Novel subtractive transcription-based amplification of mRNA (STAR) method and its application in search of rare and differentially expressed genes in AD brains

BACKGROUND: Alzheimer's disease (AD) is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels. Complementary to this approach, we described here a novel technology designed specifically to isolate rare and novel genes previously undetectable by other methods. We have used this method to identify differentially expressed genes in brains affected by AD. Our method, termed Subtractive Transcription-based Amplification of mRNA (STAR), is a combination of subtractive RNA/DNA hybridization and RNA amplification, which allows the removal of non-differentially expressed transcripts and the linear amplification of the differentially expressed genes. RESULTS: Using the STAR technology we have identified over 800 differentially expressed sequences in AD brains, both up- and down- regulated, compared to age-matched controls. Over 55% of the sequences represent genes of unknown function and roughly half of them were novel and rare discoveries in the human brain. The expression changes of nearly 80 unique genes were further confirmed by qRT-PCR and the association of additional genes with AD and/or neurodegeneration was established using an in-house literature mining tool (LitMiner). CONCLUSION: The STAR process significantly amplifies unique and rare sequences relative to abundant housekeeping genes and, as a consequence, identifies genes not previously linked to AD. This method also offers new opportunities to study the subtle changes in gene expression that potentially contribute to the development and/or progression of AD

Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification

CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5′ ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism – single crossing CPA – and provide details on alternative mechanisms

Epidemiology of Untreated Psychoses in 3 Diverse Settings in the Global South: The International Research Program on Psychotic Disorders in Diverse Settings (INTREPID II).

IMPORTANCE: Less than 10% of research on psychotic disorders has been conducted in settings in the Global South, which refers broadly to the regions of Latin America, Asia, Africa, and Oceania. There is a lack of basic epidemiological data on the distribution of and risks for psychoses that can inform the development of services in many parts of the world. OBJECTIVE: To compare demographic and clinical profiles of cohorts of cases and rates of untreated psychoses (proxy for incidence) across and within 3 economically and socially diverse settings in the Global South. Two hypotheses were tested: (1) demographic and clinical profiles of cases with an untreated psychotic disorder vary across setting and (2) rates of untreated psychotic disorders vary across and within setting by clinical and demographic group. DESIGN, SETTING, AND PARTICIPANTS: The International Research Program on Psychotic Disorders in Diverse Settings (INTREPID II) comprises incidence, case-control, and cohort studies of untreated psychoses in catchment areas in 3 countries in the Global South: Kancheepuram District, India; Ibadan, Nigeria; and northern Trinidad. Participants were individuals with an untreated psychotic disorder. This incidence study was conducted from May 1, 2018, to July 31, 2020. In each setting, comprehensive systems were implemented to identify and assess all individuals with an untreated psychosis during a 2-year period. Data were analyzed from January 1 to May 1, 2022. MAIN OUTCOMES AND MEASURES: The presence of an untreated psychotic disorder, assessed using the Schedules for Clinical Assessment in Neuropsychiatry, which incorporate the Present State Examination. RESULTS: Identified were a total of 1038 cases, including 64 through leakage studies (Kancheepuram: 268; median [IQR] age, 42 [33-50] years; 154 women [57.5%]; 114 men [42.5%]; Ibadan: 196; median [IQR] age, 34 [26-41] years; 93 women [47.4%]; 103 men [52.6%]; Trinidad: 574; median [IQR] age, 30 [23-40] years; 235 women [40.9%]; 339 men [59.1%]). Marked variations were found across and within settings in the sex, age, and clinical profiles of cases (eg, lower percentage of men, older age at onset, longer duration of psychosis, and lower percentage of affective psychosis in Kancheepuram compared with Ibadan and Trinidad) and in rates of untreated psychosis. Age- and sex-standardized rates of untreated psychoses were approximately 3 times higher in Trinidad (59.1/100 000 person-years; 95% CI, 54.2-64.0) compared with Kancheepuram (20.7/100 000 person-years; 95% CI, 18.2-23.2) and Ibadan (14.4/100 000 person-years; 95% CI, 12.3-16.5). In Trinidad, rates were approximately 2 times higher in the African Trinidadian population (85.4/100 000 person-years; 95% CI, 76.0-94.9) compared with the Indian Trinidadian (43.9/100 000 person-years; 95% CI, 35.7-52.2) and mixed populations (50.7/100 000 person-years; 95% CI, 42.0-59.5). CONCLUSIONS AND RELEVANCE: This analysis adds to research that suggests that core aspects of psychosis vary by historic, economic, and social context, with far-reaching implications for understanding and treatment of psychoses globally

Highly Parallel Genome-Wide Expression Analysis of Single Mammalian Cells

We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R(2)~0.76-0.80 between individual cells and R(2)~0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R(2)~0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input.In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology

Within-individual phenotypic plasticity in flowers fosters pollination niche shift

Authors thank Raquel Sánchez, Angel Caravante, Isabel Sánchez Almazo, Tatiana López Pérez, Samuel Cantarero, María José Jorquera and Germán Fernández for helping us during several phases of the study and Iván Rodríguez Arós for drawing the insect silhouettes. This research is supported by grants from the Spanish Ministry of Science, Innovation and Universities (CGL2015-71634-P, CGL2015-63827-P, CGL2017-86626-C2-1-P, CGL2017- 86626-C2-2-P, UNGR15-CE-3315, including EU FEDER funds), Junta de Andalucía (P18- FR-3641), Xunta de Galicia (CITACA), BBVA Foundation (PR17_ECO_0021), and a contract grant to C.A. from the former Spanish Ministry of Economy and Competitiveness (RYC-2012-12277). This is a contribution to the Research Unit Modeling Nature, funded by the Consejería de Economía, Conocimiento, Empresas y Universidad, and European Regional Development Fund (ERDF), reference SOMM17/6109/UGR.Phenotypic plasticity, the ability of a genotype of producing different phenotypes when exposed to different environments, may impact ecological interactions. We study here how within-individual plasticity in Moricandia arvensis flowers modifies its pollination niche. During spring, this plant produces large, cross-shaped, UV-reflecting lilac flowers attracting mostly long-tongued large bees. However, unlike most co-occurring species, M. arvensis keeps flowering during the hot, dry summer due to its plasticity in key vegetative traits. Changes in temperature and photoperiod in summer trigger changes in gene expression and the production of small, rounded, UV-absorbing white flowers that attract a different assemblage of generalist pollinators. This shift in pollination niche potentially allows successful reproduction in harsh conditions, facilitating M. arvensis to face anthropogenic perturbations and climate change. Floral phenotypes impact interactions between plants and pollinators. Here, the authors show that Moricandia arvensis displays discrete seasonal plasticity in floral phenotype, with large, lilac flowers attracting long-tongued bees in spring and small, rounded, white flowers attracting generalist pollinators in summer.Spanish Ministry of Science, Innovation and Universities (EU FEDER funds) CGL2015-71634-P CGL2015-63827-P CGL2017-86626-C2-1-P CGL2017-86626-C2-2-P UNGR15-CE-3315Junta de Andalucia P18-FR-3641Xunta de GaliciaBBVA Foundation PR17_ECO_0021Spanish Ministry of Economy and Competitiveness RYC-2012-12277Consejeria de Economia, Conocimiento, Empresas y Universidad SOMM17/6109/UGREuropean Union (EU) SOMM17/6109/UG