High Diversity of Cryptosporidium Subgenotypes Identified in Malaysian HIV/AIDS Individuals Targeting gp60 Gene

BACKGROUND: Currently, there is a lack of vital information in the genetic makeup of Cryptosporidium especially in developing countries. The present study aimed at determining the genotypes and subgenotypes of Cryptosporidium in hospitalized Malaysian human immunodeficiency virus (HIV) positive patients. METHODOLOGY/PRINCIPAL FINDINGS: In this study, 346 faecal samples collected from Malaysian HIV positive patients were genetically analysed via PCR targeting the 60 kDa glycoprotein (gp60) gene. Eighteen (5.2% of 346) isolates were determined as Cryptosporidium positive with 72.2% (of 18) identified as Cryptosporidium parvum whilst 27.7% as Cryptosporidium hominis. Further gp60 analysis revealed C. parvum belonging to subgenotypes IIaA13G1R1 (2 isolates), IIaA13G2R1 (2 isolates), IIaA14G2R1 (3 isolates), IIaA15G2R1 (5 isolates) and IIdA15G1R1 (1 isolate). C. hominis was represented by subgenotypes IaA14R1 (2 isolates), IaA18R1 (1 isolate) and IbA10G2R2 (2 isolates). CONCLUSIONS/SIGNIFICANCE: These findings highlighted the presence of high diversity of Cryptosporidium subgenotypes among Malaysian HIV infected individuals. The predominance of the C. parvum subgenotypes signified the possibility of zoonotic as well as anthroponotic transmissions of cryptosporidiosis in HIV infected individuals

Endogenous mouse Dicer is an exclusively cytoplasmic protein

Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion

Sequencing of a Patient with Balanced Chromosome Abnormalities and Neurodevelopmental Disease Identifies Disruption of Multiple High Risk Loci by Structural Variation

Balanced chromosome abnormalities (BCAs) occur at a high frequency in healthy and diseased individuals, but cost-efficient strategies to identify BCAs and evaluate whether they contribute to a phenotype have not yet become widespread. Here we apply genome-wide mate-pair library sequencing to characterize structural variation in a patient with unclear neurodevelopmental disease (NDD) and complex de novo BCAs at the karyotype level. Nucleotide-level characterization of the clinically described BCA breakpoints revealed disruption of at least three NDD candidate genes (LINC00299, NUP205, PSMD14) that gave rise to abnormal mRNAs and could be assumed as disease-causing. However, unbiased genome-wide analysis of the sequencing data for cryptic structural variation was key to reveal an additional submicroscopic inversion that truncates the schizophrenia- and bipolar disorder-associated brain transcription factor ZNF804A as an equally likely NDD-driving gene. Deep sequencing of fluorescent-sorted wild-type and derivative chromosomes confirmed the clinically undetected BCA. Moreover, deep sequencing further validated a high accuracy of mate-pair library sequencing to detect structural variants larger than 10 kB, proposing that this approach is powerful for clinical-grade genome-wide structural variant detection. Our study supports previous evidence for a role of ZNF804A in NDD and highlights the need for a more comprehensive assessment of structural variation in karyotypically abnormal individuals and patients with neurocognitive disease to avoid diagnostic deception

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT