Identification of differentially expressed small RNAs and prediction of target genes in Italian Large White pigs with divergent backfat deposition

The identification of the molecular mechanisms regulating pathways associated with the potential for fat deposition in pigs can lead to the detection of key genes and markers for the genetic improvement of fat traits. Interactions of microRNAs (miRNAs) with target RNAs regulate gene expression and modulate pathway activation in cells and tissues. In pigs, miRNA discovery is far from saturation, and the knowledge of miRNA expression in backfat tissue and particularly of the impact of miRNA variations is still fragmentary. Using RNA-seq, we characterized the small RNA (sRNA) expression profiles in Italian Large White pig backfat tissue. Comparing two groups of pigs divergent for backfat deposition, we detected 31 significant differentially expressed (DE) sRNAs: 14 up-regulated (including ssc-miR-132, ssc-miR-146b, ssc-miR-221-5p, ssc-miR-365-5p and the moRNA ssc-moR-21-5p) and 17 down-regulated (including ssc-miR-136, ssc-miR-195, ssc-miR-199a-5p and ssc-miR-335). To understand the biological impact of the observed miRNA expression variations, we used the expression correlation of DE miRNA target transcripts expressed in the same samples to define a regulatory network of 193 interactions between DE miRNAs and 40 DE target transcripts showing opposite expression profiles and being involved in specific pathways. Several miRNAs and mRNAs in the network were found to be expressed from backfat-related pig QTL. These results are informative for the complex mechanisms influencing fat traits, shed light on a new aspect of the genetic regulation of fat deposition in pigs and facilitate the prospective implementation of innovative strategies of pig genetic improvement based on genomic markers

Pilot study of the relationship between deck level and journey duration on plasma cortisol, epinephrine and norepinephrine levels in italian heavy pigs

The aim of this pilot study was to evaluate the relationship between journey duration, deck level and activation patterns of the hypothalamic-pituitary-adrenocortical axis (HPA) and sympathetic adrenal medullary system (SAM) in pigs. A total of 90 pigs were examined. The animals came from three different Italian farms associated with the same slaughterhouse located in Bari (Apulia region-Italy). A group of thirty animals was transported from Pordenone (11 h journey); a second group was transported from Terni (6.5 h journey); a third group was transported from Benevento (3 h journey). The animals were transported in the same vehicle, which complied with the structural characteristics indicated in the Council Regulation (EC) No. 1/2005. The truck was composed of a lorry and a trailer, each one divided into three decks. Only the animals transported in the trailer were tested for the study. Before transportation, blood samples were collected on each farm, at 6:00 a.m., from 30 pigs randomly selected out of 135 pigs ready to be transported. Blood samples were also collected during slaughter to evaluate plasma cortisol, epinephrine and norepinephrine, around 6:00 a.m. A journey duration of 11 h was associated with significantly higher plasma concentrations of stress hormones compared with shorter journeys. This increase was proportional to the journey duration, with the pigs travelling for 6.5 h displaying intermediate concentrations between those noticed after 3 h and 11 h journeys. The interaction between deck and journey distance was not significant on epinephrine, norepinephrine or cortisol levels collected at arrival. There was a significant effect of deck level on norepinephrine levels (p < 0.0001), a tendency to influence epinephrine levels (p = 0.073) but no effect on cortisol levels (p = 0.945). Overall, we observed that an 11 h-long journey seemed to impact negatively on pigs\u2019 HPA-SAM activity, likely requiring the animals to spend more time in the lairage facilities to recover

Tailoring the CRISPR system to transactivate coagulation gene promoters in normal and mutated contexts

Engineered transcription factors (TF)have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa)technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNA F7.5 )triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold)than that of an engineered TALE-TF (TF4)targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNA F7.5 combination was more efficient (~6.5-fold)in promoting factor VII (FVII)protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNA F8.1 , ~8-fold and 3-fold; sgRNA F8.2 , ~19-fold and 2-fold)with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models

A Cretaceous carbonate delta drift in the Montagna della Maiella, Italy

The Upper Cretaceous (Campanian\u2013Maastrichtian) bioclastic wedge of the Orfento Formation in the Montagna della Maiella, Italy, is compared to newly discovered contourite drifts in the Maldives. Like the drift deposits in the Maldives, the Orfento Formation fills a channel and builds a Miocene delta-shaped and mounded sedimentary body in the basin that is similar in size to the approximately 350 km 2 large coarse-grained bioclastic Miocene delta drifts in the Maldives. The composition of the bioclastic wedge of the Orfento Formation is also exclusively bioclastic debris sourced from the shallow-water areas and reworked clasts of the Orfento Formation itself. In the near mud-free succession, age-diagnostic fossils are sparse. The depositional textures vary from wackestone to float-rudstone and breccia/conglomerates, but rocks with grainstone and rudstone textures are the most common facies. In the channel, lensoid convex-upward breccias, cross-cutting channelized beds and thick grainstone lobes with abundant scours indicate alternating erosion and deposition from a high-energy current. In the basin, the mounded sedimentary body contains lobes with a divergent progradational geometry. The lobes are built by decametre thick composite megabeds consisting of sigmoidal clinoforms that typically have a channelized topset, a grainy foreset and a fine-grained bottomset with abundant irregular angular clasts. Up to 30 m thick channels filled with intraformational breccias and coarse grainstones pinch out downslope between the megabeds. In the distal portion of the wedge, stacked grainstone beds with foresets and reworked intraclasts document continuous sediment reworking and migration. The bioclastic wedge of the Orfento Formation has been variously interpreted as a succession of sea-level controlled slope deposits, a shoaling shoreface complex, or a carbonate tidal delta. Current-controlled delta drifts in the Maldives, however, offer a new interpretation because of their similarity in architecture and composition. These similarities include: (i) a feeder channel opening into the basin; (ii) an excavation moat at the exit of the channel; (iii) an overall mounded geometry with an apex that is in shallower water depth than the source channel; (iv) progradation of stacked lobes; (v) channels that pinch out in a basinward direction; and (vi) smaller channelized intervals that are arranged in a radial pattern. As a result, the Upper Cretaceous (Campanian\u2013Maastrichtian) bioclastic wedge of the Orfento Formation in the Montagna della Maiella, Italy, is here interpreted as a carbonate delta drift

Oncogenic Transformation by Inhibitor-Sensitive and -Resistant EGFR Mutants

BACKGROUND: Somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene EGFR are common in lung adenocarcinoma. The presence of mutations correlates with tumor sensitivity to the EGFR inhibitors erlotinib and gefitinib, but the transforming potential of specific mutations and their relationship to drug sensitivity have not been described. METHODS AND FINDINGS: Here, we demonstrate that EGFR active site mutants are oncogenic. Mutant EGFR can transform both fibroblasts and lung epithelial cells in the absence of exogenous epidermal growth factor, as evidenced by anchorage-independent growth, focus formation, and tumor formation in immunocompromised mice. Transformation is associated with constitutive autophosphorylation of EGFR, Shc phosphorylation, and STAT pathway activation. Whereas transformation by most EGFR mutants confers on cells sensitivity to erlotinib and gefitinib, transformation by an exon 20 insertion makes cells resistant to these inhibitors but more sensitive to the irreversible inhibitor CL-387,785. CONCLUSION: Oncogenic transformation of cells by different EGFR mutants causes differential sensitivity to gefitinib and erlotinib. Treatment of lung cancers harboring EGFR exon 20 insertions may therefore require the development of alternative kinase inhibition strategies

Proteomic analysis of urine in medication-overuse headache patients: possible relation with renal damages

Medication-overuse headache (MOH) is a chronic disorder associated with overuse of analgesic drugs, triptans, non-steroidal anti-inflammatory drugs (NSAIDs) or other acute headache compounds. Various epidemiologic investigations proved that different drug types could cause nephrotoxicity, particularly in chronic patients. The aim of the present work was to analyze, by a proteomic approach, the urinary protein profiles of MOH patients focusing on daily use of NSAIDs, mixtures and triptans that could reasonably be related to potential renal damage. We selected 43 MOH patients overusing triptans (n = 18), NSAIDs (n = 11), and mixtures (n = 14), for 2–30 years with a mean daily analgesic intake of 1.5 ± 0.9 doses, and a control group composed of 16 healthy volunteers. Urine proteins were analyzed by mono-dimensional gel electrophoresis and identified by mass spectrometry analysis. Comparing the proteomic profiles of patients and controls, we found a significantly different protein expression, especially in the NSAIDs group, in which seven proteins resulted over-secreted from kidney (OR = 49, 95% CI 2.53–948.67 vs. controls; OR = 11.6, 95% CI 0.92–147.57 vs. triptans and mixtures groups). Six of these proteins (uromodulin, α-1-microglobulin, zinc-α-2-glycoprotein, cystatin C, Ig-kappa-chain, and inter-α-trypsin heavy chain H4) were strongly correlated with various forms of kidney disorders. Otherwise, in mixtures and in triptans abusers, only three proteins were potentially associated to pathological conditions (OR = 4.2, 95% CI 0.33–53.12, vs. controls). In conclusion, this preliminary proteomic study allowed us to define the urinary protein pattern of MOH patients that is related to the abused drug. According with the obtained results, we believe that the risk of nephrotoxicity should be considered particularly in MOH patients who abuse of NSAIDs

The C. elegans Opa1 Homologue EAT-3 Is Essential for Resistance to Free Radicals

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants

Manual therapy for chronic migraine: a pragmatic randomised controlled trial study protocol

Introduction Chronic migraine is a largely refractory condition affecting between 1 and 2.2% of the overall population worldwide, with females more affected than males. There are also high health and socioeconomic costs associated both for the individual and society. The mainstay of chronic migraine management is pharmacological, but the options available have limited efficacy and there are often unwanted side effects. There is some evidence for manual therapy as a treatment option for migraine, but its effectiveness for chronic migraine is unknown. Therefore, we have designed a pragmatic randomised control trial to investigate whether adding manual therapy to the tertiary specialist treatment of chronic migraine improves patient-reported outcomes. Methods A pragmatic, randomised controlled trial in a hospital tertiary headache clinic. Participants will be randomised into one of two groups: treatment as usual or treatment as usual plus manual therapy. The primary outcome measure will be a change in the Headache Impact Test score. Secondary outcomes will also be measured over the 12-week study period including changes in headache frequency, migraine specific quality of life and reductions in relevant medicine consumption. The manual therapy group will have five treatment sessions each lasting 30 min. The recruitment target of 64 participants will allow power at 80% with p = 0.05 using minimal clinical difference for Headache Impact Test of 3.7 and includes provision for a 10% dropout rate. Recruitment will take place between August 2018 and February 2019. The results will form part of a doctoral study and be published in peer-reviewed journals and presented at national/international conferences. Discussion Current pharmacological approaches have limited effects in the management of chronic migraine and there is a requirement to improve treatment options and reduce the health and economic burden of the condition. Manual therapy has been shown to be effective in other chronic pain conditions as well as other primary headaches. This study will explore the effectiveness of manual therapy as an adjunctive approach to the management of chronic migraine. Trial registration The trial has received a favourable opinion from the UK Health Research Authority (IRAS 228901) and is registered at ClinicalTrials.gov.number NCT03395457. Registered 1st March 2018

Biocatalytic approach for direct esterification of ibuprofen with sorbitol in biphasic media

Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) introduced in the 1960s and widely used as an analgesic, anti-inflammatory, and antipyretic. In its acid form, the solubility of 21 mg/L greatly limits its bioavailability. Since the bioavailability of a drug product plays a critical role in the design of oral administration dosage, this study investigated the enzymatic esterification of ibuprofen as a strategy for hydrophilization. This work proposes an enzymatic strategy for the covalent attack of highly hydrophilic molecules using acidic functions of commercially available bioactive compounds. The poorly water-soluble drug ibuprofen was esterified in a hexane/water biphasic system by direct esterification with sorbitol using the cheap biocatalyst porcine pancreas lipase (PPL), which demonstrated itself to be a suitable enzyme for the effective production of the IBU-sorbitol ester. This work reports the optimization of the esterification reaction