High-throughput analysis of the RNA-induced silencing complex in myotonic dystrophy type 1 patients identifies the dysregulation of miR-29c and its target ASB2

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2

Black Soldier Fly live larvae as environmental enrichment in medium-growing chicken diet

Introduction. Few studies on the effects of live larvae provision in poultry have been previously conducted [1,2,3]. However, trials on the long-term provision of live larvae in chicken reared for meat consumption have never been performed before. This study evaluated the impact of Black Soldier Fly (BSF) live larvae provision on growth performance and larvae consumption behavior of intermediate-growing strains. Material and methods. A total of 240 Label naked neck birds were reared from 21 to 82 days of age, and four experimental groups (10 birds/pen, 6 replicates/treatment) were considered according to the birds’ gender and larvae provision. Experimental groups were fed 10% supplementation of BSF live larvae based on the daily feed intake. The live weight (LW), feed conversion ratio (FCR), average daily feed intake (ADFI) and average daily gain (ADG) were evaluated considering two periods: 21-35d and 35-82d. The larvae were provided daily and consumption times were analyzed considering periods of 10 days (5 time frame-T1,T2,T3,T4,T5). Data were analyzed by means of a GLMM (SPSS software, P<0.05). Results. The larvae groups displayed a lower ADFI than the control groups regardless the birds’ gender at 21-35d (P=0.01). This could be explained by the larvae nutritional contribution that led to a lower feed consumption in the experimental groups. Moreover, treated birds revealed a lower FCR than control groups (21-35d; P<0.001). Otherwise, only treated males performed a better FCR than control groups during the second period (P<0.01). Overall, time of larvae consumption at T1 and T5was respectively higher and lower than the other considered periods in both sexes (P<0.05). Such differences could be related to a progressive birds’ adaptation to larvae consumption. Significant differences between sexes were recorded only at T5, when females employed much time than males in larvae consumption (P<0.05). Conclusion. Live larvae provision ameliorated both the ADFI and FCR. Furthermore, the time of larvae consumption shrinked as birds became older. References. [1] Star L. et al. (2020). Animals. 10,216. [2] Bellezza Oddon et al. (2021). J. Anim. Physiol. Anim. 00,1–9. [3] Veldkamp T. and T.G.C.M. Van Niekerk (2019). J. Insects as Food Feed. 5,301-31

Blood chemistry of medium-growing male and female chickens supplemented black soldier fly live larvae

Effects of live larvae provision on poultry chemical blood parameters have been poorly investigated. This study aims to evaluate the changes in blood chemistry parameters in medium-growing chickens supplemented black soldier fly (BSF) live larvae. Two hundred and forty 21d old sexed Label Naked Neck birds were divided into 4 experimental groups: females fed basal organic feed (BOF), males fed BOF, females fed BOF + 10% BSF live larvae supplementation based on the expected daily feed intake (DFI) and males fed BOF + 10% BSF live larvae supplementation based on the DFI (6 replicates/diet, 10 birds/replicate). Blood samples were collected at slaughter (82d old) from 2 birds/pen (12 birds/treatment). Serum samples were used for biochemical analysis. A compact liquid chemistry analyzer system (BT 1500 vet–Futurlab) was used to determine the concentrations of alanine aminotransferase (U/I), aspartate aminotransferase (U/I), creatinine total proteins (mg/dl), uric acid (mg/dl), cholesterol (mg/dl), triglycerides (mg/dl), gamma glutamyltransferase (GGT, U/I), phosphorus (mg/dl) and magnesium (mg/dl). Data were analyzed by GLMM (SPSS software, P<0.05). Overall, the blood parameters were not affected by the live larvae supplementation (P>0.05) in both sexes, thus being indicative of a good health status of the birds. Moreover, the GGT was detected in lower concentrations in the supplemented groups than in the control groups (P<0.05), suggesting a positive effect on the hepatic function. In conclusion, the live BSF larvae provision did not negatively affect the blood parameters of medium-growing chickens and could be beneficial for bird hepatic activity

Blood chemistry of medium-growing male and female chickens supplemented black soldier fly live larvae

Effects of live larvae provision on poultry chemical blood parameters have been poorly investigated. This study aims to evaluate the changes in blood chemistry parameters in medium-growing chickens supplemented black soldier fly (BSF) live larvae. Two hundred and forty 21d old sexed Label Naked Neck birds were divided into 4 experimental groups: females fed basal organic feed (BOF), males fed BOF, females fed BOF + 10% BSF live larvae supplementation based on the expected daily feed intake (DFI) and males fed BOF + 10% BSF live larvae supplementation based on the DFI (6 replicates/diet, 10 birds/replicate). Blood samples were collected at slaughter (82d old) from 2 birds/pen (12 birds/treatment). Serum samples were used for biochemical analysis. A compact liquid chemistry analyzer system (BT 1500 vet–Futurlab) was used to determine the concentrations of alanine aminotransferase (U/I), aspartate aminotransferase (U/I), creatinine total proteins (mg/dl), uric acid (mg/dl), cholesterol (mg/dl), triglycerides (mg/dl), gamma glutamyltransferase (GGT, U/I), phosphorus (mg/dl) and magnesium (mg/dl). Data were analyzed by GLMM (SPSS software, P<0.05). Overall, the blood parameters were not affected by the live larvae supplementation (P>0.05) in both sexes, thus being indicative of a good health status of the birds. Moreover, the GGT was detected in lower concentrations in the supplemented groups than in the control groups (P<0.05), suggesting a positive effect on the hepatic function. In conclusion, the live BSF larvae provision did not negatively affect the blood parameters of medium-growing chickens and could be beneficial for bird hepatic activity

Reducing contrast agent residuals in hospital wastewater: the GREENWATER study protocol

Abstract The potential enviromental impact of iodinated (ICAs) and gadolinium-based contrast agents (GBCAs) have recently come under scrutiny, considering the current nonselective wastewater treatment. However, their rapid excretion after intravenous administration could allow their potential recovery by targeting hospital sewage. The GREENWATER study aims to appraise the effective quantities of ICAs and GBCAs retrievable from patients’ urine collected after computed tomography (CT) and magnetic resonance imaging (MRI) exams, selecting ICA/GBCA per-patient urinary excretion and patients’ acceptance rate as study endpoints. Within a prospective, observational, single-centre, 1-year framework, we will enrol outpatients aged ≥ 18 years, scheduled to perform contrast-enhanced CT or MRI, willing to collect post-examination urine in dedicated canisters by prolonging their hospital stay to 1 h after injection. Collected urine will be processed and partially stored in the institutional biobank. Patient-based analysis will be performed for the first 100 CT and 100 MRI patients, and then, all analyses will be conducted on the pooled urinary sample. Quantification of urinary iodine and gadolinium will be performed with spectroscopy after oxidative digestion. The evaluation of the acceptance rate will assess the “environmental awareness” of patients and will aid to model how procedures to reduce ICA/GBCA enviromental impact could be adapted in different settings. Key points • Enviromental impact of iodinated and gadolinium-based contrast agents represents a growing point of attention. • Current wastewater treatment is unable to retrieve and recycle contrast agents. • Prolonging hospital stay may allow contrast agents retrieval from patients’ urine. • The GREENWATER study will assess the effectively retrievable contrast agents’ quantities. • The enrolment acceptance rate will allow to evaluate patients’ “green sensitivity”

Receptor and post-receptor abnormalities contribute to insulin resistance in myotonic dystrophy type 1 and type 2 skeletal muscle

<div><p>Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant multisystemic disorders caused by expansion of microsatellite repeats. In both forms, the mutant transcripts accumulate in nuclear foci altering the function of alternative splicing regulators which are necessary for the physiological mRNA processing. Missplicing of insulin receptor (IR) gene (<i>INSR</i>) has been associated with insulin resistance, however, it cannot be excluded that post-receptor signalling abnormalities could also contribute to this feature in DM. We have analysed the insulin pathway in skeletal muscle biopsies and in myotube cultures from DM patients to assess whether downstream metabolism might be dysregulated and to better characterize the mechanism inducing insulin resistance. DM skeletal muscle exhibits alterations of basal phosphorylation levels of Akt/PKB, p70S6K, GSK3β and ERK1/2, suggesting that these changes might be accompanied by a lack of further insulin stimulation. Alterations of insulin pathway have been confirmed on control and DM myotubes expressing fetal <i>INSR</i> isoform (<i>INSR-A</i>). The results indicate that insulin action appears to be lower in DM than in control myotubes in terms of protein activation and glucose uptake. Our data indicate that post-receptor signalling abnormalities might contribute to DM insulin resistance regardless the alteration of <i>INSR</i> splicing.</p></div

Insulin receptor alternative splicing and insulin signalling in <i>biceps brachii</i> and <i>tibialis anterior</i> muscles of DM1 patients.

<p><b>(A)</b><i>INSR</i> splicing products obtained by RT-PCR amplification of RNA isolated from <i>biceps brachii</i> and <i>tibialis anterior</i> biopsies obtained from CTR and DM1 patients. Bands were quantified and proportions of isoform <i>INSR-B</i> (+exon 11) were calculated. (<b>B)</b> Representative western blot analysis of the expression of proteins involved in the insulin pathway in <i>biceps brachii</i> and <i>tibialis anterior</i> biopsies obtained from CTR and DM1 patients. Due to the high number of samples analysed, the figure panel is composed by images of bands originating from different gels. Details on how western blot experiments were performed are reported in Materials and Methods. (<b>C)</b> Quantification of proteins expression normalized to GAPDH in <i>tibialis anterior</i> biopsies obtained from CTR and DM1 patients. Histograms represent mean values and bars represent standard error of the mean (SEM). (<b>D)</b> Quantification of proteins expression normalized to GAPDH in <i>tibialis anterior</i> and <i>biceps brachii</i> biopsies obtained from two DM1 patients who performed two successive biopsies at different years of age. Data obtained from one BB and one TA sample from DM1-1 and DM1-4 were compared with mean values obtained from 2 CTR BB and 3 CTR TA. Bars represent standard error of the mean (SEM). The number of samples analysed in each group (n) is reported in histogram legends. Differences between groups have been evaluated by Student <i>t</i>-test. *p<0.05; **p<0.01.</p

Dysregulation of circular RNAs in myotonic dystrophy type 1

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by “back-splicing”, which is the linking covalently of 3′- and 5′-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients

Histopathological features of skeletal muscle in DM patients.

<p>Histopathological features of skeletal muscle in DM patients.</p

Insulin signalling activation in muscle cells.

<p>(<b>A)</b> Representative western blot analysis of the expression and activation of proteins involved in the insulin pathway. Myotubes (T5) were cultured in absence or in presence of 10 nM insulin for 5 to 30 minutes. Quantification of IRS1 activation <b>(B)</b>, of Akt/PKB <b>(C)</b>, p70S6K <b>(D)</b>, GSK3β <b>(E)</b>, ERK1 <b>(F)</b> and ERK2 <b>(G)</b>. Histograms represents mean values and bars represent standard error of the mean (SEM). The number of samples analysed in each group (n) is reported in histogram legends. The effect of time within groups was assessed by one-way ANOVA (repeated measures). Results from post-test are marked with connecting capped arcs. *Results from Student <i>t</i>-test DM1 or DM2 <i>versus</i> CTR at 0-5-15-30 minutes of insulin stimulation: *p<0.05, **p<0.01.</p