Giselda Bucca

The cold shock (CS) response of the model actinomycete bacterium, Streptomyces coelicolor A3(2) has been characterized for the first time at the transcriptome and translatome level. Key players of the CS response reside in three operons encoding a CspA homologue, a DEAD box helicase and either a cystathionine-beta-synthase domain protein, or a protein of unknown function, or both ( SCO5921–SCO5918; SCO46984-SCO4686; SCO3731-SCO3733 ). Genes in the SCO5921-SCO5918 and SCO4684-SCO4686 operons are subjected to massive transcriptional induction, up to 2,000-fold, upon cold acclimation. Moreover, SCO5920 , SCO5919 and SCO5918 are the most highly upregulated at the transcriptional and translational level (potentiated). Cold adaptation resulted in transcriptional changes in 811 genes, with enrichment for four functional groups: helicases, transcription factors, phenylalanine metabolism and extracellular functions. Major CS-induced gene products are involved in assisting transcription and translation at low temperature including RNA helicases and RNA chaperone proteins (CS proteins). The considerable induction of these gene products is presumed to reflect their critical importance to removing excessive nucleic acid secondary structure in the high %G+C Streptomyces genome (>73% G+C). Three novel CS-induced transcriptional regulators were identified: SCO1568, SCO4640 and SCO7014. Several putative cis-acting regulatory elements have been identified in the CspA-encoding cold-shock operons and other members of the CS regulon. Furthermore, two small non-coding RNAs are substantially induced by cold-shock, sRNA126 and sRNA208, and their expression correlates with that of the novel transcription factors. Other upregulated genes were identified with functions in modification of cell membrane (e.g. delta fatty acid desaturase, des ( SCO3682 )) and cell wall structure (e.g. D-alanyl-D-alanine carboxypeptidase ( SCO7050 ) and SCO617 9)). Several membrane efflux transporter pumps conferring resistance to translation targeting antibiotics were induced, including pqrB which encodes a multidrug resistance efflux pump of the major facilitator superfamily and SCO4120, conferring ciprofloxacin and chloramphenicol resistance. The phenylacetate degradation (PAA) regulon is co-ordinately CS-induced. Exclusively translationally enhanced genes are enriched in the protein phosphorylation functional category. The highly conserved gene pair, SCO0818 and SCO0819 , encoding an ABC transporter ATP-binding protein and a transmembrane protein, is in this group. The CS-induced transcriptional and translational cis-acting control regions identified in this study, within the massively induced SCO5921 and SCO4684 operons, could be exploited as novel synthetic biology tools for manipulating antibiotic biosynthetic gene clusters in this industrially important bacterial group.

Chronic wounds are widely recognized as a global health challenge, but both our understanding of wound biology and the implementation of technological innovations for their treatment have made only...

Cataract surgery removes the diseased lens of the eye replacing it with an intraocular lens, restoring visual acuity. However, accommodation, the lens’ ability to provide dynamic change in focus, is lost. A number of accommodative intraocular lens (AIOL) designs have been considered although none have provided a truly effective clinical AIOL. Two-dimensional titanium carbide (Ti₃C₂T_x) MXene has been used as a transparent conductive electrode within an AIOL feasibility study. Nevertheless, the potential for Ti₃C₂T_xto repress excessive inflammation and promote wound healing following cataract surgery has not been considered. Cataract surgery can trigger chronic inflammation and epithelial-mesenchymal transition (EMT) in residual lens epithelial cells (LECs), producing a fibrotic mass across the posterior capsule known as posterior capsule opacification (PCO). With a large surface area and capacity for surface functionalisation, MXene has properties enabling a dual purpose AIOL design with an additional therapeutic role in the repression of pathways leading to PCO development. In this study, Ti₃C₂T_xMXene was investigated to determine its impact on pathways leading to chronic inflammation and EMT using anin vitroLECs model. Ti₃C₂T_xMXene was synthesised and characterised using UV-vis spectroscopy, dynamic light scattering and scanning electron microscopy. Changes in markers linked to inflammation and EMT in Ti₃C₂T_x-treated LECs were measured using enzyme linked immunosorbent assays, quantitative polymerase chain reaction, scratch assay, RNA sequencing for whole-cell gene expression profiling and lipidomics analysis. Ti₃C₂T_xsignificantly reduced the expression of pro-inflammatory cytokines by interleukin 1 beta primed LECs and did not advocate EMT, promoting a positive resolution of the wound healing response. This study supports the role of Ti₃C₂T_xwithin an AIOL design with the potential to repress key developmental pathways leading to PCO.

Glucocorticoids (GCs) are one of the most used anti-inflammatory drugs worldwide. Despite their widespread use, our understanding of their post-transcriptional effects remains poorly understood. The tristetraprolin (TTP) RNA binding protein (RBP) family (ZFP36, ZFP36L1 and ZFP36L2) has been implicated in inflammation regulation via binding to AU-rich elements (ARE) in mRNAs, with TTP being implicated in GC modulation. We hypothesised that ZFP36L1 and ZFP36L2 are part of the GC pathway and tested this hypothesis in bronchial epithelium, which commonly encounters GC in vivo upon inhalation. Our data show that dexamethasone, a commonly used GC, modulated the levels, subcellular localisation and RNA binding of ZFP36L1/L2. Employing Frac-seq (subcellular fractionation and RNA-sequencing), we show that GC modulated distinct subsets of RNAs in a subcellular-dependent manner. In addition to their mostly known transcriptional effects (116 differentially expressed genes, DEGs), GCs modified the binding to monosomes of myriad mRNAs (83 differentially bound genes, DBGs). We also demonstrate that ZFP36L1/L2 modulated gene expression mainly at the total cytoplasmic and polyribosome binding levels. ZFP36L1/L2 down-regulation led to an increase in ARE-containing mRNAs and a pronounced modification of the effects of GC on gene expression. We observed a small overlap of genes modulated by GCs when comparing control and ZFP36L1/L2 knockdown cells, in a subcellular-dependent manner Our data also suggest a novel role for these RBPs and GCs in epithelial biology via regulation of mRNAs encoding proteins important for epithelial cell function including cellular structure. We believe that our data has further implications in how we investigate gene expression. We show the power of employing sub-cellular fractionation when analysing genome-wide effects for known ‘transcriptional modulators’ such as GCs, as well as a tool to demonstrate the extent of the effect of RBPs on gene expression modulation beyond total RNA levels.

The retention and re-migration of Chronic Lymphocytic Leukemia cells into cytoprotective and proliferative lymphoid niches is thought to contribute to the development of resistance, leading to subsequent disease relapse. The aim of this study was to elucidate the molecular processes that govern CLL cell migration to elicit a more complete inhibition of tumor cell migration. We compared the phenotypic and transcriptional changes induced in CLL cells using two distinct models designed to recapitulate the peripheral circulation, CLL cell migration across an endothelial barrier, and the lymph node interaction between CLL cells and activated T cells. Initially, CLL cells were co-cultured with CD40L-expressing fibroblasts and exhibited an activated B-cell phenotype, and their transcriptional signatures demonstrated the upregulation of pro-survival and anti-apoptotic genes and overrepresentation of the NF-κB signaling pathway. Using our dynamic circulating model, we were able to study the transcriptomics and miRNomics associated with CLL migration. More than 3000 genes were altered when CLL cells underwent transendothelial migration, with an overrepresentation of adhesion and cell migration gene sets. From this analysis, an upregulation of the FAK signaling pathway was observed. Importantly, PTK2 (FAK) gene expression was significantly upregulated in migrating CLL cells (PTK2 Fold-change = 4.9). Here we demonstrate that TLR9 agonism increased levels of p-FAK (p ≤ 0.05), which could be prevented by pharmacological inhibition of FAK with defactinib (p ≤ 0.01). Furthermore, a reduction in CLL cell migration and invasion was observed when FAK was inhibited (p ≤ 0.0001), supporting a role for FAK in both CLL migration and tissue invasion. When taken together, our data highlights the potential for combining FAK inhibition with current targeted therapies as a more effective treatment regime for CLL.

For the first time we report the relative influences of vitamin D2 and vitamin D3 on genome-wide gene expression in whole blood from healthy women, representing two ethnic groups, white European and South Asian. In this randomised placebo-controlled trial, participants were given daily physiological doses (15 µg) of either vitamin D2 or D3 for 12 weeks and changes in the transcriptome were compared relative to the transcriptome at baseline. While there was some overlap in the repertoire of differentially expressed genes after supplementation with each vitamin D source, most changes were specific to either vitamin D3 or vitamin D2, suggesting that each form of the vitamin may have different effects on human physiology. Notably, following vitamin D3 supplementation, the majority of changes in gene expression reflected a down-regulation in the activity of genes, many encoding components of the innate and adaptive immune systems. These are consistent with the emerging concept that vitamin D orchestrates a shift in the immune system towards a more tolerogenic status. Moreover, gene expression associated with type 1 and type 2 interferon activity differed following supplementation with either vitamin D2 or vitamin D3, with only vitamin D3 having a stimulatory effect. Interferons play a critical role in the innate response to infection and aberrant type 1 interferon signalling has recently been implicated in severe Covid-19 disease. The observed differences in gene expression after supplementation with vitamin D2 compared with vitamin D3 warrant a more intensive investigation of the biological effects of the two forms of vitamin D on human physiology.

For the first time, we report the influence of vitamin D ₂ and vitamin D ₃ on genome-wide gene expression in whole blood from healthy women representing two ethnic groups, white European and South Asian. In this randomised placebo-controlled trial, participants were given daily physiological doses (15 µg) of either vitamin D ₂ or D ₃ for 12 weeks and changes in the transcriptome were compared relative to the transcriptome at baseline. While there was some overlap in the repertoire of differentially expressed genes after supplementation with each vitamin D source, most changes were specific to either vitamin D ₃ or vitamin D ₂ , suggesting that each form of the vitamin may have different effects on human physiology. Notably, following vitamin D ₃ supplementation, the majority of changes in gene expression reflected a down-regulation in the activity of genes, many encoding components of the innate and adaptive immune systems. These are consistent with the emerging concept that vitamin D orchestrates a shift in the immune system towards a more tolerogenic status. Moreover, divergent changes were observed following supplementation with either vitamin D ₃ or vitamin D ₂ for gene expression associated with type 1 and type 2 interferon activity. This is particularly intriguing as interferons play a critical role in the innate response to infection and aberrant type 1 interferon signalling is implicated in severe COVID-19 disease. The observed differences in gene expression after supplementation with vitamin D ₂ compared with vitamin D ₃ warrant a more intensive investigation of the biological effects of the two forms of vitamin D on human physiology. Significance statements: This study suggests that the influence of vitamins D ₂ and D ₃ on human physiology may not be the same, as deduced from differences in gene expression within whole blood. South Asian participants were found to respond differently to vitamin D supplementation at the transcriptome level from white Europeans. The differentially expressed immune pathways identified in this study are consistent with vitamin D orchestrating a more tolerogenic immune status and this could be relevant in the context of the severity of immune response to viral infections such as Covid-19. The potential relevance of this study to severe Covid-19 disease is highlighted by our observed enhancement of type 1 interferon signalling by vitamin D ₃ , but not vitamin D ₂ .

Background: Vitamin D deficiency increases the risk of developing multiple sclerosis (MS) but it is unclear whether vitamin D supplementation improves the clinical course of MS, and there is uncertainty about the dose and form of vitamin D (D2 or D3) to be used. The mechanisms underlying the effects of vitamin D in MS are not clear. Vitamin D3 increases the rate of differentiation of primary oligodendrocyte precursor cells (OPCs), suggesting that it might help remyelination in addition to modulating the immune response. Here we analyzed the transcriptome of differentiating rat CG4 OPCs treated with vitamin D2 or with vitamin D3 at 24 h and 72 h following onset of differentiation.Methods: Gene expression in differentiating CG4 cells in response to vitamin D2 or D3 was quantified using Agilent DNA microarrays (n = 4 replicates), and the transcriptome data were processed and analysed using the R software environment. Differential expression between the experimental conditions was determined using LIMMA, applying the Benjamini and Hochberg multiple testing correction top-values, and significant genes were grouped into co-expression clusters by hierarchical clustering. The functional significance of gene groups was explored by pathway enrichment analysis using the clusterProfiler package.Results: Differentiation alone changed the expression of about 10% of the genes at 72 h compared to 24 h. Vitamin D2 and D3 exerted different effects on gene expression, with D3 influencing 1272 genes and D2 574 at 24 h. The expression of the vast majority of these genes was either not changed in differentiating cells not exposed to vitamin D or followed the same trajectory as the latter. D3-repressed genes were enriched for Gene Ontology (GO) categories including transcription factors and the Notch pathway, while D3-induced genes were enriched for the Ras pathway.Conclusions: This study shows that vitamin D3, compared with D2, changes the expression of a larger number of genes in OLs. Identification of genes affected by D3 in OLs should help to identify mechanisms mediating its action in MS.

Vitamin D deficiency increases the risk of developing multiple sclerosis (MS) but there is uncertainty about what dose and form of vitamin D could improve the clinical course of MS. The mechanisms underlying the effects of vitamin D in MS are not clear. Vitamin D3 increases the rate of differentiation of primary oligodendrocyte precursor cells (OPCs), suggesting that it might help remyelination in addition to modulating the immune response. Here we analyzed the transcriptome of differentiating rat CG4 OPCs treated with vitamin D2 or with D3 at 24 h and 72 h following onset of differentiation. Differentiation alone changed the expression of about 10% of the genes at 72 h compared to 24 h. Vitamin D2 and D3 exerted different effects on gene expression, with D3 influencing 1,272 genes and D2 574 at 24 h. The expression of the vast majority of these genes was either not changed in differentiating cells not exposed to vitamin D or followed the same trajectory as the latter. D3-repressed genes were enriched for gene ontology categories including transcription factors and the Notch pathway, while D3-induced genes were enriched for the Ras pathway. These findings should help to identify mechanisms mediating D3 action in MS.

Studying circadian rhythms in most human tissues is hampered by difficulty in collecting serial samples. Here we reveal circadian rhythms in the transcriptome and metabolic pathways of human white adipose tissue. Subcutaneous adipose tissue was taken from seven healthy males under highly controlled ‘constant routine’ conditions. Five biopsies per participant were taken at six-hourly intervals for microarray analysis and in silico integrative metabolic modelling. We identified 837 transcripts exhibiting circadian expression profiles (2% of 41619 transcript targeting probes on the array), with clear separation of transcripts peaking in the morning (258 probes) and evening (579 probes). There was only partial overlap of our rhythmic transcripts with published animal adipose and human blood transcriptome data. Morning-peaking transcripts associated with regulation of gene expression, nitrogen compound metabolism, and nucleic acid biology; evening-peaking transcripts associated with organic acid metabolism, cofactor metabolism and redox activity. In silico pathway analysis further indicated circadian regulation of lipid and nucleic acid metabolism; it also predicted circadian variation in key metabolic pathways such as the citric acid cycle and branched chain amino acid degradation. In summary, in vivo circadian rhythms exist in multiple adipose metabolic pathways, including those involved in lipid metabolism, and core aspects of cellular biochemistry.