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Bleaching is a finishing process to whiten the gray fabrics. During this process it is important to ensure the demanded results with minimum fiber damage by considering environmental aspects. As a result of this, the study focused on the wool bleaching which is also an important process for the quality of wool products. It was managed with and without ultrasound and it was found that via ultrasound better bleaching results can be obtained in addition to improvement in contact angles related to the hydrophilicity. It is expected that with the addition of ultrasound to bleaching process, same results obtained with conventional bleaching will be reached with lower treatment temperatures or lower treatment time which is now essential for the protection of environment. [ABSTRACT FROM PUBLISHER]

The aim of this study was to investigate the effects of novel treatment techniques (ozone and ultrasound) on the dyeability of Angora rabbit fiber. For this purpose, the effects of the ozonation time and fiber moisture during ozonation on the dyeing properties of Angora rabbit fiber were researched. Also, dyeing was performed conventionally and with the use of ultrasound techniques after ozonation; these were compared in terms of the color yields. Consequently, it was found that the ozonation process and dyeing with power ultrasound improved the dyeability of Angora rabbit fiber significantly. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011 [ABSTRACT FROM AUTHOR]

This paper provides a general framework for studying the yellowing tendency of ozonated cotton fabric. Yellowing tendency of ozonated cotton fabric and ways to prevent this situation was examined extensively. For this purpose, five different treatments (hot/cold rinsing, washing, reductive washing, catalase treatment) were performed after ozonation. It was found that performing any kind of treatment was significantly important for preventing yellowing of ozonated cotton fabric during storage. [ABSTRACT FROM AUTHOR]

The field of ultrasonics is still making strides towards perfection, but already many applications of ultrasonic energy have been found in science and technology. Ultrasonics is the science of sound waves above the limits of human audibility. The aim of this study was to investigate the effects of ultrasound on textile pretreatment processes especially on enzymatic processes. Use of enzymes in the textile industry has become more popular in recent years. Although enzymatic processing offers many advantages, there are a few drawbacks when compared to traditional methods, namely, expensive processing costs and relatively slow reaction rates. Introducing ultrasonic energy during enzymatic treatment of cotton fabric significantly improves enzyme efficiency without affecting the strength of the fabric. [ABSTRACT FROM AUTHOR]

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Objectives: Blunt abdominal trauma is a common cause of emergency department admission. Computed tomography (CT) scanning is the gold standard method for identifying intra-abdominal injuries in patients experiencing blunt trauma, especially those with high-energy trauma. Although the diagnostic accuracy of this imaging technique is very high, patient admission and prolonged observation protocols are still common practices worldwide. We aimed to evaluate the incidence of intra-abdominal injury in hemodynamically stable patients with high-energy blunt trauma and a normal abdominal CT scan at a Level-1 Trauma Center in Colombia, South America, to assess the relevance of a prolonged observation period., Methods: We performed a retrospective study of patients admitted to the emergency department for blunt trauma between 2021 and 2022. All consecutive patients with high-energy mechanisms of trauma and a normal CT scan at admission were included. Our primary outcomes were the incidence of intra-abdominal injury identified during a 24-hour observation period or hospital stay, ICU admission, and death., Results: We included 480 patients who met the inclusion criteria. The median age was 33 (IQR 25.5, 47), and 74.2% were male. The most common mechanisms of injury were motor vehicle accidents (64.2%), falls from height (26%), and falls from bikes (3.1%). A total of 99.2% of patients had a Revised Trauma Score of 8. Only 1 patient (0.2%) (95% CI: 0.01-1.16) presented with an abdominal injury during the observation period. No ICU admissions or deaths were reported., Conclusion: The incidence of intra-abdominal injury in patients with hemodynamically stable blunt trauma and a negative abdominal CT scan is extremely low, and prolonged observation may not be justified in these patients., (© 2024. The Author(s).)

Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO 2 . Recent research, however, demonstrated fungal conversion of 13 C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated 13 C-labeled lignin served as substrate for Agaricus bisporus , the world's most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in 13 C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in 13 C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of 13 C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin's metabolic value for fungi.

Danger-associated molecular patterns (DAMPs) are molecules that can initiate and maintain robust inflammatory responses and were investigated in the pathogenesis of graft versus host disease (GvHD). Uric acid (UA) and fibrinogen (Fib) are DAMPs released from damaged tissue during allogeneic hematopoietic stem cell transplantation (allo-HCT) and GvHD. We aimed to evaluate the effects of UA and Fib levels on survival in GvHD. One hundred seventy-four patients with grade 2-4 acute GvHD were included. UA and Fib levels were evaluated on allo-HCT day 0 and GvHD on days 0, 7, 14, and 28. Fib GvHD day 0 was the independent predictor for overall survival (OS), non-relapse mortality (NRM), and progression-free survival in multivariable models (HR 0.98, p < 0.001; HR 0.98, p = 0.001, HR 0.98, p = 0.006, respectively). Also UA GvHD day 28 was the independent predictor for OS and NRM (HR 0.77, p = 0.004; HR 0.76, p = 0.011, respectively). Our results indicated that hypouricemia and hypofibrinogenemia were associated with a significantly shorter OS and higher NRM. UA and Fib are remarkable molecules in GvHD because they are routinely utilized, readily available, can be therapeutic targets, and have DAMPs and antioxidant features., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD., (Copyright © 2023 International Society of Nephrology. All rights reserved.)

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Fungi are main lignin degraders and the edible white button mushroom, Agaricus bisporus, inhabits lignocellulose-rich environments. Previous research hinted at delignification when A. bisporus colonized pre-composted wheat straw-based substrate in an industrial setting, assumed to aid subsequent release of monosaccharides from (hemi-)cellulose to form fruiting bodies. Yet, structural changes and specific quantification of lignin throughout A. bisporus mycelial growth remain largely unresolved. To elucidate A. bisporus routes of delignification, at six timepoints throughout mycelial growth (15 days), substrate was collected, fractionated, and analyzed by quantitative pyrolysis-GC-MS, 2D-HSQC NMR, and SEC. Lignin decrease was highest between day 6 and day 10 and reached in total 42 % (w/w). The substantial delignification was accompanied by extensive structural changes of residual lignin, including increased syringyl to guaiacyl (S/G) ratios, accumulated oxidized moieties, and depleted intact interunit linkages. Hydroxypropiovanillone and hydroxypropiosyringone (HPV/S) subunits accumulated, which are indicative for β-|O-4' ether cleavage and imply a laccase-driven ligninolysis. We provide compelling evidence that A. bisporus is capable of extensive lignin removal, have obtained insights into mechanisms at play and susceptibilities of various substructures, thus we were contributing to understanding fungal lignin conversion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Despite substantial lignocellulose conversion during mycelial growth, previous transcriptome and proteome studies have not yet revealed how secretomes from the edible mushroom Agaricus bisporus develop and whether they modify lignin models in vitro . To clarify these aspects, A. bisporus secretomes collected throughout a 15-day industrial substrate production and from axenic lab-cultures were subjected to proteomics, and tested on polysaccharides and lignin models. Secretomes (day 6-15) comprised A. bisporus endo-acting and substituent-removing glycoside hydrolases, whereas β-xylosidase and glucosidase activities gradually decreased. Laccases appeared from day 6 onwards. From day 10 onwards, many oxidoreductases were found, with numerous multicopper oxidases (MCO), aryl alcohol oxidases (AAO), glyoxal oxidases (GLOX), a manganese peroxidase (MnP), and unspecific peroxygenases (UPO). Secretomes modified dimeric lignin models, thereby catalyzing syringylglycerol-β-guaiacyl ether (SBG) cleavage, guaiacylglycerol-β-guaiacyl ether (GBG) polymerization, and non-phenolic veratrylglycerol-β-guaiacyl ether (VBG) oxidation. We explored A. bisporus secretomes and insights obtained can help to better understand biomass valorization., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: Recurrent respiratory syncytial virus (RSV) infection requiring hospitalization is rare and the underlying mechanism is unknown. We aimed to determine the role of CD14-mediated immunity in the pathogenesis of recurrent RSV infection., Methods: We performed genotyping and longitudinal immunophenotyping of the first patient with a genetic CD14 deficiency who developed recurrent RSV infection. We analyzed gene expression profiles and interleukin (IL)-6 production by patient peripheral blood mononuclear cells in response to RSV pre- and post-fusion (F) protein. We generated CD14-deficient human nasal epithelial cells cultured at air-liquid interface (HNEC-ALI) of patient-derived cells and after CRISPR-based gene editing of control cells. We analyzed viral replication upon RSV infection., Results: Sanger sequencing revealed a homozygous single-nucleotide deletion in CD14, resulting in absence of the CD14 protein in the index patient. In vitro, viral replication was similar in wild-type and CD14-/- HNEC-ALI. Loss of immune cell CD14 led to impaired cytokine and chemokine responses to RSV pre- and post-F protein, characterized by absence of IL-6 production., Conclusions: We report an association of recurrent RSV bronchiolitis with a loss of CD14 function in immune cells. Lack of CD14 function led to defective immune responses to RSV pre- and post-F protein without a change in viral replication., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Maximum parsimony reconciliation is a fundamental technique for studying the evolutionary histories of pairs of entities such as genes and species, parasites and hosts, and species and their biogeographical habitats. In these contexts, reconciliation is generally performed using the duplication-transfer-loss (DTL) model in a maximum parsimony framework. While efficient maximum parsimony reconciliation algorithms are known for the DTL model, the number of such reconciliations can grow exponentially with the sizes of the two phylogenetic trees. Choosing a maximum parsimony reconciliation arbitrarily may lead to conclusions that are not supported, and may even be contradicted, by other equally optimal reconciliations. This paper addresses the fundamental problem of how well a single reconciliation can represent the entire space of optimal reconciliations.

Adult granulosa cell tumors (AGCTs) arise from the estrogen-producing granulosa cells. Treatment of recurrence remains a clinical challenge, as systemic anti-hormonal treatment or chemotherapy is only effective in selected patients. We established a method to rapidly screen for drug responses in vitro using direct patient-derived cell lines in order to optimize treatment selection. The response to 11 monotherapies and 12 combination therapies, including chemotherapeutic, anti-hormonal, and targeted agents, were tested in 12 AGCT-patient-derived cell lines and an AGCT cell line (KGN). Drug screens were performed within 3 weeks after tissue collection by measurement of cell viability 72 h after drug application. The potential synergy of drug combinations was assessed. The human maximum drug plasma concentration (Cmax) and steady state (Css) thresholds obtained from available phase I/II clinical trials were used to predict potential toxicity in patients. Patient-derived AGCT cell lines demonstrated resistance to all monotherapies. All cell lines showed synergistic growth inhibition by combination treatment with carboplatin, paclitaxel, and alpelisib at a concentration needed to obtain 50% cell death (IC50) that are below the maximum achievable concentration in patients (IC50 < Cmax). We show that AGCT cell lines can be rapidly established and used for patient-specific in vitro drug testing, which may guide treatment decisions. Combination treatment with carboplatin, paclitaxel, and alpelisib was consistently effective in AGCT cell lines and should be further studied as a potential effective combination for AGCT treatment in patients.

We report here a de novo missense variant in HIST1H4J resulting in a complex syndrome combining growth delay, microcephaly and intellectual disability. Trio whole exome sequencing (WES) revealed that the proband was heterozygous for a de novo c.274 A > G p.(K91E) variant in HIST1H4J, a gene not yet associated with human disease. The patient presented with profound intellectual disability, microcephaly, and dysmorphic facial features. Functional consequences of the identified de novo missense variant were evaluated in zebrafish embryos, where they affected general development, especially resulting in defective head organs and reduced body axis length. Our results show that the monoallelic p.K91E substitution on HIST1H4J underlies a human syndrome that is genetically and phenotypically akin to the HIST1H4C-associated neurodevelopmental disorder resulting from p.K91A and p.K91Q substitions in HIST1H4C. The highly overlapping patient phenotypes highlight functional similarities between HIST1H4J and HIST1H4C perturbations, establishing the singular importance of K91 across histone H4 genes for vertebrate development.

Cantú syndrome (CS), first described in 1982, is caused by pathogenic variants in ABCC9 and KCNJ8, which encode the regulatory and pore forming subunits of ATP-sensitive potassium (K ATP ) channels, respectively. Multiple case reports of affected individuals have described the various clinical features of CS, but systematic studies are lacking. To define the effects of genetic variants on CS phenotypes and clinical outcomes, we have developed a standardized REDCap-based registry for CS. We report phenotypic features and associated genotypes on 74 CS subjects, with confirmed ABCC9 variants in 72 of the individuals. Hypertrichosis and a characteristic facial appearance are present in all individuals. Polyhydramnios during fetal life, hyperflexibility, edema, patent ductus arteriosus (PDA), cardiomegaly, dilated aortic root, vascular tortuosity of cerebral arteries, and migraine headaches are common features, although even with this large group of subjects, there is incomplete penetrance of CS-associated features, without clear correlation to genotype., (© 2019 Wiley Periodicals, Inc.)

Cantú syndrome (CS) is caused by dominant gain-of-function mutation in ATP-dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but I KATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that I KATP inhibitors, glibenclamide and HMR1098, normalize CS channels. I KATP in response to Mg-ATP, glibenclamide and HMR1098 were measured by inside-out patch-clamp electrophysiology. Results were interpreted in view of cryo-EM I KATP channel structures. Mg-ATP IC 50 values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild-type (0.15 ± 0.01). HMR1098 dose-dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg-ATP, reaching, at 30 μmol/L, current levels displayed by wild-type and H60Y channels in the presence of 0.15 mmol/L Mg-ATP. Glibenclamide (10 μmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg-ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Stress engendered by stereotype threatening situations may facilitate encoding of negative, stereotype confirming feedback received during a performance among women in science, technology, engineering and mathematics (STEM). It is unclear, however, whether this process is comprised of the same neurophysiological mechanisms evident in any emotional memory encoding context, or if this encoding bias directly undermines positive self-perceptions in the stigmatized domain. A total of 160 men and women completed a math test that provided veridical positive and negative feedback, a memory test for feedback, and math self-enhancing and valuing measures in a stereotype threatening or neutral context while continuous electroencephalography activity and startle probe responses to positive and negative feedback was recorded. Indexing amygdala activity to feedback via startle responses and emotional memory network connectivity elicited during accurate recognition of positive and negative feedback via graph analyses, only stereotype threatened women encoded negative feedback better when they exhibited increased amygdala activity and emotional memory network connectivity in response to said feedback. Emotional memory biases, in turn, predicted decreases in women's self-enhancing, math valuing and performance. Findings provide an emotional memory encoding-based mechanism for well-established findings indicating that women have more negative math self-perceptions compared with men regardless of actual performance.

Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.

Covalent modifications of histones have an established role as chromatin effectors, as they control processes such as DNA replication and transcription, and repair or regulate nucleosomal structure. Loss of modifications on histone N tails, whether due to mutations in genes belonging to histone-modifying complexes or mutations directly affecting the histone tails, causes developmental disorders or has a role in tumorigenesis. More recently, modifications affecting the globular histone core have been uncovered as being crucial for DNA repair, pluripotency and oncogenesis. Here we report monoallelic missense mutations affecting lysine 91 in the histone H4 core (H4K91) in three individuals with a syndrome of growth delay, microcephaly and intellectual disability. Expression of the histone H4 mutants in zebrafish embryos recapitulates the developmental anomalies seen in the patients. We show that the histone H4 alterations cause genomic instability, resulting in increased apoptosis and cell cycle progression anomalies during early development. Mechanistically, our findings indicate an important role for the ubiquitination of H4K91 in genomic stability during embryonic development.

Background: The inward rectifier potassium current I K1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased I K1 , short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC 50  = 14 nM with inside-out patch clamp methodology) and specific I K1 inhibitor that interacts with the cytoplasmic pore region of the K IR 2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) K IR 2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N K IR 2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations., Methods: Molecular modelling was performed with the human K IR 2.1 closed state homology model using FlexX. WT and mutant K IR 2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. K IR 2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively., Results: PA-6 docking in the V93I/D172N double mutant homology model of K IR 2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC 50  = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC 50  = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward I K1 at -50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased K IR 2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular K IR 2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM)., Conclusions: 1) KCNJ2 gain-of-function mutations V93I and D172N in the K IR 2.1 ion channel do not impair PA-6 mediated inhibition of I K1 , 2) PA-6 elevates K IR 2.1 protein expression and induces intracellular K IR 2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

Benzoxazinoids function as defense compounds and have been suggested to possess health-promoting effects. In this work, the mass spectrometric behavior of benzoxazinoids from the classes benzoxazin-3-ones (with subclasses lactams, hydroxamic acids, and methyl derivatives) and benzoxazolinones was studied. Wheat seeds were germinated with simultaneous elicitation by Rhizopus. The seedling extract was screened for the presence of benzoxazinoid (glycosides) using reversed-phase ultra-high-performance liquid chromatography with photodiode array detection coupled in line to multiple-stage mass spectrometry (RP-UHPLC-PDA-MS(n)). Benzoxazin-3-ones from the different subclasses showed distinctly different ionization and fragmentation behaviors. These features were incorporated into a newly proposed decision guideline to aid the classification of benzoxazinoids. Glycosides of the methyl derivative 2-hydroxy-4-methoxy-1,4-benzoxazin-3-one were tentatively identified for the first time in wheat. We conclude that wheat seedlings germinated with simultaneous fungal elicitation contain a diverse array of benzoxazinoids, mainly constituted by benzoxazin-3-one glycosides.

The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.

Background: Although BRCA1-deficient tumors are extremely sensitive to DNA-damaging drugs and poly(ADP-ribose) polymerase (PARP) inhibitors, recurrences do occur and, consequently, resistance to therapy remains a serious clinical problem. To study the underlying mechanisms, we induced therapy resistance in patient-derived xenograft (PDX) models of BRCA1-mutated and BRCA1-methylated triple-negative breast cancer., Methods: A cohort of 75 mice carrying BRCA1-deficient breast PDX tumors was treated with cisplatin, melphalan, nimustine, or olaparib, and treatment sensitivity was determined. In tumors that acquired therapy resistance, BRCA1 expression was investigated using quantitative real-time polymerase chain reaction and immunoblotting. Next-generation sequencing, methylation-specific multiplex ligation-dependent probe amplification (MLPA) and Target Locus Amplification (TLA)-based sequencing were used to determine mechanisms of BRCA1 re-expression in therapy-resistant tumors., Results: BRCA1 protein was not detected in therapy-sensitive tumors but was found in 31 out of 42 resistant cases. Apart from previously described mechanisms involving BRCA1-intragenic deletions and loss of BRCA1 promoter hypermethylation, a novel resistance mechanism was identified in four out of seven BRCA1-methylated PDX tumors that re-expressed BRCA1 but retained BRCA1 promoter hypermethylation. In these tumors, we found de novo gene fusions that placed BRCA1 under the transcriptional control of a heterologous promoter, resulting in re-expression of BRCA1 and acquisition of therapy resistance., Conclusions: In addition to previously described clinically relevant resistance mechanisms in BRCA1-deficient tumors, we describe a novel resistance mechanism in BRCA1-methylated PDX tumors involving de novo rearrangements at the BRCA1 locus, demonstrating that BRCA1-methylated breast cancers may acquire therapy resistance via both epigenetic and genetic mechanisms., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Sound wave-assisted acoustic micromixing has been shown to increase the binding of molecules in small volumes (10-100 μL) where effective mixing is difficult to achieve through conventional techniques. The aim of this work is to study whether acoustic micromixing can increase the binding efficiency of antibodies to their antigens, a reaction that forms the basis of immunoassays, including enzyme-linked immunosorbent assay (ELISA). Using a procedure from a general ELISA and immobilizing an antigen on wells of 96-well plates, it was found that acoustic micromixing at 125-150 Hz increased the initial rate of antibody-antigen binding by over 80 % and the total binding at the end point (i.e., 45 min) by over 50 %. As a result, acoustic micromixing achieved a binding level in 9 min that would otherwise take 45 min on a standard platform rocking mixer. Therefore acoustic micromixing has the potential to increase the detection sensitivity of ELISA as well as shorten the antigen-antibody binding times from typically 45-60 min to 15 min.

MED13L haploinsufficiency syndrome has been described in two patients and is characterized by moderate intellectual disability (ID), conotruncal heart defects, facial abnormalities and hypotonia. Missense mutations in MED13L are linked to transposition of the great arteries and non-syndromal intellectual disability. Here we describe two novel patients with de novo MED13L aberrations. The first patient has a de novo mutation in the splice acceptor site of exon 5 of MED13L. cDNA analysis showed this mutation results in an in-frame deletion, removing 15 amino acids in middle of the conserved MED13L N-terminal domain. The second patient carries a de novo deletion of exons 6-20 of MED13L. Both patients show features of the MED13L haploinsufficiency syndrome, except for the heart defects, thus further confirming the existence of the MED13L haploinsufficiency syndrome.

Genomic rearrangements are a common cause of human congenital abnormalities. However, their origin and consequences are poorly understood. We performed molecular analysis of two patients with congenital disease who carried de novo genomic rearrangements. We found that the rearrangements in both patients hit genes that are recurrently rearranged in cancer (ETV1, FOXP1, and microRNA cluster C19MC) and drive formation of fusion genes similar to those described in cancer. Subsequent analysis of a large set of 552 de novo germline genomic rearrangements underlying congenital disorders revealed enrichment for genes rearranged in cancer and overlap with somatic cancer breakpoints. Breakpoints of common (inherited) germline structural variations also overlap with cancer breakpoints but are depleted for cancer genes. We propose that the same genomic positions are prone to genomic rearrangements in germline and soma but that timing and context of breakage determines whether developmental defects or cancer are promoted., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Ketoacidosis is a potentially lethal condition caused by the imbalance between hepatic production and extrahepatic utilization of ketone bodies. We performed exome sequencing in a patient with recurrent, severe ketoacidosis and identified a homozygous frameshift mutation in the gene encoding monocarboxylate transporter 1 (SLC16A1, also called MCT1). Genetic analysis in 96 patients suspected of having ketolytic defects yielded seven additional inactivating mutations in MCT1, both homozygous and heterozygous. Mutational status was found to be correlated with ketoacidosis severity, MCT1 protein levels, and transport capacity. Thus, MCT1 deficiency is a novel cause of profound ketoacidosis; the present work suggests that MCT1-mediated ketone-body transport is needed to maintain acid-base balance.

Recently, microarrays have replaced karyotyping as a first tier test in patients with idiopathic intellectual disability and/or multiple congenital abnormalities (ID/MCA) in many laboratories. Although in about 14-18% of such patients, DNA copy-number variants (CNVs) with clinical significance can be detected, microarrays have the disadvantage of missing balanced rearrangements, as well as providing no information about the genomic architecture of structural variants (SVs) like duplications and complex rearrangements. Such information could possibly lead to a better interpretation of the clinical significance of the SV. In this study, the clinical use of mate pair next-generation sequencing was evaluated for the detection and further characterization of structural variants within the genomes of 50 ID/MCA patients. Thirty of these patients carried a chromosomal aberration that was previously detected by array CGH or karyotyping and suspected to be pathogenic. In the remaining 20 patients no causal SVs were found and only benign aberrations were detected by conventional techniques. Combined cluster and coverage analysis of the mate pair data allowed precise breakpoint detection and further refinement of previously identified balanced and (complex) unbalanced aberrations, pinpointing the causal gene for some patients. We conclude that mate pair sequencing is a powerful technology that can provide rapid and unequivocal characterization of unbalanced and balanced SVs in patient genomes and can be essential for the clinical interpretation of some SVs.

Intra-tumor heterogeneity is a hallmark of many cancers and may lead to therapy resistance or interfere with personalized treatment strategies. Here, we combined topographic mapping of somatic breakpoints and transcriptional profiling to probe intra-tumor heterogeneity of treatment-naïve stage IIIC/IV epithelial ovarian cancer. We observed that most substantial differences in genomic rearrangement landscapes occurred between metastases in the omentum and peritoneum versus tumor sites in the ovaries. Several cancer genes such as NF1, CDKN2A, and FANCD2 were affected by lesion-specific breakpoints. Furthermore, the intra-tumor variability involved different mutational hallmarks including lesion-specific kataegis (local mutation shower coinciding with genomic breakpoints), rearrangement classes, and coding mutations. In one extreme case, we identified two independent TP53 mutations in ovary tumors and omentum/peritoneum metastases, respectively. Examination of gene expression dynamics revealed up-regulation of key cancer pathways including WNT, integrin, chemokine, and Hedgehog signaling in only subsets of tumor samples from the same patient. Finally, we took advantage of the multilevel tumor analysis to understand the effects of genomic breakpoints on qualitative and quantitative gene expression changes. We show that intra-tumor gene expression differences are caused by site-specific genomic alterations, including formation of in-frame fusion genes. These data highlight the plasticity of ovarian cancer genomes, which may contribute to their strong capacity to adapt to changing environmental conditions and give rise to the high rate of recurrent disease following standard treatment regimes.

Objectives: The aim of this study was to determine the level of middle ear pressure and alterations in middle ear pressure levels after septoplasty among the individuals having advanced degree isolated nasal septal deviation., Methods: A prospective randomized study was conducted. The study included 72 adult patients who had severely deviated septum. The middle ear pressure values at both sides of nasal obstruction and opposite side were determined using tympanometry before the surgery and at postoperative week 3. The middle ear pressure values were divided into 2 groups, side of nasal obstruction (group 1) and opposite nonaffected side (group 2). The middle ear pressure values obtained before and after septoplasty were compared., Results: Before the septoplasty, the median middle ear pressure value was -54 dPa at the side of nasal obstruction, and -46 dPa at the opposite side. Three weeks after the septoplasty, it was -38 dPa at the side of nasal obstruction, and -40 dPa at the opposite side. The middle ear pressure improved by approximately 30% at the side of nasal obstruction and by 11% at the nonaffected side; a statistically significant decrease was found at the side of nasal obstruction (P < 0.05)., Conclusions: In adult patients with isolated nasal septum deviation, the middle ear pressure is lower at the side of nasal obstruction, but it remains within reference ranges. An approximately 30% improvement occurs in the middle ear pressure after septoplasty.

Background: We present a large Dutch family with seven males affected by a novel syndrome of X-linked intellectual disability, hypogonadism, gynaecomastia, truncal obesity, short stature and recognisable craniofacial manifestations resembling but not identical to Wilson-Turner syndrome. Seven female relatives show a much milder expression of the phenotype., Methods and Results: We performed X chromosome exome (X-exome) sequencing in five individuals from this family and identified a novel intronic variant in the histone deacetylase 8 gene (HDAC8), c.164+5G>A, which disturbs the normal splicing of exon 2 resulting in exon skipping, and introduces a premature stop at the beginning of the histone deacetylase catalytic domain. The identified variant completely segregates in this family and was absent in 96 Dutch controls and available databases. Affected female carriers showed a notably skewed X-inactivation pattern in lymphocytes in which the mutated X-chromosome was completely inactivated., Conclusions: HDAC8 is a member of the protein family of histone deacetylases that play a major role in epigenetic gene silencing during development. HDAC8 specifically controls the patterning of the skull with the mouse HDAC8 knock-out showing craniofacial deformities of the skull. The present family provides the first evidence for involvement of HDAC8 in a syndromic form of intellectual disability.

Chromothripsis represents a novel phenomenon in the structural variation landscape of cancer genomes. Here, we analyze the genomes of ten patients with congenital disease who were preselected to carry complex chromosomal rearrangements with more than two breakpoints. The rearrangements displayed unanticipated complexity resembling chromothripsis. We find that eight of them contain hallmarks of multiple clustered double-stranded DNA breaks (DSBs) on one or more chromosomes. In addition, nucleotide resolution analysis of 98 breakpoint junctions indicates that break repair involves nonhomologous or microhomology-mediated end joining. We observed that these eight rearrangements are balanced or contain sporadic deletions ranging in size between a few hundred base pairs and several megabases. The two remaining complex rearrangements did not display signs of DSBs and contain duplications, indicative of rearrangement processes involving template switching. Our work provides detailed insight into the characteristics of chromothripsis and supports a role for clustered DSBs driving some constitutional chromothripsis rearrangements., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (K(ATP)) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the K(ATP) channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.

The osteogenic potential of exercise is reported to be partially a function of the magnitude of training loads. This study evaluated the ground reaction force (GRF) and rate of force development (RFD) of the eccentric and concentric phases of the back squat at 3 different loads. Twelve subjects performed the back squat on a force platform with loading conditions of 80, 100, and 120% of their 1 repetition maximum (RM). Back squats performed at 120% of the 1RM produced the highest GRF in both the eccentric and concentric conditions. No significant differences were found between RFD for any of the loading conditions. Performing the back squat at loads of 120% of the estimated 1RM, accomplished with reduced range of motion, results in higher GRF than the back squat performed at 80 or 100% of the 1RM. Thus, supermaximal back squat loads in excess of the 1RM, with decreased range of motion, may be a useful part of a resistance training program designed to maximize osteogenic potential.

Vesico-ureteral reflux (VUR) is the retrograde passage of urine from the bladder to the urinary tract and causes 8.5% of end-stage renal disease in children. It is a complex genetic developmental disorder, in which ectopic embryonal ureteric budding is implicated in the pathogenesis. VUR is part of the spectrum of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT). We performed an extensive association study for primary VUR using a two-stage, case-control design, investigating 44 candidate genes in the ureteric budding pathway in 409 Dutch VUR patients. The 44 genes were selected from the literature and a set of 567 single nucleotide polymorphisms (SNPs) capturing their genetic variation was genotyped in 207 cases and 554 controls. The 14 SNPs with p<0.005 were included in a follow-up study in 202 cases and 892 controls. Of the total cohort, ~50% showed a clear-cut primary VUR phenotype and ~25% had both a duplex collecting system and VUR. We also looked for association in these two extreme phenotype groups. None of the SNPs reached a significant p-value. Common genetic variants in four genes (GREM1, EYA1, ROBO2 and UPK3A) show a trend towards association with the development of primary VUR (GREM1, EYA1, ROBO2) or duplex collecting system (EYA1 and UPK3A). SNPs in three genes (TGFB1, GNB3 and VEGFA) have been shown to be associated with VUR in other populations. Only the result of rs1800469 in TGFB1 hinted at association in our study. This is the first extensive study of common variants in the genes of the ureteric budding pathway and the genetic susceptibility to primary VUR.

The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3-5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5-6 d for array-based enrichment and 3-4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50 pg total RNA, or 0.01-2.5 pg mRNA, per cell) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: greater numbers of genes to be analyzed per cell; more quantitative analysis of gene expression; and better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

Correlating gene expression with behavior at the single-cell level is difficult, largely because the small amount of available mRNA (<1 pg) degrades before it can be reverse transcribed into a more stable cDNA copy. This study tested the capacity for a novel acoustic microstreaming method ("micromixing"), which stirs fluid at microliter scales, to improve cDNA yields from reverse transcription (RT) reactions comprising single-cell quantities of RNA. Micromixing significantly decreased the number of qPCR cycles to detect cDNA representing mRNA for hypoxanthine phosphoribosyl-transferase (Hprt) and nuclear receptor-related 1 (Nurr1) by ~9 and ~15 cycles, respectively. The improvement was equivalent to performing RT with 10- to 100-fold more cDNA in the absence of micromixing. Micromixing enabled reliable detection of the otherwise undetectable, low-abundance transcript, Nurr1. It was most effective when RNA concentrations were low (0.1-1 pg/µL, a "single-cell equivalent") but had lesser effects at higher RNA concentrations (~1 ng/µL). This was supported by imaging experiments showing that micromixing improved mixing of a low concentration (20 pg/µL) of fluorescence-labeled RNA but not a higher concentration (1 ng/µL). We conclude that micromixing significantly increases RT yields obtainable from single-cell quantities of RNA.