Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection

Background Human cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. Some miRNAs have been shown to suppress virus replication, which could help HCMV to establish or maintain latent infection. However, HCMV miRNA expression has not been comprehensively examined and compared using cell culture systems representing permissive (lytic) and semi-permissive vs. non-permissive (latent-like) infection. Methods Viral miRNAs levels and expression kinetics during HCMV infection were determined by miRNA-specific stem-loop RT-PCR. HCMV infected THP-1 (non-permissive), differentiated THP-1 (d-THP-1, semi-permissive) and human embryo lung fibroblasts (HELs, fully-permissive) were examined. The impact of selected miRNAs on HCMV infection (gene expression, genome replication and virus release) was determined by Western blotting, RT-PCR, qPCR, and plaque assay. Results Abundant expression of 15 HCMV miRNAs was observed during lytic infection in HELs; highest peak inductions (11- to 1502-fold) occurred at 48 hpi. In d-THP-1s, fourteen mRNAs were detected with moderate induction (3- to 288-fold), but kinetics of expression was generally delayed for 24 h relative to HELs. In contrast, only three miRNAs were induced to low levels (3- to 4-fold) during quiescent infection in THP-1s. Interestingly, miR-UL70-3p was poorly induced in HEL (1.5-fold), moderately in THP-1s (4-fold), and strongly (58-fold) in d-THP-1s, suggesting a potentially specific role for miR-UL70-3p in THP-1s and d-THP-1s. MiR-US33, -UL22A and -UL70 were further evaluated for their impact on HCMV replication in HELs. Ectopic expression of miR-UL22A and miR-UL70 did not affect HCMV replication in HELs, whereas miR-US33 inhibited HCMV replication and reduced levels of HCMV US29 mRNA, confirming that US29 is a target of miR-US33. Conclusions Viral miRNA expression kinetics differs between permissive, semi-permissive and quiescent infections, and miR-US33 down-regulates HCMV replication. These results suggest that miR-US33 may function to impair entry into lytic replication and hence promote establishment of latency

TLR2 expression is increased in rosacea and stimulates enhanced serine protease production by keratinocytes.

A diverse environment challenges skin to maintain temperature, hydration, and electrolyte balance while also maintaining normal immunological function. Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. We hypothesized that abnormal function of innate immune pattern recognition could explain the enhanced sensitivity of patients with rosacea, and observed that the epidermis of patients with rosacea expressed higher amounts of Toll-like receptor 2 (TLR2) than normal patients. Increased expression of TLR2 was not seen in other inflammatory skin disorders such as atopic dermatitis or psoriasis. Overexpression of TLR2 on keratinocytes, treatment with TLR2 ligands, and analysis of TLR2-deficient mice resulted in a calcium-dependent release of kallikrein 5 from keratinocytes, a critical protease involved in the pathogenesis of rosacea. These observations show that abnormal TLR2 function may explain enhanced inflammatory responses to environmental stimuli and can act as a critical element in the pathogenesis of rosacea

Lunar Propellant Factory Mission Design To Sustain Future Human Exploration

The International Space Exploration Coordination Group (ISECG) Global Exploration Roadmap (GER) is the standard document reflecting the current focus of the leading space agencies that envision space exploration missions beyond Low Earth Orbit (LEO), returning to the Moon and going to Mars in the upcoming years. The roadmap showcases the Moon as a stepping-stone for further human space exploration, by setting up a sustainable space infrastructure on its surface an orbit. Inspired from this vision, we present the result of a phase A study about a lunar propellant factory near the Shackleton south-pole crater relying on In-Situ Resources Utilization (ISRU) to produce and sell Liquid Oxygen (LOX) on the moon surface and in orbit. The overall timeline of the mission is in line with the ISECG exploration roadmap Moon phase, based on realistic technologies of advanced-enough Technology Readiness Levels (TRL). It is a second iteration on the Lunar Propellant Outpost (LUPO) mission architecture, presented during IAC 2018. We preserved and reviewed the original building blocks (Habitats, Crew Mobility Elements, ISRU Facilities, and Lunar Spaceport) of the LUPO mission architecture, and further improved the mission design, supported by trade-off analysis on different mission scenarios. An extensive analysis and optimisation have been performed on ISRU processes and surface electrical power management, the core of our infrastructure. The mission architecture also includes crew on the lunar surface, so life support systems and habitat, as well as operations concepts, have been studied in-depth, and a synthesis of all results is presented. The main aim of this iteration was to improve and refine the baseline infrastructural and technological design architecture of LUPO and reflect on missions going beyond the Moon by providing refuelling services, with sustainability and economic viability in mind

Beneficial autoimmunity at body surfaces – immune surveillance and rapid type 2 immunity regulate tissue homeostasis and cancer

Epithelial cells line body surface tissues and provide a physicochemical barrier to the external environment. Frequent microbial and non-microbial challenges such as those imposed by mechanical disruption, injury or exposure to noxious environmental substances including chemicals, carcinogens, ultraviolet-irradiation or toxins cause activation of epithelial cells with release of cytokines and chemokines as well as alterations in the expression of cell surface ligands. Such display of epithelial stress is rapidly sensed by tissue resident immunocytes, which can directly interact with self-moieties on epithelial cells and initiate both local and systemic immune responses. Epithelial cells are thus key drivers of immune surveillance at body surface tissues. However, epithelial cells have a propensity to drive type 2 immunity (rather than type 1) upon non-invasive challenge or stress – a type of immunity whose regulation and function still remain enigmatic. Here we review the induction and possible role of type 2 immunity in epithelial tissues and propose that rapid immune surveillance and type 2 immunity are key regulators of tissue homeostasis and carcinogenesis

Long-Range Enhancer Associated with Chromatin Looping Allows AP-1 Regulation of the Peptidylarginine Deiminase 3 Gene in Differentiated Keratinocyte

Transcription control at a distance is a critical mechanism, particularly for contiguous genes. The peptidylarginine deiminases (PADs) catalyse the conversion of protein-bound arginine into citrulline (deimination), a critical reaction in the pathophysiology of multiple sclerosis, Alzheimer's disease and rheumatoid arthritis, and in the metabolism of the major epidermal barrier protein filaggrin, a strong predisposing factor for atopic dermatitis. PADs are encoded by 5 clustered PADI genes (1p35-6). Unclear are the mechanisms controlling the expression of the gene PADI3 encoding the PAD3 isoform, a strong candidate for the deimination of filaggrin in the terminally differentiating epidermal keratinocyte. We describe the first PAD Intergenic Enhancer (PIE), an evolutionary conserved non coding segment located 86-kb from the PADI3 promoter. PIE is a strong enhancer of the PADI3 promoter in Ca2+-differentiated epidermal keratinocytes, and requires bound AP-1 factors, namely c-Jun and c-Fos. As compared to proliferative keratinocytes, calcium stimulation specifically associates with increased local DNase I hypersensitivity around PIE, and increased physical proximity of PIE and PADI3 as assessed by Chromosome Conformation Capture. The specific AP-1 inhibitor nordihydroguaiaretic acid suppresses the calcium-induced increase of PADI3 mRNA levels in keratinocytes. Our findings pave the way to the exploration of deimination control during tumorigenesis and wound healing, two conditions for which AP-1 factors are critical, and disclose that long-range transcription control has a role in the regulation of the gene PADI3. Since invalidation of distant regulators causes a variety of human diseases, PIE results to be a plausible candidate in association studies on deimination-related disorders or atopic disease

Human cytomegalovirus infection is associated with increased expression of the lissencephaly gene PAFAH1B1 encoding LIS1 in neural stem cells and congenitally infected brains

peer reviewedCongenital infection of the central nervous system by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae, including mental retardation or neurodevelopmental abnormalities. The most severe complications include smooth brain or polymicrogyria, which are both indicative of abnormal migration of neural cells, although the underlying mechanisms remain to be determined. To gain better insight on the pathogenesis of such sequelae, we assessed the expression levels of a set of neurogenesis-related genes, using HCMV-infected human neural stem cells derived from embryonic stem cells (NSCs). Among the 84 genes tested, we found dramatically increased expression of the gene PAFAH1B1, encoding LIS1 (lissencephaly-1), in HCMV-infected versus uninfected NSCs. Consistent with these ndings, western blotting and immunouorescence analyses conrmed the increased levels of LIS1 in HCMV-infected NSCs at the protein level. We next assessed the migratory abilities of HCMV-infected NSCs and observed that infection strongly impaired the migration of NSCs, without detectable effect on their proliferation. Moreover, we observed increased immunostaining for LIS1 in brains of congenitally infected fetuses, but not in control samples, highlighting the clinical relevance of our ndings. Of note, PAFAH1B1 mutations (resulting in either haploinsufciency or gain of function) are primary causes of hereditary neurodevelopmental diseases. Notably, mutations resulting in PAFAH1B1 haploinsufciency cause classic lissencephaly. Taken together, our ndings suggest that PAFAH1B1 is a critical target of HCMV infection. They also shine a new light on the pathophysiological basis of the neurological outcomes of congenital HCMV infection, by suggesting that defective neural cell migration might contribute to the pathogenesis of the neurodevelopmental sequelae of infectio

A functional polymorphism in the SPINK5 gene is associated with asthma in a Chinese Han Population

<p>Abstract</p> <p>Background</p> <p>Mutation in <it>SPINK5 </it>causes Netherton syndrome, a rare recessive skin disease that is accompanied by severe atopic manifestations including atopic dermatitis, allergic rhinitis, asthma, high serum IgE and hypereosinophilia. Recently, single nucleotide polymorphism (SNP) of the <it>SPINK5 </it>was shown to be significantly associated with atopy, atopic dermatitis, asthma, and total serum IgE. In order to determine the role of the <it>SPINK5 </it>in the development of asthma, a case-control study including 669 asthma patients and 711 healthy controls in Han Chinese was conducted.</p> <p>Methods</p> <p>Using PCR-RFLP assay, we genotyped one promoter SNP, -206G>A, and four nonsynonymous SNPs, 1103A>G (Asn368Ser), 1156G>A (Asp386Asn), 1258G>A (Glu420Lys), and 2475G>T (Glu825Asp). Also, we analyzed the functional significance of -206G>A using the luciferase reporter assay and electrophoresis mobility shift assay.</p> <p>Results</p> <p>we found that the G allele at SNP -206G>A was associated with increased asthma susceptibility in our study population (p = 0.002, odds ratio 1.34, 95% confidence interval 1.11–1.60). There was no significant association between any of four nonsynonymous SNPs and asthma. The A allele at -206G>A has a significantly higher transcriptional activity than the G allele. Electrophoresis mobility shift assay also showed a significantly higher binding efficiency of nuclear protein to the A allele compared with the G allele.</p> <p>Conclusion</p> <p>Our findings indicate that the -206G>A polymorphism in the <it>SPINK5 </it>is associated with asthma susceptibility in a Chinese Han population.</p

Peptidylarginine deiminases as drug targets in neonatal hypoxic-ischemic encephalopathy

Oxygen deprivation and infection are major causes of perinatal brain injury leading to cerebral palsy and other neurological disabilities. The identification of novel key factors mediating white and grey matter damage are crucial to allow better understanding of the specific contribution of different cell types to the injury processes and pathways for clinical intervention. Recent studies in the Rice-Vannucci mouse model of neonatal hypoxic ischaemia (HI) have highlighted novel roles for calcium-regulated peptidylarginine deiminases (PADs) and demonstrated neuro-protective effects of pharmacological PAD inhibition following HI and synergistic infection mimicked by LPS stimulation

An SF1 affinity model to identify branch point sequences in human introns

Splicing factor 1 (SF1) binds to the branch point sequence (BPS) of mammalian introns and is believed to be important for the splicing of some, but not all, introns. To help identify BPSs, particularly those that depend on SF1, we generated a BPS profile model in which SF1 binding affinity data, validated by branch point mapping, were iteratively incorporated into computational models. We searched a data set of 117 499 human introns for best matches to the SF1 Affinity Model above a threshold, and counted the number of matches at each intronic position. After subtracting a background value, we found that 87.9% of remaining high-scoring matches identified were located in a region upstream of 3′-splice sites where BPSs are typically found. Since U2AF65 recognizes the polypyrimidine tract (PPT) and forms a cooperative RNA complex with SF1, we combined the SF1 model with a PPT model computed from high affinity binding sequences for U2AF65. The combined model, together with binding site location constraints, accurately identified introns bound by SF1 that are candidates for SF1-dependent splicing