Analysis of FGF-Dependent and FGF-Independent Pathways in Otic Placode Induction

The inner ear develops from a patch of thickened cranial ectoderm adjacent to the hindbrain called the otic placode. Studies in a number of vertebrate species suggest that the initial steps in induction of the otic placode are regulated by members of the Fibroblast Growth Factor (FGF) family, and that inhibition of FGF signaling can prevent otic placode formation. To better understand the genetic pathways activated by FGF signaling during otic placode induction, we performed microarray experiments to estimate the proportion of chicken otic placode genes that can be up-regulated by the FGF pathway in a simple culture model of otic placode induction. Surprisingly, we find that FGF is only sufficient to induce about 15% of chick otic placode-specific genes in our experimental system. However, pharmacological blockade of the FGF pathway in cultured chick embryos showed that although FGF signaling was not sufficient to induce the majority of otic placode-specific genes, it was still necessary for their expression in vivo. These inhibitor experiments further suggest that the early steps in otic placode induction regulated by FGF signaling occur through the MAP kinase pathway. Although our work suggests that FGF signaling is necessary for otic placode induction, it demonstrates that other unidentified signaling pathways are required to co-operate with FGF signaling to induce the full otic placode program.     

The Evidence for Association of ATP2B2 Polymorphisms with Autism in Chinese Han Population

Background

Autism is a neurodevelopmental disorder with a high estimated heritability. ATP2B2, located on human chromosome 3p25.3, encodes the plasma membrane calcium-transporting ATPase 2 which extrudes Ca²⁺ from cytosol into extracellular space. Recent studies reported association between ATP2B2 and autism in samples from Autism Genetic Resource Exchange (AGRE) and Italy. In this study, we investigated whether ATP2B2 polymorphisms were associated with autism in Chinese Han population.

Methods

We performed a family based association study between five SNPs (rs35678 in exon, rs241509, rs3774180, rs3774179, and rs2278556 in introns) in ATP2B2 and autism in 427 autism trios of Han Chinese descent. All SNPs were genotyped using the Sequenom genotyping platform. The family-based association test (FBAT) program was used to perform association test for SNPs and haplotype analyses.

Results

This study demonstrated a preferential transmission of T allele of rs3774179 to affected offsprings under an additive model (T>C, Z = 2.482, p = 0.013). While C allele of rs3774179 showed an undertransmission from parents to affected children under an additive and a dominant model, respectively (Z = −2.482, p = 0.013; Z = −2.591, p = 0.0096). Haplotype analyses revealed that three haplotypes were significantly associated with autism. The haplotype C-C (rs3774180–rs3774179) showed a significant undertransmission from parents to affected offsprings both in specific and global haplotype FBAT (Z = −2.037, p = 0.042; Global p = 0.03). As for the haplotype constructed by rs3774179 and rs2278556, C-A might be a protective haplotype (Z = −2.206, p = 0.027; Global p = 0.04), while T-A demonstrated an excess transmission from parents to affected offsprings (Z = 2.143, p = 0.032). These results were still significant after using the permutation method to obtain empirical p values.

Conclusions

Our research suggested that ATP2B2 might play a role in the etiology of autism in Chinese Han population.     

Mid-Neolithic Exploitation of Mollusks in the Guanzhong Basin of Northwestern China: Preliminary Results

Mollusk remains are abundant in archaeological sites in the Guanzhong Basin of Northwestern China, providing good opportunities for investigations into the use of mollusks by prehistoric humans. Here we report on freshwater gastropod and bivalve mollusks covering the time interval from about 5600 to 4500 cal. yrs BP from sites of Mid-Late Neolithic age. They are identified as Cipangopaludina chinensis and Unio douglasiae, both of which are currently food for humans. The shells are well preserved and have no signs of abrasion. They are all freshwater gastropods and bivalves found in pits without water-reworked deposits and have modern representatives which can be observed in rivers, reservoirs, and paddy fields in the studied region. Mollusk shells were frequently recovered in association with mammal bones, lithic artifacts, and pottery. These lines of evidence indicate that the mollusks are the remains of prehistoric meals. The mollusk shells were likely discarded into the pits by prehistoric humans after the flesh was eaten. However, these mollusk remains may not have been staple food since they are not found in large quantities. Mollusk shell tools and ornaments are also observed. Shell tools include shell knives, shell reaphooks and arrowheads, whereas shell ornaments are composed of pendants and loops. All the shell tools and ornaments are made of bivalve mollusks and do not occur in large numbers. The finding of these freshwater mollusk remains supports the view that the middle Holocene climate in the Guanzhong Basin may have been warm and moist, which was probably favorable to freshwater mollusks growing and developing in the region.     

Silencing Mutant Ataxin-3 Rescues Motor Deficits and Neuropathology in Machado-Joseph Disease Transgenic Mice

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.     

Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer

Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

Evaluating GWAS-Identified SNPs for Age at Natural Menopause among Chinese Women

Background

Age at natural menopause (ANM) is a complex trait with high heritability and is associated with several major hormonal-related diseases. Recently, several genome-wide association studies (GWAS), conducted exclusively among women of European ancestry, have discovered dozens of genetic loci influencing ANM. No study has been conducted to evaluate whether these findings can be generalized to Chinese women.

Methodology/Principal Findings

We evaluated the index single nucleotide polymorphisms (SNPs) in 19 GWAS-identified genetic susceptibility loci for ANM among 3,533 Chinese women who had natural menopause. We also investigated 3 additional SNPs which were in LD with the index SNP in European-ancestry but not in Asian-ancestry populations. Two genetic risk scores (GRS) were calculated to summarize SNPs across multiple loci one for all SNPs tested (GRSall), and one for SNPs which showed association in our study (GRSsel). All 22 SNPs showed the same association direction as previously reported. Eight SNPs were nominally statistically significant with P≤0.05: rs4246511 (RHBDL2), rs12461110 (NLRP11), rs2307449 (POLG), rs12611091 (BRSK1), rs1172822 (BRSK1), rs365132 (UIMC1), rs2720044 (ASH2L), and rs7246479 (TMEM150B). Especially, SNPs rs4246511, rs365132, rs1172822, and rs7246479 remained significant even after Bonferroni correction. Significant associations were observed for GRS. Women in the highest quartile began menopause 0.7 years (P = 3.24×10⁻⁹) and 0.9 years (P = 4.61×10⁻¹¹) later than those in the lowest quartile for GRSsel and GRSall, respectively.

Conclusions

Among the 22 investigated SNPs, eight showed associations with ANM (P<0.05) in our Chinese population. Results from this study extend some recent GWAS findings to the Asian-ancestry population and may guide future efforts to identify genetic determination of menopause.     

Massive Sorghum Collection Genotyped with SSR Markers to Enhance Use of Global Genetic Resources

Large ex situ collections require approaches for sampling manageable amounts of germplasm for in-depth characterization and use. We present here a large diversity survey in sorghum with 3367 accessions and 41 reference nuclear SSR markers. Of 19 alleles on average per locus, the largest numbers of alleles were concentrated in central and eastern Africa. Cultivated sorghum appeared structured according to geographic regions and race within region. A total of 13 groups of variable size were distinguished. The peripheral groups in western Africa, southern Africa and eastern Asia were the most homogeneous and clearly differentiated. Except for Kafir, there was little correspondence between races and marker-based groups. Bicolor, Caudatum, Durra and Guinea types were each dispersed in three groups or more. Races should therefore better be referred to as morphotypes. Wild and weedy accessions were very diverse and scattered among cultivated samples, reinforcing the idea that large gene-flow exists between the different compartments. Our study provides an entry to global sorghum germplasm collections. Our reference marker kit can serve to aggregate additional studies and enhance international collaboration. We propose a core reference set in order to facilitate integrated phenotyping experiments towards refined functional understanding of sorghum diversity.