Phasing DNA Markers Using Digital PCR.

Methods Mol Biol. 2018;1768:489-512

Authors: Regan J, Karlin-Neumann G

Abstract
Besides quantifying the absolute number of copies of known DNA targets, digital PCR can also be used to assess whether two nonpolymorphic gene sequences or two heterozygous markers reside on the same DNA molecule (i.e., are physically linked). Some useful linkage applications include: phasing variants to define a haplotype; genotyping of inversions; determining the presence of multimarker pathogenic bacteria in a metagenomic sample; and assessing DNA integrity. This chapter describes an efficient and cost-effective method for analyzing linkage of any two genetic sequences up to at least 200 Kb apart, including phasing of heterozygous markers such as that which occur abundantly in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

PMID: 29717461 [PubMed - in process]

A phosphatidic acid binding protein is important for lipid homeostasis and adaptation to anaerobic biofilm conditions in Pseudomonas aeruginosa.

Biochem J. 2018 May 01;:

Authors: Groenewold MK, Massmig M, Hebecker S, Danne L, Magnowska Z, Nimtz M, Narberhaus F, Jahn D, Heinz DW, Jänsch L, Moser J

Abstract
A quantitative Pseudomonas aeruginosa proteomics approach revealed increased abundance of the so far uncharacterized protein PA3911 in anaerobic biofilms grown under conditions of the cystic fibrosis lung. Physiological relevance of ORF PA3911 was demonstrated, inter alia , using phenotype microarray experiments. The mutant strain showed increased susceptibility in the presence of antimicrobials (minocycline, nafcillin, oxacillin, chloramphenicol, thiamphenicol), enhanced twitching motility and significantly impaired biofilm formation. PA3911 is a soluble, cytoplasmic protein in P. aeruginosa In protein-lipid overlay experiments, purified PA3911 bound specifically to phosphatidic acid (PA), the central hub of phospholipid metabolism. Structure-guided site-directed mutagenesis was used to explore the proposed ligand binding cavity of PA3911. Proteins variant of Leu56, Leu58, Val69 and Leu114 were shown to impair PA interaction. A comparative shotgun lipidomics approach demonstrated a multifaceted response of P. aeruginosa to anaerobic conditions at the lipid head group and fatty acid level. Lipid homeostasis in the PA3911 mutant strain was imbalanced with respect to lysophosphatidylcholine, phosphatidylcholine and diacylglycerol under anaerobic and/or aerobic conditions. The impact of the newly identified PA binding protein on lipid homeostasis and the related macroscopic phenotypes of P. aeruginosa are discussed.

PMID: 29717024 [PubMed - as supplied by publisher]

[The lung microbiota. Review].

Arch Pediatr. 2017 Jul;24(7):667-674

Authors: Ploton MC, Abakka S, Amouyal E, Besnard C, Dufour L, El Harrif S, Kipnis C, Prim B, Vignot J, Houdouin V

Abstract
In the last 20years, culture-independent DNA-based techniques ("shotgun sequencing") demonstrated that complex microbial communities reside on most epithelial surfaces, including the lower airways. Until the amniotic sac ruptures, a fetus is considered to be essentially sterile. Many factors affect the composition of the lung microbiota: inheritance, mode of delivery, diet, and age-related changes in adults. It interacts with the digestive and oropharyngeal microbiotas. Animal models show that these interactions play a role in innate pulmonary immunity and modulation of the inflammatory response. The microbial composition of the airway microbiota differs between healthy children and those with chronic lung disease. The advances in the comprehension of microbiome changes have resulted in new approaches concerning the microbiota for treatment and prevention of disease.

PMID: 28576586 [PubMed - indexed for MEDLINE]

Involvement of Cl(-)/HCO3(-) exchanger SLC26A3 and SLC26A6 in preimplantation embryo cleavage.

Sci Rep. 2016 06 27;6:28402

Authors: Lu YC, Yang J, Fok KL, Ye YH, Jin L, Chen ZY, Zhang XM, Huang HF, Chan HC

Abstract
Bicarbonate (HCO3(-)) is essential for preimplantation embryo development. However, the mechanism underlying the HCO3(-) transport into the embryo remains elusive. In the present study, we examined the possible involvement of Cl(-)/HCO3(-) exchanger in mediating HCO3(-) transport into the embryo. Our results showed that depletion of extracellular Cl(-), even in the presence of HCO3(-), suppressed embryo cleavage in a concentration-dependent manner. Cleavage-associated HCO3(-)-dependent events, including increase of intracellular pH, upregulation of miR-125b and downregulation of p53, also required Cl(-). We further showed that Cl(-)/HCO3(-) exchanger solute carrier family 26 (SLC26) A3 and A6 were expressed at 2-cell through blastocyst stage. Blocking individual exchanger's activity by inhibitors or gene knockdown differentially decreased embryo cleavage and inhibited HCO3(-)-dependent events, while inhibiting/knocking down both produced an additive effect to an extent similar to that observed when CFTR was inhibited. These results indicate the involvement of SLC26A3 and A6 in transporting HCO3(-) essential for embryo cleavage, possibly working in concert with CFTR through a Cl(-) recycling pathway. The present study sheds light into our understanding of molecular mechanisms regulating embryo cleavage by the female reproductive tract.

PMID: 27346053 [PubMed - indexed for MEDLINE]

Identification of a Novel Keratin 9 Missense Mutation in a Chinese Family with Epidermolytic Palmoplantar Keratoderma.

Cell Physiol Biochem. 2018 Apr 26;46(5):1919-1929

Authors: Xiao H, Guo Y, Yi J, Xia H, Xu H, Yuan L, Hu P, Yang Z, He Z, Lu H, Deng H

Abstract
BACKGROUND/AIMS: Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis. It is characterized by diffuse yellow keratoses on the palmoplantar epidermis, with an erythematous border. The keratin 9 gene (KRT9) and less frequently the keratin 1 gene (KRT1) are responsible for EPPK. This study aims to identify and analyse genetic defects responsible for EPPK in a Han Chinese pedigree.
METHODS: A four-generation Han Chinese pedigree containing five individuals affected with EPPK was recruited. Exome sequencing, Sanger sequencing, and bioinformatics tools were conducted to identify the mutation in this pedigree. HaCaT cells were transfected with either wild-type or mutated KRT9. Confocal laser immunofluorescence assay, imaging processing, and statistical analysis were performed to evaluate wild-type and mutant KRT9 groups.
RESULTS: A novel heterozygous c.1369C>T transition (p.Leu457Phe) in exon 6 of the KRT9 gene was identified in four patients. It co-segregated with the disorder in the family. Functional analysis showed that withdrawal of the filament network from the cell periphery and particle formation were present in about 10% of Leu457Phe-transfected HaCaT cells, while approximately 3% of cells transfected with wild-type KRT9 showed this phenotype. The particles in mutant group were larger than that in wild-type group (P-value < 0.05).
CONCLUSION: The variant may be the disease-causing missense mutation and produce dominant negative effects by interrupting keratin network formation. This study indicates the pathogenic role of the KRT9 gene mutation in this pedigree with EPPK, and may be helpful in genetic counseling, prenatal diagnosis and gene-targeted therapies of EPPK.

PMID: 29719290 [PubMed - as supplied by publisher]

CEP250 mutations associated with mild cone-rod dystrophy and sensorineural hearing loss in a Japanese family.

Ophthalmic Genet. 2018 May 02;:1-8

Authors: Kubota D, Gocho K, Kikuchi S, Akeo K, Miura M, Yamaki K, Takahashi H, Kameya S

Abstract
BACKGROUND: CEP250 encodes the C-Nap1 protein which belongs to the CEP family of proteins. C-Nap1 has been reported to be expressed in the photoreceptor cilia and is known to interact with other ciliary proteins. Mutations of CEP250 cause atypical Usher syndrome which is characterized by early-onset sensorineural hearing loss (SNHL) and a relatively mild retinitis pigmentosa. This study tested the hypothesis that the mild cone-rod dystrophy (CRD) and SNHL in a non-consanguineous Japanese family was caused by CEP250 mutations.
METHODS: Detailed ophthalmic and auditory examinations were performed on the proband and her family members. Whole exome sequencing (WES) was used on the DNA obtained from the proband.
RESULTS: Electrophysiological analysis revealed a mild CRD in two family members. Adaptive optics (AO) imaging showed reduced cone density around the fovea. Auditory examinations showed a slight SNHL in both patients. WES of the proband identified compound heterozygous variants c.361C>T, p.R121*, and c.562C>T, p.R188* in CEP250. The variants were found to co-segregate with the disease in five members of the family.
CONCLUSIONS: The variants of CEP250 are both null variants and according to American College of Medical Genetics and Genomics (ACMG) standards and guideline, these variants are classified into the very strong category (PVS1). The criteria for both alleles will be pathogenic. Our data indicate that mutations of CEP250 can cause mild CRD and SNHL in Japanese patients. Because the ophthalmological phenotypes were very mild, high-resolution retinal imaging analysis, such as AO, will be helpful in diagnosing CEP250-associated disease.

PMID: 29718797 [PubMed - as supplied by publisher]

DNAp: A Pipeline for DNA-seq Data Analysis.

Sci Rep. 2018 May 01;8(1):6793

Authors: Causey JL, Ashby C, Walker K, Wang ZP, Yang M, Guan Y, Moore JH, Huang X

Abstract
Next-generation sequencing is empowering genetic disease research. However, it also brings significant challenges for efficient and effective sequencing data analysis. We built a pipeline, called DNAp, for analyzing whole exome sequencing (WES) and whole genome sequencing (WGS) data, to detect mutations from disease samples. The pipeline is containerized, convenient to use and can run under any system, since it is a fully automatic process in Docker container form. It is also open, and can be easily customized with user intervention points, such as for updating reference files and different software or versions. The pipeline has been tested with both human and mouse sequencing datasets, and it has generated mutations results, comparable to published results from these datasets, and reproducible across heterogeneous hardware platforms. The pipeline DNAp, funded by the US Food and Drug Administration (FDA), was developed for analyzing DNA sequencing data of FDA. Here we make DNAp an open source, with the software and documentation available to the public at http://bioinformatics.astate.edu/dna-pipeline/ .

PMID: 29717215 [PubMed - in process]

Exome sequencing identifies targets in the treatment-resistant ophthalmoplegic subphenotype of myasthenia gravis.

Neuromuscul Disord. 2017 Sep;27(9):816-825

Authors: Nel M, Jalali Sefid Dashti M, Gamieldien J, Heckmann JM

Abstract
Treatment-resistant ophthalmoplegia (OP-MG) is not uncommon in individuals with African genetic ancestry and myasthenia gravis (MG). To identify OP-MG susceptibility genes, extended whole exome sequencing was performed using extreme phenotype sampling (11 OP-MG vs 4 control-MG) all with acetylcholine receptor-antibody positive MG. This approach identified 356 variants that were twice as frequent in OP-MG compared to control-MG individuals. After performing probability test estimates and filtering variants according to those 'suggestive' of association with OP-MG (p < 0.05), only three variants remained which were expressed in extraocular muscles. Validation in 25 OP-MG and 50 control-MG cases supported the association of DDX17delG (p = 0.014) and SPTLC3insACAC (p = 0.055) with OP-MG, but ST8SIA1delCCC could not be verified by Sanger sequencing. A parallel approach, using a semantic model informed by current knowledge of MG-pathways, identified an African-specific interleukin-6 receptor (IL6R) variant, IL6R c.*3043 T>C, that was more frequent in OP-MG compared to control-MG cases (p = 0.069) and population controls (p = 0.043). A weighted genetic risk score, derived from the odds ratios of association of these variants with OP-MG, correlated with the OP-MG phenotype as opposed to control MG. This unbiased approach implicates several potentially functional gene variants in the gangliosphingolipid and myogenesis pathways in the development of the OP-MG subphenotype.

PMID: 28673556 [PubMed - indexed for MEDLINE]

Utilizing next-generation sequencing in the management of multiple myeloma.

Expert Rev Mol Diagn. 2017 Jul;17(7):653-663

Authors: Lionetti M, Neri A

Abstract
INTRODUCTION: Multiple myeloma (MM) is a bone marrow plasma cell malignancy characterized by wide clinical presentation and heterogeneous genetic background. Despite the recent advances in patient outcome, new markers are needed for improving risk prediction and choice of a more appropriate therapy. In this perspective, the genetic makeup of MM cells is being better characterized by means of next-generation sequencing (NGS) technologies. Areas covered: The authors discuss how the application of NGS has improved our knowledge of MM biology by discovering its mutational landscape, identifying the operating mutational processes, and revealing the clonal composition of tumors and the dynamics of its evolution; and how this can have important clinical implications in terms of prognostication, therapeutic choices, and response assessment. Finally, the authors provide a quick outlook of future applications of these technologies that could help in the management of the disease in the next years. Expert commentary: The clinical exploitation of NGS-based characterization of MM patients has as its ultimate goal the precision medicine. Considerable obstacles to its implementation in myeloma management exist; therefore, the concerted effort of all involved stakeholders is mandatory to ensure that it will become a reality in routine clinical practice in the next future.

PMID: 28524737 [PubMed - indexed for MEDLINE]

iFish: predicting the pathogenicity of human nonsynonymous variants using gene-specific/family-specific attributes and classifiers.

Sci Rep. 2016 08 16;6:31321

Authors: Wang M, Wei L

Abstract
Accurate prediction of the pathogenicity of genomic variants, especially nonsynonymous single nucleotide variants (nsSNVs), is essential in biomedical research and clinical genetics. Most current prediction methods build a generic classifier for all genes. However, different genes and gene families have different features. We investigated whether gene-specific and family-specific customized classifiers could improve prediction accuracy. Customized gene-specific and family-specific attributes were selected with AIC, BIC, and LASSO, and Support Vector Machine classifiers were generated for 254 genes and 152 gene families, covering a total of 5,985 genes. Our results showed that the customized attributes reflected key features of the genes and gene families, and the customized classifiers achieved higher prediction accuracy than the generic classifier. The customized classifiers and the generic classifier for other genes and families were integrated into a new tool named iFish (integrated Functional inference of SNVs in human, http://ifish.cbi.pku.edu.cn). iFish outperformed other methods on benchmark datasets as well as on prioritization of candidate causal variants from whole exome sequencing. iFish provides a user-friendly web-based interface and supports other functionalities such as integration of genetic evidence. iFish would facilitate high-throughput evaluation and prioritization of nsSNVs in human genetics research.

PMID: 27527004 [PubMed - indexed for MEDLINE]

Higher versus Lower Dose of Cefotetan or Cefoxitin for Surgical Prophylaxis in Patients Weighing One Hundred Twenty Kilograms or More.

Surg Infect (Larchmt). 2018 May 02;:

Authors: Banoub M, Curless MS, Smith JM, Jarrell AS, Cosgrove SE, Rock C, Avdic E

Abstract
BACKGROUND: Clinical practice guidelines recommend a 2-g dose of cefotetan and cefoxitin for surgical prophylaxis. Pharmacokinetic data suggest benefit from higher cefotetan and cefoxitin dosing in obese patients. However, clinical studies examining higher dosing strategies in this at-risk population are lacking. The purpose of this study was to determine whether 3 g of cefotetan or cefoxitin administered pre-operatively for patients who weigh 120 kg or more is associated with a lower proportion of surgical site infection (SSI) compared with 2 g.
PATIENTS AND METHODS: Medical records of patients weighing 120 kg or more who had received cefotetan or cefoxitin (2 or 3 g) as surgical prophylaxis for intra-abdominal procedures between July 2012 and August 2015 were reviewed for the development of an SSI (primary outcome), study drug-related adverse events, and re-admissions attributed to SSIs (secondary outcomes). Relative risk calculations were performed for analysis of the primary and secondary outcomes.
RESULTS: One-hundred seventy-five procedures in 169 patients were included in the study. Cefotetan was used in 81% (141/175) of procedures. Three grams of cefotetan or cefoxitin was used in 20% (35/175) of procedures. The median body mass index (BMI) in both dosing groups was 42 kg/m2 and patients who received 3 g more often weighed more than 130 kg (relative risk [RR] 1.36, 1.01-1.76; p = 0.04). Surgical site infections occurred in 20.7% within the 2-g group and 22.9% in the 3-g group (RR 1.10, 0.55-2.20; p = 0.78). There was no difference in the number of study drug-related adverse effects in the 3-g compared with the 2-g group. Thirty-day re-admissions because of SSI also did not differ between the 2-g and 3-g groups (7.9% vs. 17.1%, respectively; p = 0.11).
CONCLUSION: This small retrospective study did not find a difference in SSI rates between 3-g and 2-g surgical prophylaxis dosing for patients 120 kg or more with a median BMI >40 kg/m2.

PMID: 29717917 [PubMed - as supplied by publisher]

Anabolic Steroid Effect on the Liver.

Curr Sports Med Rep. 2018 Mar;17(3):97-102

Authors: Niedfeldt MW

Abstract
Anabolic steroids are synthetic derivatives of testosterone shown to increase muscle size and strength. Chemical substitutions on the testosterone molecule cause increased potency and duration of action. The 17-α-alkylation modification allows steroids to be taken orally, but the slower clearance in the liver makes them more hepatotoxic. The frequency and severity of side effects depends on several factors including the formulation of the drug, route of administration, dosage, duration of use, and individual sensitivity and response. Anabolic steroid users tend to take supraphysiologic doses or multiple steroids and other drugs simultaneously which increases risk of adverse effects. Hepatotoxicity can be seen as elevated liver transaminases, acute cholestatic syndrome, chronic vascular injury, hepatic tumors, and toxicant-associated fatty liver disease, as well as significant changes in lipoproteins. Many of these changes will stabilize or reverse with cessation of steroid use, but some can be life-threatening. Over-the-counter supplements can be contaminated with anabolic steroids, causing hepatotoxicity in unsuspecting consumers.

PMID: 29521706 [PubMed - indexed for MEDLINE]

Pulmonary toxicity following bleomycin use: A single-center experience.

J Cancer Res Ther. 2017 Jul-Sep;13(3):466-470

Authors: Madabhavi I, Modi G, Patel A, Anand A, Panchal H, Parikh S

Abstract
BACKGROUND: Bleomycin-induced pulmonary (BIP) toxicity is a notorious entity and cropped up in roughly 10% of cases. The aim of the study is to evaluate BIP at our tertiary care cancer center.
PATIENTS AND METHODS: This is a retrospective, analytical study conducted at a tertiary care center from January 1998 to December 2012. Records of all the patients who were offered bleomycin chemotherapy as an integral part of adriamycin, bleomycin, vinblastine, and dacarbazine or bleomycin, etoposide, and cisplatin regimen in Hodgkin disease (HD) or germ cell tumor (GCT) were studied for the study inclusion criteria. Twenty-two patients treated with bleomycin who had respiratory symptoms and/or abnormal high-resolution computed tomography (HRCT) findings, suggestive of bleomycin-induced lung injury were included in this study. Results and Statistical Analysis: A total of 22 patients met the inclusion criteria for the study cohort. Of 22 patients, 8 were of HD and 14 were of GCT (nonseminomatous GCT [NSGCT] = 10 and seminomatous GCT = 4). Of 22 patients, 14 had symptoms of nonproductive cough, dyspnea and showed HRCT findings of ground glass opacities, diffuse alveolar damage, extensive reticular markings, traction bronchiectasis, and/or nodular densities. Two patients had fever and pleuritic pain. Eight patients were asymptomatic. Symptomatic patients were treated with prednisone at the dose of 0.75-1 mg/kg 4-8 weeks then gradually tapered. Four patients required noninvasive ventilatory support and managed with oxygen, nebulization, and antibiotics. Two patients required mechanical ventilatory support (HD = 1 and NSGCT = 1) and developed multiorgan failure subsequently succumbed to death.
CONCLUSION: BIP is noteworthy lung toxicity as subsequent mortality ranges from 10% to 20% and shrinks survival rate in patients with highly curable malignant conditions. Physicians should be vigilant concerning this impending side effect.

PMID: 28862210 [PubMed - indexed for MEDLINE]

Computational approaches to understand the adverse drug effect on potassium, sodium and calcium channels for predicting TdP cardiac arrhythmias.

J Mol Graph Model. 2017 Sep;76:152-160

Authors: Sharifi M

Abstract
Ion channels play a crucial role in the cardiovascular system. Our understanding of cardiac ion channel function has improved since their first discoveries. The flow of potassium, sodium and calcium ions across cardiomyocytes is vital for regular cardiac rhythm. Blockage of these channels, delays cardiac repolarization or tend to shorten repolarization and may induce arrhythmia. Detection of drug risk by channel blockade is considered essential for drug regulators. Advanced computational models can be used as an early screen for torsadogenic potential in drug candidates. New drug candidates that are determined to not cause blockage are more likely to pass successfully through preclinical trials and not be withdrawn later from the marketplace by manufacturer. Several different approved drugs, however, can cause a distinctive polymorphic ventricular arrhythmia known as torsade de pointes (TdP), which may lead to sudden death. The objective of the present study is to review the mechanisms and computational models used to assess the risk that a drug may TdP.
KEY POINTS: There is strong evidence from multiple studies that blockage of the L-type calcium current reduces risk of TdP. Blockage of sodium channels slows cardiac action potential conduction, however, not all sodium channel blocking antiarrhythmic drugs produce a significant effect, while late sodium channel block reduces TdP. Interestingly, there are some drugs that block the hERG potassium channel and therefore cause QT prolongation, but they are not associated with TdP. Recent studies confirmed the necessity of studying multiple distinctionic ion channels which are responsible for cardiac related diseases or TdP, to obtain an improved clinical TdP risk prediction of compound interactions and also for designing drugs.

PMID: 28756335 [PubMed - indexed for MEDLINE]

Adverse event management in patients with relapsed and refractory multiple myeloma taking pomalidomide plus low-dose dexamethasone: A pooled analysis.

Eur J Haematol. 2017 Sep;99(3):199-206

Authors: Moreau P, Dimopoulos MA, Richardson PG, Siegel DS, Cavo M, Corradini P, Weisel K, Delforge M, O'Gorman P, Song K, Chen C, Bahlis N, Oriol A, Hansson M, Kaiser M, Anttila P, Raymakers R, Joao C, Cook G, Sternas L, Biyukov T, Slaughter A, Hong K, Herring J, Yu X, Zaki M, San-Miguel J

Abstract
OBJECTIVES: Heavily pretreated patients with relapsed and refractory multiple myeloma are susceptible to treatment-related adverse events (AEs). Managing AEs are important to ensure patients continue therapy long enough to receive the best clinical benefit. Data from the MM-002, MM-003, and MM-010 trials were pooled to further characterize the safety profile of pomalidomide plus low-dose dexamethasone and AE management.
METHODS: This analysis included 1088 patients who received ≥ 2 prior therapies, including lenalidomide and bortezomib, and progressed ≤ 60 days of last therapy. Patients received 28-day cycles of pomalidomide 4 mg/day on days 1-21 and low-dose dexamethasone 40 mg (20 mg if aged > 75 years) weekly until disease progression or unacceptable toxicity. Thromboprophylaxis was required.
RESULTS: The most common grade 3/4 AEs were neutropenia (56.2%), anemia (32.3%), and thrombocytopenia (25.8%), which occurred within the first few cycles of treatment. Grade 3/4 infections occurred in 33.7% patients, of whom 13.9% had pneumonia, and 40.3% had neutropenia. Pomalidomide dose reductions or interruptions were reported in 24.2% and 66.0% of patients, respectively. AEs were managed by dose modifications and/or supportive care.
CONCLUSIONS: Pomalidomide plus low-dose dexamethasone showed an acceptable safety profile, and AEs were well managed according to study protocols and established guidelines.

PMID: 28504846 [PubMed - indexed for MEDLINE]

Compact Magnetic Resonance Imaging Systems-Novel Cost-Effective Tools for Preclinical Drug Safety and Efficacy Evaluation.

Toxicol Sci. 2017 05 01;157(1):3-7

Authors: Ramot Y, Schiffenbauer YS, Maronpot R, Nyska A

Abstract
Practical magnetic resonance imaging for use in investigative and preclinical toxicology studies is now feasible. Newly developed, self-containing imaging systems provide an efficient and cost-effective means to rapidly obtain in vivo and ex vivo magnetic resonance imaging images to improve how we perform toxicology and toxicologic pathology.

PMID: 28329801 [PubMed - indexed for MEDLINE]

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