Sandhya Kulkarni

Membrane array–based differential profiling of platelets during storage for 52 miRNAs associated with apoptosis

BACKGROUND: Enucleated platelets (PLTs) utilize posttranscriptional gene (mRNA) regulation (PTGR) for their normal morphologic and physiologic functions, which are altered in their ex vivo storage, also collectively referred to as storage lesions. While cellular micro‐RNAs (miRNAs) play a significant role in posttranscriptional gene (mRNA) regulation by binding to their target mRNAs, comprehensive analysis of apoptosis‐associated miRNAs and global changes in their profiles during PLT storage have not been evaluated to date.

Rubella virus P90 associates with the cytokinesis regulatory protein Citron-K kinase and the viral infection and constitutive expression of P90 protein both induce cell cycle arrest following S phase in cell culture

Summary.In utero infection of developing fetus by Rubella virus (RV) causes cell division inhibition of critical precursor cells in organogenesis, CNS-associated birth defects and induction of apoptosis in cell culture. The underlying mechanisms of RV-induced congenital abnormalities are not known. Here, we identified a novel interaction between RV replicase P90 protein and a cytokinesis-regulatory protein, the Citron-K kinase (CK), in a yeast two-hybrid cDNA library screen. Aberrations in cytokinesis and subsequent apoptosis do occur in specific cell types when the CK gene is knocked out or, its regulatory function is perturbed. Our analysis found that full-length P90 binds CK and in RV-infected cells P90 colocalizes with CK in the cytoplasm. Furthermore, during RV infection as well as cellular expression of P90 alone, we identified a discrete subpopulation of cells containing 4N DNA content, indicating that these cells are arrested in the cell cycle following S phase, suggesting that cellular expression of viral P90 during RV infection perturbs cytokinesis. Previous reports by others established that RV infection leads to apoptosis in cell culture. These observations together taken to the fetal organogenesis level, favor the idea that RV P90, by binding to cellular CK, invokes cell cycle aberrations resulting in the cell- and organ-specific growth inhibition and programmed cell death during RV infection in utero, which commonly is referred to as RV-induced teratogenesis.

A Human Vaccine Strain of Lamb Rotavirus (Chinese) NSP4 Gene: Complete Nucleotide Sequence and Phylogenetic Analyses

A lamb strain of rotavirus has recently been licensed for use in China as a live vaccine to prevent rotavirus diarrhea in children. As rotavirus NSP4, especially the cytotoxic domain alone is considered to be associated with diarrhea, we sequenced gene segment 10, which encodes NSP4, of lamb rotavirus. Comparative analyses was performed to identify differences from human rotavirus strains, that might be associated with attenuation, and to ascertain whether the lamb rotavirus gene fits among the NSP4 of other sequenced rotavirus strains. Our comparative nucleotide sequence analysis suggests its close identity (91.17% homology) with that of group-A equine rotavirus (strain HI23). Multiple alignment of the deduced amino acid sequence of lamb NSP4 with that of other group A rotaviruses demonstrated homology ranging from 63.42% with that of porcine YM strain to 93.71% with equine HI23 strain of rotavirus. A group A-specific NSP4 monoclonal antibody recognized the glycosylated and unglycosylated forms of the protein from virus-infected lysates, suggesting a well-conserved group-specificity of the lamb NSP4. Phylogenetic analysis of the lamb rotavirus gene, with 60 other NSP4 gene sequences of human and animal rotavirus strains, demonstrated that the lamb rotavirus strain belongs to genotype A. Comparative analysis also revealed that although it is a vaccine strain, the NSP4 cytotoxic domain of lamb strain demonstrated an overall amino acid conservation similar to that of other strains, whose NSP4 alone causes diarrhea in animal models. These results taken together with our previous observations clearly reaffirm the idea that the attenuation phenotype of rotaviruses does not involve NSP4 cytotoxic domain, perhaps due to the suppression of NSP4 cytotoxic activity by other rotaviral proteins.

miR-570 interacts with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets

Abstract Loss of platelet quality during ex vivo storage is a major concern in the transfusion medicine field and it has been known that platelet mitochondrial dysfunction is associated with storage time. In the last decade, small noncoding RNAs also known as microRNAs (miRNAs) have been reported to regulate key cellular processes through their target sequence interactions with selected mRNAs. In this study, we focused on understanding the mechanisms of platelet mitochondrial dysfunction during storage through miRNA regulation of mRNAs. RNA was isolated from day 0, day 5, and day 9 of stored human leukocyte-depleted platelets and subjected to differential miRNA and mRNA profiling. The miRNA profiling identified several miRNAs at low levels including a set of 12 different miR-548 family members (miR-548a-3p, miR-548aa, miR-548x, miR-548ac, miR-548c-3p, miR-603, miR-548aj, miR-548ae, miR-548z, miR-548u, miR-548al, and miR-570-3p). The mRNA profiling identified, among many, the mitochondrial ATP synthase subunit g (ATP5L) mRNA at high levels during storage. Target Scan algorithm for potential targets of miR-570-3p also identified ATP5L as one of its targets. We further identified two target sites for miR-570-3p in the 3′ untranslated region (3′UTR) of ATP5L mRNA. While ATP5L is a subunit of F0ATPase complex, its function is not established yet. Overexpression of miR-570-3p in platelets resulted in reduced levels of ATP5L mRNA and concomitant ATP loss. These experimental results provide first-time insights into the miRNA–mRNA interactions underlying mitochondrial dysfunction in ex vivo stored platelets and warrants further investigation.

Analysis of Argonaute 2–microRNA complexes in ex vivo stored red blood cells

Human enucleated mature red blood cells (RBCs) contain both mature microRNAs (miRNAs) and mRNAs, and we have previously correlated RBC storage lesion processes such as eryptosis, adenosine 5′‐triphosphate loss, and RBC indices with differentially expressed miRNAs. Here we have characterized Argonaute 2 (AGO2)–miRNA complexes in stored mature RBCs as a first step toward understanding their role, if any.