Fabrication of inhaled hybrid silver/ciprofloxacin nanoparticles with synergetic effect against Pseudomonas aeruginosa.

Eur J Pharm Biopharm. 2018 Apr 11;:

Authors: Al-Obaidi H, Kalgudi R, Gulrez Zariwala M

Abstract
Ciprofloxacin (CFX) is a fluroquinolone antibiotic used as a first line treatment against infections caused by Pseudomonas aeruginosa and Streptococcus pneumonia that are commonly acquired by cystic fibrosis (CF) patients. However, no inhalation formulation is currently available for ciprofloxacin. Hybrid silica coated silver nanoparticles were prepared using Stöber reaction and the optimum ratio of chitosan and sodium tripolyphosphate was used to encapsulate CFX. Particle deposition was assessed in vitro using twin stage impinger while antimicrobial activity was evaluated based on the planktonic growth of P. aeruginosa as well as against P. aeruginosa sp biofilm formation. In vitro deposition results showed significant deposition in stage 2 using twin stage impinger (TSI) (∼70%). Compared to CFX, the formed hybrid nanoparticles were 3-4 folds more effective against inhibiting growth and biofilm formation by P. aeruginosa PAO1 and P. aeruginosa NCTC 10662.

PMID: 29654885 [PubMed - as supplied by publisher]

Volumetric dynamic oxygen-enhanced MRI (OE-MRI): comparison with CT Brody score and lung function in cystic fibrosis patients.

Eur Radiol. 2018 Apr 13;:

Authors: Martini K, Gygax CM, Benden C, Morgan AR, Parker GJM, Frauenfelder T

Abstract
OBJECTIVES: To demonstrate, in patients with cystic fibrosis (CF), the correlation between three-dimensional dynamic oxygen-enhanced magnetic resonance imaging (OE-MRI) measurements and computed tomography Brody score (CF-CT) and lung function testing (LFT).
METHODS: Twenty-one patients (median age, 25 years; female, n = 8) with a range of CF lung disease and five healthy volunteers (median age, 31 years; female, n = 2) underwent OE-MRI performed on a 1.5-T MRI scanner. Coronal volumes were acquired while patients alternately breathed room air and 100% oxygen. Pre-oxygen T1 was measured. Dynamic series of T1-weighted volumes were then obtained while breathing oxygen. T1-parameter maps were generated and the following OE-MRI parameters were measured: oxygen uptake (ΔPO2max), wash-in time and wash-out time. High-resolution CT and LFT were performed. The relationship between CF-CT, LFT and OE-MRI parameters were evaluated using Pearson correlation for the whole lung and regionally.
RESULTS: Mean CF-CT was 24.1±17.1. Mean ΔPO2max and mean wash-in as well as skewness of wash-out showed significant correlation with CF-CT (ΔPO2max: r = -0.741, p < 0.001; mean wash-in: r = 0.501, p = 0.017; skewness of wash-out: r = 0.597, p = 0.001). There was significant correlation for the whole lung and regionally between LFT parameters and OE-MR (ΔPO2max: r = 0.718, p < 0.001; wash-in: r = -0.576, p = 0.003; wash-out skewness: r = -0.552, p = 0.004).
CONCLUSIONS: Functional lung imaging using OE-MRI has the capability to assess the severity of CF lung disease and shows a significant correlation with LFT and CF-CT.
KEY POINTS: • Oxygen-enhanced MRI might play a future role in evaluation and follow-up of cystic fibrosis. • Heterogeneity of parameter maps reflects localised functional impairment in cystic fibrosis. • Avoidance of cumulative radiation burden in CF is feasible using OE-MRI.

PMID: 29654559 [PubMed - as supplied by publisher]

A European regulatory perspective on cystic fibrosis: current treatments, trends in drug development and translational challenges for CFTR modulators.

Eur Respir Rev. 2018 Jun 30;27(148):

Authors: Ponzano S, Nigrelli G, Fregonese L, Eichler I, Bertozzi F, Bandiera T, Galietta LJV, Papaluca M

Abstract
In this article we analyse the current authorised treatments and trends in early drug development for cystic fibrosis (CF) in the European Union for the time period 2000-2016. The analysis indicates a significant improvement in the innovation and development of new potential medicines for CF, shifting from products that act on the symptoms of the disease towards new therapies targeting the cause of CF. However, within these new innovative medicines, results for CF transmembrane conductance regulator (CFTR) modulators indicate that one major challenge for turning a CF concept product into an actual medicine for the benefit of patients resides in the fact that, although pre-clinical models have shown good predictability for certain mutations, a good correlation to clinical end-points or biomarkers (e.g. forced expiratory volume in 1 s and sweat chloride) for all mutations has not yet been achieved. In this respect, the use of alternative end-points and innovative nonclinical models could be helpful for the understanding of those translational discrepancies. Collaborative endeavours to promote further research and development in these areas as well as early dialogue with the regulatory bodies available at the European competent authorities are recommended.

PMID: 29653946 [PubMed - in process]

Prognostic Impact of Tumor Mutation Burden in Patients with Completely Resected Non-Small Cell Lung Cancer: Brief Report.

J Thorac Oncol. 2018 Apr 11;:

Authors: Owada-Ozaki Y, Muto S, Takagi H, Inoue T, Watanabe Y, Fukuhara M, Yamaura T, Okabe N, Matsumura Y, Hasegawa T, Ohsugi J, Hoshino M, Shio Y, Nanamiya H, Imai JI, Isogai T, Watanabe S, Suzuki H

Abstract
INTRODUCTION: Tumor mutation burden (TMB), is thought to be associated with the amount of neoantigen in the tumor and to have an important role in predicting the effect of immune checkpoint inhibitors. However, the relevance of TMB to prognosis is not yet fully understood. In this study, we investigated the clinical significance of TMB in patients with non-small cell lung cancer (NSCLC) and examined the relationship between TMB and prognosis.
METHODS: We calculated TMB within individual tumors by whole exome sequencing analysis using next-generation sequencing. We included 90 patients with NSCLC who underwent surgery in the Hospital of Fukushima Medical University from 2013 to 2016. No patients received chemotherapy or immunotherapy before surgery. We assessed the correlation between TMB and prognosis.
RESULTS: TMB >62 was associated with worse overall survival (OS) of patients with NSCLC (hazard ratio [HR] = 6.633, P = 0.0003). Multivariate analysis showed poor prognosis with high TMB (HR = 12.31, P = 0.019). In patients with stage I NSCLC, higher TMB was associated with worse prognosis for both OS (HR = 7.582, P = 0.0018) and disease-free survival (HR = 6.07, P = 0.0072).
CONCLUSION: High TMB in NSCLC is a poor prognostic factor. If high TMB is a predictor of the efficacy of immune checkpoint inhibitors, postoperative adjuvant therapy with immune checkpoint inhibitors may contribute to improvement of recurrence and OS.

PMID: 29654927 [PubMed - as supplied by publisher]

Advances in Evaluation of Chronic Diarrhea in Infants.

Gastroenterology. 2018 Apr 11;:

Authors: Thiagarajah JR, Kamin DS, Acra S, Goldsmith JD, Roland JT, Lencer WI, Muise AM, Goldenring JR, Avitzur YA, Martín MG, PediCODE consortium

Abstract
Diarrhea is common in infants (children less than 2 years of age), usually acute, and if chronic, commonly caused by allergies and occasionally by infectious agents. Congenital diarrheas and enteropathies (CODEs) are rare causes of devastating chronic diarrhea in infants. Evaluation of CODEs is a lengthy process and infrequently leads to a clear diagnosis. However, genomic analyses and the development of model systems have increased our understanding of CODE pathogenesis. With these advances, a new diagnostic approach is needed. We propose a revised approach to determine causes of diarrhea in infants, including CODEs, based on stool analysis, histologic features, responses to dietary modifications, and genetic tests. After exclusion of common causes of diarrhea in infants, the evaluation proceeds through analyses of stool characteristics (watery, fatty, or bloody) and histologic features such as the villus to crypt ratio in intestinal biopsies. Infants with CODEs resulting from defects in digestion, absorption, transport of nutrients and electrolytes, or enteroendocrine cell development or function have normal villi to crypt ratios; defects in enterocyte structure or immune-mediated conditions result in an abnormal villus to crypt ratios and morphology. Whole-exome and genome sequencing in the early stages of evaluation can reduce the time required for a definitive diagnosis of CODEs, or lead to identification of new variants associated with these enteropathies. The functional effects of gene mutations can be analyzed in model systems such as enteroids or induced pluripotent stem cells and are facilitated by recent advances in gene editing procedures. Characterization and investigation of new CODE disorders will improve management of patients and advance our understanding of epithelial cells and other cells in the intestinal mucosa.

PMID: 29654747 [PubMed - as supplied by publisher]

Muscle Weakness, Cardiomyopathy, and L-2-Hydroxyglutaric Aciduria Associated with a Novel Recessive SLC25A4 Mutation.

JIMD Rep. 2018 Apr 14;:

Authors: von Renesse A, Morales-Gonzalez S, Gill E, Salomons GS, Stenzel W, Schuelke M

Abstract
BACKGROUND: Mutations in SLC25A4 (syn. ANT1, Adenine nucleotide translocase, type 1) are known to cause either autosomal dominant progressive external ophthalmoplegia (adPEO) or recessive mitochondrial myopathy, hypertrophic cardiomyopathy, and lactic acidosis.
METHODS AND RESULTS: Whole exome sequencing in a young man with myopathy, subsarcolemmal mitochondrial aggregations, cardiomyopathy, lactic acidosis, and L-2-hydroxyglutaric aciduria (L-2-HGA) revealed a new homozygous mutation in SLC25A4 [c.653A>C, NM_001151], leading to the replacement of a highly conserved glutamine by proline [p.(Q218P); NP_001142] that most likely affects the folding of the ANT1 protein. No pathogenic mutation was found in L2HGDH, which is associated with "classic" L-2-HGA. Furthermore, L-2-HGDH enzymatic activity in the patient fibroblasts was normal. Long-range PCR and Southern blot confirmed absence of mtDNA-deletions in blood and muscle.
CONCLUSION: The disturbed ADP/ATP transport across the inner mitochondrial membrane may lead to an accumulation of different TCA-cycle intermediates such as 2-ketoglutarate (2-KG) in our patient. As L-2-HG is generated from 2-KG we hypothesize that the L-2-HG increase is a secondary effect of 2-KG accumulation. Hence, our report expands the spectrum of laboratory findings in ANT1-related diseases and hints towards a connection with organic acidurias.

PMID: 29654543 [PubMed - as supplied by publisher]

Identification of somatic mutations in monozygotic twins discordant for psychiatric disorders.

NPJ Schizophr. 2018 Apr 13;4(1):7

Authors: Nishioka M, Bundo M, Ueda J, Yoshikawa A, Nishimura F, Sasaki T, Kakiuchi C, Kasai K, Kato T, Iwamoto K

Abstract
Monozygotic twins are assumed to have identical genomes. Based on this assumption, phenotypic discordance in monozygotic twins has been previously attributed to environmental factors. However, recent genomic studies have identified characteristic somatic mutations in monozygotic twins discordant for Darier disease, Van der Woude syndrome, and Dravet syndrome. Here, we explored somatic mutations in four pairs of monozygotic twins discordant for schizophrenia or delusional disorder. We analyzed whole exome sequence data obtained from blood samples and identified seven somatic mutations in one twin pair discordant for delusional disorder. All seven of these mutations were validated by independent amplicon sequencing, and five of them were further validated by pyrosequencing. One somatic mutation in the patient with delusional disorder showed a missense variant in ABCC9 with an allele fraction of 7.32%. Although an association between the somatic mutations and phenotypic discordance could not be established conclusively in this study, our results suggest that somatic mutations in monozygotic twins may contribute to the development of psychiatric disorders, and can serve as high-priority candidates for genetic studies.

PMID: 29654278 [PubMed]

Functional Assays to Screen and Dissect Genomic Hits: Doubling Down on the National Investment in Genomic Research.

Circ Genom Precis Med. 2018 Apr;11(4):e002178

Authors: Musunuru K, Bernstein D, Cole FS, Khokha MK, Lee FS, Lin S, McDonald TV, Moskowitz IP, Quertermous T, Sankaran VG, Schwartz DA, Silverman EK, Zhou X, Hasan AAK, Luo XJ

Abstract
The National Institutes of Health have made substantial investments in genomic studies and technologies to identify DNA sequence variants associated with human disease phenotypes. The National Heart, Lung, and Blood Institute has been at the forefront of these commitments to ascertain genetic variation associated with heart, lung, blood, and sleep diseases and related clinical traits. Genome-wide association studies, exome- and genome-sequencing studies, and exome-genotyping studies of the National Heart, Lung, and Blood Institute-funded epidemiological and clinical case-control studies are identifying large numbers of genetic variants associated with heart, lung, blood, and sleep phenotypes. However, investigators face challenges in identification of genomic variants that are functionally disruptive among the myriad of computationally implicated variants. Studies to define mechanisms of genetic disruption encoded by computationally identified genomic variants require reproducible, adaptable, and inexpensive methods to screen candidate variant and gene function. High-throughput strategies will permit a tiered variant discovery and genetic mechanism approach that begins with rapid functional screening of a large number of computationally implicated variants and genes for discovery of those that merit mechanistic investigation. As such, improved variant-to-gene and gene-to-function screens-and adequate support for such studies-are critical to accelerating the translation of genomic findings. In this White Paper, we outline the variety of novel technologies, assays, and model systems that are making such screens faster, cheaper, and more accurate, referencing published work and ongoing work supported by the National Heart, Lung, and Blood Institute's R21/R33 Functional Assays to Screen Genomic Hits program. We discuss priorities that can accelerate the impressive but incomplete progress represented by big data genomic research.

PMID: 29654098 [PubMed - in process]

Phenotype variability and allelic heterogeneity in KMT2B-Associated disease.

Parkinsonism Relat Disord. 2018 Apr 05;:

Authors: Kawarai T, Miyamoto R, Nakagawa E, Koichihara R, Sakamoto T, Mure H, Morigaki R, Koizumi H, Oki R, Montecchiani C, Caltagirone C, Orlacchio A, Hattori A, Mashimo H, Izumi Y, Mezaki T, Kumada S, Taniguchi M, Yokochi F, Saitoh S, Goto S, Kaji R

Abstract
BACKGROUND: Mutations in Lysine-Specific Histone Methyltransferase 2B gene (KMT2B) have been reported to be associated with complex early-onset dystonia. Almost all reported KMT2B mutations occurred de novo in the paternal germline or in the early development of the patient. We describe clinico-genetic features on four Japanese patients with novel de novo mutations and demonstrate the phenotypic spectrum of KMT2B mutations.
METHODS: We performed genetic studies, including trio-based whole exome sequencing (WES), in a cohort of Japanese patients with a seemingly sporadic early-onset generalized combined dystonia. Potential effects by the identified nucleotide variations were evaluated biologically. Genotype-phenotype correlations were also investigated.
RESULTS: Four patients had de novo heterozygous mutations in KMT2B, c.309delG, c.1656dupC, c.3325_3326insC, and c.5636delG. Biological analysis of KMT2B mRNA levels showed a reduced expression of mutant transcript frame. All patients presented with motor milestone delay, microcephaly, mild psychomotor impairment, childhood-onset generalized dystonia and superimposed choreoathetosis or myoclonus. One patient cannot stand due to axial hypotonia associated with cerebellar dysfunction. Three patients had bilateral globus pallidal deep brain stimulation (DBS) with excellent or partial response.
CONCLUSIONS: We further demonstrate the allelic heterogeneity and phenotypic variations of KMT2B-associated disease. Haploinsufficiency is one of molecular pathomechanisms underlying the disease. Cardinal clinical features include combined dystonia accompanying mild psychomotor disability. Cerebellum would be affected in KMT2B-associated disease.

PMID: 29653907 [PubMed - as supplied by publisher]

Pharmacokinetic and pharmacodynamic profile of the sodium-glucose co-transporter-2 inhibitor empagliflozin in young people with Type 2 diabetes: a randomized trial.

Diabet Med. 2018 Apr 14;:

Authors: Laffel LMB, Tamborlane WV, Yver A, Simons G, Wu J, Nock V, Hobson D, Hughan KS, Kaspers S, Marquard J

Abstract
AIMS: To assess the pharmacokinetic and pharmacodynamic profile of a single dose of empagliflozin in young people with Type 2 diabetes to identify the appropriate doses for further paediatric development.
METHODS: We conducted a single-dose, open-label, randomized, parallel-group study with empagliflozin 5 mg, 10 mg and 25 mg in young people with Type 2 diabetes aged 10-17 years.
RESULTS: Of 39 participants screened, 27 were randomized and completed the study; their mean (± sd) age was 14.1±2.0 years and body weight was 96.7±23.5 kg. Compared with similar studies in adults with Type 2 diabetes, the maximum observed plasma concentrations were slightly lower with the 10-mg and 25-mg doses, and the area under the plasma concentration-time curve was slightly lower with the 10-mg but slightly higher with the 25-mg dose. The adjusted mean increases in urinary glucose excretion were 53 g/24 h (95% CI 32,74), 73 g/24 h (95% CI 52,94) and 87 g/24 h (95% CI 68,107), and the adjusted mean decreases in fasting plasma glucose were 0.9 mmol/l (95% CI -1.6,-0.1), 0.9 mmol/l (95% CI -1.7,-0.2) and 1.1 mmol/l (95% CI -1.8,-0.5) for the 5- 10- and 25-mg doses, respectively. There were no serious adverse events and one investigator-reported drug-related event (dehydration).
CONCLUSIONS: After a single oral dose of empagliflozin, adults and young people with Type 2 diabetes had similar exposure-response relationships after adjusting for significant covariates. These data support testing 10-mg and/or 25-mg doses of empagliflozin in an upcoming paediatric phase III Type 2 diabetes trial. This article is protected by copyright. All rights reserved.

PMID: 29655290 [PubMed - as supplied by publisher]

Cardiovascular safety signals with dipeptidyl peptidase-4 inhibitors: A disproportionality analysis among high-risk patients.

Pharmacoepidemiol Drug Saf. 2018 Apr 14;:

Authors: Baksh SN, McAdams-DeMarco M, Segal JB, Alexander GC

Abstract
PURPOSE: In 2008, the US Food and Drug Administration (FDA) issued Draft Guidance on investigating cardiovascular risk with oral diabetic drugs, including dipeptidyl peptidase-4 inhibitors (DPP-4i). In 2014, underpowered, post hoc analyses of clinical trials suggested an increased risk of heart failure with the use of these products. As such, we assessed disproportionate reporting of major adverse cardiac events (MACE) among reports for DPP-4i submitted to the FDA Adverse Event Reporting System (FAERS) from 2006 to 2015.
METHODS: We assessed the empirical Bayes geometric mean (EBGM) and its lower bound (EB05) of the relative reporting ratio for MACE among DPP-4i reports in the full FAERS database and in a subset of reports limited to cardiovascular and diabetic drugs. We then compared the EB05 in these 2 analyses and calculated the percent positive agreement for signals of disproportional reporting (SDRs) involving MACE.
RESULTS: Of 180.3 million adverse event reports, 13.4 million were for diabetic and cardiovascular drugs. In the cardiovascular subset, there was an SDR for heart failure with linagliptin (EB05 = 2782.47) and saxagliptin (EB05 = 2.40), myocardial infarction with alogliptin (EB05 = 290.11), and cerebral infarction with sitagliptin (EB05 = 2.80). Of the 14 MACE, 8 had a percent positive agreement ≥50% for an SDR in both analyses. Overall, the cardiovascular subset elicited 11 more SDRs for DPP-4i than the full dataset.
CONCLUSIONS: Postmarketing surveillance of DPP-4i through FAERS suggest increased reporting of MACE, supporting the current FDA warning of heart failure risk. This suggests the need for additional longitudinal, observational research into the association of DPP-4i and other MACE.

PMID: 29655237 [PubMed - as supplied by publisher]

Factors affecting adherence to antiretroviral therapy among pregnant women in the Eastern Cape, South Africa.

BMC Infect Dis. 2018 Apr 13;18(1):175

Authors: Adeniyi OV, Ajayi AI, Ter Goon D, Owolabi EO, Eboh A, Lambert J

Abstract
BACKGROUND: Context-specific factors influence adherence to antiretroviral therapy (ART) among pregnant women living with HIV. Gaps exist in the understanding of the reasons for the variable outcomes of the prevention of mother-to-child transmission (PMTCT) programme at the health facility level in South Africa. This study examined adherence levels and reasons for non-adherence during pregnancy in a cohort of parturient women enrolled in the PMTCT programme in the Eastern Cape, South Africa.
METHODS: This was a mixed-methods study involving 1709 parturient women in the Eastern Cape, South Africa. We conducted a multi-centre retrospective analysis of the mother-infant pair in the PMTCT electronic database in 2016. Semi-structured interviews of purposively selected parturient women with self-reported poor adherence (n = 177) were conducted to gain understanding of the main barriers to adherence. Binary logistic regression was used to determine the independent predictors of ART non-adherence.
RESULTS: A high proportion (69.0%) of women reported perfect adherence. In the logistic regression analysis, after adjusting for confounding factors, marital status, cigarette smoking, alcohol use and non-disclosure to a family member were the independent predictors of non-adherence. Analysis of the qualitative data revealed that drug-related side-effects, being away from home, forgetfulness, non-disclosure, stigma and work-related demand were among the main reasons for non-adherence to ART.
CONCLUSIONS: Non-adherence to the antiretroviral therapy among pregnant women in this setting is associated with lifestyle behaviours, HIV-related stigma and ART side-effects. In order to eliminate mother-to-child transmission of HIV, clinicians need to screen for these factors at every antenatal clinic visit.

PMID: 29653510 [PubMed - in process]

Genome-wide and candidate gene approaches of clopidogrel efficacy using pharmacodynamic and clinical end points-Rationale and design of the International Clopidogrel Pharmacogenomics Consortium (ICPC).

Am Heart J. 2018 Apr;198:152-159

Authors: Bergmeijer TO, Reny JL, Pakyz RE, Gong L, Lewis JP, Kim EY, Aradi D, Fernandez-Cadenas I, Horenstein RB, Lee MTM, Whaley RM, Montaner J, Gensini GF, Cleator JH, Chang K, Holmvang L, Hochholzer W, Roden DM, Winter S, Altman RB, Alexopoulos D, Kim HS, Déry JP, Gawaz M, Bliden K, Valgimigli M, Marcucci R, Campo G, Schaeffeler E, Dridi NP, Wen MS, Shin JG, Simon T, Fontana P, Giusti B, Geisler T, Kubo M, Trenk D, Siller-Matula JM, Ten Berg JM, Gurbel PA, Hulot JS, Mitchell BD, Schwab M, Ritchie MD, Klein TE, Shuldiner AR, ICPC Investigators

Abstract
RATIONALE: The P2Y12 receptor inhibitor clopidogrel is widely used in patients with acute coronary syndrome, percutaneous coronary intervention, or ischemic stroke. Platelet inhibition by clopidogrel shows wide interpatient variability, and high on-treatment platelet reactivity is a risk factor for atherothrombotic events, particularly in high-risk populations. CYP2C19 polymorphism plays an important role in this variability, but heritability estimates suggest that additional genetic variants remain unidentified. The aim of the International Clopidogrel Pharmacogenomics Consortium (ICPC) is to identify genetic determinants of clopidogrel pharmacodynamics and clinical response.
STUDY DESIGN: Based on the data published on www.clinicaltrials.gov, clopidogrel intervention studies containing genetic and platelet function data were identified for participation. Lead investigators were invited to share DNA samples, platelet function test results, patient characteristics, and cardiovascular outcomes to perform candidate gene and genome-wide studies.
RESULTS: In total, 17 study sites from 13 countries participate in the ICPC, contributing individual patient data from 8,829 patients. Available adenosine diphosphate-stimulated platelet function tests included vasodilator-stimulated phosphoprotein assay, light transmittance aggregometry, and the VerifyNow P2Y12 assay. A proof-of-principle analysis based on genotype data provided by each group showed a strong and consistent association between CYP2C19*2 and platelet reactivity (P value=5.1 × 10-40).
CONCLUSION: The ICPC aims to identify new loci influencing clopidogrel efficacy by using state-of-the-art genetic approaches in a large cohort of clopidogrel-treated patients to better understand the genetic basis of on-treatment response variability.

PMID: 29653637 [PubMed - in process]

New suggestion title: Bone Marrow - Mesenchymal Stem Cells Impacts on the U937 Cells in the Presence of Staphylococcal Enterotoxin B (SEB).

Clin Exp Pharmacol Physiol. 2018 Apr 14;:

Authors: Ejtehadifar M, Halabian R, Ghazavi A, Khansarinejad B, Mosayebi G, Imani Fooladi AA

Abstract
The growing resistance against conventional chemotherapy in acute myeloid leukemia (AML) is a noticeable clinical concern. Therefore, many researchers are looking for novel substances to overcome drug resistance in cancer.Staphylococcal enterotoxin B (SEB) is a superantigen (SAg) and a promising compound whichhas lethal effects on malignant cells. Inthis unprecedented study, SEB was used against U937 cells in a co-culture system in the presence of human bone marrow-mesenchymal stem cells (hBM-MSCs). The effects of hBM-MSCs on the proliferation and survival of U937 cell line with SEB was assessed using MTT assay and AnnexinV/PI flowcytometry, respectively. Moreover, the expression of IL-6, IL-10, TGF-β, and IKKb was evaluated by real-time PCR technique. Same experiments were also carried out using hBM-MSCs-conditioned medium (hBM-MSCs-CM).The results showed that SEB reduced the proliferation and survival of U937 cell line, but hBM-MSCs or hBM-MSCs-CM suppressed the effects of SEB. Furthermore, real-timePCR demonstrated that SEB could decrease the expression of IL-6, IL-10, and TGF-β in hBM-MSCs (P<0.05), while the production of IKKb was increased in comparison with the control group.These findings help us to have a broader understanding ofthe usage of SEB in the treatment of hematological malignancies, especially if it is targeted against hBM-MSCs to disrupt their supportive effects on malignant cells. This article is protected by copyright. All rights reserved.

PMID: 29655181 [PubMed - as supplied by publisher]

Proteomic approach and expression analysis revealed the differential expression of predicted leptospiral proteases capable of ECM degradation.

Biochim Biophys Acta. 2018 Apr 11;:

Authors: Gunasekaran D, Sikha T, Pinto SM, Kiran Kumar M, Patel K, Kumar M, Kumar V, Tennyson J, Satheeshkumar PK, Gowda H, Keshava Prasad TS, Madanan MG

Abstract
Leptospira, the causative agent of leptospirosis is known to have many proteases with potential to degrade extracellular matrix. However, a multipronged approach to identify, classify, characterize and elucidate their role has not been attempted. Our proteomic approach using high-resolution LC-MS/MS analysis of Triton X-114 fractions of Leptospira interrogans resulted in the identification of 104 proteases out of 130 proteases predicted by MEROPS. In Leptospira approximately 3.5% of the genome complements for proteases, which include catalytic types of metallo-, serine-, cysteine-, aspartic-, threonine- and asparagine- peptidases. Comparison of proteases from different serovars revealed that M04, M09B, M14A, M75, M28A, A01 and U73 protease families are exclusively present in pathogenic form. The M23 and S33 protease families are represented with >14 members in Leptospira. The differential expression under physiological temperature (37 °C) and osmolarity (300 mOsM) showed that proteases belonging to the catalytic type of Metallo-peptidases are upregulated significantly in pathogenic conditions. In silico prediction and characterization of the proteases revealed that several proteases are membrane anchored and secretory, classical as well as non-classical system. The study demonstrates the diversity and complexity of proteases, while maintaining conservation across the serovars in Leptospira and their differential expression under pathogenic conditions.

PMID: 29654978 [PubMed - as supplied by publisher]

FOXO1 inhibition potentiates endothelial angiogenic functions in diabetes via suppression of ROCK1/Drp1-mediated mitochondrial fission.

Biochim Biophys Acta. 2018 Apr 11;:

Authors: Shi Y, Fan S, Wang D, Huyan T, Chen J, Chen J, Su J, Li X, Wang Z, Xie S, Yun C, Li X, Tie L

Abstract
Diabetes-induced endothelial cell (EC) dysfunction and neovascularization impairment constitute vascular complications with limited treatment regimens. Transcription factor FOXO1 is a key angiogenic regulator and plays a pathologic role in progression of diabetes. The present study was designed to determine the involvement of FOXO1 in impaired EC function and post-ischemic neovascularization in diabetes and investigate underlying mechanisms. We found that FOXO1-selective inhibitor AS1842856 improved blood flow recovery and capillary density in ischemic hindlimb, and rescued the delay of wound closure with a concomitant augmentation of mean perfusion rate in diabetic mice. In vitro, treatment with AS1842856 or FOXO1 siRNA abrogated high glucose-induced apoptosis and ameliorated capillary tube formation in human umbilical vein endothelial cells (HUVECs). FOXO1 inhibition relieved alterations in mitochondrial networks and significantly suppressed the overproduction of mitochondrial reactive oxygen species (mtROS) induced by high glucose in ECs. Expression of dynamin-relatedprotein-1 (Drp1) and phosphorylation at Ser616, a protein required for mitochondrial fission, were enhanced by hyperglycemia, which could be neutralized by FOXO1 inhibition. Moreover, the transcription of Rho-associated coiled-coil containing protein kinase 1 (ROCK1), which phosphorylates Drp1 at Ser616, was shown by luciferase assay to be directly regulated by FOXO1. These findings suggested that FOXO1 is critical to preserve mitochondrial quantity and function in ECs, and FOXO1 may serve as a therapeutic target for microvascular complications of diabetes.

PMID: 29654945 [PubMed - as supplied by publisher]

Characterization of therapeutic antibodies in the presence of human serum proteins by AU-FDS analytical ultracentrifugation.

Anal Biochem. 2018 Apr 11;:

Authors: Wright RT, Hayes DB, Stafford WF, Sherwood PJ, Correia JJ

Abstract
The preclinical characterization of biopharmaceuticals seeks to determine the stability, state of aggregation, and interaction of the antibody/drug with other macromolecules in serum. Analytical ultracentrifugation is the best experimental method to understand these factors. Sedimentation velocity experiments using the AU-FDS system were performed in order to quantitatively characterize the nonideality of fluorescently labeled therapeutic antibodies in high concentrations of human serum proteins. The two most ubiquitous serum proteins are human serum albumin, HSA and γ-globulins, predominantly IgG. Tracer experiments were done pairwise as a function of HSA, IgG, and therapeutic antibody concentration. The sedimentation coefficient for each fluorescently labeled component as a function of the concentration of the unlabeled component yields the hydrodynamic nonideality (ks). This generates a 3x3 matrix of ks values that describe the nonideality of each pairwise interaction. The ks matrix is validated by fitting both 2:1 mixtures of HSA (1-40 mg/ml) and IgG (0.5-20 mg/ml) as serum mimics, and human serum dilutions (10-100%). The data are well described by SEDANAL global fitting with the ks nonideality matrix. The ks values for antibodies are smaller than expected and appear to be masked by weak association. Global fitting to ks and K2 models significantly improve the fits.

PMID: 29654743 [PubMed - as supplied by publisher]

Untargeted GC-MS Metabolomics.

Methods Mol Biol. 2018;1738:133-147

Authors: Papadimitropoulos MP, Vasilopoulou CG, Maga-Nteve C, Klapa MI

Abstract
Untargeted metabolomics refers to the high-throughput analysis of the metabolic state of a biological system (e.g., tissue, biological fluid, cell culture) based on the concentration profile of all measurable free low molecular weight metabolites. Gas chromatography-mass spectrometry (GC-MS), being a highly sensitive and high-throughput analytical platform, has been proven a useful tool for untargeted studies of primary metabolism in a variety of applications. As an omic analysis, GC-MS metabolomics is a multistep procedure; thus, standardization of an untargeted GC-MS metabolomics protocol requires the integrated optimization of pre-analytical, analytical, and computational steps. The main difference of GC-MS metabolomics compared to other metabolomics analytical platforms, including liquid chromatography-MS, is the need for the derivatization of the metabolite extracts into volatile and thermally stable derivatives, the latter being quantified in the metabolic profiles. This analytical step requires special care in the optimization of the untargeted GC-MS metabolomics experimental protocol. Moreover, both the derivatization of the original sample and the compound fragmentation that takes place in GC-MS impose specialized GC-MS metabolomic data identification, quantification, normalization and filtering methods. In this chapter, we describe the integrated protocol of untargeted GC-MS metabolomics with both the analytical and computational steps, focusing on the GC-MS specific parts, and provide details on any sample depending differences.

PMID: 29654587 [PubMed - in process]

DRUG-NEM: Optimizing drug combinations using single-cell perturbation response to account for intratumoral heterogeneity.

Proc Natl Acad Sci U S A. 2018 Apr 13;:

Authors: Anchang B, Davis KL, Fienberg HG, Williamson BD, Bendall SC, Karacosta LG, Tibshirani R, Nolan GP, Plevritis SK

Abstract
An individual malignant tumor is composed of a heterogeneous collection of single cells with distinct molecular and phenotypic features, a phenomenon termed intratumoral heterogeneity. Intratumoral heterogeneity poses challenges for cancer treatment, motivating the need for combination therapies. Single-cell technologies are now available to guide effective drug combinations by accounting for intratumoral heterogeneity through the analysis of the signaling perturbations of an individual tumor sample screened by a drug panel. In particular, Mass Cytometry Time-of-Flight (CyTOF) is a high-throughput single-cell technology that enables the simultaneous measurements of multiple ([Formula: see text]40) intracellular and surface markers at the level of single cells for hundreds of thousands of cells in a sample. We developed a computational framework, entitled Drug Nested Effects Models (DRUG-NEM), to analyze CyTOF single-drug perturbation data for the purpose of individualizing drug combinations. DRUG-NEM optimizes drug combinations by choosing the minimum number of drugs that produce the maximal desired intracellular effects based on nested effects modeling. We demonstrate the performance of DRUG-NEM using single-cell drug perturbation data from tumor cell lines and primary leukemia samples.

PMID: 29654148 [PubMed - as supplied by publisher]

Deregulation of LIMD1-VHL-HIF-1α-VEGF pathway is associated with different stages of cervical cancer.

Biochem J. 2018 Apr 13;:

Authors: Chakraborty C, Mitra S, Roychowdhury A, Samadder S, Dutta S, Roy A, Das P, Mandal RK, Sharp TV, Roychoudhury S, Panda CK

Abstract
To understand the mechanism of cellular stress in basal-parabasal layers of normal-cervix and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy-number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease free normal-cervix (n=9), adjacent normal-cervix of tumors (n=70), CIN (n=32), CACX (n=174) samples and two CACX cell lines. In basal-parabasal layers of normal-cervix, LIMD1 showed high protein-expression while low protein expression of VHL was concordant with high expression of HIF-1α and VEGF irrespective of HPV16 infection. This was in concordance with the low promoter methylation of LIMD1 and high in VHL in the basal-parabasal layers of normal-cervix. LIMD1 expression was significantly reduced while VHL expression was unchanged during different stages of cervical carcinoma. This was in concordance with their frequent methylation during different stages of this tumor. In different stages of cervical carcinoma, the expression pattern of HIF-1α and VEGF was high as seen in basal-parabasal layers and inversely correlated with the expression of LIMD1 and VHL. This was validated by demethylation experiments using 5-aza-dC in CACX cell lines. Additional deletion of LIMD1 and VHL in CIN/CACX provided an additional growth advantage during cervical carcinogenesis through reduced expression of genes and associated with poor prognosis of patients. Our data showed that overexpression of HIF-1α and its target gene VEGF in the basal-parabasal layers of normal-cervix was due to frequent inactivation of VHL by its promoter methylation. This profile was maintained during different stages of cervical carcinoma with additional methylation/deletion of VHL and LIMD1.

PMID: 29654110 [PubMed - as supplied by publisher]