Reconstituting Mouse Lungs with Conditionally Reprogrammed Human Bronchial Epithelial Cells.

Tissue Eng Part A. 2017 Jul 20;:

Authors: LaRanger R, Peters-Hall JR, Coquelin M, Alabi BR, Chen C, Wright WE, Shay J

Abstract
We developed methods for conditionally reprogramming (CR) primary human bronchial epithelial cells (HBECs) to extend their functional life span and permit their differentiation into both upper and lower airway lung epithelium. We also developed a bioreactor to support vascular perfusion and rhythmic breathing of decellularized mouse lungs reconstituted with CR HBECs isolated from patients with and without cystic fibrosis (CF). While conditionally reprogrammed cells only differentiate into an upper airway epithelium after 35 days at the air liquid interface, in reconstituted lungs these cells differentiate into upper airway bronchial epithelium and lower airway alveolar structures after 12 days. Rapid scale-up and the ability to obtain clonal derivatives of primary patient-derived HBECs without the need for genetic manipulation may permit rapid reconstitution of the lung epithelium; facilitating the study of lung disease in tissue engineered models.

PMID: 28726588 [PubMed - as supplied by publisher]

A PRECLINICAL STUDY IN RHESUS MACAQUES FOR CYSTIC FIBROSIS TO ASSESS GENE TRANSFER AND TRANSDUCTION BY AAV1 AND AAV5 WITH A DUAL-LUCIFERASE REPORTER SYSTEM.

Hum Gene Ther Clin Dev. 2017 Jul 20;:

Authors: Guggino W, Benson J, Seagrave J, Yan Z, Engelhardt JF, Gao G, Conlon TJ, Cebotaru L

Abstract
Cystic fibrosis (CF) is an autosomal recessive disease that is potentially treatable by gene therapy. Since the identification of the gene encoding CFTR, a number of preclinical and clinical trials have been conducted using the first generation of adeno-associated virus, AAV2. All these studies showed that AAV gene therapy for CF is safe, but clinical benefit was not clearly demonstrated. Thus, a new generation of AAV Vectors based on other serotypes is needed to move the field forward. Here we have tested two AAV serotypes (AAV1 and AAV5), using a dual-luciferase reporter system with firefly and Renilla luciferase genes packaged into AAV1 or AAV5, respectively. Two male and two female Rhesus macaques were each instilled in their lungs with both serotypes using a Penn-Century microsprayer. Both AAV1 and AAV5 vector genomes were detected in all the lung samples when measured at the time of necropsy, 45 days after instillation. However, the vector genome number for AAV1 was at least 10-fold higher than for AAV5. Likewise, luciferase activity was also detected in the same samples at 45 days. AAV1-derived activity was not statistically greater than that derived from AAV5. These data suggest that gene transfer is greater for AAV1 than for AAV5 in macaque lungs. Serum neutralizing antibodies were increased dramatically against both serotypes but were less abundant with AAV1 than with AAV5. No adverse events were noted, again indicating that AAV gene therapy is safe. Our results suggest that with more lung-tropic serotypes such as AAV1, new clinical studies of gene therapy using AAV are warranted.

PMID: 28726496 [PubMed - as supplied by publisher]

Genomic diagnostics within a medically underserved population: efficacy and implications.

Genet Med. 2017 Jul 20;:

Authors: Strauss KA, Gonzaga-Jauregui C, Brigatti KW, Williams KB, King AK, Van Hout C, Robinson DL, Young M, Praveen K, Heaps AD, Kuebler M, Baras A, Reid JG, Overton JD, Dewey FE, Jinks RN, Finnegan I, Mellis SJ, Shuldiner AR, Puffenberger EG

Abstract
PurposeWe integrated whole-exome sequencing (WES) and chromosomal microarray analysis (CMA) into a clinical workflow to serve an endogamous, uninsured, agrarian community.MethodsSeventy-nine probands (newborn to 49.8 years) who presented between 1998 and 2015 remained undiagnosed after biochemical and molecular investigations. We generated WES data for probands and family members and vetted variants through rephenotyping, segregation analyses, and population studies.ResultsThe most common presentation was neurological disease (64%). Seven (9%) probands were diagnosed by CMA. Family WES data were informative for 37 (51%) of the 72 remaining individuals, yielding a specific genetic diagnosis (n = 32) or revealing a novel molecular etiology (n = 5). For five (7%) additional subjects, negative WES decreased the likelihood of genetic disease. Compared to trio analysis, "family" WES (average seven exomes per proband) reduced filtered candidate variants from 22 ± 6 to 5 ± 3 per proband. Nineteen (51%) alleles were de novo and 17 (46%) inherited; the latter added to a population-based diagnostic panel. We found actionable secondary variants in 21 (4.2%) of 502 subjects, all of whom opted to be informed.ConclusionCMA and family-based WES streamline and economize diagnosis of rare genetic disorders, accelerate novel gene discovery, and create new opportunities for community-based screening and prevention in underserved populations.GENETICS in MEDICINE advance online publication, 20 July 2017; doi:10.1038/gim.2017.76.

PMID: 28726809 [PubMed - as supplied by publisher]

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Beta-Ketothiolase Deficiency Presenting with Metabolic Stroke After a Normal Newborn Screen in Two Individuals.

JIMD Rep. 2017 Jul 20;:

Authors: Wojcik MH, Wierenga KJ, Rodan LH, Sahai I, Ferdinandusse S, Genetti CA, Towne MC, Peake RWA, James PM, Beggs AH, Brownstein CA, Berry GT, Agrawal PB

Abstract
Beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase) deficiency is a genetic disorder characterized by impaired isoleucine catabolism and ketone body utilization that predisposes to episodic ketoacidosis. It results from biallelic pathogenic variants in the ACAT1 gene, encoding mitochondrial beta-ketothiolase. We report two cases of beta-ketothiolase deficiency presenting with acute ketoacidosis and "metabolic stroke." The first patient presented at 28 months of age with metabolic acidosis and pallidal stroke in the setting of a febrile gastrointestinal illness. Although 2-methyl-3-hydroxybutyric acid and trace quantities of tiglylglycine were present in urine, a diagnosis of glutaric acidemia type I was initially suspected due to the presence of glutaric and 3-hydroxyglutaric acids. A diagnosis of beta-ketothiolase deficiency was ultimately made through whole exome sequencing which revealed compound heterozygous variants in ACAT1. Fibroblast studies for beta-ketothiolase enzyme activity were confirmatory. The second patient presented at 6 months of age with ketoacidosis, and was found to have elevations of urinary 2-methyl-3-hydroxybutyric acid, 2-methylacetoacetic acid, and tiglylglycine. Sequencing of ACAT1 demonstrated compound heterozygous presumed causative variants. The patient exhibited choreoathethosis 2 months after the acute metabolic decompensation. These cases highlight that, similar to a number of other organic acidemias and mitochondrial disorders, beta-ketothiolase deficiency can present with metabolic stroke. They also illustrate the variability in clinical presentation, imaging, and biochemical evaluation that make screening for and diagnosis of this rare disorder challenging, and further demonstrate the value of whole exome sequencing in the diagnosis of metabolic disorders.

PMID: 28726122 [PubMed - as supplied by publisher]

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Partial uniparental isodisomy of chromosome 16 unmasks a deleterious biallelic mutation in IFT140 that causes Mainzer-Saldino syndrome.

Hum Genomics. 2017 Jul 19;11(1):16

Authors: Helm BM, Willer JR, Sadeghpour A, Golzio C, Crouch E, Vergano SS, Katsanis N, Davis EE

Abstract
BACKGROUND: The ciliopathies represent an umbrella group of >50 clinical entities that share both clinical features and molecular etiology underscored by structural and functional defects of the primary cilium. Despite the advances in gene discovery, this group of entities continues to pose a diagnostic challenge, in part due to significant genetic and phenotypic heterogeneity and variability. We consulted a pediatric case from asymptomatic, non-consanguineous parents who presented as a suspected ciliopathy due to a constellation of retinal, renal, and skeletal findings.
RESULTS: Although clinical panel sequencing of genes implicated in nephrotic syndromes yielded no likely causal mutation, an oligo-SNP microarray identified a ~20-Mb region of homozygosity, with no altered gene dosage, on chromosome 16p13. Intersection of the proband's phenotypes with known disease genes within the homozygous region yielded a single candidate, IFT140, encoding a retrograde intraflagellar transport protein implicated previously in several ciliopathies, including the phenotypically overlapping Mainzer-Saldino syndrome (MZSDS). Sanger sequencing yielded a maternally inherited homozygous c.634G>A; p.Gly212Arg mutation altering the exon 6 splice donor site. Functional studies in cells from the proband showed that the locus produced two transcripts: a majority message containing a mis-splicing event that caused a premature termination codon and a minority message homozygous for the p.Gly212Arg allele. Zebrafish in vivo complementation studies of the latter transcript demonstrated a loss of function effect. Finally, we conducted post-hoc trio-based whole exome sequencing studies to (a) test the possibility of other causal loci in the proband and (b) explain the Mendelian error of segregation for the IFT140 mutation. We show that the proband harbors a chromosome 16 maternal heterodisomy, with segmental isodisomy at 16p13, likely due to a meiosis I error in the maternal gamete.
CONCLUSIONS: Using clinical phenotyping combined with research-based genetic and functional studies, we have characterized a recurrent IFT140 mutation in the proband; together, these data are consistent with MZSDS. Additionally, we report a rare instance of a uniparental isodisomy unmasking a deleterious mutation to cause a ciliary disorder.

PMID: 28724397 [PubMed - in process]

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Low T-cell Receptor Diversity, High Somatic Mutation Burden, and High Neoantigen Load as Predictors of Clinical Outcome in Muscle-invasive Bladder Cancer.

Eur Urol Focus. 2016 Oct;2(4):445-452

Authors: Choudhury NJ, Kiyotani K, Yap KL, Campanile A, Antic T, Yew PY, Steinberg G, Park JH, Nakamura Y, O'Donnell PH

Abstract
BACKGROUND: The success of cancer immunotherapies has highlighted the potent ability of local adaptive immune responses to eradicate cancer cells by targeting neoantigens generated by somatic alterations. However, how these factors interact to drive the natural history of muscle-invasive bladder cancer (MIBC) is not well understood.
OBJECTIVE: To investigate the role of immune regulation in MIBC disease progression, we performed massively parallel T-cell receptor (TCR) sequencing of tumor-infiltrating T cells (TILs), in silico neoantigen prediction from exome sequences, and expression analysis of immune-related genes.
DESIGN, SETTING, AND PARTICIPANTS: We analyzed 38 MIBC tissues from patients who underwent definitive surgery with a minimum clinical follow-up of 2 yr.
OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Recurrence-free survival (RFS) was determined. TCR diversity was quantified using Simpson's diversity index. The main analyses involved the Mann-Whitney U test, Kaplan-Meier survival analysis, and Cox proportional hazards models.
RESULTS AND LIMITATIONS: Low TCRβ chain diversity, correlating with oligoclonal TIL expansion, was significantly correlated with longer RFS, even after adjustment for pathologic tumor stage, node status, and receipt of adjuvant chemotherapy (hazard ratio 2.67, 95% confidence interval 1.08-6.60; p=0.03). Patients with both a high number of neoantigens and low TCRβ diversity had longer RFS compared to those with fewer neoantigens and high TCR diversity (median RFS 275 vs 30 wk; p=0.03). Higher expression of immune cytolytic genes was associated with nonrecurrence among patients with low TCR diversity or fewer neoantigens. Limitations include the sample size and the inability to distinguish CD8(+) and CD4(+) T cells using TCR sequencing.
CONCLUSIONS: These findings are the first to show that detailed tumor immune-genome analysis at definitive surgery can identify molecular patterns of antitumor immune response contributing to better clinical outcomes in MIBC.
PATIENT SUMMARY: We discovered that clonal expansion of certain T cells in tumor tissue, possibly targeting cancer-specific antigens, contributes to prevention of bladder cancer recurrence.

PMID: 28723478 [PubMed]

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Neuronal deficiency of ARV1 causes an autosomal recessive epileptic encephalopathy.

Hum Mol Genet. 2016 Jul 15;25(14):3042-3054

Authors: Palmer EE, Jarrett KE, Sachdev RK, Al Zahrani F, Hashem MO, Ibrahim N, Sampaio H, Kandula T, Macintosh R, Gupta R, Conlon DM, Billheimer JT, Rader DJ, Funato K, Walkey CJ, Lee CS, Loo C, Brammah S, Elakis G, Zhu Y, Buckley M, Kirk EP, Bye A, Alkuraya FS, Roscioli T, Lagor WR

Abstract
We report an individual who presented with severe neurodevelopmental delay and an intractable infantile-onset seizure disorder. Exome sequencing identified a homozygous single nucleotide change that abolishes a splice donor site in the ARV1 gene (c.294 + 1G > A homozygous). This variant completely prevented splicing in minigene assays, and resulted in exon skipping and an in-frame deletion of 40 amino acids in primary human fibroblasts (NP_073623.1: p.(Lys59_Asn98del). The p.(Lys59_Asn98del) and previously reported p.(Gly189Arg) ARV1 variants were evaluated for protein expression and function. The p.(Gly189Arg) variant partially rescued the temperature-dependent growth defect in arv1Δ yeast, while p.(Lys59-Asn98del) completely failed to rescue at restrictive temperature. In contrast to wild type human ARV1, neither variant expressed detectable levels of protein in mammalian cells. Mice with a neuronal deletion of Arv1 recapitulated the human phenotype, exhibiting seizures and a severe survival defect in adulthood. Our data support ARV1 deficiency as a cause of autosomal recessive epileptic encephalopathy.

PMID: 27270415 [PubMed - indexed for MEDLINE]

Related Articles

Differential burden of rare protein truncating variants in Alzheimer's disease patients compared to centenarians.

Hum Mol Genet. 2016 Jul 15;25(14):3096-3105

Authors: Freudenberg-Hua Y, Li W, Abhyankar A, Vacic V, Cortes V, Ben-Avraham D, Koppel J, Greenwald B, Germer S, T2D-GENES Consortium, Darnell RB, Barzilai N, Freudenberg J, Atzmon G, Davies P

Abstract
We compared coding region variants of 53 cognitively healthy centenarians and 45 patients with Alzheimer's disease (AD), all of Ashkenazi Jewish (AJ) ancestry. Despite the small sample size, the known AD risk variant APOE4 reached genome-wide significance, indicating the advantage of utilizing 'super-controls'. We restricted our subsequent analysis to rare variants observed at most once in the 1000 Genomes database and having a minor allele frequency below 2% in our AJ sample. We compared the burden of predicted protein altering variants between cases and controls as normalized by the level of rare synonymous variants. We observed an increased burden among AD subjects for predicted loss-of-function (LoFs) variants defined as stop-gain, frame shift, initiation codon (INIT) and splice site mutations (n = 930, OR = 1.3, P = 1.5×E-5). There was no enrichment across all rare protein altering variants defined as missense plus LoFs, in frame indels and stop-loss variants (n = 13 014, OR = 0.97, P = 0.47). Among LoFs, the strongest burden was observed for INIT (OR = 2.16, P = 0.0097) and premature stop variants predicted to cause non-sense-mediated decay in the majority of transcripts (NMD) (OR = 1.98, P = 0.02). Notably, this increased burden of NMD, INIT and splice variants was more pronounced in a set of 1397 innate immune genes (OR = 4.55, P = 0.0043). Further comparison to additional exomes indicates that the difference in LoF burden originated both from the AD and centenarian sample. In summary, we observed an overall increased burden of rare LoFs in AD subjects as compared to centenarians, and this enrichment is more pronounced for innate immune genes.

PMID: 27260402 [PubMed - indexed for MEDLINE]

Type 1 diabetes mellitus caused by treatment with low-dose interferon-α in a melanoma patient.

Melanoma Res. 2017 Jul 19;:

Authors: Sossau D, Kofler L, Eigentler T

Abstract
Interferon-α (INF-α) is used as an adjuvant treatment for high-risk cutaneous melanoma. It has a large variety of potentially severe and irreversible side effects and can contribute toward the development of autoimmune disease. We report a case of a 59-year-old woman who developed type 1 diabetes following the use of low-dose IFN-α for the adjuvant treatment of stage IIB melanoma. Fifteen months after initiating IFN-α, she presented with blood glucose of 1126 mg/dl, hyponatremia, and microalbuminuria. Antibodies to glutamic acid decarboxylase and islet antigen-2 were negative and C-peptide was markedly reduced. There was no personal or family history of any autoimmune conditions. Reinforced insulin treatment and volume substitution with saline and glucose as a counter-regulation was started. To the best of our knowledge, this is the first reported case of low-dose IFN-α-induced type 1 diabetes. Clinicians should closely evaluate the pros and cons of IFN-α treatment in an adjuvant setting and remain mindful of the possibility of drug-induced autoimmune disease.

PMID: 28727675 [PubMed - as supplied by publisher]

Related Articles

Epidural steroid injection-related events requiring hospitalisation or emergency room visits among 52,935 procedures performed at a single centre.

Eur Radiol. 2017 Jul 19;:

Authors: Lee JW, Lee E, Lee GY, Kang Y, Ahn JM, Kang HS

Abstract
OBJECTIVES: To analyse the incidence and type of epidural steroid injection (ESI)-related adverse events, including procedure-related complications and drug-related systemic effects requiring hospitalisation or emergency room (ER) visits.
METHODS: This study included 52,935 ESI procedures performed in 22,059 patients in our department from March 2004 to February 2016. Of these, we retrospectively reviewed the cases of 1570 patients (1713 procedures) who were hospitalised or visited the ER within 1 month after ESI. ESI-related events were classified as procedure-related complications, drug-related systemic effects, or of uncertain relationship. Descriptive data are provided; no statistical analysis was performed.
RESULTS: There were 244 ESI-related events in 235 patients (males:females = 102:133; mean age: 65.7 years; range: 20-93 years). The incidence of ESI-related events was 0.46% per procedure, including 14 procedure-related complications, 56 drug-related systemic effects, and 174 events of uncertain cause. Of the 52,935 patients, 6 (0.011%) experienced major complications (two spine haematomas and four infections), 1 patient died, and 1 experienced neurological sequelae.
CONCLUSIONS: Although major procedure-related complications and drug-related systemic effects of ESI requiring hospitalisation are very rare, infection and haematoma can occur, resulting in serious outcomes. Hence, ESI should be carefully considered in high-risk patients.
KEY POINTS: • The incidence of ESI-related events requiring hospitalisation was 0.46%. • The incidence of procedure-related complications was 0.026%. • The incidence of drug-related systemic effects was 0.11%. • The incidence of major complication of ESI was 0.011%. • The major complications were spine infection, haematoma, and sepsis.

PMID: 28726118 [PubMed - as supplied by publisher]

Related Articles

Medication Literacy in a Cohort of Chinese Patients Discharged with Acute Coronary Syndrome.

Int J Environ Res Public Health. 2016 Jul 15;13(7):

Authors: Zhong Z, Zheng F, Guo Y, Luo A

Abstract
This study aims at investigating medication literacy of discharged patients with acute coronary syndrome (ACS) in China, and the important determinants of medication literacy among them. For this purpose, we conducted a prospective cohort study. Patient's demographic and clinical data were retrieved from hospital charts and medication literacy was measured by instructed interview using the Chinese version of Medication Literacy Questionnaire on Discharged Patient between 7 and 30 days after the patient was discharged from the hospital. The results show that medication literacy for the surveyed patients was insufficient: >20% did not have adequate knowledge on the types of drugs and the frequency that they need to take the drugs, >30% did not know the name of and the dosage of the drugs they are taking, and >70% did not have adequate knowledge on the effects and side effects of the drugs they are taking. Our research indicated that medication literacy scores decreased with age but increased with education. The number of medicines the discharged patient took with them and days between discharge and interview were not associated with medication literacy levels.

PMID: 27428990 [PubMed - indexed for MEDLINE]

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Funding Opportunity RFA-HD-18-012 from the NIH Guide for Grants and Contracts. This funding opportunity announcement (FOA) will use the specialized research center mechanism (P50) to call for multidisciplinary centers to serve as the CAPSTONE for research on child abuse and neglect and to serve as a national resource for the field. The Center(s) will conduct innovative and high quality research including: 1) trials testing the efficacy and effectiveness of clinical interventions; 2) longitudinal prospective studies examining the long term effects of specific and understudied types of maltreatment including abusive head trauma, medical child abuse and neglect, chronic sexual abuse; 3) studies examining the neurobiology of abuse and neglect and implications for health outcomes; and 4) studies testing the development of screening tools and clinical assessment measures for early identification and treatment of specific types of abuse and neglect to decrease morbidity and mortality and to identify potential comorbidities. The Centers are also required to propose a community engagement core which will provide opportunities for students and faculty at all levels, to be exposed to cutting edge educational tools and technologies, research, and expertise within the field of child maltreatment. A central goal of this core will be to engage the scientific and lay professional communities in participatory activities such as technical assistance, evidence-based practice, participation in grand rounds, conferences and seminars, or webinars based on information emanating from the other cores. The format for the community engagement activities will be tailored to the expertise of the Center and the needs of the community.

Notice NOT-OD-17-093 from the NIH Guide for Grants and Contracts

Notice NOT-OD-17-092 from the NIH Guide for Grants and Contracts

Notice NOT-OD-17-091 from the NIH Guide for Grants and Contracts

Funding Opportunity RFA-OD-17-012 from the NIH Guide for Grants and Contracts. The purpose of this Funding Opportunity Announcement (FOA) is to invite R03 applications to support biomedical and behavioral research that will provide scientific data to inform regulation of tobacco products to protect public health. Research Projects must address the research priorities related to the regulatory authority of the Food and Drug Administration (FDA) Center for Tobacco Products (CTP). The awards under this FOA will be administered by NIH using funds that have been made available through FDA CTP and the Family Smoking Prevention and Tobacco Control Act (P.L. 111-31). Research results from this FOA are expected to generate findings and data that are directly relevant in informing the FDA's regulation of the manufacture, distribution, and marketing of tobacco products to protect public health.

Funding Opportunity RFA-OD-17-014 from the NIH Guide for Grants and Contracts. The purpose of this Funding Opportunity Announcement (FOA) is to invite research project applications to support biomedical and behavioral research that will provide scientific data to inform regulation of tobacco products to protect public health. Research Projects must address the research priorities related to the regulatory authority of the Food and Drug Administration (FDA) Center for Tobacco Products (CTP). The awards under this FOA will be administered by NIH using funds that have been made available through FDA CTP and the Family Smoking Prevention and Tobacco Control Act (P.L. 111-31). Research results from this FOA are expected to generate findings and data that are directly relevant in informing the FDA's regulation of the manufacture, distribution, and marketing of tobacco products to protect public health.

Funding Opportunity RFA-OD-17-013 from the NIH Guide for Grants and Contracts. The purpose of this Funding Opportunity Announcement (FOA) is to invite R01 applications to support biomedical and behavioral research that will provide scientific data to inform regulation of tobacco products to protect public health. Research Projects must address the research priorities related to the regulatory authority of the Food and Drug Administration (FDA) Center for Tobacco Products (CTP). The awards under this FOA will be administered by NIH using funds that have been made available through FDA CTP and the Family Smoking Prevention and Tobacco Control Act (P.L. 111-31). Research results from this FOA are expected to generate findings and data that are directly relevant in informing the FDA's regulation of the manufacture, distribution, and marketing of tobacco products to protect public health.

Funding Opportunity PAR-17-332 from the NIH Guide for Grants and Contracts. This Funding Opportunity Announcement (FOA) will support small grants on primary immunodeficiency diseases focusing on ex vivo studies with human specimens and on studies with current or new animal models including novel clinical strategies for detecting, identifying the molecular basis of, or developing innovative therapies for primary immunodeficiency diseases. In addition, this FOA aims to encourage analyses of clinical data and samples maintained in primary immunodeficiency registries, consortium databases and repositories to address questions relevant to primary immunodeficiency research. The R03 grant supports different types of projects including pilot and feasibility studies; secondary analysis of existing data; small, self-contained research projects; development of research methodology; and development of new research technology. The R03 is intended to support small research projects that can be carried out in a short period of time with limited resources. Investigators who have not received independent NIH funding or independent NIH funding in this field are encouraged to apply to this FOA.