Time related variations in stem cell harvesting of umbilical cord blood.

Umbilical cord blood (UCB) contains hematopoietic stem cells and multipotent mesenchymal cells useful for treatment in malignant/nonmalignant hematologic-immunologic diseases and regenerative medicine. Transplantation outcome is correlated with cord blood volume (CBV), number of total nucleated cells (TNC), CD34+ progenitor cells and colony forming units in UCB donations. Several studies have addressed the role of maternal/neonatal factors associated with the hematopoietic reconstruction potential of UCB, including: gestational age, maternal parity, newborn sex and birth weight, placental weight, labor duration and mode of delivery. Few data exist regarding as to how time influences UCB collection and banking patterns. We retrospectively analyzed 17.936 cord blood donations collected from 1999 to 2011 from Tuscany and Apulia Cord Blood Banks. Results from generalized multivariable linear mixed models showed that CBV, TNC and CD34+ cell were associated with known obstetric and neonatal parameters and showed rhythmic patterns in different time domains and frequency ranges. The present findings confirm that volume, total nucleated cells and stem cells of the UCB donations are hallmarked by rhythmic patterns in different time domains and frequency ranges and suggest that temporal rhythms in addition to known obstetric and neonatal parameters influence CBV, TNC and CD34+ cell content in UBC units.

Circadian transcriptome analysis in human fibroblasts from Hunter syndrome and impact of iduronate-2-sulfatase treatment.

BACKGROUND: Hunter syndrome (HS) is a lysosomal storage disease caused by iduronate-2-sulfatase (IDS) deficiency and loss of ability to break down and recycle the glycosaminoglycans, heparan and dermatan sulfate, leading to impairment of cellular processes and cell death. Cell activities and functioning of intracellular organelles are controlled by the clock genes (CGs), driving the rhythmic expression of clock controlled genes (CCGs). We aimed to evaluate the expression of CGs and downstream CCGs in HS, before and after enzyme replacement treatment with IDS. METHODS: The expression levels of CGs and CCGs were evaluated by a whole transcriptome analysis through Next Generation Sequencing in normal primary human fibroblasts and fibroblasts of patients affected by HS before and 24 h/144 h after IDS treatment. The time related expression of CGs after synchronization by serum shock was also evaluated by qRT-PCR before and after 24 hours of IDS treatment. RESULTS: In HS fibroblasts we found altered expression of several CGs and CCGs, with dynamic changes 24 h and 144 h after IDS treatment. A semantic hypergraph-based analysis highlighted five gene clusters significantly associated to important biological processes or pathways, and five genes, AHR, HIF1A, CRY1, ITGA5 and EIF2B3, proven to be central players in these pathways. After synchronization by serum shock and 24 h treatment with IDS the expression of ARNTL2 at 10 h (p = 0.036), PER1 at 4 h (p = 0.019), PER2 at 10 h (p = 0.041) and 16 h (p = 0.043) changed in HS fibroblasts. CONCLUSION: CG and CCG expression is altered in HS fibroblasts and IDS treatment determines dynamic modifications, suggesting a direct involvement of the CG machinery in the physiopathology of cellular derangements that characterize HS.

Differential patterns in the periodicity and dynamics of clock gene expression in mouse liver and stomach.

The rhythmic recurrence of biological processes is driven by the functioning of cellular circadian clocks, operated by a set of genes and proteins that generate self-sustaining transcriptional-translational feedback loops with a free-running period of about 24 h. In the gastrointestinal apparatus, the functioning of the biological clocks shows distinct patterns in the different organs. The aim of this study was to evaluate the time-related variation of clock gene expression in mouse liver and stomach, two components of the digestive system sharing vascular and autonomic supply, but performing completely different functions. The authors analyzed the periodicity by cosinor analysis and the dynamics of variation by computing the fractional variation to assess the rate of change in gene expression. Five-week-old male Balb/c mice were exposed to 2 wks of 12-h light/12-h dark cycles, then kept in complete darkness for 3 d as a continuation of the dark span of the last light-dark cycle. The authors evaluated the expression of Bmal1, Clock, Cry1, Cry2, Per1, Per2, Per3, Rev-erbα, Rev-erbß, Npas2, Timeless, Dbp, Csnk1d, and Csnk1e by using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in mouse liver and stomach. A significant 24-h rhythmic component was found for 10 genes in the liver (Bmal1, Clock, Cry1, Per1, Per2, Per3, Rev-erbα, Rev-erbß, Npas2, and Dbp), and for 9 genes in the stomach (Bmal1, Cry1, Per1, Per2, Per3, Rev-erbα, Rev-erbß, Npas2, and Dbp). In particular, Clock showed marked rhythm differences between liver and stomach, putatively due to some compensation by Npas2. The acrophase of the original values of Bmal1, Per2, Per3, Rev-erbα, Rev-erbß, Npas2, and Dbp expression was delayed in the stomach, and the average delay expressed as mean ± SD was 14.30 ± 7.94 degrees (57.20 ± 31.78 minutes). A statistically significant difference was found in the acrophases of Bmal1 (p = .015) and Npas2 (p = .011). Fractional variations provided significant circadian rhythms for nine genes in the liver (Bmal1, Per1, Per2, Per3, Rev-erbα, Rev-erbß, Npas2, Timeless, and Dbp), and for seven genes in the stomach (Bmal1, Clock, Per2, Rev-erbα, Npas2, Dbp, and Csnk1e). The acrophase of the fractional variations of Bmal1, Per2, Per3, Rev-erbα, Rev-erbß, and Dbp expression was delayed in the stomach, and the average delay expressed as mean ± SD was 19.10 ± 9.39 degrees (76.40 ± 37.59 minutes). A significantly greater fractional variation was found in the liver for Clock at 06:00 h (p = .034), Per1 at 02:00 h (p = .037), and Per3 at 02:00 h (p = .029), whereas the fractional variation was greater in the stomach for Clock at 10:00 h (p = .016), and for Npas2 at 02:00 h (p = .029) and at 06:00 h (p = .044). In conclusion, liver and stomach show different phasing and dynamics of clock gene expression, which are probably related to prevailing control by different driving cues, and allow them to keep going the various metabolic pathways and diverse functional processes that they manage.

Time-Qualified Patterns of Variation of PPARγ, DNMT1, and DNMT3B Expression in Pancreatic Cancer Cell Lines.

Carcinogenesis is related to the loss of homeostatic control of cellular processes regulated by transcriptional circuits and epigenetic mechanisms. Among these, the activities of peroxisome proliferator-activated receptors (PPARs) and DNA methyltransferases (DNMTs) are crucial and intertwined. PPARγ is a key regulator of cell fate, linking nutrient sensing to transcription processes, and its expression oscillates with circadian rhythmicity. Aim of our study was to assess the periodicity of PPARγ and DNMTs in pancreatic cancer (PC). We investigated the time-related patterns of PPARG, DNMT1, and DNMT3B expression monitoring their mRNA levels by qRT-PCR at different time points over a 28-hour span in BxPC-3, CFPAC-1, PANC-1, and MIAPaCa-2 PC cells after synchronization with serum shock. PPARG and DNMT1 expression in PANC-1 cells and PPARG expression in MIAPaCa-2 cells were characterized by a 24 h period oscillation, and a borderline significant rhythm was observed for the PPARG, DNMT1, and DNMT3B expression profiles in the other cell lines. The time-qualified profiles of gene expression showed different shapes and phase relationships in the PC cell lines examined. In conclusion, PPARG and DNMTs expression is characterized by different time-qualified patterns in cell lines derived from human PC, and this heterogeneity could influence cell phenotype and human disease behaviour.