Genomic tools and sex determination in the extremophile brine shrimp Artemia franciscana

The aim of this study was the construction of a genomic Artemia toolkit. Sex-specific AFLP-based genetic maps were constructed based on 433 AFLP markers segregating in a 112 full-sib family, revealing 21 male and 22 female linkage groups (2n = 42). Fifteen putatively homologous linkage groups, including the sex linkage groups, were identified between the female and male linkage maps. Eight sex-linked markers, heterozygous in female animals, mapped to a single locus on a female linkage group, supporting the hypothesis of a WZ/ZZ genetic sex-determining system and showing primary sex determination is likely directed by a single gene. To fine-map the sex locus, bulked segregant analysis was performed. Candidate primary sex-determining genes were identified, including Cytochrome P450 which, through transcriptomic studies, is already known as a candidate sex-determining gene for Macrobrachium nipponense. The 1,310-Mbp Artemia draft genome sequence (N50 = 14,784 bp; GC-content = 35%; 176,667 scaffolds) was annotated, predicting 188,101 genes with an average length of 692 bp. Ninety-two percent of the transcriptome reads of Artemia in different conditions were present in the Artemia genome, indicating that the functional part of the genome under the RNAseq sampling conditions is virtually fully represented in the assembly. Several steps were taken in this study to introduce Artemia as a new genomic model for crustaceans. Although the functional part of the Artemia genome under the RNAseq sampling conditions is virtually fully represented in the assembly, thus making it useful for qualitative research, genome finishing strategies will still be necessary to complete the genome project. The further development of genomic resources for Artemia will add a completely new dimension to Artemia research and its use as live food in aquaculture

Faecal leukocyte esterase activity is an alternative biomarker in inflammatory bowel disease

Background: Leukocyte cytosolic proteins (e.g., calprotectin) are emerging biomarkers for inflammatory bowel disease. Leukocyte aryl esterase activity has been commonly used for sensitive detection of leukocytes in human body fluids such as urine. Urine test strip results are generally reported in categories. As automated strip readers allow quantitative data to be reported, sensitive quantitative detection of leukocytes in body fluids has become possible. Here, we explored the use of leukocyte esterase as a potential alternative faecal biomarker for inflammatory bowel disease. Methods: We evaluated leukocyte esterase activity in faecal extracts and compared Cobas u 411 (Roche) quantitative reflectance data with calprotectin concentration for 107 routine samples. Stability of leukocyte esterase for trypsin digestion was carried out by adding trypsin to the extract. Incubation occurred at 37 ° C for 24 h or 48 h. Results: Reproducibility of the reflectance signal was good (within-run imprecision: 6.1%; between-run imprecision: 6.2%). Results were linear in the range 10 3 – 10 6 WBC/100 mg faeces. The lower limit of detection was 4 WBC/ μ L and the lower limit of quantification was 5 WBC/ μ L. Stability of LE activity in stool and faecal matrix was good. An adequate correlation was obtained between leukocyte esterase activity and the faecal calprotectin concentration: log(y)  =  4.28 + 0.29log(x). In vitro experiments monitored the digestion of leukocyte esterase and faecal calprotectin. Leukocyte esterase activity was significantly less affected by trypsin activity than calprotectin immunoreactivity. Conclusions: Quantitative leukocyte esterase activity of faecal extracts provides information about the leukocyte count in the gut lumen. Leukocyte esterase is a promising and affordable alternative biomarker for monitoring inflammatory bowel disease

Infliximab treatment in pediatric IBD patients: a single centre experience

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CT in a Neonate with Nasal Obstruction

A three-day-old girl was referred to the radiology department by the otorhinolaryngologist for work-up of nasal obstruction as she presented with loud nasal respiration and respiratory distress during feeding. Nasal endoscopy revealed a bilateral intranasal mass. CT of the sinonasal region showed a bilateral soft-tissue mass at the medial canthus, with bilateral widening of the nasolacrimal canal, and revealed the presence of a bilateral endonasal mass below the inferior turbinate (Figures 1–3). This triad of imaging findings is diagnostic for a congenital dacryocystocoele. Endonasal wide marsupialisation using a microdebrider was performed, restoring patency of the nasolacrimal apparatus and resolving the symptoms

The chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) controls cellular quiescence by hyperpolarizing the cell membrane during diapause in the crustacean Artemia

Cellular quiescence, a reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance. However, the physiological mechanisms underlying cellular quiescence remain largely unknown. In the present study, we used embryos of the crustacean Artemia in the diapause stage, in which these embryos remain quiescent for prolonged periods, as a model to explore the relationship between cell-membrane potential (V-mem) and quiescence. We found that V-mem is hyperpolarized and that the intracellular chloride concentration is high in diapause embryos, whereas V-mem is depolarized and intracellular chloride concentration is reduced in postdiapause embryos and during further embryonic development. We identified and characterized the chloride ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR) of Artemia (Ar-CFTR) and found that its expression is silenced in quiescent cells of Artemia diapause embryos but remains constant in all other embryonic stages. Ar-CFTR knockdown and GlyH-101-mediated chemical inhibition of Ar-CFTR produced diapause embryos having a high V-mem and intracellular chloride concentration, whereas control Artemia embryos released free-swimming nauplius larvae. Transcriptome analysis of embryos at different developmental stages revealed that proliferation, differentiation, and metabolism are suppressed in diapause embryos and restored in postdiapause embryos. Combined with RNA sequencing (RNA-Seq) of GlyH-101-treated MCF-7 breast cancer cells, these analyses revealed that CFTR inhibition down-regulates the Wnt and Aurora Kinase A (AURKA) signaling pathways and up-regulates the p53 signaling pathway. Our findings provide insight into CFTR-mediated regulation of cellular quiescence and V-mem in the Artemia model

Sequence and expression analysis of HSP70 family genes in Artemia franciscana

Thus far, only one gene from the heat shock protein 70 (HSP70) family has been identified in Artemia franciscana. Here, we used the draft Artemia transcriptome database to search for other genes in the HSP70 family. Four novel HSP70 genes were identified and designated heat shock cognate 70 (HSC70), heat shock 70 kDa cognate 5 (HSC70-5), lmmunoglobulin heavy-chain binding protein (BIP), and hypoxia up-regulated protein I (HYOUI). For each of these genes, we obtained nucleotide and deduced amino acid sequences, and reconstructed a phylogenetic tree. Expression analysis revealed that in the juvenile state, the transcription of HSP70 and HSC70 was significantly (P 0.05) induction. Gene expression analysis demonstrated that not all members of the HSP70 family are involved in the response to heat stress and selection and that especially altered expression of HSC70 plays a role in a population selected for increased thermotolerance

The complete mitochondrial genome of Artemia sinica Cai, 1989 (Crustacea : Anostraca) using next-generation sequencing

The complete mitochondrial genome of Artemia sinica was obtained using the next-generation sequencing (NGS) method. The mitochondrial genome is a circular molecule of 15,689 bp in length, with the typical structure of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and 2 ribosomal RNA genes, and a non-coding control region (CR). The base composition is 31.53% A, 18.99% C, 16.50% G, and 32.98% T, with an A + T content of 64.51%. All tRNAs have a cloverleaf structure excepting tRNA-Ser(1), that represents the D-loop structure

Translational research into the effects of cigarette smoke on inflammatory mediators and epithelial TRPV1 in Crohn’s disease

Crohn's disease is a pathological condition of the gastro-intestinal tract, causing severe transmural inflammation in the ileum and/or colon. Cigarette smoking is one of the best known environmental risk factors for the development of Crohn's disease. Nevertheless, very little is known about the effect of prolonged cigarette smoke exposure on inflammatory modulators in the gut. We examined the effect of cigarette smoke on cytokine profiles in the healthy and inflamed gut of human subjects and in the trinitrobenzene sulphonic acid mouse model, which mimics distal Crohn-like colitis. In addition, the effect of cigarette smoke on epithelial expression of transient receptor potential channels and their concurrent increase with cigarette smoke-augmented cytokine production was investigated. Active smoking was associated with increasedIL-8transcription in ileum of controls (p < 0,001; n = 18-20/group). In the ileum, TRPV1 mRNA levels were decreased in never smoking Crohn's disease patients compared to healthy subjects (p <0,001; n = 20/group). In the colon, TRPV1 mRNA levels were decreased (p = 0,046) in smoking healthy controls (n = 20/group). Likewise, healthy mice chronically exposed to cigarette smoke (n = 10/group) showed elevated ilealCxcl2(p = 0,0075) and colonicKcmRNA levels (p = 0,0186), whereas TRPV1 mRNA and protein levels were elevated in the ileum (p = 0,0315). Although cigarette smoke exposure prior to trinitrobenzene sulphonic acid administration did not alter disease activity, increased pro-inflammatory cytokine production was observed in the distal colon (Kc: p = 0,0273; Cxcl2: p = 0,104; Il1-beta: p = 0,0796), in parallel with the increase ofTrpv1mRNA (p < 0,001). We infer that CS affects pro-inflammatory cytokine expression in healthy and inflamed gut, and that the simultaneous modulation of TRPV1 may point to a potential involvement of TRPV1 in cigarette smoke-induced production of inflammatory mediators

Exploring the Correlation Between Fibrosis Biomarkers and Clinical Disease Severity in PLN p.Arg14del Patients

Background: Pathogenic variants in phospholamban (PLN, like p. Arg14del), are found in patients diagnosed with arrhythmogenic (ACM) and dilated cardiomyopathy (DCM). Fibrosis formation in the heart is one of the hallmarks in PLN p.Arg14del carriers. During collagen synthesis and breakdown, propeptides are released into the circulation, such as procollagen type I carboxy-terminal propeptide (PICP) and C-terminal telopeptide collagen type I (ICTP). Aim: To investigate if PICP/ICTP levels in blood are correlative biomarkers for clinical disease severity and outcome in PLN p.Arg14del variant carriers. Methods: Serum and EDTA blood samples were collected from 72 PLN p.Arg14del carriers (age 50.5 years, 63% female) diagnosed with ACM (n = 12), DCM (n = 14), and preclinical variant carriers (n = 46). PICP levels were measured with an enzyme-linked immune sorbent assay and ICTP with a radio immuno-assay. Increased PICP/ICTP ratios suggest a higher collagen deposition. Clinical data including electrocardiographic, and imaging results were adjudicated from medical records. Results: No correlation between PICP/ICTP ratios and late gadolinium enhancement (LGE) was found. Moderate correlations were found between the PICP/ICTP ratio and end-diastolic/systolic volume (both r(s) = 0.40, n = 23, p = 0.06). PICP/ICTP ratio was significantly higher in patients with T wave inversion (TWI), especially in leads V4–V6, II, III, and aVF (p < 0.022) and in patients with premature ventricular contractions (PVCs) during an exercise tolerance test (p = 0.007). Conclusion: High PICP/ICTP ratios correlated with clinical parameters, such as TWI and PVCs. Given the limited size and heterogeneity of the patient group, additional studies are required to substantiate the incremental prognostic value of these fibrosis biomarkers in PLN p.Arg14del patients

The effect of cigarette smoke exposure on the development of inflammation in lungs, gut and joints of TNFΔARE mice

The inflammatory cytokine TNF-alpha is a central mediator in many immune-mediated diseases, such as Crohn's disease (CD), spondyloarthritis (SpA) and chronic obstructive pulmonary disease (COPD). Epidemiologic studies have shown that cigarette smoking (CS) is a prominent common risk factor in these TNF-dependent diseases. We exposed TNF Delta ARE mice; in which a systemic TNF-alpha overexpression leads to the development of inflammation; to 2 or 4 weeks of air or CS. We investigated the effect of deregulated TNF expression on CS-induced pulmonary inflammation and the effect of CS exposure on the initiation and progression of gut and joint inflammation. Upon 2 weeks of CS exposure, inflammation in lungs of TNF Delta ARE mice was significantly aggravated. However, upon 4 weeks of CS-exposure, this aggravation was no longer observed. TNF Delta ARE mice have no increases in CD4+ and CD8+ T cells and a diminished neutrophil response in the lungs after 4 weeks of CS exposure. In the gut and joints of TNF Delta ARE mice, 2 or 4 weeks of CS exposure did not modulate the development of inflammation. In conclusion, CS exposure does not modulate gut and joint inflammation in TNF Delta ARE mice. The lung responses towards CS in TNF Delta ARE mice however depend on the duration of CS exposure