigate gene expression. Results PDLIM5 was classified as the top candidate. An interesting heterozygous variant (189_190delinsGG) was found in a DCM patient with a known pathogenic truncating TTN‐variant. The PDLIM5 loss‐of‐function (LoF) variant affected all cardiac‐specific isoforms of PDLIM5 and no LoF variants were detected in the same region in a control cohort of 26,000 individuals. RNA expression of PDLIM5 and its direct interactors (MYOT, LDB3, and MYOZ2) was increased in cardiac tissue of this patient, indicating a possible compensatory mechanism. The PDLIM5 variant cosegregated with the TTN‐variant and the phenotype, leading to a high disease penetrance in this family. A second patient was an infant with a homozygous 10 kb‐deletion of exon 2 in PDLIM5 resulting in early‐onset cardiac disease, showing the importance of PDLIM5 in cardiac function. Conclusions Heterozygous PDLIM5 variants are rare and therefore will not have a major contribution in DCM. Although they likely play a role in disease development as this gene plays a major role in contracting cardiomyocytes and homozygous variants lead to early‐onset cardiac disease. Other environmental and/or genetic factors are probably necessary to unveil the cardiac phenotype in PDLIM5 mutation carriers.

Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines.

Chromodomain Helicase DNA-binding (CHD) proteins have been implicated in neurodevelopmental processes. Here, the authors identify missense variants in CHD3 that disturb its chromatin remodeling activities and cause a neurodevelopmental disorder with macrocephaly and speech and language impairment.

A proportion of neurodevelopmental disorder and congenital anomaly cases remain without a genetic diagnosis. Here, the authors study aberrations of DNA methylation in such cases and find that epivariations might provide an explanation for some of these undiagnosed patients.

Synovial haemangiomas are rare benign vascular proliferations arising in synovium-lined surfaces. While the knee is by far the joint most commonly involved, this condition can also occur in the elbow. We report an eight-year-old boy who presented to the National University of Malaysia Medical Centre, Kuala Lumpur, Malaysia, in 2016 with a left elbow swelling of one year’s duration. Magnetic resonance imaging showed a lobulated intraarticular mass with intermediate signal intensity on T1-weighted imaging and low signal punctate and linear structures within the hyperintense mass on T2-weighted imaging. In addition, there was heterogeneous yet avid contrast enhancement on post-gadolinium contrast images. The mass had juxta-articular extension and bony erosion to the coronoid process and the head of the radius. Synovial haemangiomas present a diagnostic dilemma. This report highlights certain imaging characteristics to distinguish this entity from other differential diagnoses.

Cellular self-digestion is an evolutionarily conserved process occurring in prokaryotic cells that enables survival under stressful conditions by recycling essential energy molecules. Self-digestion, which is triggered by extracellular stress conditions, such as nutrient depletion and overpopulation, induces degradation of intracellular components. This self-inflicted damage renders the bacterium less fit to produce building blocks and resume growth upon exposure to fresh nutrients. However, self-digestion may also provide temporary protection from antibiotics until the self-digestion-mediated damage is repaired. In fact, many persistence mechanisms identified to date may be directly or indirectly related to self-digestion, as these processes are also mediated by many degradative enzymes, including proteases and ribonucleases (RNases). In this review article, we will discuss the potential roles of self-digestion in bacterial persistence.

Although ubiquitously present, the relevance of cilia for vertebrate development and health has long been underrated. However, the aberration or dysfunction of ciliary structures or components results in a large heterogeneous group of disorders in mammals, termed ciliopathies. The majority of human ciliopathy cases are caused by malfunction of the ciliary dynein motor activity, powering retrograde intraflagellar transport (enabled by the cytoplasmic dynein-2 complex) or axonemal movement (axonemal dynein complexes). Despite a partially shared evolutionary developmental path and shared ciliary localization, the cytoplasmic dynein-2 and axonemal dynein functions are markedly different: while cytoplasmic dynein-2 complex dysfunction results in an ultra-rare syndromal skeleto-renal phenotype with a high lethality, axonemal dynein dysfunction is associated with a motile cilia dysfunction disorder, primary ciliary dyskinesia (PCD) or Kartagener syndrome, causing recurrent airway infection, degenerative lung disease, laterality defects, and infertility. In this review, we provide an overview of ciliary dynein complex compositions, their functions, clinical disease hallmarks of ciliary dynein disorders, presumed underlying pathomechanisms, and novel developments in the field.

Mingfeng Shao,1 Wenfang Yang,2 Guangying Han1 1Department of Cardiology, Linyi People’s Hospital, Linyi, Shandong, People’s Republic of China; 2Department of Internal Medicine, Linyi Hot Spring Hospital of Shandong Coal Mine, Linyi, Shandong, People’s Republic of China Purpose: Schisandrin B (Sch B) is clinically applied for the treatment of hepatitis and ischemic disease. However, its clinical efficacy is limited due to the poor solubility and low bioavailability. This study aimed to develop matrix metalloproteinase (MMP)-sensitive peptide-modified, polyethylene glycol (PEG)-modified (PEGylated) solid lipid nanoparticles (SLNs) for loading Sch B (MMP-Sch B SLNs), and to evaluate the therapeutic effect in the myocardial infarction model.Methods: PEG lipid and MMP-targeting peptide conjugate were synthesized. MMP-Sch B SLNs were prepared by solvent displacement technique. The physicochemical properties and pharmacokinetics of SLNs were investigated. In vivo effects on infarct size was evaluated in rats.Results: The successful synthesis of lipid-peptide conjugate was confirmed. MMP-Sch B SLNs had a particle size of 130 nm, a zeta potential of 18.3 mV, and a sustained-release behavior. Higher heart drug concentration and longer blood circulation times were achieved by Sch B loaded SLNs than the drug solution according to the pharmacokinetic and biodistribution results. The best therapeutic efficacy was exhibited by MMP-Sch B SLNs by reducing the infarction size to the greatest extent.Conclusion: The modified SLNs may be a good choice for delivery of Sch B for the treatment of myocardial infarction. Keywords: cardiovascular diseases, CVDs, schisandrin B, matrix metalloproteinase, lipid nanoparticles

The hippocampus in mammalian brain varies in size across individuals. Here, Hibar and colleagues perform a genome-wide association meta-analysis to find six genetic loci with significant association to hippocampus volume.

The clinical utility of computational phenotyping for both genetic and rare diseases is increasingly appreciated; however, its true potential is yet to be fully realized. Alongside the growing clinical and research availability of sequencing technologies, precise deep and scalable phenotyping is required to serve unmet need in genetic and rare diseases. To improve the lives of individuals affected with rare diseases through deep phenotyping, global big data interrogation is necessary to aid our understanding of disease biology, assist diagnosis, and develop targeted treatment strategies. This includes the application of cutting-edge machine learning methods to image data. As with most digital tools employed in health care, there are ethical and data governance challenges associated with using identifiable personal image data. There are also risks with failing to deliver on the patient benefits of these new technologies, the biggest of which is posed by data siloing. The Minerva Initiative has been designed to enable the public good of deep phenotyping while mitigating these ethical risks. Its open structure, enabling collaboration and data sharing between individuals, clinicians, researchers and private enterprise, is key for delivering precision public health.

IntroductionAnorectal malformations (ARM) are rare congenital malformations, resulting from disturbed hindgut development. A genetic etiology has been suggested, but evidence for the involvement of specific genes is scarce. We evaluated the contribution of rare and low-frequency coding variants in ARM etiology, assuming a multifactorial model.MethodsWe analyzed 568 Caucasian ARM patients and 1,860 population-based controls using the Illumina HumanExome Beadchip array, which contains >240,000 rare and low-frequency coding variants. GenomeStudio clustering and calling was followed by re-calling of 'no-calls' using zCall for patients and controls simultaneously. Single variant and gene-based analyses were performed to identify statistically significant associations, applying Bonferroni correction. Following an extra quality control step, candidate variants were selected for validation using Sanger sequencing.ResultsWhen we applied a MAF of ≥1.0%, no variants or genes showed statistically significant associations with ARM. Using a MAF cut-off at 0.4%, 13 variants initially reached statistical significance, but had to be discarded upon further inspection: ten variants represented calling errors of the software, while the minor alleles of the remaining three variants were not confirmed by Sanger sequencing.ConclusionOur results show that rare and low-frequency coding variants with large effect sizes, present on the exome chip do not contribute to ARM etiology.

[This corrects the article DOI: 10.1371/journal.pgen.1002114.].

Regulatory T cells (Treg cells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Treg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Treg cells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal Treg cells to regulate helper T17 cell (TH17 cell) immunity. Selectively increased intestinal TH17 cell responses in mice with Treg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+CD62Llo Treg cell subset that is distinct from previously characterized intestinal Treg cell populations as the main IL-27 producers. Collectively, our study uncovers a new Treg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific Treg cell-mediated immune regulation.

Gang Han, Zhenwei Yu, Ke Ma Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China Purpose: The aim of the study reported here was to assess the use of parenteral nutrition (PN) and enteral nutrition (EN), and the prevalence of PN and EN formulas, in the People’s Republic of China. Methods: Fifty-nine hospitals in the People’s Republic of China participated in a nutrition survey. The resulting information on nutritional support was analyzed. Results: We received 379,584 nutritional-support prescriptions over 40 days in 2013. PN provided approximately 63.2% and EN provided approximately 36.8% of nitrogen intake. PN provided 63.5% and EN provided 36.5% of lipid intake. There were obvious differences in nitrogen and lipid intake between PN and EN in different regions, departments, and diseases. The percentage of nourishment provided by PN in different regions was highest in Chengdu, followed by the Beijing, Guangzhou, and Hangzhou areas. The percentage of nourishment provided by PN in different departments was highest in general surgery, followed by gastroenterology and the intensive care unit. The percentage of nourishment provided by PN in different diseases/conditions was highest in acute pancreatitis, followed by cancer, and burns. The main source of nitrogen in PN was balanced amino-acid preparations, and in EN, it was protein. The main source of lipids in PN was long- and medium-chain triglyceride lipid emulsion injection. Conclusion: Despite recent improvements in the application of nutritional support in the People’s Republic of China, a much higher percentage of nitrogen and lipids is delivered through PN than through EN. Furthermore, there are marked regional, departmental, and disease-based differences in the selection of PN versus EN. The rationale for use of nutritional support needs to be improved. Keywords: nutrition survey, prescription, nutritional support, amino-acid preparations, lipid emulsion

Copy number variation (CNV) is a common source of genetic variation that has been implicated in many genomic disorders, Mendelian diseases, and common/complex traits. Genomic microarrays are often employed for CNV detection. More recently, whole-exome sequencing (WES) has enabled detection of clinically relevant point mutations and small insertion—deletion exome wide. We evaluated (de Ligt et al. 2013) [1] the utility of short-read WES (SOLiD 5500xl) to detect clinically relevant CNVs in DNA from 10 patients with intellectual disability and compared these results to data from three independent high-resolution microarray platforms. Calls made by the different platforms and detection software are available at dbVar under nstd84.