Essential tremor (ET) is one of the most common movement disorders in human adults. It can be characterized as a progressive neurological disorder of which the most recognizable feature is a tremor of the arms or hands that is apparent during voluntary movements such as eating and writing. The pathology of ET remains unclear. Resting-state fMRI (RS-fMRI), as a non-invasive imaging technique, was employed to investigate abnormalities of functional connectivity in ET in the brain. Regional homogeneity (ReHo) was used as a metric of RS-fMRI to assess the local functional connectivity abnormality in ET with 20 ET patients and 20 age- and gender-matched healthy controls (HC). The ET group showed decreased ReHo in the anterior and posterior bilateral cerebellar lobes, the bilateral thalamus and the insular lobe, and increased ReHo in the bilateral prefrontal and parietal cortices, the left primary motor cortex and left supplementary motor area. The abnormal ReHo value of ET patients in the bilateral anterior cerebellar lobes and the right posterior cerebellar lobe were negatively correlated with the tremor severity score, while positively correlated with that in the left primary motor cortex. These findings suggest that the abnormality in cerebello-thalamo-cortical motor pathway is involved in tremor generation and propagation, which may be related to motor-related symptoms in ET patients. Meanwhile, the abnormality in the prefrontal and parietal regions may be associated with non-motor symptoms in ET. These findings suggest that the ReHo could be utilized for investigations of functional-pathological mechanism of ET.
Current genome-wide association studies still heavily rely on a single-marker strategy, in which each single nucleotide polymorphism (SNP) is tested individually for association with a phenotype. Although methods and software packages that consider multimarker models have become available, they have been slow to become widely adopted and their efficacy in real data analysis is often questioned. Based on conducting extensive simulations, here we endeavor to provide more insights into the performance of simple multimarker association tests as compared to single-marker tests. The results reveal the power advantage as well as disadvantage of the two- vs. the single-marker test. Power differentials depend on the correlation structure among tag SNPs, as well as that between tag SNPs and causal variants. A two-marker test has relatively better performance than single-marker tests when the correlation of the two adjacent markers is high. However, using HapMap data, two-marker tests tended to have a greater chance of being less powerful than single-marker tests, due to constraints on the number of actual possible haplotypes in the HapMap data. Yet, the average power difference was small whenever the one-marker test is more powerful, while there were many situations where the two-marker test can be much more powerful. These findings can be useful to guide analyses of future studies.
Asymptotic power; single-marker test; two-marker test; genome-wide association
Toll-like receptor (TLR) ligands have been explored as vaccine adjuvants for tumor and virus immunotherapy, but few TLR ligands affecting schistosoma vaccines have been characterized. Previously, we developed a partially protective DNA vaccine encoding the 26-kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST).
In this study, we evaluated a TLR7/8 ligand (R848) and a TLR9 ligand (CpG oligodeoxynucleotides, or CpG) as adjuvants for pVAX1-Sj26GST and assessed their effects on the immune system and protection against S. japonicum. We show that combining CpG and R848 with pVAX1-Sj26GST immunization significantly increases splenocyte proliferation and IgG and IgG2a levels, decreases CD4+CD25+Foxp3+ regulatory T cells (Treg) frequency in vivo, and enhances protection against S. japonicum. CpG and R848 inhibited Treg-mediated immunosuppression, upregulated the production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, IL-2, and IL-6, and decreased Foxp3 expression in vitro, which may contribute to prevent Treg suppression and conversion during vaccination and allow expansion of antigen-specific T cells against pathogens.
Our data shows that selective TLR ligands can increase the protective efficacy of DNA vaccines against schistosomiasis, potentially through combined antagonism of Treg-mediated immunosuppression and conversion.
There is evidence that TLR activation can block Treg cell responses and thereby break tolerance to self-antigens. It is expected that the use of TLR ligands as vaccine adjuvants will induce potent anti-pathogen immune responses and simultaneously overcome immune inhibition mediated by Tregs. However, the impact of TLR ligands on schistosomiasis vaccines is unclear. Here, we demonstrate that the use of a TLR7/8 ligand (R848) and a TLR9 ligand (CpG) as adjuvants in combination with the S. japonicum vaccine pVAX1-Sj26GST improves disease protection. The combination of CpG and R848 administered after vaccination causes an immune response marked by an upregulation of splenocyte proliferation and IgG and IgG2a levels that also coincides with a decreased proportion of CD4+CD25+ Tregs in mice. We also show that combined adjuvant use of CpG and R848 may impair Treg development and function by promoting the secretion of proinflammatory cytokines and reducing Foxp3 expression. Our findings suggest that in combination with the vaccine, TLR ligands may protect the effector response from Treg-mediated suppression, thereby eliciting the appropriate immune response to improve vaccine efficacy. Immunization combined with the TLR ligands CpG and R848 thus represents a promising new approach for the design of schistosoma vaccines.
Incorporating family data in genetic association studies has become increasingly appreciated, especially for its potential value in testing rare variants. We introduce here a variance-component based association test that can test multiple common or rare variants jointly using both family and unrelated samples.
The proposed approach implemented in our R package aggregates or collapses the information across a region based on genetic similarity instead of genotype scores, which avoids the power loss when the effects are in different directions or have different association strengths. The method is also able to effectively leverage the LD information in a region and it can produce a test statistic with an adaptively estimated number of degrees of freedom. Our method can readily allow for the adjustment of non-genetic contributions to the familial similarity, as well as multiple covariates.
We demonstrate through simulations that the proposed method achieves good performance in terms of Type I error control and statistical power. The method is implemented in the R package “fassoc”, which provides a useful tool for data analysis and exploration.
Association studies; Family data; Score test; Multi-marker test
New-generation antiepileptic drugs (AEDs) tend to replace traditional AEDs as the first-line choice for epilepsy. However, whether this change results in better outcome, especially in China, remains unknown.
Two broad spectrum AEDs, the traditional drug of sustained-release formulation of valproate (SRVPA) and the new-generation drug of topiramate, were compared in patients with epilepsy as monotherapy in this multi-centre, observational cohort study from 2000 to 2011. The primary outcome was time to treatment failure. The secondary outcomes included time to first seizure, time to 12-month remission, and time to 24-month remission. Drug tolerability was assessed. Cox proportional hazard models (95% confidence interval [CI]) were used to analyse the relative risks expressed as hazard ratios (HR).
Of the 1008 recruited patients, 519 received SRVPA and 489 received topiramate. SRVPA was better than topiramate (28.3% vs. 41.5%; HR = 0.62, [95% CI 0.49–0.77]; p<0.0001) in primary outcome, and in time to first seizure (56.1% vs. 69.3%; HR = 0.73, [95% CI 0.62–0.86]; p = 0.0002). No significant difference was observed between two groups in time to 12-month remission (52.6% vs. 42.5%; HR = 1.01, [95% CI 0.84–1.23]; p = 0.88) and time to 24-month remission (34.7% vs. 25.2%; HR = 1.11, [95% CI 0.88–1.42]; p = 0.38). 36 patients (6.9%) in SRVPA group and 37 patients (7.6%) in topiramate group presented treatment failure associated with intolerable adverse events, there was no significant difference between the two groups (p = 0.70).
The SRVPA is more suitable than topiramate for Chinese epileptic patients, and our results support the viewpoint that traditional AEDs should be the first-line choice for epilepsy rather than new-generation AEDs.
Iron deficiency anemia is an extra-stomach disease experienced in H. pylori carriers. Individuals with type A blood are more prone to suffering from H. pylori infection than other individuals. To clarify the molecular mechanisms underlying H. pylori-associated anemia, we collected erythrocytes from A, B, O, and AB blood donors and analyzed morphology, the number of erythrocytes with H. pylori colonies attached to them, and iron contents in erythrocytes and H. pylori (NCTC11637 and SS1 strains) by means of optical microscopy, scanning electron microscopy, and synchrotron radiation soft X-ray imaging. The number of type A erythrocytes with H. pylori attached to them was significantly higher than that of other erythrocytes (P<0.05). Far more iron distribution was observed in H. pylori bacteria using dual energy analysis near the iron L2, 3 edges by soft X-ray imaging. Iron content was significantly reduced in host erythrocytes after 4 hours of exposure to H. pylori. H. pylori are able to adhere more strongly to type A erythrocytes, and this is related to iron shift from the host to the bacteria. This may explain the reasons for refractory iron deficiency anemia and elevated susceptibility to H. pylori infection in individuals with type A blood.
We have built a rat's model to investigate whether the hypothermia induced by adenosine 5′-monophosphate (5′-AMP) (AIH) could attenuate acute lung injury induced by LPS in rats. We detected the inflammatory cytokine levels in the plasma and bronchoalveolar lavage fluid samples, and we analyzed the pathological changes in the lungs. We have found that AIH can effectively inhibit acute inflammatory reactions and protect the lung from acute injury induced by LPS in rats.
The universal conductance fluctuations (UCFs), one of the most important manifestations of mesoscopic electronic interference, have not yet been demonstrated for the two-dimensional surface state of topological insulators (TIs). Even if one delicately suppresses the bulk conductance by improving the quality of TI crystals, the fluctuation of the bulk conductance still keeps competitive and difficult to be separated from the desired UCFs of surface carriers. Here we report on the experimental evidence of the UCFs of the two-dimensional surface state in the bulk insulating Bi2Te2Se microflakes. The solely-B⊥-dependent UCF is achieved and its temperature dependence is investigated. The surface transport is further revealed by weak antilocalizations. Such survived UCFs of the surface states result from the limited dephasing length of the bulk carriers in ternary crystals. The electron-phonon interaction is addressed as a secondary source of the surface state dephasing based on the temperature-dependent scaling behavior.
CD4+CD25+ regulatory T cells (Tregs) do
not only influence self-antigen specific immune responses, but also dampen
the protective effect induced by a number of vaccines. The impact of CD4+CD25+
Tregs on vaccines against schistosomiasis, a neglected tropical disease that
is a major public health concern, however, has not been examined. In this
study, a DNA vaccine encoding a 26 kDa glutathione S-transferase of Schistosoma
japonicum (pVAX1-Sj26GST) was constructed and its potential effects
were evaluated by depleting CD25+ cells prior to pVAX1-Sj26GST
immunization. This work shows that removal of CD25+ cells
prior to immunization with the pVAX1-Sj26GST schistosomiasis DNA vaccine significantly
increases the proliferation of splenocytes and IgG levels. However, CD25+
cell-depleted mice immunized with pVAX1-Sj26GST show no improved protection
against S. japonicum. Furthermore, depletion of CD25+
cells causes an increase in both pro-inflammatory cytokines (e.g. IFN-γ,
GM-CSF and IL-4) and an anti-inflammatory cytokine (e.g. IL-10), with CD4+CD25-
T cells being one of the major sources of both IFN-γ and IL-10. These
findings indicate that partial CD25+ cell depletion fails
to enhance the effectiveness of the schistosome vaccine, possibly due to IL-10
production by CD4+CD25- T cells, or other cell
types, after CD25+ cell depletion during vaccination.
Although recent studies have attempted to dispel the confusion that exists in regard to the definition, analysis and interpretation of interaction in genetics, there still remain aspects that are poorly understood by non-statisticians. After a brief discussion of the definition of gene-gene interaction, the main part of this study addresses the fundamental meaning of statistical interaction and its relationship to measurement scale, disproportionate sample sizes in the cells of a two-way table and gametic phase disequilibrium.
Epistasis; Gametic phase disequilibrium; Interaction; Transformation
Huanglongbing (HLB) is a highly destructive disease of citrus production worldwide. 'Candidatus Liberibacter asiaticus', an unculturable alpha proteobacterium, is a putative pathogen of HLB. Information about the biology and strain diversity of 'Ca. L. asiaticus' is currently limited, inhibiting the scope of HLB research and control.
A genomic region (CLIBASIA_05640 to CLIBASIA_05650) of 'Ca. L. asiaticus' showing hyper-sequence variation or locus mosaicism was identified and investigated using 262 bacterial strains (188 from China and 74 from Florida). Based on the characteristic electrophoretic profiles of PCR amplicons generated by a specific primer set, eight electrophoretic types (E-types) were identified, six E-types (A, B, C, D, E, and F) in China and four E-types (A, C, G, and H) in Florida. The 'Ca. L. asiaticus' strains from China consisted predominately of E-type A (71.3%) and E-type B (19.7%). In contrast, the 'Ca. L. asiaticus' strains from Florida was predominated by E-type G (82.4%). Diversity of 'Ca. L. asiaticus' in China was also evidenced. Strains from the high altitude Yunnan Province consisted of five E-types with E-type B being the majority (62.8%), whereas strains from the low altitude coastal Guangdong Province consisted of only two E-types with E-type A as the majority (97.0%). Sequence analyses revealed that variation of DNA amplicons was due to insertion/deletion events at CLIBASIA_05650 and the downstream intergenic region.
This study demonstrated the genomic mosaicism of 'Ca. L. asiaticus' resulted from active DNA insertion/deletion activities. Analyses of strain variation depicted the significant inter- and intra-continent diversity of 'Ca. L. asiaticus'.
Gene-based and single-nucleotide polymorphism (SNP) set association studies provide an important complement to SNP analysis. Kernel-based nonparametric regression has recently emerged as a powerful and flexible tool for this purpose. Our goal is to explore whether this approach can be extended to incorporate and test for interaction effects, especially for genes containing rare variant SNPs. Here, we construct nonparametric regression models that can be used to include a gene-environment interaction effect under the framework of the least-squares kernel machine and examine the performance of the proposed method on the Genetic Analysis Workshop 17 unrelated individuals data set. Two hundred simulated replicates were used to explore the power for detecting interaction. We demonstrate through a genome scan of the quantitative phenotype Q1 that the simulated gene-environment interaction effect in the data can be detected with reasonable power by using the least-squares kernel machine method.
The current knowledge of immunological responses to schistosomiasis, a major tropical helminthic disease, is insufficient, and a better understanding of these responses would support vaccine development or therapies to control granuloma-associated immunopathology. CD4+ T cells play critical roles in both host immune responses against parasitic infection and immunopathology in schistosomiasis. The induction of T helper (Th)1, Th2 and T regulatory (Treg) cells and their roles in schistosome infections are well-illustrated. However, little in vivo data are available on the dynamics of Th17 cells, another important CD4+ T cell subset, after Schistosoma japonicum infection or whether these cells and their defining IL-17 cytokine mediate host protective responses early in infection.
Levels of Th17 and the other three CD4+ T cell subpopulations and the cytokines related to induction or repression of Th17 cell generation in different stages of S. japonicum infection were observed. Contrary to reported in vitro studies, our results showed that the Th17 cells were induced along with the Th1, Th2, Treg cells and the IFN-γ and IL-4 cytokines in S. japonicum infected mice. The results also suggested that S. japonicum egg antigens but not adult worm antigens preferentially induced Th17 cell generation. Furthermore, decreasing IL-17 with a neutralizing anti-IL-17 monoclonal antibody (mAb) increased schistosome-specific antibody levels and partial protection against S. japonicum infection in mice.
Our study is the first to report the dynamics of Th17 cells during S. japonicum infection and indicate that Th17 cell differentiation results from the integrated impact of inducing and suppressive factors promoted by the parasite. Importantly, our findings suggest that lower IL-17 levels may result in favorable host protective responses. This study significantly contributes to the understanding of immunity to schistosomiasis and may aid in developing interventions to protect hosts from infection or restrain immunopathology.
Th17 immune cells secrete the IL-17 cytokine and contribute to host defenses against certain infections. Recent studies linked IL-17 with the severity of liver inflammation and suggested that Th17 cells contribute to the pathology in schistosomiasis, a serious disease caused by parasitic worms such as Schistosoma japonicum widespread in vertebrates including humans. However, the role of Th17 cells in protection against S. japonicum infection is still unclear. For the first time, we describe here the changes in Th17 cell levels during S. japonicum infection and suggest that the schistosome egg antigens are primarily responsible for stimulating the generation of host Th17 cells after S. japonicum infection. We further show that the level of Th17 cells in the host is determined by a combination of factors, namely exposure to complex parasitic antigens that either induce or suppress their generation. We also suggest that lowering IL-17 levels may favor the host's protective responses against S. japonicum infection. Our findings help to better understand the relationship between the host and parasite in terms of immune protection and pathology in schistosomiasis and may contribute to the future development of vaccination and therapeutic strategies.
Since the isolation of Helicobacter species in biliary system, a hypothetical question was raised about the role of these agents in the development of cholelithiasis. This meta-analysis is to explore the association between the Helicobacter infection and biliary lithiasis.
A systematic literature search was performed to identify all eligible articles. Meta-analysis which was carried out using odds ratio and random effect model, 95% confidence intervals for odds ratio was calculated. Quantitative assessment of heterogeneity was explored by chi-square test with significance set at P value 0.10 and was measured using I2 statistic. Eighteen studies published between 1998 and 2011 were finally eligible for meta-analysis. H. Pylori, H. Bilis, H. Hepaticus, H. Pullorum and H. Ganmani were studied. With heterogeneity (I2 = 69.5%, P<0.0001), significantly higher pooled infection rates of H. Pylori (OR: 2.59, 35.82% versus 26.75%, P = 0.01) and H. Hepaticus (OR: 3.13, 31.30% versus 12.12%, P = 0.02) were observed in lithiasis group. Higher prevalence of H. Pylori in cholelithiasis patients were reported by studies from East Asia, South Asia and South America. Evidences supporting the higher presence of H. Pylori in cholelithiasis patients could be found by PCR for detecting 16s rRNA in bile, 26kDa protein gene in biliary tissue and immunohistochemistry. Using multiple detection tests could increase the detection rate of H. Pylori.
Our meta-analysis suggests a trend of higher presence of H. Pylori in cholelithiasis patients than control group and this trend was significant in the regions with higher prevalence of this agent. Evidences supporting the association between Helicobacter and cholelithiasis could be found by using different tests but the gold standard for the identification of these bacteria in biliary system has yet to be established. Considering obvious heterogeneity, a large multi-center study will facilitate us to further clarify the association between the Helicobacter infection and cholelithiasis.
The purpose of this study is to investigate the distribution and antimicrobial susceptibility of pathogenic bacteria in inpatients with pulmonary infection in the neurological intensive care unit (NICU).
A total of 947 sputum specimens of 428 inpatients from May 2007 to May 2008 in the NICU were enrolled in the study, and bacterial identification and antibiotic susceptibility tests were analyzed using a VITEK 2 system.
A total of 400 positive bacterial strains were separated from 947 sputum specimens, with Gram-negative bacteria accounting for 69.0% of the total strains collected. The most common strain of Gram-negative bacteria was Klebsiella pneumoniae (20.5%). Gram-positive bacteria accounted for 10.0% of the total strains, with the most common strain being Staphylococcus aureus (2.5%). Fungal species accounted for 21.0% of the total strains, and the most common strain collected was Candida albicans (12.25%). Imipenem was the most effective antibiotic against Gram-positive and Gram-negative bacteria. The drug resistance rate of Gram-positive bacteria to penicillin G was 100%, and the Gram-positive bacteria were 100% sensitive to teicoplanin, vancomycin, and linezolid.
Gram-negative bacterial infections account for the majority of pulmonary infections in the NICU, with fungal infections being the second most common infection type observed. In addition, fungal infections seem to be related to mortality in the NICU.
nosocomial infection; pulmonary infection; drug resistance; neurological intensive care unit
Schistosomiasis remains a major public health problem in endemic countries and is caused by infections with any one of three primary schistosome species. Although there are no vaccines available to date, this strategy appears feasible since natural immunity develops in individuals suffering from repeated infection during a lifetime. Since vaccinations resulting in both Th1- and Th2-type responses have been shown to contribute to protective immunity, a vaccine formulation with the capacity for stimulating multiple arms of the immune response will likely be the most effective. Previously we developed partially protective, single Th- and B cell-epitope-based peptide-DNA dual vaccines (PDDV) (T3-PDDV and B3-PDDV, respectively) capable of eliciting immune responses against the Schistosoma japonicum 22.6 kDa tegument antigen (Sj22.6) and a 62 kDa fragment of myosin (Sj62), respectively.
In this study, we developed PDDV cocktails containing multiple epitopes of S. japonicum from Sj22.6, Sj62 and Sj97 antigens by predicting cytotoxic, helper, and B-cell epitopes, and evaluated vaccine potential in vivo. Results showed that mice immunized with a single-epitope PDDV elicited either Tc, Th, or B cell responses, respectively, and mice immunized with either the T3- or B3- single-epitope PDDV formulation were partially protected against infection. However, mice immunized with a multicomponent (3 PDDV components) formulation elicited variable immune responses that were less immunoprotective than single-epitope PDDV formulations.
Our data show that combining these different antigens did not result in a more effective vaccine formulation when compared to each component administered individually, and further suggest that immune interference resulting from immunizations with antigenically distinct vaccine targets may be an important consideration in the development of multicomponent vaccine preparations.
Mass transport of analyte to surface-immobilized affinity reagents is the fundamental bottleneck for sensitive detection in solid-support microarrays and biosensors. Analyte depletion in the volume adjacent to the sensor causes deviation from ideal association, significantly slows down reaction kinetics, and causes inhomogeneous binding across the sensor surface. In this paper we use high-resolution molecular interferometric imaging (MI2), a label-free optical interferometry technique for direct detection of molecular films, to study the inhomogeneous distribution of intra-spot binding across 100 micron-diameter protein spots. By measuring intra-spot binding inhomogeneity, reaction kinetics can be determined accurately when combined with a numerical three-dimensional finite element model. To ensure homogeneous binding across a spot, a critical flow rate is identified in terms of the association rate ka and the spot diameter. The binding inhomogeneity across a spot can be used to distinguish high-affinity low-concentration specific reactions from low-affinity high-concentration non-specific binding of background proteins.
In chronic infectious diseases, such as schistosomiasis, pathogen growth and immunopathology are affected by the induction of a proper balanced Th1/Th2 response to the pathogen and by antigen-triggered activation-induced T cell death. Here, by using S. japonicum infection or schistosome antigens-immunized mouse model, or antigens in vitro stimulation, we report that during the early stage of S. japonicum infection, nonegg antigens trigger Th2 cell apoptosis via the granzyme B signal pathway, contributing to Th1 polarization, which is thought to be associated with worm clearance and severe schistosomiasis. Meanwhile, after the adult worms lay their eggs, the egg antigens trigger Th1 cell apoptosis via the caspase pathway, contributing to Th2 polarization, which is associated with mild pathology and enhanced survival of both worms and their hosts. Thus, our study suggests that S. japonicum antigen-induced Th1 and Th2 cell apoptosis involves the Th1/Th2 shift and favorites both hosts and parasites.
The Metabolic Syndrome (MetSyn), which is a clustering of traits including insulin resistance, obesity, hypertension and dyslipidemia, is estimated to have a substantial genetic component, yet few specific genetic targets have been identified. Factor analysis, a sub-type of structural equation modeling (SEM), has been used to model the complex relationships in MetSyn. Therefore, we aimed to define the genetic determinants of MetSyn in the Framingham Heart Study (Offspring Cohort, Exam 7) using the Affymetrix 50 k Human Gene Panel and three different approaches: 1) an association-based "one-SNP-at-a-time" analysis with MetSyn as a binary trait using the World Health Organization criteria; 2) an association-based "one-SNP-at-a-time" analysis with MetSyn as a continuous trait using second-order factor scores derived from four first-order factors; and, 3) a multivariate SEM analysis with MetSyn as a continuous, second-order factor modeled with multiple putative genes, which were represented by latent constructs defined using multiple SNPs in each gene. Results were similar between approaches in that CSMD1 SNPs were associated with MetSyn in Approaches 1 and 2; however, the effects of CSMD1 diminished in Approach 3 when modeled simultaneously with six other genes, most notably CETP and STARD13, which were strongly associated with the Lipids and MetSyn factors, respectively. We conclude that modeling multiple genes as latent constructs on first-order trait factors, most proximal to the gene's function with limited paths directly from genes to the second-order MetSyn factor, using SEM is the most viable approach toward understanding overall gene variation effects in the presence of multiple putative SNPs.
While recently performed genome-wide association studies have advanced the identification of genetic variants predisposing to type 2 diabetes (T2D), the potential application of these novel findings for disease prediction and prevention has not been well studied. Diabetes prediction and prevention have become urgent issues owing to the rapidly increasing prevalence of diabetes and its associated mortality, morbidity, and health care cost. New prediction approaches using genetic markers could facilitate early identification of high risk sub-groups of the population so that appropriate prevention methods could be effectively applied to delay, or even prevent, disease onset.
This paper assessed 18 recently identified T2D loci for their potential role in diabetes prediction. We built a new predictive genetic test for T2D using the Framingham Heart Study dataset. Using logistic regression and 15 additional loci, the new test was slightly improved over the existing test using just three loci. A formal comparison between the two tests suggests no significant improvement. We further formed a predictive genetic test for identifying early onset T2D and found higher classification accuracy for this test, not only indicating that these 18 loci have great potential for predicting early onset T2D, but also suggesting that they may play important roles in causing early-onset T2D.
To further improve the test's accuracy, we applied a newly developed nonparametric method capable of capturing high order interactions to the data, but it did not outperform a logistic regression that only considers single-locus effects. This could be explained by the absence of gene-gene interactions among the 18 loci.
Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a β-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.
Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10–100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery.