Knockdown of plakophilin 2 downregulates MIR-184 through CpG hypermethylation and suppression of the E2F1 pathway and leads to enhanced adipogenesis in vitro

Rationale: PKP2, encoding plakophilin 2 (PKP2), is the most common causal gene for arrhythmogenic cardiomyopathy. Objective: To characterize miRNA expression profile in PKP2-deficient cells. Methods and results: Control and PKP2-knockdown HL-1 (HL-1(Pkp2-shRNA)) cells were screened for 750 miRNAs using low-density microfluidic panels. Fifty-nine miRNAs were differentially expressed. MiR-184 was the most downregulated miRNA. Expression of miR-184 in the heart and cardiac myocyte was developmentally downregulated and was low in mature myocytes. MicroRNA-184 was predominantly expressed in cardiac mesenchymal progenitor cells. Knockdown of Pkp2 in cardiac mesenchymal progenitor cells also reduced miR-184 levels. Expression of miR-184 was transcriptionally regulated by the E2F1 pathway, which was suppressed in PKP2-deficient cells. Activation of E2F1, on overexpression of its activator CCND1 (cyclin D1) or knockdown of its inhibitor retinoblastoma 1, partially rescued miR-184 levels. In addition, DNA methyltransferase-1 was recruited to the promoter region of miR-184, and the CpG sites at the upstream region of miR-184 were hypermethylated. Treatment with 5-aza-2'-deoxycytidine, a demethylation agent, and knockdown of DNA methyltransferase-1 partially rescued miR-184 level. Pathway analysis of paired miR-184:mRNA targets identified cell proliferation, differentiation, and death as the main affected biological processes. Knockdown of miR-184 in HL-1 cells and mesenchymal progenitor cells induced and, conversely, its overexpression attenuated adipogenesis. Conclusions: PKP2 deficiency leads to suppression of the E2F1 pathway and hypermethylation of the CpG sites at miR-184 promoter, resulting in downregulation of miR-184 levels. Suppression of miR-184 enhances and its activation attenuates adipogenesis in vitro. Thus, miR-184 contributes to the pathogenesis of adipogenesis in PKP2-deficient cells

Aloe arborescens supplementation in cat diet: Evaluation of effects by in vitro gas production technique

The aim of the present study was to evaluate the effects of Aloe arborescens on organic matter digestibility (OMD), cumulative gas (OMCV) and short chain fatty acids (SCFA) production, using the in vitro gas production technique (IVGPT). Three adult cats were fed with a commercial diet (CP 31.21; EE 16.64% as fed) for 20 days before the collection of their faeces used as inoculum. The same diet, used as substrate, was incubated in vitro supplemented with different amounts (0, 0.7, 1.6 and 3.2%) of lyophilised Aloe arborescens. OMD, OMCV and SCFA significantly decreased with the increase of Aloe addition; an increase of L-lactic acid production was detected, even if pH was within physiological range. A potential prebiotic role of the Aloe arborescens car- bohydrates was hypothesised in cats, but it needs further investigations. As a whole, our results show that IVGPT can represent a useful tool for nutritional evaluation of novel ingredient and/or additive also in cats

Influence of Feeding Linseed on SCD Activity in Grazing Goat Mammary Glands

The effects of linseed feeding on the stearoyl-CoA desaturase (SCD) activity were evaluated on grazing dairy goats divided into two homogeneous groups (C, control, and L, treated) fed the same amount of concentrate which, for group L was supplemented with linseed. Milk yield was unaffected by the treatment. Group L showed significantly higher milk fat (4.10% vs 2.94%, p < 0.01) than group S. Within milk fatty acids, group C showed significantly higher levels of saturated fatty acids and lower values of mono-unsaturated and polyunsaturated fatty acids. In group L, total CLAs were higher than in group S (0.646% vs 0.311%; p < 0.01) mainly because of the differences in CLA cis9 trans 11 (0.623% vs 0.304%; p < 0.01). In treated animals, SCD activity, measured as cis9 C14:1/C14:0, was lower than in the control group, mainly in July and August

Nutritional Characterization of Hay Produced in Campania Region: Analysis by the near Infrared Spectroscopy (NIRS) Technology

: Since the dietary characteristics of hays can be very variable, it is of great importance for nutritionists to know their chemical composition in order to formulate adequate rations for the animals. Laboratory analyses are time-consuming and expensive while the Near Infrared Spectroscopy offers several advantages, including obtaining information on feeds nutritional characteristics very quickly and in situ at the farm, thanks to portable NIRS. In this trial, over 400 hay samples collected in the Campania region (Italy) were analyzed with portable NIRS device. The final aim was to analyze the differences in hay quality in different production areas, highlighting the possible factors involved and suggesting possible corrective measures. All the analyzed hays (polyphite, Gramineae and alfalfa) were significantly (p < 0.05) influenced by the area of cultivation/preservation. In particular, the polyphite and Gramineae hays produced in most of the areas of Campania region showed poor nutritional value due to the low protein content and high structural carbohydrate that significantly reduced its digestibility. The use of high-quality forages in the ration represents a pivotal factor to allow the production of high-quality products of animal origin. The use of NIRS seems to be a valuable strategy to select potential treatments that can increase feed digestibility and to avoid long chemical analysis

Small fiber neuropathy is a common feature of Ehlers-Danlos syndromes

To investigate the involvement of small nerve fibers in Ehlers-Danlos syndrome (EDS). Patients diagnosed with EDS underwent clinical, neurophysiologic, and skin biopsy assessment. We recorded sensory symptoms and signs and evaluated presence and severity of neuropathic pain according to the Douleur Neuropathique 4 (DN4) and ID Pain questionnaires and the Numeric Rating Scale (NRS). Sensory action potential amplitude and conduction velocity of sural nerve was recorded. Skin biopsy was performed at distal leg and intraepidermal nerve fiber density (IENFD) obtained and referred to published sex- and age-adjusted normative reference values. Our cohort included 20 adults with joint hypermobility syndrome/hypermobility EDS, 3 patients with vascular EDS, and 1 patient with classic EDS. All except one patient had neuropathic pain according to DN4 and ID Pain questionnaires and reported 7 or more symptoms at the Small Fiber Neuropathy Symptoms Inventory Questionnaire. Pain intensity was moderate (NRS ≥4 and <7) in 8 patients and severe (NRS ≥7) in 11 patients. Sural nerve conduction study was normal in all patients. All patients showed a decrease of IENFD consistent with the diagnosis of small fiber neuropathy (SFN), regardless of the EDS type. SFN is a common feature in adults with EDS. Skin biopsy could be considered an additional diagnostic tool to investigate pain manifestations in EDS