Understanding the molecular basis of Chinese noodle quality

Tese de doutoramento em Ciências da Saúde (Pré-Bolonha), Ramo de Ciências Biomédicas, apresentada à Faculdade de Medicina da Universidade de CoimbraA terapia fotodinâmica (PDT, do inglês, photodynamic therapy) é uma terapêutica que depende da administração de um fotossensibilizador que, por si só, não tem efeito farmacológico, e da sua posterior ativação por irradiação, com luz visível de comprimento de onda adequado do local a tratar. Como consequência da fotoativação do fotossensibilizador, ocorre produção de espécies reativas de oxigénio (ROS, do inglês, reactive oxygen species) que determinam a morte celular. Pelo seu modo de ação, a PDT está associada a poucos efeitos secundários. Esta reduzida manifestação de efeitos secundários, deve-se não só ao facto do efeito citotóxico ocorrer apenas no local de ativação pela luz, mas também devido ao local de acumulação ser principalmente o tumor, dado que os fotossensibilizadores tipicamente se acumulam nas células tumorais, pelo que a PDT apresenta elevada seletividade. No contexto clínico, a PDT é utilizada em oftalmologia, em dermatologia e também em oncologia. No entanto, a sua utilização no tratamento de cancro mantem-se limitada, possivelmente, devido ao baixo número de fármacos aprovados e também ao baixo número de aprovações para terapêutica oncológica. Este trabalho teve como objetivo principal desenvolver novos fotossensibilizadores para PDT. Pretendeu-se desenvolver um fotossensibilizador com elevado potencial terapêutico, caracteriza-lo do ponto de vista fotoquímico, verificar o seu efeito fotodinâmico em várias linhas celulares tumorais, contribuir para esclarecer as suas vias moleculares de atuação e estudar o seu potencial terapêutico in vivo e biodistribuição. Paralelamente, pretendeu-se desenvolver outro fotossensibilizador passível de ser complexado com tecnécio-99 metastável (99mTc), otimizar um procedimento de marcação simples e reprodutível e estudar a biodistribuição do complexo. Esta vertente, de associação da PDT à medicina nuclear, pode proporcionar a maximização do efeito terapêutico pela identificação do momento de captação máxima pelo tumor.Para a concretização destes objetivos utilizaram-se diversas metodologias desde a síntese química, estudos in vitro, em que se recorreu a diversas metodologias das quais são exemplo, a microscopia confocal, a citometria de fluxo, o western blot e marcação radioativa e a microcromatografia ascendente em camada fina, até aos estudos in vivo. Foi possível sintetizar o fotossensibilizador BBr2HPC, com caraterísticas fotoquímicas adequadas à utilização em terapêutica, nomeadamente, uma banda com elevada absorção a 645 nm. O valor de IC50 deste fotossensibilizador nas linhas celulares humanas de carcinoma colorretal, carcinoma do esófago e melanoma melanocítico foi na ordem de nanomolar, o que aponta para um efeito terapêutico promissor. Verificou-se que o fotossensibilizador BBr2HPC é captado pelas células de carcinoma colorretal e de carcinoma do esófago e que se acumula nas mitocôndrias, nos lisossomas e no retículo endoplasmático, mas não penetra no núcleo celular. Após a fotossensibilização verificou-se que o tipo de morte celular ativado, apoptose ou necrose, é influenciado pela concentração do fotossensibilizador e tipo de célula tumoral. Várias ROS estão implicadas no efeito fotodinâmico do fotossensibilizador BBr2HPC e os mecanismos citoprotetores não parecem ser suficientes para inibir os danos celulares. Os estudos in vivo comprovaram o efeito terapêutico significativo do tratamento fotodinâmico, baseado no fotossensibilizador BBr2HPC, particularmente quando administrada uma concentração de 2 mg/kg e irradiação após 24 horas, em que se observaram casos de resposta patológica completa. A eliminação deste fotossensibilizador é hepatobiliar e urinária e o tempo de eliminação aponta para um perfil de segurança favorável numa fase pré-clínica. Na perspetiva do desenvolvimento de um fotossensibilizador com propriedades adequadas à aplicação diagnóstica, sintetizou-se e caracterizou-se o 2CPP. Este fotossensibilizador foi caracterizado e complexado com 99mTc, após o desenvolvimento de uma metodologia de marcação radioquímica simples e reprodutível que permitiu obter uma formulação com uma eficiência de marcação superior a 92%, estável no tempo in vitro e em soro sanguíneo humano. Este fotossensibilizador, também apresenta um perfil de eliminação hepatobiliar e urinário e apresenta potencial para estudos de imagem. Com este trabalho, foi possível concretizar o objetivo de desenvolver um fotossensibilizador com intuito terapêutico, caracterizar as suas vias subcelulares de atuação e verificar o seu desempenho pré-clínico que se revelou extremamente promissor. Paralelamente, a utilização de fotossensibilizadores complexados com radioisótopos constituiu uma abordagem de medicina personalizada em PDT, através da medicina nuclear, com potencialidade teranóstica e de seguimento

Hepatitis C Seroprevalence and Associated Risk Factors, Anyang, China

Hepatitis C virus screening was conducted among 8,226 residents 25–65 years of age in 4 counties of China; virus prevalence was 0.9%. A subsequent case–control study indicated blood transfusion (odds ratio [OR] 4.55), esophageal balloon examination (OR 3.78), and intravenous injection (OR 5.83) were associated with infection

QTL Mapping for Grain Zinc and Iron Concentrations in Bread Wheat

Deficiency of micronutrient elements, such as zinc (Zn) and iron (Fe), is called “hidden hunger,” and bio-fortification is the most effective way to overcome the problem. In this study, a high-density Affymetrix 50K single-nucleotide polymorphism (SNP) array was used to map quantitative trait loci (QTL) for grain Zn (GZn) and grain Fe (GFe) concentrations in 254 recombinant inbred lines (RILs) from a cross Jingdong 8/Bainong AK58 in nine environments. There was a wide range of variation in GZn and GFe concentrations among the RILs, with the largest effect contributed by the line × environment interaction, followed by line and environmental effects. The broad sense heritabilities of GZn and GFe were 0.36 ± 0.03 and 0.39 ± 0.03, respectively. Seven QTL for GZn on chromosomes 1DS, 2AS, 3BS, 4DS, 6AS, 6DL, and 7BL accounted for 2.2–25.1% of the phenotypic variances, and four QTL for GFe on chromosomes 3BL, 4DS, 6AS, and 7BL explained 2.3–30.4% of the phenotypic variances. QTL on chromosomes 4DS, 6AS, and 7BL might have pleiotropic effects on both GZn and GFe that were validated on a germplasm panel. Closely linked SNP markers were converted to high-throughput KASP markers, providing valuable tools for selection of improved Zn and Fe bio-fortification in breeding

Genetic analysis of phytoene synthase 1 (Psy1) gene function and regulation in common wheat

Transcriptome details for three transgenic lines with the most significantly reduced YPC and non-transformed controls. (DOCX 18 kb

The Anyang Esophageal Cancer Cohort Study: Study Design, Implementation of Fieldwork, and Use of Computer-Aided Survey System

Background: Human papillomavirus (HPV) has been observed repeatedly in esophageal squamous cell carcinoma (ESCC) tissues. However, the causal relationship between HPV infection and the onset of ESCC remains unknown. A large cohort study focusing on this topic is being carried out in rural Anyang, China. Methodology/Principal Findings: The Anyang Esophageal Cancer Cohort Study (AECCS) is a population-based prospective endoscopic cohort study designed to investigate the association of HPV infection and ESCC. This paper provides information regarding the design and implementation of this study. In particular we describe the recruitment strategies and quality control procedures which have been put into place, and the custom designed computer-aided survey system (CASS) used for this project. This system integrates barcode technology and unique identification numbers, and has been developed to facilitate real-time data management throughout the workflow using a wireless local area network. A total of 8,112 (75.3%) of invited subjects participated in the baseline endoscopic examination; of those invited two years later to take part in the first cycle of follow-up, 91.9 % have complied. Conclusions/Significance: The AECCS study has high potential for evaluating the causal relationship between HPV infection and the occurrence of ESCC. The experience in setting up the AECCS may be beneficial for others planning to initiate simila

Allelic effects and variations for key bread-making quality genes in bread wheat using high-throughput molecular markers

We developed and validated high-throughput Kompetitive Allele-Specific PCR (KASP) assays for key genes underpinning bread-making quality, including the wbm gene on chromosome 7AL and over-expressed glutenin Bx7 (Glu-B1al) gene. Additionally, we used pre-existing KASP assay for Sec1 (1B.1R translocation) gene on chromosome 1B. The newly developed KASP assays were compared with gel-based markers for reliability and phenotypically validated in a diversity panel for Mixograph, Rapid Visco Analyzer (RVA) and Mixolab traits. Genotypes carrying the 1B.1R translocation had significantly lower Mixolab parameters than those without the translocation. Similarly, superior effects of the wbm+ and Bx7 alleles on Mixograph and RVA properties and their extremely low frequencies in global wheat collections supported the idea of using these genes for bread-making quality improvement. The allele frequencies of wbm+ and Bx7 were extremely low in historical Chinese and CIMMYT wheat germplasm, but were relatively higher in synthetic hexaploid wheats and their breeding derivatives. In both the Vavilov and Watkins global landrace collections, the frequency of wbm+ was 6.4 and 3.5%, and frequency of Bx7 was 3.2% and 7.0%, respectively. The high-throughput marker resources and large-scale global germplasm screening provided further opportunities to exploit these genes in wheat breeding to enhance bread-making quality

An evaluation of EQ-5D-3L health utility scores using five country-specific tariffs in a rural population aged 45-69 years in Hua county, Henan province, China.

BACKGROUND: This study aims to compare the performance of the recently developed Chinese (city) tariff of the EQ-5D-3L against the UK, US, Japanese and Korean tariffs in a general rural population in China. METHODS: From November 2015 to September 2016, 12,085 permanent residents aged 45-69 from 257 villages randomly selected from Hua County, Henan Province, China, were interviewed using EQ-5D-3L, and a one-on-one questionnaire investigation was used to collect data on factors associated with HRQOL. The health utility scores were calculated using the UK, US, Japanese, Korean and Chinese (city) tariffs. The agreement, known-groups validity and sensitivity of these five tariffs were evaluated. Transition scores for pairs of observed EQ-5D-3L health states were calculated and compared. RESULTS: The Korean tariff yielded the highest mean health utility score (0.963), followed by the Chinese (city) (0.948), US (0.943), UK (0.930) and Japanese (0.921) tariffs, but the differences in the scores of any two tariffs did not exceed the MCID. The Chinese (city) tariff showed higher ICC values (ICCs> 0.89, 95% CI:0.755-0.964) and narrower limits of agreement (0.099-0.167) than the Korean tariff [(ICCs> 0.71, 95% CI:0.451-0.955); (0.146-0.253)]. The Chinese (city) tariff had a higher relative efficiency and effect size statistics in 10 out of 11 variables as compared to the UK, US and Japanese tariffs. The Chinese (city) tariff (0.215) was associated with moderate mean absolute transition scores compared with the UK (0.342), US (0.230), Japanese (0.149) and Korean (0.189) tariffs for 1485 observed pairs of the EQ-5D-3L health states. CONCLUSIONS: Health utility scores derived from the five tariffs differed. The Chinese (city) tariff was the most suitable of these tariffs and was without obvious weakness. We recommend adopting the Chinese (city) tariff when applying EQ-5D-3L to assess quality of life among the elderly in China's agricultural region with socio-economic status similar to Hua County. Results of this study had provided a crucial basis for health surveys, health promotion projects, health intervention trials, and health economic evaluation taking HRQOL as a target in rural areas of China

Genetic diversity and selection signatures in synthetic-derived wheats and modern spring wheat

Synthetic hexaploid wheats and their derived advanced lines were subject to empirical selection in developing genetically superior cultivars. To investigate genetic diversity, patterns of nucleotide diversity, population structure, and selection signatures during wheat breeding, we tested 422 wheat accessions, including 145 synthetic-derived wheats, 128 spring wheat cultivars, and 149 advanced breeding lines from Pakistan. A total of 18,589 high-quality GBS-SNPs were identified that were distributed across the A (40%), B (49%), and D (11%) genomes. Values of population diversity parameters were estimated across chromosomes and genomes. Genome-wide average values of genetic diversity and polymorphic information content were estimated to be 0.30 and 0.25, respectively. Neighbor-joining (NJ) tree, principal component analysis (PCA), and kinship analyses revealed that synthetic-derived wheats and advanced breeding lines were genetically diverse. The 422 accessions were not separated into distinct groups by NJ analysis and confirmed using the PCA. This conclusion was validated with both relative kinship and Rogers' genetic distance analyses. EigenGWAS analysis revealed that 32 unique genome regions had undergone selection. We found that 50% of the selected regions were located in the B-genome, 29% in the D-genome, and 21% in the A-genome. Previously known functional genes or QTL were found within the selection regions associated with phenology-related traits such as vernalization, adaptability, disease resistance, and yield-related traits. The selection signatures identified in the present investigation will be useful for understanding the targets of modern wheat breeding in Pakistan