Differential expression of genes influencing mitotic processes in cord blood mononuclear cells after a pre-conceptional micronutrient-based randomised controlled trial: Pune Rural Intervention in Young Adolescents (PRIYA).

In The Pune Maternal Nutrition Study, vitamin B12 deficiency was seen in 65% of pregnant women, folate deficiency was rare. Maternal total homocysteine concentrations were inversely associated with offspring birthweight, and low vitamin B12 and high folate concentrations predicted higher offspring adiposity and insulin resistance. These findings guided a nested pre-conceptional randomised controlled trial 'Pune Rural Intervention in Young Adolescents'. The interventions included: (1) vitamin B12+multi-micronutrients as per the United Nations International Multiple Micronutrient Antenatal Preparation, and proteins (B12+MMN), (2) vitamin B12 (B12 alone), and (3) placebo. Intervention improved maternal pre-conceptional and in-pregnancy micronutrient nutrition. Gene expression analysis in cord blood mononuclear cells in 88 pregnancies revealed 75 differentially expressed genes between the B12+MMN and placebo groups. The enriched biological processes included G2/M phase transition, chromosome segregation, and nuclear division. Enriched pathways included, mitotic spindle checkpoint and DNA damage response while enriched human phenotypes were sloping forehead and decreased head circumference. Fructose-bisphosphatase 2 (FBP2) and Cell Division Cycle Associated 2 (CDCA2) genes were under-expressed in the B12 alone group. The latter, involved in chromosome segregation was under-expressed in both intervention groups. Based on the role of B-complex vitamins in the synthesis of nucleotides and S-adenosyl methionine, and the roles of vitamins A and D on gene expression, we propose that the multi-micronutrient intervention epigenetically affected cell cycle dynamics. Neonates in the B12+MMN group had the highest ponderal index. Follow-up studies will reveal if the intervention and the altered biological processes influence offspring diabesity.

Global Transcriptomics of Congenital Hepatic Fibrosis in Autosomal Recessive Polycystic Kidney Disease using PCK rats.

Congenital hepatic fibrosis / Autosomal recessive polycystic kidney disease (CHF/ARPKD) is an inherited neonatal disease induced by mutations in the PKHD1 gene and characterized by cysts, and robust pericystic fibrosis in liver and kidney. The PCK rat is an excellent animal model which carries a Pkhd1 mutation and exhibits similar pathophysiology. We performed RNA-Seq analysis on liver samples from PCK rats over a time course of postnatal day (PND) 15, 20, 30, and 90 using age-matched Sprague-Dawley (SD) rats as controls to characterize molecular mechanisms of CHF/ARPKD pathogenesis. A comprehensive differential gene expression (DEG) analysis identified 1298 DEGs between PCK and SD rats. The genes overexpressed in the PCK rats at PND 30 and 90 were involved cell migration (e.g. Lamc2, Tgfb2 , and Plet1 ), cell adhesion (e.g. Spp1, Adgrg1 , and Cd44 ), and wound healing (e.g. Plat, Celsr1, Tpm1 ). Connective tissue growth factor ( Ctgf ) and platelet-derived growth factor ( Pdgfb ), two genes associated with fibrosis, were upregulated in PCK rats at all time-points. Genes associated with MHC class I molecules (e.g. RT1-A2 ) or involved in ribosome assembly (e.g. Pes1 ) were significantly downregulated in PCK rats. Upstream regulator analysis showed activation of proteins involved tissue growth (MTPN) and inflammation (STAT family members) and chromatin remodeling (BRG1), and inhibition of proteins involved in hepatic differentiation (HNF4α) and reduction of fibrosis (SMAD7). The increase in mRNAs of four top upregulated genes including Reg3b, Aoc1, Tm4sf20 , and Cdx2 was confirmed at the protein level using immunohistochemistry. In conclusion, these studies indicate that a combination of increased inflammation, cell migration and wound healing, and inhibition of hepatic function, decreased antifibrotic gene expression are the major underlying pathogenic mechanisms in CHF/ARPKD.

Snail induces epithelial cell extrusion by regulating RhoA contractile signalling and cell-matrix adhesion.

Cell extrusion is a morphogenetic process that is implicated in epithelial homeostasis and elicited by stimuli ranging from apoptosis to oncogenic transformation. To explore whether the morphogenetic transcription factor Snail (SNAI1) induces extrusion, we inducibly expressed a stabilized Snail6SA transgene in confluent MCF-7 monolayers. When expressed in small clusters (less than three cells) within otherwise wild-type confluent monolayers, Snail6SA expression induced apical cell extrusion. In contrast, larger clusters or homogenous cultures of Snail6SA cells did not show enhanced apical extrusion, but eventually displayed sporadic basal delamination. Transcriptomic profiling revealed that Snail6SA did not substantively alter the balance of epithelial and mesenchymal genes. However, we identified a transcriptional network that led to upregulated RhoA signalling and cortical contractility in cells expressing Snail6SA Enhanced contractility was necessary, but not sufficient, to drive extrusion, suggesting that Snail collaborates with other factors. Indeed, we found that the transcriptional downregulation of cell-matrix adhesion cooperates with contractility to mediate basal delamination. This provides a pathway for Snail to influence epithelial morphogenesis independently of classic epithelial-to-mesenchymal transition.

Neutrophils generated in vitro from hematopoietic stem cells isolated from apheresis samples and umbilical cord blood form neutrophil extracellular traps.

Neutrophils release neutrophil extracellular traps (NET) comprising of decondensed chromatin that immobilizes and kills pathogens. In vitro generation of neutrophils on a large scale from hematopoietic stem cells (HSCs) may be a useful strategy for treating neutropenic patients in future, though it is not in clinical practice yet. Microbial infections lead to major cause of morbidity and mortality in these patients. Despite the importance of NET in preventing infection, efficacy of in vitro-generated neutrophils from HSCs to form NET is not tested. We show that functional neutrophils could be generated in vitro from HSCs/MNCs isolated from umbilical cord blood (UCB) and apheresis-derived peripheral blood (APBL). Neutrophils generated from UCB showed properties comparable to those isolated from peripheral blood. We also show that isolation of HSCs is not absolutely essential for in vitro neutrophil generation. Further, we show that neutrophils generated from HSCs express PADI4 enzyme and their NET-forming ability is comparable to peripheral blood neutrophils. Taken together, our data show that fully functional neutrophils can be generated in vitro from HSCs. NET-forming ability of in vitro-generated neutrophils is an important parameter to determine their functionality and thus, should be studied along with other standard functional assays.

Cinnamate-CoA ligase is involved in biosynthesis of benzoate-derived biphenyl phytoalexin in Malus × domestica 'Golden Delicious' cell cultures.

Apple (Malus sp.) and other genera belonging to the sub-tribe Malinae of the Rosaceae family produce unique benzoic acid-derived biphenyl phytoalexins. Cell cultures of Malus domestica cv. 'Golden Delicious' accumulate two biphenyl phytoalexins, aucuparin and noraucuparin, in response to the addition of a Venturia inaequalis elicitor (VIE). In this study, we isolated and expressed a cinnamate-CoA ligase (CNL)-encoding sequence from VIE-treated cell cultures of cv. 'Golden Delicious' (M. domestica CNL; MdCNL). MdCNL catalyses the conversion of cinnamic acid into cinnamoyl-CoA, which is subsequently converted to biphenyls. MdCNL failed to accept benzoic acid as a substrate. When scab-resistant (cv. 'Shireen') and moderately scab-susceptible (cv. 'Golden Delicious') apple cultivars were challenged with the V. inaequalis scab fungus, an increase in MdCNL transcript levels was observed in internodal regions. The increase in MdCNL transcript levels could conceivably correlate with the pattern of accumulation of biphenyls. The C-terminal signal in the MdCNL protein directed its N-terminal reporter fusion to peroxisomes in Nicotiana benthamiana leaves. Thus, this report records the cloning and characterisation of a cinnamoyl-CoA-forming enzyme from apple via a series of in vivo and in vitro studies. Defining the key step of phytoalexin formation in apple provides a biotechnological tool for engineering elite cultivars with improved resistance.

Overexpression of histone variant H2A.1 and cellular transformation are related in N-nitrosodiethylamine-induced sequential hepatocarcinogenesis.

Histones through a complex repertoire of non-allelic variants and their post-translational modifications regulate gene expression. Though alterations in histone-modifying enzymes and post-translational modifications of histones have been studied in cancer, expression of histone variants has not been clearly associated with dedifferentiation and malignant transformation of hepatocyte in vivo. In the present work, the pattern of variants of histones was investigated during N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis. Our studies show for the first time in vivo overexpression of a major histone H2A variant H2A.1 and a decrease in H2A.2 at protein and mRNA levels by sodium dodecyl sulfate-Acetic acid-Urea-Triton (SDS-AUT) two-dimensional gel electrophoresis followed by matrix-assisted-laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometry and reverse transcriptase-polymerase chain reaction analysis during sequential development of hepatocellular carcinoma (HCC). H2A.1 and H2A.2 are highly homologous, replication-dependent, non-allelic variants of histone H2A differing at only three amino acid positions. Our results of increase in proliferating cell nuclear antigen expression indicate that with increase in replicating population of transformed cells in HCC, H2A.1 expression increases, suggesting association of H2A.1 overexpression with hyper-proliferation of hepatocytes during cellular dedifferentiation and progressive transformation of normal liver to preneoplastic and neoplastic stages of HCC.