Accurate protein function annotation is a severe bottleneck when utilizing the deluge of high-throughput, next generation sequencing data. Keeping database annotations up-to-date has become a major scientific challenge that requires the development of reliable automatic predictors of protein function. The CAFA experiment provided a unique opportunity to undertake comprehensive 'blind testing' of many diverse approaches for automated function prediction. We report on the methodology we used for this challenge and on the lessons we learnt.
Our method integrates into a single framework a wide variety of biological information sources, encompassing sequence, gene expression and protein-protein interaction data, as well as annotations in UniProt entries. The methodology transfers functional categories based on the results from complementary homology-based and feature-based analyses. We generated the final molecular function and biological process assignments by combining the initial predictions in a probabilistic manner, which takes into account the Gene Ontology hierarchical structure.
We propose a novel scoring function called COmbined Graph-Information Content similarity (COGIC) score for the comparison of predicted functional categories and benchmark data. We demonstrate that our integrative approach provides increased scope and accuracy over both the component methods and the naïve predictors. In line with previous studies, we find that molecular function predictions are more accurate than biological process assignments.
Overall, the results indicate that there is considerable room for improvement in the field. It still remains for the community to invest a great deal of effort to make automated function prediction a useful and routine component in the toolbox of life scientists. As already witnessed in other areas, community-wide blind testing experiments will be pivotal in establishing standards for the evaluation of prediction accuracy, in fostering advancements and new ideas, and ultimately in recording progress.
Medulloblastomas are the most common malignant brain tumors in children1. Identifying and understanding the genetic events that drive these tumors is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma based on transcriptional and copy number profiles2–5. Here, we utilized whole exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas exhibit low mutation rates consistent with other pediatric tumors, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR, and LDB1, novel findings in medulloblastoma. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant but not wild type beta-catenin. Together, our study reveals the alteration of Wnt, Hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic beta-catenin signaling in medulloblastoma.
To test the safety and efficacy of exenatide once weekly (EQW) compared with metformin (MET), pioglitazone (PIO), and sitagliptin (SITA) over 26 weeks, in suboptimally treated (diet and exercise) drug-naive patients with type 2 diabetes.
RESEARCH DESIGN AND METHODS
Patients were randomized to subcutaneous (SC) EQW 2.0 mg + oral placebo (n = 248), MET 2,000 mg/day + SC placebo (n = 246), PIO 45 mg/day + SC placebo (n = 163), or SITA 100 mg/day + SC placebo (n = 163) for 26 weeks. MET and PIO therapies were increased to maximum-tolerated dosages. Injections with EQW or placebo were administered weekly, while oral medication or placebo was administered daily.
Baseline characteristics were as follows: 59% men, 67% Caucasian, mean age 54 years, HbA1c 8.5%, fasting serum glucose 9.9 mmol/L, body weight 87.0 kg, and diabetes duration 2.7 years. HbA1c reductions (%) at 26 weeks (least-squares means) with EQW versus MET, PIO, and SITA were −1.53 vs. −1.48 (P = 0.620), −1.63 (P = 0.328), and −1.15 (P < 0.001), respectively. Weight changes (kg) were −2.0 vs. −2.0 (P = 0.892), +1.5 (P < 0.001), and −0.8 (P < 0.001), respectively. Common adverse events were as follows: EQW, nausea (11.3%) and diarrhea (10.9%); MET, diarrhea (12.6%) and headache (12.2%); PIO, nasopharyngitis (8.6%) and headache (8.0%); and SIT, nasopharyngitis (9.8%) and headache (9.2%). Minor (confirmed) hypoglycemia was rarely reported. No major hypoglycemia occurred.
EQW was noninferior to MET but not PIO and superior to SITA with regard to HbA1c reduction at 26 weeks. Of the agents studied, EQW and MET provided similar improvements in glycemic control along with the benefit of weight reduction and no increased risk of hypoglycemia.
Ornithine transcarbamylase deficiency (OTCD), the most common and severe urea cycle disorder, is an excellent model for developing liver-directed gene therapy. No curative therapy exists except for liver transplantation which is limited by available donors and carries significant risk of mortality and morbidity. Adeno-associated virus 8 (AAV8) has been shown to be the most efficient vector for liver-directed gene transfer and is currently being evaluated in a clinical trial for treating hemophilia B. In this study, we generated a clinical candidate vector for a proposed OTC gene therapy trial in humans based on a self-complementary AAV8 vector expressing codon-optimized human OTC (hOTCco) under the control of a liver-specific promoter. Codon-optimization dramatically improved the efficacy of OTC gene therapy. Supraphysiological expression levels and activity of hOTC were achieved in adult spfash mice following a single intravenous injection of hOTCco vector. Vector doses as low as 1×1010 genome copies (GC) achieved robust and sustained correction of the OTCD biomarker orotic aciduria and clinical protection against an ammonia challenge. Functional expression of hOTC in 40% of liver areas was found in mice treated with a low vector dose of 1×109 GC. We suggest that the clinical candidate vector we have developed has the potential to achieve therapeutic effects in OTCD patients.
Adeno-associated viruses (AAV); liver gene therapy; ornithine transcarbamylase deficiency (OTCD); codon optimization
The HIV-1 capsid (CA) protein plays essential roles in both early and late stages of virl replication and has emerged as a novel drug target. We report hybrid structure-based virtual screening to identify small molecules with the potential to interact with the N-terminal domain (NTD) of HIV-1 CA and disrupt early, preintegration steps of the HIV-1 replication cycle. The small molecule 4,4′-[dibenzo[b,d]furan-2,8-diylbis(5-phenyl-1H-imidazole-4,2-diyl)]dibenzoic acid (CK026), which had anti-HIV-1 activity in single- and multiple-round infections but failed to inhibit viral replication in peripheral blood mononuclear cells (PBMCs), was identified. Three analogues of CK026 with reduced size and better drug-like properties were synthesized and assessed. Compound I-XW-053 (4-(4,5-diphenyl-1H-imidazol-2-yl)benzoic acid) retained all of the antiviral activity of the parental compound and inhibited the replication of a diverse panel of primary HIV-1 isolates in PBMCs, while displaying no appreciable cytotoxicity. This antiviral activity was specific to HIV-1, as I-XW-053 displayed no effect on the replication of SIV or against a panel of nonretroviruses. Direct interaction of I-XW-053 was quantified with wild-type and mutant CA protein using surface plasmon resonance and isothermal titration calorimetry. Mutation of Ile37 and Arg173, which are required for interaction with compound I-XW-053, crippled the virus at an early, preintegration step. Using quantitative PCR, we demonstrated that treatment with I-XW-053 inhibited HIV-1 reverse transcription in multiple cell types, indirectly pointing to dysfunction in the uncoating process. In summary, we have identified a CA-specific compound that targets and inhibits a novel region in the NTD-NTD interface, affects uncoating, and possesses broad-spectrum anti-HIV-1 activity.
Genomic rearrangements are thought to occur progressively during tumor development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome rearrangements in a one-step catastrophic event termed chromothripsis. We report the whole-genome sequencing-based analysis of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals a striking association between TP53 mutation and chromothripsis in SHH-MBs. Analysis of additional tumor entities substantiates a link between TP53 mutation and chromothripsis, and indicates a context-specific role for p53 in catastrophic DNA rearrangements. Among these, we observed a strong association between somatic TP53 mutations and chromothripsis in acute myeloid leukemia. These findings connect p53 status and chromothripsis in specific tumor types, providing a genetic basis for understanding particularly aggressive subtypes of cancer.
S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.
lung cancer; S-nitrosoglutathione reductase; Ras
Tc-99m ethyl cysteinate diethylester (ECD) and Tc-99m hexamethyl propylene amine oxime (HMPAO) are commonly used for single-photon emission computed tomography (SPECT) studies of a variety of neurologic disorders. Although these tracers have been very helpful in diagnosing and guiding treatment of neurologic disease, data describing the distribution and laterality of these tracers in normal resting brain are limited. Advances in quantitative functional imaging have demonstrated the value of using resting studies from control populations as a baseline to account for physiologic fluctuations in cerebral perfusion. Here, we report results from 30 resting Tc-99m ECD SPECT scans and 14 resting Tc-99m HMPAO scans of normal volunteers with no history of neurologic disease. Scans were analyzed with regions of interest and with statistical parametric mapping, with comparisons performed laterally (left vs. right), as well as for age, gender, and handedness. The results show regions of significant asymmetry in the normal controls affecting widespread areas in the cerebral hemispheres, but most marked in superior parietotemporal region and frontal lobes. The results have important implications for the use of normal control SPECT images in the evaluation of patients with neurologic disease.
brain asymmetry; brain imaging; SPECT
Genome3D, available at http://www.genome3d.eu, is a new collaborative project that integrates UK-based structural resources to provide a unique perspective on sequence–structure–function relationships. Leading structure prediction resources (DomSerf, FUGUE, Gene3D, pDomTHREADER, Phyre and SUPERFAMILY) provide annotations for UniProt sequences to indicate the locations of structural domains (structural annotations) and their 3D structures (structural models). Structural annotations and 3D model predictions are currently available for three model genomes (Homo sapiens, E. coli and baker’s yeast), and the project will extend to other genomes in the near future. As these resources exploit different strategies for predicting structures, the main aim of Genome3D is to enable comparisons between all the resources so that biologists can see where predictions agree and are therefore more trusted. Furthermore, as these methods differ in whether they build their predictions using CATH or SCOP, Genome3D also contains the first official mapping between these two databases. This has identified pairs of similar superfamilies from the two resources at various degrees of consensus (532 bronze pairs, 527 silver pairs and 370 gold pairs).
We determined the efficacy, biological activity, pharmacokinetics and safety of the hypomethylating agent 5-azacitidine (Celgene Corp., Summit, New Jersey) in dogs with naturally occurring invasive urothelial carcinoma.
Materials and Methods
We performed a preclinical phase I trial in dogs with naturally occurring invasive urothelial carcinoma to examine once daily subcutaneous administration of 5-azacitidine in 28-day cycles at doses of 0.10 to 0.30 mg/kg per day according to 2 dose schedules, including days 1 to 5 (28-day cohort) or days 1 to 5 and 15 to 19 (14-day cohort). Clinical efficacy was assessed by serial cystosonography, radiography and cystoscopy. Urinary 5-azacitidine pharmacokinetic analysis was also done. Pretreatment and posttreatment peripheral blood mononuclear cell and invasive urothelial carcinoma DNA, respectively, was analyzed for global and gene specific [CDKN2A (p14ARF)] methylation changes.
Enrolled in the study were 19 dogs with naturally occurring invasive urothelial carcinoma. In the 28-day cohort the maximum tolerated dose was 0.20 mg/kg per day with higher doses resulting in grade 3 or 4 neutropenia in 4 of 6 dogs. In the 14-day cohort the maximum tolerated dose was 0.10 mg/kg per day with grade 3 or 4 neutropenia seen in 2 of 3 dogs treated at higher doses. No grade 3 or 4 nonhematological toxicity was observed during either dosing schedule. Of 18 dogs evaluable for tumor response partial remission, stable disease and progressive disease were observed in 4 (22.2%), 9 (50.0%) and 4 (22.2%), respectively. Consistent 5-azacitidine levels (205 to 857 ng/ml) were detected in urine. Pretreatment and posttreatment methylation analysis revealed no significant correlation with clinical response.
Subcutaneous 5-azacitidine showed promising clinical activity in a canine invasive urothelial carcinoma model, thus meriting further development in humans with urothelial carcinoma.
urinary bladder; urothelium; carcinoma; azacitidine; dogs
Embryonal tumor with multilayered rosettes (ETMR, previously known as ETANTR) is a highly aggressive embryonal CNS tumor, which almost exclusively affects infants and is associated with a dismal prognosis. Accurate diagnosis is of critical clinical importance because of its poor response to current treatment protocols and its distinct biology. Amplification of the miRNA cluster at 19q13.42 has been identified previously as a genetic hallmark for ETMR, but an immunohistochemistry-based assay for clinical routine diagnostics [such as INI-1 for atypical teratoid rhabdoid tumor (AT/RT)] is still lacking. In this study, we screened for an ETMR-specific marker using a gene-expression profiling dataset of more than 1,400 brain tumors and identified LIN28A as a highly specific marker for ETMR. The encoded protein binds small RNA and has been implicated in stem cell pluripotency, metabolism and tumorigenesis. Using an LIN28A specific antibody, we carried out immunohistochemical analysis of LIN28A in more than 800 childhood brain-tumor samples and confirmed its high specificity for ETMR. Strong LIN28A immunoexpression was found in all 37 ETMR samples tested, whereas focal reactivity was only present in a small (6/50) proportion of AT/RT samples. All other pediatric brain tumors were completely LIN28A-negative. In summary, we established LIN28A immunohistochemistry as a highly sensitive and specific, rapid, inexpensive diagnostic tool for routine pathological verification of ETMR.
ETMR; Pediatric brain tumor; LIN28A; Diagnostic marker
The survival of patients with non–small-cell lung cancer (NSCLC), even when resectable, remains poor. Several small studies suggest that occult metastases (OMs) in pleura, bone marrow (BM), or lymph nodes (LNs) are present in early-stage NSCLC and are associated with a poor outcome. We investigated the prevalence of OMs in resectable NSCLC and their relationship with survival.
Patients and Methods
Eligible patients had previously untreated, potentially resectable NSCLC. Saline lavage of the pleural space, performed before and after pulmonary resection, was examined cytologically. Rib BM and all histologically negative LNs (N0) were examined for OM, diagnosed by cytokeratin immunohistochemistry (IHC). Survival probabilities were estimated using the Kaplan-Meier method. The log-rank test and Cox proportional hazards regression model were used to compare survival of groups of patients. P < .05 was considered significant.
From July 1999 to March 2004, 1,047 eligible patients (538 men and 509 women; median age, 67.2 years) were entered onto the study, of whom 50% had adenocarcinoma and 66% had stage I NSCLC. Pleural lavage was cytologically positive in only 29 patients. OMs were identified in 66 (8.0%) of 821 BM specimens and 130 (22.4%) of 580 LN specimens. In univariate and multivariable analyses OMs in LN but not BM were associated with significantly worse disease-free survival (hazard ratio [HR], 1.50; P = .031) and overall survival (HR, 1.58; P = .009).
In early-stage NSCLC, LN OMs detected by IHC identify patients with a worse prognosis. Future clinical trials should test the role of IHC in identifying patients for adjuvant therapy.
Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1HI cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24LOCD44HI cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDHHI and dye effluxHI cells, and increasing Oct1 increases the proportion of ALDHHI cells. Normal ALDHHI cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function.
Understanding the mechanisms that control stem cell function is a fundamental prerequisite both for the full application of stem cells to regenerative medicine and for a full understanding of the relationship between stem cells and cancer. In this study we show that a transcription factor known as Oct1 is a central regulator of normal and cancer stem cell function. We show that high Oct1 levels are associated with stem cells in multiple normal and malignant settings. Altering Oct1 expression, up or down, correspondingly alters multiple stem cell parameters, as well as stem cell function. We highlight known and identify new target genes Oct1 binds to that are consistent with a role in stem cell function.
Using vital records and census data representing 165,136 singleton births from 2003–2006, geospatial filtering and density estimates enabled the calculation of preterm birth rates at each geographical point within three urban Ohio counties. Adjusted attributable risk calculations were used to identify risk factors associated with regions of high and low rates of preterm birth. Among the three counties, affected populations varied in size as well as in demographic composition. Variation in the risk factors from one region to another suggests that a single one size fits all intervention strategy would be unlikely to efficiently or effectively impact the complex preterm birth problem. Although more useful in areas with a heterogeneous distribution of preterm birth, application of the presented approach supports the development of efficient community-level health intervention strategies by identifying communities with the highest potential impact and allowing for the prioritization of efforts on specific risk factors within those communities.
Sublobar resection (SR) is commonly used for patients considered high-risk for lobectomy. Non-operative therapies are increasingly being reported for similar risk patients because of perceived lower morbidity. We report 30 and 90 day adverse events (AEs) from ACOSOG Z4032; a multicenter phase III study for high-risk stage I non-small cell lung cancer (NSCLC) patients.
Data from 222 evaluable patients randomized to SR (n=114) or SR with brachytherapy (SRB) (n=108) are reported. AEs were recorded using the Common Terminology Criteria for Adverse Events, Version 3.0 at 30 and 90 days post surgery. Risk factors (age, baseline DLCO%, and FEV1%, upper lobe versus lower lobe resections, performance status, surgery approach; VATS versus open and extent ; wedge versus segmentectomy) were analyzed using a multivariable logistic model for their impact on the incidence of Grade 3 (G3+) and higher AEs. Respiratory AEs were also specifically analyzed
Median age, FEV1% and DLCO% were similar for the two treatment groups. There was no difference in the location of resection (upper versus lower lobe) or in the use of segmental or wedge resections. There were no differences between the groups with respect to “respiratory” G3+ (30 days: 14.9% vs. 19.4%; p=0.35; 0–90 days: 19.3% vs. 25%; p=0.31) and “any” G3+AEs (30 days: 25.4% vs. 30.6%; p=0.37; 0–90 days: 29.8% vs. 37%; p=0.25). Further analysis combined the two groups. Mortality occurred in 3 (1.4%) patients by 30 days and in 6 (2.7%) patients by 90 days. Four of the six deaths were felt to be attributable to surgery. When considered as continuous variables, FEV1% was associated with “any” grade 3 + AE at days 0–30 (p=0.03; OR=0.98), and days 0–90 (p=0.05; OR=0.98) respectively; and DLCO% was associated with “respiratory” grade 3+AE at days 0–30 (p=0.03; OR=0.97), and days 0–90 (p=0.05; OR=0.98) respectively. Segmental resection was associated with a higher incidence of any G3+ AE compared to wedge at days 0–30(40.3% versus 22.7%; OR=2.56; p<0.01) and days 0–90 (41.5% versus 29.7%; OR=1.96; p=0.04). The median FEV1% was 50% and the median DLCO% was 46%. Using these median values as potential cutpoints, only a DLCO% of less than 46% was significantly associated with an increased risk of “respiratory” and “any” grade 3+ AE for days 0–30, and 0–90.
In a multicenter setting, SRB was not associated with increased morbidity compared to SR alone. SR/SRB can be performed safely in high-risk patients with NSCLC with low 30 and 90 day mortality and acceptable morbidity. Segmental resection was associated with increased “any” G3+ AE, and DLCO% less than 46% was associated with “any” G3+AE as well as “respiratory” G3+ AE at both 30 and 90 days.
The shortage in organ donation is a major limiting factor for patients with end-stage lung disease. Expanding the donor pool would be beneficial. We investigated the importance of geographic distance between the donor and recipient and hypothesized that it would not be a critical determinant of outcomes after lung transplantation.
We retrospectively reviewed the United Network for Organ Sharing lung transplant database from 2000 to 2005 to allow sufficient time for bronchiolitis obliterans syndrome (BOS) development. Allograft recipients were stratified by geographic distance from their donors (local, regional, and national) and had yearly follow-up. The primary end points were the development of BOS and 1-year and 3-year mortality. Posttransplant outcomes were compared using a multivariable Cox proportional hazard model. Kaplan-Meier curves were compared by log-rank test.
Of 6,055 allograft recipients, donors were local in 59%, regional in 19.3%, and national in 21.7%. BOS-free survival did not differ by geographic distance. Geographic distance did not independently predict BOS (hazard ratio, 1.03; 95% confidence interval, 0.96 to 1.10). Similarly, Kaplan-Meier survival curves were not significantly worse for recipients with national donors. Geographic distance did not independently predict 3-year mortality (hazard ratio, 0.95; 95% confidence interval, 0.89 to 1.01).
With appropriate donor selection, moderately long geographic distance (average ischemic time < 6 hours) between the donor and recipient is not associated with the development of BOS or increased death after lung transplantation. By placing less emphasis on distance, more donors could potentially be used to expand the donor pool.
Virtual screening targeting the urokinase receptor (uPAR) led to (3R)-4-cyclohexyl-3-(hexahydrobenzo[d][1,3]dioxol-5-yl)-N-((hexahydrobenzo[d][1,3]dioxol-5-yl)methyl)butan-1-aminium 1 (IPR-1) and 4-(4-((3,5-dimethylcyclohexyl)carbamoyl)-2-(4-isopropylcyclohexyl)pyrazolidin-3-yl)piperidin-1-ium 3 (IPR-69). Synthesis of an analog of 1, namely 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC50 near 30 μM. Both compounds blocked angiogenesis with IC50 of 3 μM. Compounds 2 and 3 inhibited cell growth with IC50 of 6 and 18 μM and induced apoptosis. Biochemical assays revealed lead-like properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 hours. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.
The Pacific population of leatherback sea turtles (Dermochelys coriacea) has drastically declined in the last 25 years. This decline has been linked to incidental capture by fisheries, egg and meat harvesting, and recently, to climate variability and resource limitation. Here we couple growth rates with feeding experiments and food intake functions to estimate daily energy requirements of leatherbacks throughout their development. We then estimate mortality rates from available data, enabling us to raise food intake (energy requirements) of the individual to the population level. We place energy requirements in context of available resources (i.e., gelatinous zooplankton abundance). Estimated consumption rates suggest that a single leatherback will eat upward of 1000 metric tonnes (t) of jellyfish in its lifetime (range 924–1112) with the Pacific population consuming 2.1×106 t of jellyfish annually (range 1.0–3.7×106) equivalent to 4.2×108 megajoules (MJ) (range 2.0–7.4×108). Model estimates suggest 2–7 yr-old juveniles comprise the majority of the Pacific leatherback population biomass and account for most of the jellyfish consumption (1.1×106 t of jellyfish or 2.2×108 MJ per year). Leatherbacks are large gelatinous zooplanktivores with consumption to biomass ratios of 96 (up to 192 if feeding strictly on low energy density Cnidarians); they, therefore, have a large capacity to impact gelatinous zooplankton landscapes. Understanding the leatherback's needs for gelatinous zooplankton, versus the availability of these resources, can help us better assess population trends and the influence of climate induced resource limitations to reproductive output.
Mutations in the four-and-a-half LIM domain 1 (FHL1) gene, which encodes a 280-amino-acid protein containing four LIM domains and a single zinc-finger domain in the N-terminal region, have been associated with a broad clinical spectrum of X-linked muscle diseases encompassing a variety of different phenotypes. Patients might present with a scapuloperoneal myopathy, a myopathy with postural muscle atrophy and generalized hypertrophy, an Emery–Dreifuss muscular dystrophy, or an early onset myopathy with reducing bodies. It has been proposed that the phenotypic variability is related to the position of the mutation within the FHL1 gene. Here, we report on three British families with a heterogeneous clinical presentation segregating a single FHL1 gene mutation and haplotype, suggesting that this represents a founder mutation. The underlying FHL1 gene mutation was detected by direct sequencing and the founder effect was verified by haplotype analysis of the FHL1 gene locus. A 3-bp insertion mutation (p.Phe127_Thr128insIle) within the second LIM domain of the FHL1 gene was identified in all available affected family members of the three families. Haplotype analysis of the FHL1 region on Xq26 revealed that the families shared a common haplotype. The p.Phe127_Thr128insIle mutation in the FHL1 gene therefore appears to be a British founder mutation and FHL1 gene screening, in particular of exon 6, should therefore be indicated in British patients with a broad phenotypic spectrum of X-linked muscle diseases.
FHL1; myopathy; founder mutation; British
Ornithine transcarbamylase deficiency (OTCD) is the most common inborn error of urea synthesis. Complete OTCD can result in hyperammonemic coma in the neonatal period which can rapidly become fatal. Current acute therapy involves dialysis; chronic therapy involves the stimulation of alternate nitrogen clearance pathways; and the only curative approach is liver transplantation. AAV vector based gene therapy would add to current treatment options provided the vector delivers high level and stable transgene expression in liver without dose limiting toxicity. In this study, we employed an AAV2/8-based self-complementary (sc) vector expressing the murine OTC gene under a liver-specific thyroxine-binding globulin (TBG) promoter and examined the therapeutic effects in a mouse model of OTCD, the spf ash mouse. Seven days after a single intravenous injection of vector, treated mice showed complete normalization of urinary orotic acid, a measure of OTC activity. We further improved vector efficacy by incorporating a Kozak or Kozak-like sequence into mOTC cDNA which increased the OTC activity by 5- or 2-fold and achieved sustained correction of orotic aciduria for up to 7 months. Our results demonstrate that vector optimizations can significantly improve the efficacy of gene therapy.
Adeno-associated viruses (AAV); liver gene therapy; OTC deficiency; self-complementary
aurophilicity; geminal diauration; gold complexes; protodemetalation
Z4032 is a randomized clinical trial conducted by the American College of Surgeons Oncology Group that compared sublobar resection alone (SR) to sublobar resection with brachytherapy (SRB) for high-risk operable patients with non-small cell lung cancer (NSCLC). This current report evaluate the early impact that adjuvant brachytherapy has on pulmonary function tests (PFT), dyspnea and perioperative (30-day) respiratory complications on this impaired patient population.
Eligible stage I NSCLC patients with tumors 3cm or less were randomized to SR or SRB. The outcomes measured included the % predicted forced expiratory volume (FEV1%), % predicted carbon monoxide diffusion capacity (DLCO%), dyspnea score using the UC San Diego Shortness of Breath Questionnaire. Pulmonary morbidity was assessed using the Common Terminology Criteria for Adverse Events (AE) Version 3.0 (CTCAE). Outcomes were measured at baseline, and at 3-months. A 10% change in PFT or a 10-point change in dyspnea score was deemed clinically meaningful.
Z4032 permanently closed to patient accrual in January 2010 with a total of 224 patients. At 3-month follow-up, PFT data is currently available on 148 (74 SR/74 SRB) patients described in this report. There were no differences in baseline characteristics between the arms. In the SR arm, 9 (12%) patients reported grade-3 respiratory AE compared to 12 (16%) in the SRB arm (p=0.49). There was no significant change in the percent change in DLCO%, or dyspnea score from baseline to 3-month within either arm. In the case of FEV1%, the percent change from baseline to 3-month was significant within SR arm (p=0.03), with patients reporting an improvement in the FEV1% at month 3. Multivariable regression analysis (adjusted for baseline values) showed no significant impact of treatment arm, tumor location (upper versus other lobe), or surgical approach (VATS versus thoracotomy) on the 3-month values for FEV1%, DLCO% and dyspnea scores. There was no significant difference in the incidence of clinically meaningful (10% PFT change, or 10-point dyspnea score) change between the two arms. Twenty-two percent of patients with lower lobe tumors compared to 9% with upper lobe tumors demonstrated a 10% decline in FEV1% (odds ratio 2.79; 95 CI=1.07 – 7.25; p=0.04).
Adjuvant intraoperative brachytherapy performed in conjunction with sublobar resection does not significantly worsen pulmonary function, or dyspnea at 3-months in a high-risk population with NSCLC. SRB was not associated with increased perioperative pulmonary AE. Lower-lobe resection was the only factor that was significantly associated with a clinically meaningful decline in FEV1%.
Progressive supranuclear palsy (PSP) is associated with pathological changes along the dentatorubrothalamic tract and in premotor cortex. We aimed to assess whether functional neural connectivity is disrupted along this pathway in PSP, and to determine how functional changes relate to changes in structure and diffusion. Eighteen probable PSP subjects and 18 controls had resting-state (task-free) fMRI, diffusion tensor imaging and structural MRI. Functional connectivity was assessed between thalamus and the rest of the brain, and within the basal ganglia, salience and default mode networks (DMN). Patterns of atrophy were assessed using voxel-based morphometry, and patterns of white matter tract degeneration were assessed using tract-based spatial statistics. Reduced in-phase functional connectivity was observed between the thalamus and premotor cortex including supplemental motor area (SMA), striatum, thalamus and cerebellum in PSP. Reduced connectivity in premotor cortex, striatum and thalamus were observed in the basal ganglia network and DMN, with subcortical salience network reductions. Tract degeneration was observed between cerebellum and thalamus and in superior longitudinal fasciculus, with grey matter loss in frontal lobe, premotor cortex, SMA and caudate. SMA functional connectivity correlated with SMA volume and measures of cognitive and motor dysfunction, while thalamic connectivity correlated with degeneration of superior cerebellar peduncles. PSP is therefore associated with disrupted thalamocortical connectivity that is associated with degeneration of the dentatorubrothalamic tract and the presence of cortical atrophy.
Resting state fMRI; functional connectivity; white matter tracts; atrophy; dentatorubrothalamic tract