Importin Alpha Subtypes Determine Differential Transcription Factor Localization in Embryonic Stem Cells Maintenance

SummaryWe recently demonstrated that the expression of the importin α subtype is switched from α2 to α1 during neural differentiation in mouse embryonic stem cells (ESCs) and that this switching has a major impact on cell differentiation. In this study, we report a cell-fate determination mechanism in which importin α2 negatively regulates the nuclear import of certain transcription factors to maintain ESC properties. The nuclear import of Oct6 and Brn2 was inhibited via the formation of a transport-incompetent complex of the cargo bound to a nuclear localization signal binding site in importin α2. Unless this dominant-negative effect was downregulated upon ESC differentiation, inappropriate cell death was induced. We propose that although certain transcription factors are necessary for differentiation in ESCs, these factors are retained in the cytoplasm by importin α2, thereby preventing transcription factor activity in the nucleus until the cells undergo differentiation

Gene Knock-Outs of Inositol 1,4,5-Trisphosphate Receptors Types 1 and 2 Result in Perturbation of Cardiogenesis

Rs in cardiogenesis remain unclear.Rs mimicked the phenotype of the AV valve defect that result from the inhibition of calcineurin, and it could be rescued by constitutively active calcineurin.Rs in cardiogenesis in part through the regulation of calcineurin-NFAT signaling

Christopher Simpson The Division-Viol, or, The Art of PLAYING Extempore upon a GROUND. EDITIO SECVNDA Dedication & Recommendation

本訳稿はChristopher Simpson (1605頃－1669) 著 The Division-Viol, or, The Art of PLAYING Ex tempore upon a GROUND. DIVIDED INTO THREE PARTS. EDITIO SECVNDA, London, 1665 の著者による献辞および楽譜出版権所有者による推薦文の全訳である

Carotenoid composition and carotenogenic gene expression during Ipomoea petal development

Japanese morning glory (Ipomoea nil) is a representative plant lacking a yellow-flowered cultivar, although a few wild Ipomoea species contain carotenoids in their petals such as Ipomoea sp. (yellow petals) and I. obscura (pale-yellow petals). In the present study, carotenoid composition and the expression patterns of carotenogenic genes during petal development were compared among I. nil, I. obscura, and Ipomoea sp. to identify the factors regulating carotenoid accumulation in Ipomoea plant petals. In the early stage, the carotenoid composition in petals of all the Ipomoea plants tested was the same as in the leaves mainly showing lutein, violaxanthin, and β-carotene (chloroplast-type carotenoids). However, in fully opened flowers, chloroplast-type carotenoids were entirely absent in I. nil, whereas they were present in trace amounts in the free form in I. obscura. At the late stage of petal development in Ipomoea sp., the majority of carotenoids were β-cryptoxanthin, zeaxanthin, and β-carotene (chromoplast-type carotenoids). In addition, most of them were present in the esterified form. Carotenogenic gene expression was notably lower in I. nil than in Ipomoea sp. In particular, β-ring hydroxylase (CHYB) was considerably suppressed in petals of both I. nil and I. obscura. The CHYB expression was found to be significantly high in the petals of Ipomoea sp. during the synthesis of chromoplast-type carotenoids. The expression levels of carotenoid cleavage genes (CCD1 and CCD4) were not correlated with the amount of carotenoids in petals. These results suggest that both I. obscura and I. nil lack the ability to synthesize chromoplast-type carotenoids because of the transcriptional down-regulation of carotenogenic genes. CHYB, an enzyme that catalyses the addition of a hydroxyl residue required for esterification, was found to be a key enzyme for the accumulation of chromoplast-type carotenoids in petals

First molecular identification of Trypanosoma evansi from cattle in Syria

Trypanosoma evansi, the “surra” disease-causing agent, is a blood protozoan parasite that infects a wide range of mammalian species within an unlimited geographical region. It causes anemia, weight loss, and even death of the infected livestock that heavily affect animal husbandry. However, the full epidemiological information of T. evansi is lacking, especially in developing countries, and the risk of the disease is largely underestimated. In this study, 207 samples of blood DNA collected from Holstein Friesian crossbred cattle in the central region of Syria in May 2010 were screened for T. evansi, aiming to determine the prevalence of the parasite. T. evansi was screened by PCR targeting the internal transcribed spacer (ITS) 1 region, and 27 samples were found positive out of 207 (13%), which is relatively high considering that no clinical symptoms were observed. The ITS1 amplicons were later subjected to RoTat1.2-PCR for detection of T. evansi type A. This is the first report of molecular detection of T. evansi in Syria. Our study suggests that advanced investigations in cattle and other domestic animals are necessary in Syria

Development and validation of direct dry loop mediated isothermal amplification for diagnosis of Trypanosoma evansi

Non-tsetse transmitted Trypanosoma evansi infection (Surra) is one of the most important diseases of camels in north and east Africa and of buffalo and cattle in Asia. Early, accurate and feasible diagnosis is a crucial step towards the control of Surra. Dry format of loop-mediated isothermal amplification (LAMP) diagnostics for the detection of T. evansi was developed, where the detection limit was determined as to equivalent to one parasite per reaction. The assay was validated by testing blood from 48 camels clinically diagnosed to have Surra, which all tested negative microscopically and revealed 43 (89.6%) to be positive for T. evansi when tested by the dry-LAMP. Furthermore, DNA extracted from a randomly selected subset of 20 of these blood samples were then subjected to RoTat1.2-PCR (TaKara Ex Taq), with 14 matching results, with six that were positive by dry-LAMP and negative by PCR. The kappa value of dry-LAMP applied to direct blood was 0.4211, indicating moderate agreement to RoTat 1.2-PCR. In addition, 103 genomic DNA extracted from camels' blood were tested by both dry-LAMP and RoTat1.2-PCR revealed 67 matching results and 31 positive by dry-LAMP and negative by PCR and a further five positives by PCR and negative by dry-LAMP. This novel dry-LAMP method is more sensitive than conventional PCR, direct (without DNA extraction step), is user friendly and does not require cold chain or highly trained personnel

Whole-genome assembly of Babesia ovata and comparative genomics between closely related pathogens

Background: Babesia ovata, belonging to the phylum Apicomplexa, is an infectious parasite of bovids. It is not associated with the manifestation of severe symptoms, in contrast to other types of bovine babesiosis caused by B. bovis and B. bigemina; however, upon co-infection with Theileria orientalis, it occasionally induces exacerbated symptoms. Asymptomatic chronic infection in bovines is usually observed only for B. ovata. Comparative genomic analysis could potentially reveal factors involved in these distinguishing characteristics; however, the genomic and molecular basis of these phenotypes remains elusive, especially in B. ovata. From a technical perspective, the current development of a very long read sequencer, MinION, will facilitate the obtainment of highly integrated genome sequences. Therefore, we applied next-generation sequencing to acquire a high-quality genome of the parasite, which provides fundamental information for understanding apicomplexans.　Results: The genome was assembled into 14,453,397 bp in size with 5031 protein-coding sequences (91 contigs and N50 = 2,090,503 bp). Gene family analysis revealed that ves1 alpha and beta, which belong to multigene families in B. bovis, were absent from B. ovata, the same as in B. bigemina. Instead, ves1a and ves1b, which were originally specified in B. bigemina, were present. The B. ovata and B. bigemina ves1a configure one cluster together even though they divided into two sub-clusters according to the spp. In contrast, the ves1b cluster was more dispersed and the overlap among B. ovata and B. bigemina was limited. The observed redundancy and rapid evolution in sequence might reflect the adaptive history of these parasites. Moreover, same candidate genes which potentially involved in the distinct phenotypes were specified by functional analysis. An anamorsin homolog is one of them. The human anamorsin is involved in hematopoiesis and the homolog was present in B. ovata but absent in B. bigemina which causes severe anemia.　Conclusions: Taking these findings together, the differences demonstrated by comparative genomics potentially explain the evolutionary history of these parasites and the differences in their phenotypes. Besides, the draft genome provides fundamental information for further characterization and understanding of these parasites

A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of internal transcribed spacer 1 amplicons

To improve our knowledge on the epidemiological status of African trypanosomiasis, better tools are required to monitor Trypanosome genotypes circulating in both mammalian hosts and tsetse fly vectors. This is important in determining the diversity of Trypanosomes and understanding how environmental factors and control efforts affect Trypanosome evolution. We present a single test approach for molecular detection of different Trypanosome species and subspecies using newly designed primers to amplify the Internal Transcribed Spacer 1 region of ribosomal RNA genes, coupled to Illumina sequencing of the amplicons. The protocol is based on Illumina's widely used 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. Results from analysis of wild tsetse flies collected from Zambia and Zimbabwe show that conventional methods for Trypanosome species detection based on band size comparisons on gels is not always able to accurately distinguish between T. vivax and T. godfreyi. Additionally, this approach shows increased sensitivity in the detection of Trypanosomes at species level with the exception of the Trypanozoon subgenus. We identified subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi without the need for additional tests. Results show T. congolense Kilifi subspecies is more closely related to T. simiae than to other T. congolense subspecies. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any subspecies for T. godfreyi, we observed two distinct clusters for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from other T. simiae Tsavo sequences suggesting the Nannomonas group is more divergent than currently thought thus the need for better classification criteria. This method presents a simple but comprehensive way of identification of Trypanosome species and subspecies-specific using one PCR assay for molecular epidemiology of trypanosomes. Author summary Tsetse flies are central actors in the transmission of Trypanosomes to vertebrate hosts. Therefore, detection of Trypanosomes in the tsetse flies is important for understanding the epidemiology of African trypanosomiasis as a component of new control or surveillance strategies. We have developed a method that combines multiplex PCR and next-generation sequencing for the detection of different Trypanosome species and subspecies. Similar to the widely used bacterial metagenomic analysis protocol, this method uses a modular, two-step PCR process followed by sequencing of all amplicons in a single run, making sequencing of amplicons more efficient and cost-effective when dealing with large sample sizes. As part of this approach, we designed novel Internal Transcribed Spacer 1 primers optimized for short read sequencing and have slightly better sensitivity than conventional primers. Taxonomic identification of amplicons is based on BLAST searches against the constantly updated NCBI's nt database. Our approach is more accurate than traditional gel-based analyses which are prone to misidentification of species. It is also able to discriminate between subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi species. This method has the potential to provide new insights into the epidemiology of different Trypanosome genotypes and the discovery of new ones

Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies

Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied