Lepeophtheirus salmonis  and systematically characterised its function and complex abnormalities emerging after LsalMS knockdown by RNAi in all developmental stages in this species. Immunohistochemistry analysis localized the LsalMS mainly to the central nervous system, but also to the vital organs within the alimentary tract and the reproductive system. The most striking feature of LsalMS deficiency during lice development was severe reduction of the muscle content, with abnormalities detected in both the visceral and skeletal muscles. Moreover, down-regulation of LsalMS affects moulting, spermatophore deposition and feeding by affecting development of the intestinal wall and increasing its contraction frequency.Muscle activity is regulated by stimulatory and inhibitory neuropeptides allowing for contraction and relaxation. In Arthropods, one of the important myoinhibitors is Myosuppressin, belonging to FMRFamide-like peptides, that was shown to have inhibitory effects on visceral muscle contraction and to regulate vital physiological processes including reproduction or feeding. We have identified myosuppressin in salmon louse  Lepeophtheirus salmonis, (Copepoda: Crustacea), is a marine ecto-parasite of Atlantic salmon  that causes large economic loss to the farmed salmonids1 and constitute a major threat to the wild fish populations2. Salmon louse feeds on the host’s mucus, skin and blood, decreasing fish growth and causing lesions that constitute the gateway for other pathogens3. Until now, fight against salmon louse has been based mainly on the medicinal treatment and various physical methods4. However, the medicine-based delousing strategy has progressively lost the efficacy, since the lice have developed resistance against the various chemotherapeutics5, while, physical methods cause stress and damage to the fish, leading to high post-treatment mortality. Therefore, there is an urgent need to develop new and efficient means to control salmon louse infections.The salmon louse, 8. Neuropeptides, their receptors and enzymes involved in their metabolism, have long been considered as potential targets for the development of new chemotherapeutics, due to their various physiological and behavioural functions, conserved structure and widespread distribution in Arthropods, and the possibility that such targets could deliver specific and environment friendly treatment13. However, until now, no commercial insecticide utilising neuropeptide’s agonists or antagonists has been discovered or used, mainly due to the missing information and lack of full understanding of neuropeptide and their receptors function and structure. To strengthen the development of such pest control strategy, knowledge about neuropeptide composition, their biological activities and receptor-neuropeptide interactions is of critical importance13.In recent years, increased interest has been aimed at endocrine-based pest control strategy that involves development of drugs targeting neuropeptides in invertebratesNeuropeptides are an important class of messengers and hormones acting as critical signal transducers and modulators in a wide range of developmental, physiological and behavioural processes. The variety of functions neuropeptides play in the animals suggest that alternations in peptide expression, synthesis, or signalling could result in abnormalities or impaired function resulting in decreased fitness or death of parasite such as the salmon louse.19. The first myosuppressin was isolated from the extracts of the nervous tissues of the cockroach Leucophaea maderae 21, and subsequently identical decapeptides were isolated from other cockroaches23. Similar decapeptides, which differ from LMS by only one amino acid residue in the N-terminal end of the peptide, have been isolated and sequenced in the flies Neobellieria bullata24 and Drosophila melanogaster 25, the beetle Zophobas atratus14, and the locusts Schistocerca gregaria and Locusta migratoria31.One of the important neuropeptides found in Arthropods’ genomes is Myosuppressin (MS). Numerous myosuppressin precursors have been identified through cloned DNA or predicted from the genomes in a wide range of invertebrates1DVX4HX6FLRFamide 34. The last six amino acids of the decapeptide constitute the active core that is identical in all Arthropods studied so far and sufficient for antimyotropic activity30. Myosuppressin decapeptides mediate their function by binding to myosuppressin receptors that belong to the G-protein coupled receptors36.Myosuppressin belongs to the FMRFamide-like peptides family with the consensus decapeptide sequence at the C-terminal end of X39. Apparently, it plays an important role in digestion by inhibiting peristaltic contraction of the alimentary tract42, and food intake23. It stimulates invertase and α-amylase release in the midgut44, and increases free sugar level in the haemolymph45. It plays an important role in reproduction by inhibiting muscles responsible for ovulation and egg-laying49, and the contractions of the ejaculatory duct14. Furthermore, myosuppressin decreases cardiac contractility39 in a dose-dependent manner50. Recently, it was shown that myosuppressin from Mamestra brassicae is involved in regulation of prothoracic glands activity and plays an important role in the initiation and maintenance of pupal diapause51.Myosuppressin belongs to the myoinhibitory peptides and was shown to have pleiotropic functions in regulating activity of the visceral musclesLepeophtheirus salmonis , from free-living planktonic stages to parasitic stages. We knocked-down LsalMS by RNA interference at various developmental time points and looked for developmental and behavioural abnormalities. Using MS-specific antisera and quantitative qRT-PCR we mapped gene expression and peptide localization to nervous system, reproductive system and alimentary tract.The majority of publications describe structure-activity relationship and function of myosuppressin in controlling various muscles, whereas, myosuppressin’s role in development, and effects of its deficiency or dysfunction are poorly investigated. Thus, we investigated myosuppressin function in the animal development in a number of developmental stages of salmon louse Lepeophtheirus salmonis  was identified by the standard Blastp search of the collection of protein predictions from expressed sequence tags (EST)  using Drosophila melanogaster myosuppressin peptide (AAF56283.1) as the query sequences. The result revealed one candidate with low e-value (e = 7e-08). To obtain the full cDNA sequences of the identified gene, we carried out 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) using PCR primers specific for exon sequences obtained from sealouse.imr.no database . The full cDNA sequence of LsalMS is 763 nucleotides long. It contains a 5′-UTR of 154 bp and a 3′-UTR of 195 bp, including a polyA. The deduced open reading frame is 414 nucleotides long and encoded a prepropeptide of 137 amino acid residues. A signal peptide was identified with the cleavage site located between the 39 and 40 amino acid (S/R), giving a precursor peptide of 98 aa. The deduced amino acid sequence of the decapeptide, identified based on multiple sequence alignment, is flanked by abnormal proteolytic processing sites, tribasic (KRK) and dibasic (KR) sites located before and after the decapeptide52  and ends with a phenylalanine residue (F), and it follows the consensus pattern for myosuppressin peptides  or glutamine (Q), and both amino acids can be changed to pyroglutamic acid during post-translational modifications54. The glycine residue at the end of the decapeptide is necessary for amidation by peptidylglycine α-amidating monooxygenase55. The final sequence of L. salmonis mature peptide is predicted to be pEDVDHVFLRFamide. No transmembrane regions, specific functional domain, or N-glycosylation sites were detected in the predicted peptide sequence.The gene encoding myosuppressin in AAF56283. as the qe52 Fig. . Thus, tdes Figs  and 2a. LsalMS cDNA sequence with the genomic sequence of L. salmonis showed the presence of four exons in the LsalMS gene  by only one amino acid residue at the N-terminal end.The LsalMS groups together with insects, the sister group to the Crustaceans.The propeptide sequences were used to construct a phylogenetic tree  and five parasitic stages, attached to the fish 56. The level of LsalMS in nauplius I was set to 1 and considered as a baseline for quantification of expression in other stages. The lowest expression level of LsalMS transcripts was detected in adult females, whereas the copepodids showed the highest expression levels , which indicates that LsalMS transcripts detected in the genital segment were localised mainly in the oviduct  of LsalMS in larvae treated with dsLsalMS in comparison to the control animals . Development of lice was followed daily by visual observation. While, the control animals developed normally, dsLsalMS treated animals were able to reach chalimus II stage, but none of them were able to reach preadult I stage. The experiment was terminated after 30 days when all dsLsalMS treated lice had disappeared from the fish.While animals treated with dsLsalMS treatment. In total, nine fish were infected with lice from each treatment group. Lice were collected after 8, 12 and 18 days post infection , when control lice were in the middle of chalimus I, chalimus II and preadult I/II stage, respectively. Animals were counted, photographed and carefully examined for potential morphological abnormalities. Similar infection rates were observed in both treatment groups, where in average 18 animals were recovered from each fish at day 8  vs. dsLsalMS 17,7 ) and 27 animals at day 12 post infection  vs. dsLsalMS 26 (±6))  in the number of collected lice was obvious at day 18 post infection, where 96% less dsLsalMS treated lice were recovered from fish than controls, and only 34% of these were at the preadult stage ) Fig. . While cage Fig. .LsalMS transcript level in dsLsalMS treated lice was done with controls in the corresponding developmental stage. Since copepodids were not present in the control group, they were excluded from analysis. To obtain sufficient number of preadult animals, infection was repeated , and all lice were collected on day 18 post infection. On day 8, transcript level was tested in chalimus I (n = 10 for each group), on day 12 in chalimus II (n = 10 for each group) and on day 18 in preadult I males and females (n = 5♀/5♂ for each group). While majority of dsLsalMS treated chalimus I and II revealed significant down-regulation of transcript level, few animals  showed LsalMS transcript levels comparable to controls. On the other hand, all recovered preadult I lice from dsLsalMS treatment group showed expression level similar to control group . Animals were placed on the fish and observed daily. Lost animals were collected from the filters at the water outlet. The experiment was ended after 16 days, when all dsLsalMS treated females were lost from the fish and only a few males were still visible. In average, 70% of control females and 90% of control males were recovered from the fish, while only 10% of dsLsalMS treated males were still on the fish at the termination. The majority of female lice were lost at the preadult II stage or during moulting to adult stage  the intestine wall appeared thinner, with numerous granules and gut content was reduced or absent. In adult males, spermatophores were smaller and vasa deferentia appeared swollen    Fig. .LsalMS or control dsRNA, and placed together with non-injected females. The experiment was terminated after 6 weeks when all males reached adult stages and all control females had developed egg strings. In total, four males (13.3%) were recovered from the dsLsalMS group in comparison to 13 (43.3%) in the control group. Whereas all control females were mated, had spermatophores attached to the genital segment, and extruded normal, fertilized egg strings, with viable offspring, none of the females placed together with dsLsalMS injected males had spermatophore attached  were sectioned and stained with toluidine blue. All animals treated with dssue Fig. . The intsue Fig. . Not allsue Fig. . Numerouzed Fig. . The celzed Fig. .Figure 7LsalMS had smaller testes, with tubules tightly compressed. In the posterior zone of testes, spermatogonia were visible and the midzone contained primary spermatocytes. However, the anterior zone with spermatids contained a higher number of “A” granules than in control animals  with the decapeptide sequence following the general pattern characteristic for myosuppressins identified in other Arthropod species. Protein sequence analysis revealed that the putative decapeptide of L. salmonis, pEDVDHVFLRFamide, is flanked by abnormal proteolytic cleavage sites of tribasic and dibasic amino acids, therefore, unusual processing into the active substance could be involved59. While the final mature sequence of LsalMS needs to be experimentally validated in the future, the predicted one varies with only one amino acid on the N-terminal end of the peptide from leucomyosuppressin, LMS – QDVDHVFLRFamide21, and it is identical to one of the peptides from the beetle Zophobas atratus14.The sequence analysis of 30. However, despite of the single copy of the MS gene, in a few species additional copies of MS peptide products have been detected, with distinct tissue-specific distribution and function61. The exact mechanism of their production is unclear in most of the cases but post-transcriptional modifications and alternative processing sites could play a role60. In our study, only one gene encoding myosuppressin was found in salmon louse, with a single putative decapeptide released from the C-terminal end of the precursor. No alternative splicing or post-transcriptional modifications were detected, thus it seems that a single molecular form could account for all MS activity in L. salmonis tissues.Only one gene encoding MS has been identified in Arthropod species, and in the majority of these only one peptide product has been found65, but to a lesser extent in larvae62. However, none of these studies have investigated the MS levels during the whole life cycle for any species. LsalMS expression was detected in all developmental stages. LsalMS transcripts can be detected just after hatching, in the nauplius I stage, with slightly higher expression in nauplii II stage, to reach the highest (13-fold higher than in nauplii I) expression in the copepodids  stimulated by prothoracicotropic hormone leading to diapause51. It is therefore possible that LsalMS could possess a similar function in copepodid stage of L. salmonis.The significant variation in the transcript level between copepodid and remaining stages suggests the increased requirement for LsalMS plays a role in feeding/digestion and reproduction in L. salmonis. The presence of the myosuppressin transcripts in the tissue surrounding vital organs collected from the adult lice was also validated by quantitative RT-PCR. As expected, the highest transcript level was identified in the extracts from the brain, followed by mouth, intestine, and the reproductive organs: testes and ovaries. The observed LsalMS distribution is consistent with previously reported from other Arthropod species, where the MS peptide was detected in the central nervous system, peripheral neurons and endocrine glands65. Similarly to our results in L. salmonis, the MS-positive nerves were identified in close proximity of the crop muscle, in the crop nerve and in ramifying nerve fibres on the surface of the crop in Drosophila suzukii64. Furthermore, in Rhodnius prolixus, MS-positive nerves were detected in the endocrine-like cells in the midgut50. However, previous studies did not reveal MS-positive neurons or endocrine-like cells in the reproductive system. In L. salmonis, MS-positive cells were detected in the lumen of tubules of the ovaries. This suggests that in various species MS may play its role in distinct routes, acting as a neurotransmitter directly delivered to the target tissue and/or as a circulatory hormone, released from endocrine-like cells. Nevertheless, the localisation of the LsalMS was consistent with previously described physiological function in regulating contractions of the various visceral muscles and support claims that LsalMS, as myosuppressins in other species, is likely the neuromodulator in the nervous system and plays a role in regulating feeding and reproduction.The analysis of the tissue specific peptide distribution suggests that B. germanica, RNAi mediated knockdown of LMS caused increase in the heartbeat frequencies66, whereas, nothing is known about the influence of the myosuppressin deficiency on the development and growth. Our RNAi studies indicate that myosuppressin directly or indirectly influences proper development, moulting and survival. The RNAi treated L. salmonis larvae, with absence of myosuppressin, were able to settle on the fish in the same rates as control animals, whereas, the development and further growth were affected significantly, with very few animals reaching the pre-adult stage. The survival was significantly reduced and only single animals, those where RNAi mediated knock-down was insufficient, survived to adulthood. Similar results were obtained when pre-adult lice were injected with dsLsalMS. Visual inspection of the intestine of dsLsalMS treated animals in all evaluated stages revealed significantly reduced or no gut content, indicating disturbance of the feeding process. In fact, close examination of the intestinal peristalsis in the recovered dsLsalMS treated preadult II females revealed significant increase in the contraction frequency. Moreover, lice had difficulties with moulting to the next stage, which could be connected to impaired coordination of muscle contractions. The observed phenotype was most severe in the female lice, which were unable to reach the adult stage, therefore, the exact effect of myosuppressin deficiency on reproduction of female lice could not be examined. However, very few male lice reach the adult stage and the complex malformations could be observed, mainly in the genital segment. Adult dsLsalMS treated males placed together with non-injected adult females were not able to complete mating. This effect clearly indicates that lack of LsalMS, apart from other defects, significantly affects mating and reproduction in L. salmonis males. These results are consistent with previously reported function of myosuppressin in reproduction and feeding67.Very few data from RNA interference experiments assess effects of the myosuppressin deficiency in Arthropods. In females of 31. The results from the study in the cockroach Periplaneta americana indicates that the muscles of the legs receive innervation by FMRFamide-related peptide-containing neurons and that may be an additional method of modulating the mechanics of skeletal muscle contraction69. These findings indicate that myosuppressin has distinct effects on the different types of muscles, inhibiting contractions of the visceral muscles and enhancing the force of contractions in the skeletal muscles.The histological analysis of the adult males and preadult II females indicated developmental abnormalities in several tissues. The most striking feature was severe reduction of the muscle content, with a significant reduction of circular muscles in the intestine, as well as the visceral muscles associated with the vasa deferentia in the genital segment. Moreover, abnormalities in the skeletal muscles were apparent, with discontinuous fibres disturbed by the ectopic tissue. While the function of the myosuppressin on the activity of visceral muscles is well investigated, the influence of deficiency of myosuppressin on muscles development is poorly understood. Moreover, there is very scarce information available regarding myosuppressin function in the skeletal muscles, suggesting that it enhances the force of neurally induced contractions in skeletal muscle72, suggesting that the significant reduction in the content of the muscle tissue in myosuppressin deficient animals could be potentially linked with a lack of the proper neuronal stimulation. In contrary, LsalMS deficiency affects intestinal morphology and function, likely leading to the reduced nutrient uptake, causing decreased tissue development and growth. Inability to acquire nutrients, malnutrition and starvation are well known causes of muscle atrophy in various species75, manifested as decrease in the body size and weight, cell death, intracellular vacuolation and increased intracellular spaces74. Due to various potential reasons of the observed phenotype in LsalMS deficiency L. salmonis, the exact reasons of muscle mass decline need to be study further.It was previously shown that muscle mass can dramatically decline in response to pathological signals or lack of normal neuronal signallingLsalMS plays an important role in various physiological processes that are crucial for development, growth and behaviour in the salmon louse. Considering the importance this peptide has in function of vital organs in arthropods, it is not surprising that lack of LsalMS causes major phenotype. The knock-down of LsalMS affects several processes that are critical for survival, like feeding and reproduction. The myosuppressin peptide could be a valuable target for the development of the new and effective anti-lice control method, that could effectively reduce feeding success and reproduction rate of lice and decrease the overall growth of sea lice populations in salmon cages, while targeting adult stages, and significantly decrease the number of lice reaching the adulthood, while targeting myosuppressin signalling in early developmental stages. However, further studies are required in order to unravel the complex roles played by LsalMS in L. salmonis development, peptide processing and maturation pathways and interactions with specific receptors to find out how this peptide could be successfully targeted.Data presented here suggests that the Lepeophtheirus salmonis, whereas fish Atlantic salmon  were used only as a host for the experimental animals. All procedures involving animals were performed according to The Norwegian Animal Welfare Legislation. The Animal Ethics Committee by The Norwegian Food Safety Authority approved all experiments.The experiments were carried out on salmon louse LsGulen strain of salmon lice  was maintained on farmed Atlantic salmon 76. Salmon were hand fed on a commercial diet and reared in seawater with a salinity of 33.5‰ and a temperature of 12 °C. Eggs, nauplii and copepodids were kept in seawater from the same supply. Nauplii were obtained from hatching eggs and kept in single wells in a flow through incubators76.A laboratory L. salmonis myosuppressin peptide gene was identified by blasting (BLASTP) the known propeptide sequence of Drosophila melanogaster (AAF56283.1) against collection of predicted protein sequences from the salmon louse . Only one high confidence hit was identified . Full-length cDNA sequence was obtained by rapid amplification of cDNA ends (RACE) using SMARTer®RACE cDNA Amplification Kit (Clontech) according to the manufacturer’s protocol. RACE primers . A precursor peptide sequence was predicted by identification of a signal peptide cleavage site using SignalP 4.1 software 77. The mature peptide sequence was identified by multiple sequence alignment of the amino acid sequences  and their accession numbers are listed in Table LsalMS were predicted and examined by Pfam (http://pfam.xfam.org/). Multiple alignment of the propeptides amino acid sequences was performed with ClustalOmega78, and converted to nexus format by Mesquite v. 3.279. LG + G + I model of substitution was adopted as the optimal evolutionary model, which was predicted by ProtTest 3.480. Phylogenetic analysis was conducted using MrBayes v.3.281. Markov Chain Monte Carlo (MCMC) algorithm was used with four chains running for 1,000,000 generations and sampled every 1000 generations. Output data were analysed with Tracer v1.582, and tree was obtained using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).The full length of The following numbers and groups of animals were collected in quadruple samples. Stages: nauplius I, nauplius II and copepodid (free-living) – approximately 200 larvae per sample, chalimus I – 30 animals, chalimus II – 20 animals, preadult I, II and adult stages – single animals. Specific tissue and organs were dissected from 50–100 animals per sample from both sexes  or only from one corresponding sex .LsalMS peptide, the following immunohistochemistry protocol was used. Briefly, slides with paraffin sections were incubated at 65 °C for 30 min and afterwards cleared in Histo-Clear II  three times for 10 min, following by rehydration in decreasing ethanol series  for 3 min each and then put into distilled water. After a heat-induced antigen retrieval (HIER) )83, slides were washed two times for 10 min in PBST  on the shaker. 2% NGS  and 2% BSA  in PBST was used to block unspecific antiserum binding for 2 h at room temperatures. Primary antiserum was diluted 1:500 in PBST with 2% BSA/2% NGS and left on the sections overnight at 4 °C, in the dark. Polyclonal rabbit anti-MS, the gift form Dr. A. Urbański, were previously used in65. Incubating tissues without peptide antiserum was performed as a control. After five washes in PBST (10 min each) and one overnight wash at 4 °C, the secondary antibody –Alkaline Phosphatase antibody produced in goat, Sigma Aldrich) was applied for 1 h at room temperature. After five 10 minutes washes in PBST on the shaker, the slides were washed three times 10 minutes with processing buffer  and then incubated in NBT-BCIP solution (Sigman Aldrich). As soon as staining became visible, the reaction was stopped by incubation in distilled water. Sections were mounted using ImmunoHistomount (Sigma Aldrich) and photographed using Zeiss Axicam 105 color (Zeiss) camera mounted with to Zeiss Axio Scope.A1 (Zeiss) microscope.For detection of LsalMS gene sequence was too short to test two independent dsRNA fragments. Therefore, only one dsRNA fragment was tested in this study, complementary to the gene sequence that shows lowest similarity to other salmon louse sequences. To test it, selected sequence was blasted (BLASTN) against sea lice genomic sequence and collection of predicted transcripts . Templates for dsRNA were generated in two steps. First, PCR products were obtained from copepodids cDNA for LsalMS and plasmid containing cod CPY185 gene for control fragment with the specific pairs of primers 85. Animals were incubated overnight. When moulting was finished, all animals within each treatment group were pooled and transferred to flow through incubators, where they were kept until sampling on day 7 post hatching. Before sampling, lice were inspected under a binocular microscope . Photographs of copepodids were taken using a Zeiss Axicam 105 color (Zeiss) camera mounted to Zeiss Axio Scope.A1 (Zeiss) microscope. For fish infection, 12 groups of 100 copepodids from each treatment group were separated. For total RNA extraction copepodids were divided into five parallels with around 100 animals per sample and preserved in RNAlater (Invitrogen). For histological analysis, copepodids were collected in Karnovsky fixative.RNAi on nauplius I larvae was performed as described earlier85. dsRNA fragments were diluted to a concentration of 600 ng/μl and bromophenol blue was added to visualise dsRNA solution during injection. On the day of injection, preadults I of both sexes were collected from the fish and injected with 0,5 μl of dsRNA solution in cephalothorax, with care not to disturb the intestine. After injection, the animals were incubated in seawater for around 4 h and placed back on fish. For each fragment, in total eight fish were used, each carrying 20 injected preadult I females and 20 injected preadult I males. A cod trypsin dsRNA CPY185 fragment was used as a control. The experiment was terminated after 3 weeks, when all dsLsalMS treated female were lost from the fish. To evaluate mating potential of adult males, a total of 30 pre-adult I male lice were injected with dsLsalMS, and placed together with non-injected females . The same experimental set up was repeated for males injected with control dsRNA. The experiment was terminated after 6 weeks, when all control female lice had developed at least their first pair of egg strings. At termination, all recovered lice were subjected to a gross morphological inspection before lice were sampled for RNA extraction in RNAlater or histological analysis in Karnovsky fixative. Photographs were taken with Canon EOS 600D camera mounted with an adaptor (LMscope) to an Olympus SZX9 dissecting microscope.RNAi on preadult I females and males was performed as previously describedFor evaluation of the intestine contraction rate, preadult II females were placed on petri dish in the drop of seawater and intestinal movement was recorder for 1 minute at a time, using Canon EOS 600D camera mounted with an adaptor (LMscope) to an Olympus SZX9 dissecting microscope. Each louse was recorded twice. Number of contractions was counted on each movie.  were placed in single fish tanks (70 litres) with constant flow of seawater76. Seawater temperature was maintained at 12 °C during the whole experiment. 12 fish were infected with copepodids treated with dsLsalMS and 12 with control copepodids treated with dsCPY185. During infection period, water level was decreased to around 15 cm, to enhance contact between fish and copepodids. Fish were washed by 100 copepodids (7–9 days post hatching) and left without water flow for 10 minutes, with water manually mixed after 5 minutes. After that time, the water flow was restarted. Lice were collected from four fish from each group after 8, 12 and 18 days post infection, when control lice were in chalimus I, chalimus II and preadult stages, respectively. At termination, all recovered lice were subjected to a gross morphological inspection, counted and staged. Photographs were taken with Canon EOS 600D camera mounted with an adaptor (LMscope) to an Olympus SZX9 dissecting microscope. Lice were sampled for RNA extraction in RNAlater or histological analysis in Karnovsky fixative.Atlantic salmon  and the final concentration was evaluated with spectrometry (NanoDrop Technologies Inc.). From each sample 2000 ng of total RNA was DNAse treated in 10 μl reaction . 3 μl of each DNAse treated total RNA was used in cDNA synthesis with oligo(dT) and random primers, using program recommended by the manufacturer: primers annealing: 5 min −25 °C, synthesis: 45 min −42 °C, termination: 5 min −95 °C . cDNA was diluted four times before use in qRT-PCR or storage at −20 °C. 2 μl of final dilution was used in each PCR reaction.Total RNA was extracted as described earlierLsalMS transcript level in chalimus I and chalimus II Direct zol RNA Microprep Plus (ZYMO Research) kit was used, according to the manufacturer instructions. TotalRNA from single preadult I lice was extracted using Tri Reagent (Sigman Aldrich) as described before86.For evaluation of elongation factor 1 alpha (EF1α)87 and filamin A88 as reference genes. Both genes are considered to be good references, with stable expression rate in all developmental stages and under RNAi treatment conditions88. All primers and probes are listed in Supp. Table ef1α – 93,8%, filamin A – 105,7%, LsalMS – 104,6%). Relative expression was calculated using 2−ΔΔCT formula, with amplification efficiency correction for each gene (Pfaffl Method), utilizing the controls of each experiment as a standard. Independent-Samples T-tests were used to determine if control and test groups were differently expressed. A p-value of 0.05 was chosen as threshold.qRT-PCR was performed and analysed using the Animals were fixed in Karnovsky fixative, washed twice in PBS, dehydrated in a graded ethanol series, pre-infiltrated with Technovit7100/ethanol (50/50) for 4 hours  and infiltrated with Technovit7100/hardener overnight before embedding in plastic. Two μm thick sections were produced using a microtome (Leica RM 2165) and stained with toluidine blue (1% toluidine blue in 2% borax) for 30 s. The stained sections were mounted using DPX New Mounting Medium (Merck). Photographs were taken using a Zeiss Axicam 105 color (Zeiss) camera mounted to Zeiss Axio Scope.A1 (Zeiss) microscope.Statistical evaluation was performed by Independent-Samples T-Test.Primer sequences used in this studyContractions of the intestinal wall in female lice after RNAi experiment


We used multivariable regression models to assess associations between sexual harassment in the past 6 months and the following outcomes: 1) substance use in past 30 days ; 2) other drug use ever; and 3) feelings of depression and anxiety, respectively, in past 30 days.Almost two-thirds of girls (65.4%) reported experiencing sexual harassment in the past 6 months. Among those reporting recent harassment (n = 104), the abuse most frequently occurred in public spaces  and most often in the form of sexual comments, jokes, or gestures (57.2%), although 12.6% were “touched, grabbed, or pinched in a sexual way.” The vast majority (82.7%) did not know the perpetrators (82.7%). Experiences of sexual harassment in the past 6 months were significantly associated with alcohol use , binge drinking , and marijuana use  in the past 30 days; other drug use ever ; and feelings of depression  and anxiety  in the past 30 days. Just over half (55.3%) reported sexual harassment in more than one place, which was also associated with poor substance use and mental health outcomes.Findings suggest that sexual harassment experiences are pervasive, occur in girls' immediate and everyday environments, and are associated with girls’ substance use and adverse mental health outcomes. •We found a high prevalence of recent experiences of sexual harassment (65%) among this clinic-based sample of girls.•Victimization is largely occurring in girls’ immediate environments and predominantly perpetrated by strangers.•Sexual harassment was associated with substance use, as well as depression and anxiety among adolescent girls. Young wSexual harassment is important to study because it not only carries social implications as a form of gender discrimination and sexual aggression but it also poses a serious public health concern for adolescent girls in the United States and worldwide . The detFew studies have sought to examine the full range of perpetrators, types of harassment, and places in which girls experience harassment. Many studies on sexual harassment among adolescent girls have either not asked about the location of the event or focused specifically on sexual harassment occurring at school , at workmore than one context and relation to substance use and mental health outcomes.In summary, future research on sexual harassment among adolescent girls is needed that focuses on those residing in low-income/high-poverty communities, assesses sexual harassment experiences that occur across various places, identifies the perpetrators involved, and examines associations with poor mental health and substance use outcomes. This study aims to address the current gaps in the literature by 1) assessing experiences of sexual harassment among adolescent girls by type , place , and perpetrator ; 2) assessing girls’ experiences of sexual harassment in relation to substance use and poor mental health outcomes; and 3) assessing the proportion of girls reporting experiences of sexual harassment in 22.1The current study involves cross-sectional data on sexual harassment and substance use and mental health outcomes, collected as part of a larger study on adolescent girls’ spatial mobility and risk for sexually transmitted infections (STIs) in a low-resource community on the US side of the California-Mexico border. More than one-quarter of residents in the study neighborhood live below the national federal poverty line . High raParticipants were recruited from an adolescent health clinic, where clinic providers and research staff from the study approached them in the waiting room. In order to be eligible for the study, participants had to: a) be female; b) be aged 15–19 years; c) report being sexually active in the past 6 months (given that the focus of the larger study was on STI risk); d) speak English; and e) be able to provide consent. Of the participants who were approached and eligible (n = 182), 87% (n = 159) participated. The most common reason for nonparticipation was lack of time. All participants responded to survey questions on sexual harassment and were included in our analyses (n = 159).2.2Upon establishing eligibility, participants completed the informed consent procedures administered by research assistants. Participants then completed a 45- to 60-min self-guided questionnaire. The questionnaire was administered on tablets and collected general information on sociodemographic factors , sexual and reproductive health , sexual behavior , alcohol/substance use , mental health , experiences of violence and harassment , and social media use. Participants received a $20 gift card upon completion of the survey. The Institutional Review Board (IRB) at the University of California, San Diego (UCSD) approved this study.2.3Sexual harassment victimization was assessed using items from the American Association of University Women (AAUW) survey , which were not at the level of collinearity but precluded assessment of their independent associations with our outcomes of interest.Sexual harassment. Participants were asked about various experiences of sexual harassment that occurred in the past 6 months, including harassment type, places where the harassment occurred, and perpetrators of the harassment. ) survey . These 4) survey . This selocations of sexual harassment. The item asked: “In the past, when boys or men have done any of these things to you, where did it happen?” Responses were “at school,” “at home,” “at work,” “on public transportation,” “my neighborhood,” “another neighborhood nearby,” or “other place,” and responses were not mutually exclusive. Participants were able to write in any “other place” they experienced sexual harassment. Our qualitative research that guided this question highlighted that sexual harassment across diverse locations was indicative of the behavior as normative in the girls’ social environment. We developed a variable that represented whether participants reported sexual harassment in more than one place in the previous 6 months (yes/no). This also served as an independent variable of focus.Based on prior research documenting that sexual harassment occurs across diverse locations in public and in schools  as well Again, based on our formative research, we found that girls were often familiar with those who sexually harass them, and some are even viewed as friends. Thus, we created for this survey an additional item on relationship to the perpetrator assessed for the subsample reporting sexual harassment. We asked, “When boys or men have done any of these things to you, who did this?” Responses were: “boys/men I did not know,” “boys/men I knew of or have seen but am not friendly with,” and “boys/men I knew pretty well or who are my friends.” Participants could choose more than one response . This sexual harassment variable was used for descriptive purposes to understand the nature of the harassment experienced.marijuana within the past 30 days (yes/no). Separately, we summated responses on the other 11 drugs and created a dichotomized variable of other drug use within the past 30 days (yes/no).Substance use. To assess drug use behaviors, we asked about lifetime use and use in the past 30 days of marijuana and 11 different substances, including cocaine, stimulants, and prescription drugs, using items adapted from the Texas Christian University Drug Screen 2 (TCU-DS2) . For eacever drank an alcoholic beverage and b) drank an alcoholic beverage in the past 30 days (yes/no). Participants who reported alcohol use in the past 30 days were then asked about binge drinking with the one item measure: “How often in the past 30 days did you have 5 or more alcoholic drinks in one occasion?” on a 0 (never) to 5 (everyday) Likert-type scale. A variable was created to reflect participants who reported any binge drinking in the past 30 days. Based on our qualitative assessments with this population to refine survey measures, we asked about 5 drinks in one occasion rather than 4 drinks, which is typically used to define binge drinking among girls  to 4 (nearly every day). A variable was created to reflect participants who reported feeling anxious or depressed at all in the previous 30 days.Mental health. Single-item measures were used to ask participants whether they felt a)  30 days  and/or b 30 days , using aDemographics. Demographic variables such as age, race , and living situation  were used to characterize the sample. Except age, all demographic variables were categorical.2.4P < .05 were included in all adjusted models. Findings from logistic regression models were presented as odds ratios (ORs) and exact ORs with associated 95% confidence intervals (CIs), and significance of individual variables was evaluated using Wald chi-square and exact conditional tests. All analyses were conducted using SAS® version 9.4.Descriptive statistics were used to summarize girls' demographic characteristics, types of sexual harassment, perpetrators involved, and the places in which these events occur. A t-test and chi-square and Fisher's exact tests were used to examine: 1) the association of demographic variables with any sexual harassment in the past 6 months and 2) sexual harassment in relation to each study outcome, including participants' alcohol use in the past 30 days, drinking 5 or more alcoholic drinks at once in the past 30 days, marijuana use in the past 30 days, other drug use ever, feeling depressed in the past 30 days, and feeling anxious in the past 30 days. Crude and adjusted logistic regression/exact conditional logistic regression models were used to examine sexual harassment  in relation to each outcome. Demographic variables associated with any outcome at 33.1N = 159). The average age of the sample was 17 years. Most participants identified as Latina (76.7%), were born in the United States (77.2%), and lived with their parents (83.5%).X2 12.3; P = .0005). In addition, among those experiencing sexual harassment, those born in the United States (62.0%) were more likely to report sexual harassment in more than one place than those not born in the United States (33.3%) .Of the 159 adolescent girls, 65% (n = 104) reported experiencing any sexual harassment during the past 6 months. Among those experiencing sexual harassment in the past 6 months, 55.3% (n = 57) experienced sexual harassment in more than one location. There were no statistically significant differences in experiencing sexual harassment by demographic variables. However, among those who experienced any sexual harassment, Latina adolescents appeared less likely to report experiencing sexual harassment in more than one location (44.6%) than non-Latinas (82.8%) (3.2not known to the girls (82.7%).The most frequent type of sexual harassment experienced involved sexual comments, jokes, or gestures (57.2%), followed by being touched with any part of a perpetrator's body in an unwanted sexual way (12.6%), being touched, grabbed, or pinched in a sexual way (12.6%), and experiences with men exposing themselves sexually in public (3.8%). More than 30% of girls reported experiencing two or more types of sexual harassment during the past 6 months (data not shown in tables). Girls reported that, for the most part, harassment took place on public transportation (50.5%), at school (42.7%), and in their or surrounding neighborhoods (39.8%). In addition, 5.8% of girls reported experiencing sexual harassment at work, 3.0% reported experiencing sexual harassment at home, and another 41.8% reported experiencing sexual harassment in some other place. More than half (55.3%) of the girls who experienced sexual harassment reported experiencing it in more than one place; 18.4% reported experiencing sexual harassment in 2 places, 20.4% in 3 places, 9.7% in 4 places, and 6.8% in 5 or more places. The perpetrators of sexual harassment were predominantly boys/men who were 3.3Less than half (38.3%) of participants reported alcohol use or binge drinking in the past 30 days. Slightly more than one-third (34.4%) reported marijuana use in the past 30 days. Slightly less than one-quarter (22.6%) reported having ever used drugs. High proportions reported experiencing depression (60.1%) and anxiety (73.9%) in the past 30 days. .Table 2C3.4Adjusted logistic regression models show that, compared with girls who had not been harassed, girls who experienced any type of sexual harassment during the past 6 months had statistically significantly greater odds of alcohol use , binge drinking , and marijuana use  within the past 30 days . Girls wExperiencing sexual harassment in more than one location was also associated with alcohol use in the past 30 days , ever using drugs , and feeling anxious in the past 30 days . Notably, we also found evidence of a dose-response relationship when looking at the number of places girls’ experience sexual harassment (as a continuous variable) and relation to alcohol use, drug use, and feeling anxious in the past 30 days. Although not statistically significant, trends were observed when looking at other study outcomes as well but were likely limited because of the small sample size.4The current study documents that this urban, predominantly racial/ethnic minority sample of girls is experiencing a very high rate of sexual harassment, with almost two-thirds of girls (65.4%) reporting harassment in just the past 6 months. We found that victimization largely occurrs in public spaces, including on public transportation and at school, and is predominantly perpetrated by strangers. Findings also demonstrate that sexual harassment is associated with increased risk for substance use, depression, and anxiety among adolescent girls from a low-income, predominantly Hispanic community near the US-Mexico border. Our study builds on previous research by documenting the multiple settings and perpetrators that characterize girls’ experiences of sexual harassment, reporting the proportion of girls who experience sexual harassment in more than one setting and its relation to poor health outcomes, and focusing these assessments on a sample of adolescent girls from a community where violence, including sexual violence and harassment, occurs in high proportions.The proportion of girls who reported experiencing sexual harassment (65% in the past 6 months) among this sample of girls aged 15–19 years was higher than those reported in many other studies . Many stNotably, we found some differences in experiences of sexual harassment by sociodemographic variables: Latina and non–US-born adolescent girls were less likely to report experiencing sexual harassment in more than one location than non-Latina and US-born adolescents. Although more research is needed to understand these findings, adolescent girls who are recent immigrants may be more likely to go to places with their families , making them less exposed to sexual harassment. In our study, girls who were non-Latina were predominantly Filipina—and in a predominantly Latina community, Filipina girls may be considered the minority, which may put them at greater risk for being targeted for harassment, including sexual harassment. More research is needed to further assess and better understand these findings.Experiencing sexual harassment at school has been shown to compromise girls' perceptions of safety at school  and is lConsistent with other studies , our finas a result of being pressured or coerced, which is more commonly measured in sexual violence studies. Thus, future measures of sexual harassment may need to broaden their definition to include sexual pressure/coercion and assess relationship and sexual partners as possible perpetrators. Unfortunately, our measure of perpetrators was limited in that we could not assess whether perpetrator characteristics vary by the type of sexual harassment reported or the place in which it occurred. In addition to assessing the relationship of the perpetrator to the victim , future research may also benefit from assessing the sociodemographic characteristics of the perpetrator .Perpetration of sexual harassment was largely by those unknown to female participants, which is aligned with our finding that the majority of sexual harassment was reported in public places, such as public transport and neighborhoods. In contrast, previous studies have found that sexual harassment occurring in school is often perpetrated by those known to the victim . Thus, f4.1Although our findings offer important insight into the role of sexual harassment and its potential health consequences for adolescent girls, they must be considered in light of certain limitations. The study relies on self-reported data and is subject to recall and social desirability biases. To reduce recall bias, we focused on recent experiences, from the past 30 days to 1 year, and to reduce social desirability bias, we used self-administered surveys . Future 4.2Despite limitations, our findings suggest that sexual harassment is pervasive, occurs in adolescent girls' immediate environments , and is associated with adverse substance use and mental health outcomes. Our findings suggest that experiences of sexual harassment create a stressful environment and lead to poor health-related outcomes for girls. Given that most girls in our study have experienced sexual harassment and its link to poor health outcomes, sexual harassment should be considered a major public health concern. Our findings suggest that sexual harassment is not only pervasive but has similar health outcomes as other forms of sexual violence  experienced by adolescent girls . The intAuthors do not have any potential conflicts of interest or financial disclosures to report.Eunice Kennedy Shriver National Institute on Child Health and Development . Elizabeth Reed was also supported by the National Institute of Mental Health . Marissa Salazar was supported by the National Institute of Drug Abuse .This research was funded by the The Institutional Review Board (IRB) at the University of California, San Diego (UCSD) approved this study.


Stressful conditions can play an important role in affecting welfare, productive performances and meat quality of lambs. The relation between nutrition and immune response has been investigated in the livestock production, particularly in dairy cattle and sheep. Due to costs related to animal feed it is useful to evaluate the proper feeding strategy supplementation for improving animal welfare and lamb meat quality. The present study aimed to evaluate the effects of supplementation with linseed, quinoa seed and their combination on metabolic profile, immune system, and cortisol response in blood and on meat quality of merinos derived lambs. Both linseed and quinoa supplementation enhanced the immune responses of lambs, and showed a hypo cholesterol effect on blood of lambs. Moreover, lambs supplemented with linseed resulted in the lowest level of cortisol secretion during the loading test demonstrating the link between stress and the immune system. In addition, data from the present experiment highlighted that linseed supplementation in lambs enhance meat quality producing a better meat tenderness. These findings should be considered for development of specific strategies aimed at improving the quality of meat and sustaining lambs’ welfare.In the last years several studies have investigated the strong relation between nutrition and immune response in the livestock production, particularly in dairy cattle and sheep. The aim of this study was to evaluate the effects of supplementation based on linseed, quinoa seeds and their combination on welfare, productivity and quality of meat from merinos derived lambs. 32 weaned lambs were divided into 4 experimental groups: quinoa (Q), linseed (LS) and combination of quinoa and linseed (LS + Q) that received the respective supplementation and control group (C) without supplementation. Lambs from all supplemented groups showed lower plasma urea, creatinine and cholesterol than control. Both linseed and quinoa supplementation enhanced the cell-mediated immune responses of lambs, furthermore, linseed supplementation resulted in the lowest level of cortisol secretion after handling, loading and transport. Meat from lambs supplemented with linseed and LS + Q showed the highest pH, at 1 and 3 h post-mortem, while, meat from all supplemented groups was more tender than meat from control. Results indicated that linseed and quinoa seeds supplementation can help the animal to cope with stressful events due to the close link between stress responses and the immune system and for improving meat quality in terms of better tenderness. Consumer perception and preference for meat products are affected by many factors that contribute to define meat quality, defining direct factors linked to the muscle, nutritional, sensorial and technologic factors, and some factor related to the image of the product such as the welfare of the animals . FeedingIn the last two decades several studies investigated the strong relation between nutrition and immune response . ParticuMoreover, stressful conditions such as human–animal relationship can play an important role in affecting welfare, productive performances and meat quality of lambs . To respAlthough the effect of dietary linseed sources on productive performances and lamb meat quality has been studied, little information is available on the effect on welfare parameters, including cortisol production, cellular and humoral immune responses. In addition, the effect of the inclusion of quinoa in ruminant’s diet on animal welfare and on meat quality have not been previously investigated.Our hypothesis was that PUFA enriched-diet could be able to sustain immune and metabolic responses of lambs, promoting welfare, and, as a consequence, increase the productivity, and the whole quality of meat from merinos derived lambs. Therefore, the aim of this study was to evaluate the effects of supplementation based on linseed, quinoa seeds and their combination on immune responses, productivity and quality of meat from merinos derived lambs.The experimental design and all animal procedures were developed in accordance with the Foggia University Institutional Animal Care and Use Committee (protocol number 003-2016). The experiment was conducted at the research station of the Council for Agricultural Research and Economics (CREA-ZA) (latitude: 41°27′6′′ and longitude: 15°33′5′′) located in Apulia Region . A total of 32 Italian Merino male lambs after weaning (42 ± 2 days of age), with average initial live weight 14.30 ± 1.22 kg, were used in this experiment. Lambs were randomly allotted to four dietary groups (8 per treatment group): control (C), fed with vetch, oat hay and pelleted concentrate; quinoa (Q), fed with vetch, oat hay and pelleted concentrate and supplemented with quinoa seed; linseed (LS), fed with vetch, oat hay and pelleted concentrate and supplemented with linseed; quinoa and linseed (LS + Q), fed with vetch, oat hay and pelleted concentrate and supplemented with of quinoa seed and linseed. During the 50-day feeding experiment, lambs were housed in individual pens equipped with water dispenser. Ingredients and chemical composition of the diets are shown in Analysis on feed were carried out on pooled feed samples collected throughout the trial period. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to Van Soest et al.  while thquadriceps femoris muscle. After 24 h post-mortem from each half carcass quadriceps femoris muscle was removed; chemical composition, Warner Bratzler shear force (WBSF) and colorimetric parameters were evaluated.Animals were individually weighed at weekly intervals to estimate the average daily gain . At the end of the experiment, animals were fasted for 12 h, weighed and transported to a local slaughterhouse where they were slaughtered, according to industrial routines used in Italy and to the EU rule n. 1099/2009 . CarcassBlood samples (7 mL) were collected by jugular venipuncture at d 0, 15, 25, 35, 49 (5 recordings) at 7 am using vacutainer tubes containing Sodium–heparin. Blood samples were immediately centrifuged (3000 rpm for 15 min), then plasma samples were collected and immediately frozen at −20 °C until analysis. Plasma metabolites  and enzymatic activities  were determined using an automated analyzer for biochemical chemistry , and its dedicated kits. Plasma globulin concentration was obtained as the difference between total protein and albumin.3)1.15 + 1.18, R2 = 99.8%) obtained using scalar dilution of ovine IgG . Data were expressed as µg/mL. The assay was optimized in our laboratory for dilution of plasma, concentration of coating antigen and detector antibody.To determine the lambs’ humoral response during the trial, the animals received a subcutaneous injection of the antigen KLH , to which the animals had not previously been exposed. At the start of the experiment, 0 day, 0.5 mg of KLH dissolved in 1 mL of sterile saline solution and 1 mL of incomplete Freund’s adjuvant  were injected subcutaneously . A subseAt 0, 25, and 35 day the in vivo cell-mediated immune response after intradermal injection of the mitogen phytohemoagglutinin (PHA)  suspended in sterile saline solution (1 mL) was evaluated on each lamb by measuring lymphocyte proliferation. The lymphocyte proliferation was measured as the changes in skin-fold thickness after 24 h of PHA injection according to Caroprese et al. .3 vacuum tube  to determine the cortisol level. Hormone concentration was determined by colorimetric competitive sheep ELISA  according to the manufacturer’s instruction. The sensibility of the assay was 0.1 ng/mL. Data were expressed as ng/mL of cortisol.At 45 day of the experiment a loading/unloading test in order to simulate stress related to handling, loading, transport and unloading and to evaluate cortisol secretions. Before loading (0 min), after transport and unloading at 10 min, and 60 min after unloading, blood samples were collected into EDTA-KMeat sample was ground to homogeneous consistency using a food processor before chemical analysis. Moisture, protein, fat and ash content were determined according to methods recommended by the AOAC .Color was measured using a color meter Minolta CR 400  on 1 cm thick steaks after storage at 3 ± 1 °C for 1 h. Color measurements were expressed as L* (lightness), a* (redness) and b* (yellowness) according to the standard conditions of the Commission International d’Eclairage (CIE). Results were reported as the average of five measurements taken from random locations on the surface of each slice.The instrumental tenderness was estimated by using Warner–Bratzler shear force (WBSF) test on cooked meat as described by Marino et al. . Measurep-value lower than 0.05 were considered statistically significant.Data of immunological parameters, metabolic profile and cortisol concentrations were checked for normality in SAS and analyzed using ANOVA for mixed models using the MIXED procedure of SAS  with thep < 0.05) and significant effects were reported in tables. When significant differences were found (p < 0.05), the Student t-test was used to locate significant differences between means.Data of growth, carcass characteristics, pH values, chemical composition, colour and WBSF were subjected to a one-way ANOVA with diet as fixed effect. Individual lamb was the experimental unit. All effects were tested for statistical significance (p < 0.001), creatinine (p < 0.05), cholesterol (p < 0.05), and higher NEFA and triglyceride plasma concentration (p < 0.01) than control group. Lambs supplemented with linseed or in combination showed higher plasma levels of total protein (p < 0.05) than C and Q groups, while the highest plasma albumin concentration (p < 0.01) was found in lambs fed with LS diet respect to all other groups.The effects of dietary treatments and sampling time on metabolic profile in blood plasma of lambs are shown in p < 0.01), while, lambs supplemented with quinoa seed shows the highest plasma glucose values (p < 0.05). GGT enzyme plasma level of LS group was found higher (p < 0.001) than all other groups. Referred to sampling time effect, urea (p < 0.001), total protein (p < 0.05) and ALP (p < 0.01) activity showed higher values at the end of trial, while glucose (p < 0.05) and cholesterol (p < 0.05) values decrease during time with the lowest values at the end of trial.In addition, lambs from LS group showed the lowest glucose level (The effects of dietary treatments and time sampling on antibody titers to KLH are showed in p < 0.001). Each of experimental group registered an increase from starting time, to 15 and to 25 day of the experiment on the anti-KLH IgG produced in plasma lambs. Moreover, an increase between from 15 and to 25 day in all experimental groups was registered.Anti-KLH IgG production was affected by time of sampling (p < 0.001) and of diet (p < 0.01) was found. Q, LS and LS + Q showed an increase from 0 day to 15 day and from 15 to 35 day; whereas, C group displayed a reduction of cell-mediated immune response from 15 to 35 day. At 15 day and 35 day both Q and LS lambs displayed higher skinfold thickness than C and LS + Q lambs . On average, the LS group displayed the lowest cortisol concentration in comparison to C and Q lambs during the loading test (p < 0.001). At 10 min after loading test LS lambs displayed lower cortisol secretion than C; whereas, at 60 min after loading test no differences emerged between groups in cortisol secretion returning to basal level of starting test.Plasma cortisol production of loading test is depicted in post-mortem pH of quadriceps femoris muscle is shown in The effect of dietary supplementation on growth performance, carcass characteristics and p < 0.05) at 1 and 3 h post-mortem in LS and LS + Q meat compared to Q and control meat. The effect of dietary supplementation with linseed, quinoa seed and their combination on intramuscular fat content, WBSF and colorimetric parameters of quadriceps femoris muscle from lambs is reported in No significant differences in the average daily gain, slaughter weight, carcass weight and dressing percentage were observed among all experimental groups. On the contrary, muscle pH was affected by diet supplementation showing higher values (p < 0.05) showing the highest values in meat from lambs supplemented with linseed, quinoa seed and their combination. Regarding to organoleptic properties of meat, lambs subjected to dietary supplementation with linseed or/and quinoa seed showed a greater meat tenderness in terms of lower WBSF values (p < 0.01) than meat from control group. Dietary supplementation affected the meat color parameters, in particular higher values of lightness, redness and yellowness (p < 0.01) were detected in meat from lambs of all supplemented groups.Diet had a significant effect on intramuscular fat content (Metabolic profile of all experimental groups were in normal range for the young animals of the sheep specie ,28. ResuCortisol concentration in blood after a stressor, such as transport, increases according to the proportional perception of stress from the animal. In the present experiment, as expected, cortisol levels picked up after 10 min post loading test in all groups returning to basal level when stress was over. Stress response changes according to the type of stress acting and to the animals’ physiological state. In our study the low levels of cortisol secretion in plasma of lambs fed linseed could be responsible for the enhanced cellular immune responses. In a previous study lambs displaying an increased secretion of cortisol during open field test exhibited a reduced cellular response to skin test . In sheeA tentative explanation for the absence of effects of linseed supplementation on humoral response of lambs in the present study could be the different nature of experimental procedure as compared to previous experiments by Caroprese et al. ,10,23. FThe lack of significant effect on performance in vita among the different dietary treatments could be due to the similar crude protein and metabolizable energy level of diets, in agreement with previous studies ,37 reporStressful conditions can increase the release of adrenaline leading to an ante mortem glycogen breakdown and, as a consequence, to a lower pH values . PreviouIn addition, the major tenderness of meat from lambs supplemented with LS could be due to the highest pH found after slaughtering meaning optimal conditions for the activation of endogenous enzymes responsible for postmortem tenderization. It has been demonstrated that the rate of glycolysis and consequently the rate of pH decline could improve tenderness by altering the optimal conditions of enzyme activity. In the previous study, della Malva et al.  highlighColor is an important factor determining the consumer purchase of meat . It is kThe present results confirm that the improvement of animal health can affect lamb’s meat quality in agreement with Napolitano et al. .This study evaluated the effects of supplementation with linseed, quinoa seed and their combination on metabolic profile, immune system, and cortisol response in blood, and on meat quality of merinos derived lambs. Both linseed and quinoa supplementation enhanced the cell-mediated immune responses of lambs, and showed a hypo cholesterol effect on blood of lambs. Moreover, lambs supplemented with linseed resulted in the lowest level of cortisol secretion after 10 min post loading test. The reduced reactivity to stress by linseed fed lambs could be responsible for the enhanced cellular immune responses highlighting the link between stress and the immune system. This result provides an important contribution to sustain the immune response of lambs. In addition, data from the present experiment highlighted that linseed supplementation in lambs enhance meat quality producing better meat tenderness. These findings should be considered for development of specific strategies aimed at improving the value and quality of meat from merinos derived lambs and sustaining lambs’ welfare.


To understand the mechanism of action by which the inhibitor FK866 works, we used Raman imaging microscopy and identified that proteins and lipids are decreased upon treatment with the drug. Raman imaging showed a different distribution of lipids throughout the cell in the presence of the drug compared with the untreated cells. We employed nuclear magnetic resonance NMR spectroscopy and mass spectrometry to identify the classes of lipids altered. Our combined analyses point to a decrease in cell division due to loss of lipid content that contributes to membrane formation in the in vitro setting. However, the FK866 drug did not have the same potency in vivo. The use of Raman imaging microscopy indicated an opposite trend of lipid distribution in the tissue collected from treated versus untreated mice when compared with the cells. These results demonstrate the role of Raman imaging microscopy to identify and quantify metabolic changes in cancer cells and tissue.Isocitrate dehydrogenase 1 (IDH1) mutations in gliomas, fibrosarcoma, and other cancers leads to a novel metabolite, D-2-hydroxyglutarate, which is proposed to cause tumorigenesis. The production of this metabolite also causes vulnerabilities in cellular metabolism, such as lowering NADPH levels. To exploit this vulnerability, we treated glioma and fibrosarcoma cells that harbor an IDH1 mutation with an inhibitor of nicotinamide adenine dinucleotide (NAD Raman spectroscopy has been used for decades to determine vibrational models linked with structural changes of small molecules . Due to mut) enzyme produces a novel metabolite D-2-hydroxyglutarate (D-2HG), which is proposed to contribute to tumorigenesis via alteration of the epigenome [mut cancer cells is the depletion of reduced nicotinamide adenine dinucleotide NADH, which triggers autophagy and cytotoxicity selectively for IDH1mut gliomas [+ salvage pathway has been tested for lower grade gliomas with the goal to be added to clinical trials [+ were not fully explored. Although the reported mechanisms by which depletion of NAD+ leads to cell death was found to be autophagy; however, these investigations left a lot of unanswered questions related to the metabolic consequences of reducing such an important cofactor.The majority of lower grade glioma possess an activating mutation in a metabolic gene called isocitrate dehydrogenase I (IDH1). The IDH1 mutant . For tissue experiments, unstained quartz slides of 5 µm tissue from mice brain (vehicle or treated) were obtained from Histoserv., Inc. . The Raman acquisition was done under the same general conditions but using a 100X air/dry objective .Samples used for Raman spectroscopy were transferred into 35 mm sterile glass bottom dishes (Ibidi) and cultured for 72 h in the presence and absence of 12.5 nM FK866. Raman spectra were acquired using a DXR2xi Raman microscope  with 9.9 mW of 532 nm laser at the sample through a 60X water immersed confocal objective  at 0.5 s exposure time for a 1 μm pixel size, for the 50 to 3200 cm6 per cell culture dish with 12.5 nM FK866 dissolved in DMSO or an equivalent volume DMSO. After 72 h, cells were quenched and collected as follows. Media was aspirated from adherent cells, washed with PBS, and then 1 mL of ice-cold water was added to the flask and immediately placed onto dry ice. These frozen cell layers were then scraped and transferred to a 15 mL tube and placed into dry ice until metabolites extraction. For cell viability, 0.5 million cells were incubated with FK866 for 72 h. Following treatment, cells were trypsinzed and counted by mixing with Trypan Blue and using a Vi-CELL XR  cell viability analyzer.HT1080 were maintained in Dulbecco minimal essential medium (DMEM) supplemented with 10% FBS  and Pen/Strep. Cells were plated at 2 × 10Cell suspensions in water were lysed via three cycles of freeze-thawing, including a 5 min sonication process in an ice-water bath after samples were thawed. Fifty microliters of this cell lysate were put aside for further protein quantification using the Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific). Subsequently, 2 mL of ice-cold methanol were added to the cell lysates and incubated in ice with agitation for 10 min. Two milliliters of ice-cold chloroform were then added and also incubated in ice with agitation for 10 min. Then, samples were centrifuged at 12,000 rpm for 20 min at 4 °C. Bottom (lipophilic) layer was transferred to a glass vial and dried under a gentle stream of N2. The sediment was reconstituted in 180 µL of CDCl3 (0.03% v/v tetramethylsilane (TMS):CD3OD (2:1) and transferred to a 3 mm-NMR tube. 1H NMR spectra were acquired on a Bruker Avance III 600 MHz spectrometer  operating at a probe temperature of 298 K. Pulse sequence employed was zgpr  for suppression of the residual water resonance. For each spectrum, 64 scans were acquired, with a relaxation delay of 3 s, a spectral width of 10,800 Hz, and a time domain of 32 K points. Spectra were referenced to the TMS internal standard signal , zero-filled to 64 K points, phased, and baseline-corrected using an ACD Labs Spectrus Processor (2016) , and an exponential line broadening function of 0.30 Hz was applied. Resonances arising from lipids were normalized to the TMS resonance peak and to the protein content.Single-pulse HT1080 cells were grown in DMEM supplemented with 5% FBS and treated with 12.7 nM FK866 drug or DMSO as control. After 72 h of incubation, the media was removed, and cells were washed with cold PBS. Cells were scrapped on dry-ice and the cell lysate was subjected to freeze though and sonication for subsequent metabolite extraction. Cell samples were centrifuged at 400 rpm for 10 min and the supernatant was discarded. Resulting pellets were quenched in 0.6 µL MilliQ water, centrifuged at 400 rpm for 5 min to discard the supernatant, and snap-frozen on dry ice prior to storage at −80 °C. Each cell pellet was placed on ice, resuspended in 450 µL of chilled MilliQ water, and then lysed for 30 s with sonicator probe set at 20 A. Samples were placed on dry ice for 3 min and returned to a sonicator ice bath for 10 min. A total of 10% of the resulting lysate was collected to perform Bradford protein quantification. The Bligh and Dyer biphasic liquid extraction was performed on remaining lysates using a ratio of 2:2:1.5 chloroform/water/methanol . The chiMass spectrometry data analysis was performed following the logical binning of mass features using the XCMS software package in R Studio  as described previously . LIPID MIntracranial orthotopic mouse models with an IDH1mut glioma cell line were established according to an approved animal study proposal NOB-008 by NCI-Animal Use and Care Committee (ACUC). Briefly, cells were harvested, washed with PBS, and counted. The resulting pellet was resuspended in Hank’s Balanced Salt Solution (HBSS) and 5 μL of the cell suspension were injected stereotactically into the striatum of female severe combined immunodeficiency (SCID) mice  using a stereotactic device . Tumor growth was monitored for neurological symptoms daily. Mice were randomized into two groups  and FK866 treatment  started on day 68 via gavage and ended on day 82 (lasted for 14 days). When the animals reached end-points, they were euthanized via perfusion with 4% paraformaldehyde (PFA) under anesthesia and their brains were dissected for histopathological examination and immunochemistry analysis. For comparison of survival curves, a log-rank (Mantel–Cox) test has been used. Haematoxylin and eosin( H&E) stained, and unstained tissue slides were obtained from Histoserv. Inc. . Adjacent slides were cut for Raman analysis, therefore each slide analyzed was confirmed to have tumor via histology.−1, which was assigned to lipids that were used as a proxy for lipid content. Assignments used for displaying distribution of nucleic acids, proteins, and lipids are shown in −1), we noticed marked differences in the lipid and protein specific bands (2845–2930 cm−1)  d. Throug30 cm−1) . When ra30 cm−1) g. To tes30 cm−1) d. Since + availability. Since NAD+ was contributing significantly to lipids synthesis it was not surprising to find these results; however, the specific lipids altered and their contribution to cell viability could not be discerned via Raman imaging alone [Since the lipid distribution was variable inside the cytoplasmic region of the cells as shown in the ng alone .Using NMR, we observed downregulation of phosphatidylcholines (PCs) phosphatidylethanolamine (PEs), phospholipids (PLs), monounsaturated fatty acids (MUFAs), triglycerides (TGs), monoglycerides as well as sphingomyelin (SM), and plasmalogen . In the To test the efficacy of FK866 in vivo, we designed an animal experiment in which we injected 0.5 million BT142 glioma cells intracranially in SCID mice, randomized the mice into two groups and start treatment after 68 days. Treating mice with a dose of 30 mg/kg for 14 days did not improve the survival of mice a, althou−1 that was increased in the FK866-treated mice tissue and an overlap with a resonance from the FK866 drug, which we tentatively assigned to the Raman frequency coming from C=C of the drug [−1 in the absence of the FK866 drug. However, in the treated tissue, we could not distinguish the contribution drug-induced tissue changes that would lead to increases in the peak intensity at 1595 cm−1 and the signal coming from the drug itself. The intensity changes at 1595 cm−1 could have been due to the presence of FK866, or due to FK866-induced tissue changes. By accounting for the local effect of such a drug, one could at least indirectly map the presence of FK866. Therefore, using the 1595 cm−1 peak’s intensity, we looked at the distribution of FK866 in tissue  polymerase (PARP) and sirtuin (SIRT) enzymes. PARP enzymes are involved in DNA repair, gene expression, genomic stability and cell cycle amongst other processes [+-dependent enzymes are more diverse than the abovementioned classes and are involved in many biological processes from DNA damage repair to gene expression and calcium signaling [+. An enzyme that uses NAD+ as a cofactor is pyruvate dehydrogenase, the TCA cycle enzyme that performs pyruvate decarboxylation towards acetyl coenzyme A(acetyl-CoA). Since acetyl-CoA is the primary precursor of lipids synthesis, it is not surprising that a depletion in NAD+ would lead to inhibition of this enzyme and decreased lipids synthesis. However, additional experiments need to be performed to validate the inhibition of this enzyme.FK866 is as an inhibitor of the NADrocesses ,26, whilrocesses ,28. Howeignaling . Herein,+ could affect NADPH pools leading to a reduction in the catecholamine synthesis pathway with consequences in proper neuronal function and signaling. Catecholamines are present in some regions of the brain where they control the activity of certain neurons via opening and closing of ion channels. Not surprisingly, defects in this pathway are linked with neurologic disorders [Using NMR and mass spectrometry we validated the decrease in lipids observed via Raman imaging microscopy. In addition, we examined the classes of lipids that are altered the most and rationalized their contribution to cell growth and proliferation. Few classes of lipids were strikingly decreased in cells treated with FK866. Amongst them is arachidyl oleate, which is a long-chain fatty alcohol which is primarily located in the membrane, and its decrease would lead to a decrease in membrane and therefore cell division. PCs, PEs, and PLs are all decreased in both NMR and mass spectrometry experiments suggesting decreased membrane integrity and cellular division . Catechoisorders . Mice caisorders .18F-FDG PET) analysis for live animals or humans. The use of 18F-FDG PET in the clinic is a standard practice in any oncology field; however, it is limited to the tumors that consume large amounts of glucose and that are larger than 7 mm [Tracing metabolites or chemicals has attracted major interest since the days of fludeoxyglucose positron emission tomography (han 7 mm . Herein,han 7 mm . Secondlhan 7 mm ,35,36 (Shan 7 mm . We havehan 7 mm ,39,40. IThe use of Raman imaging microscopy for quantification of subtitle changes due to drug treatments is demonstrated. Herein, we identified and quantified changes in lipid content upon addition of the FK866 drug, which was not expected to decrease lipid content. The power of this imaging technique stands in the spatial distribution and the wealth of information that can be obtained from a single scan of a live cell. Therefore, in the future, this technique could be applied in determining the heterogeneity of the cells, or to identify tumor infiltrating cells located within the normal tissue. Other Raman-based methods could be developed to look at the synthesis of lipids, proteins and nucleic acids at the single cell level. Raman imaging microscopy has the potential to become the next single cell technology similar to single cell RNA-sequencing. The ability to obtain information at the single cell level could be exploited for the cases where material is sparse, such as immune cells. However, the limitations in mapping chemicals were highlighted as well and are related to the detection of low concentration of the drug and the identification of significantly altered peaks coming either from the drug without overlap from the sample or from drug-induced changes.


Acidic beverages, such as soft drinks, can produce erosion of resin composites. The purpose of the present study was to investigate mechanical properties of different esthetic restorative materials after exposure to acidic drink. Nine different composites were tested: nanofilled , microfilled hybrid , nanohybrid Ormocer , microfilled , microfilled hybrid , nanoceramic , supranano spherical hybrid , flowable microfilled hybrid , and bulk fill flowable . Thirty specimens of each esthetic restorative material were divided into 3 subgroups (n=10): specimens of subgroup 1 were used as control, specimens of subgroup 2 were immersed in 50 ml of Coca Cola for 1 week, and specimens of subgroup 3 were immersed in 50 ml of Coca Cola for 1 month. Flexural strength and elastic modulus were measured for each material with an Instron Universal Testing Machine. Data were submitted to statistical analysis. After distilled water immersion, nanofilled composite showed the highest value of both flexural strength and elastic modulus, but its flexural values decreased after acidic drink immersion. No significant differences were reported between distilled water and acidic drink immersion for all other materials tested both for flexural and for elastic modulus values. Even if nanofilled composite showed highest results, acidic drink immersion significantly reduced flexural values. Dental caries is an infective process that causes the fade of the tooth's hydroxyapatite. This process is mainly due to acid degradation of tooth structures. In order to stop the progression of this disease, the infected tissue has to be removed and it has to be replaced with a filling material . In the Composites are also used to treat other noncarious lesions of dental tissues, such as in case of dental erosion. This process is an irreversible loss of dental hard tissue by a chemical process without the involvement of microorganisms and this is due to either extrinsic  or intrinsic  sources , 6. In fThe purpose of the present investigation was to evaluate and compare deflection strengths and elastic modulus of various composites tested after acidic drink immersion.The 9 different composites were divided into groups : (1) nan−1. Once the composite was packed into the device, a mylar strip was used to make a flat surface of the specimen. Then all the specimens were light-cured for 3 minutes [2, Wavelength: 430-480 nm, Lamp socket: R7s, Lamp Diameter: 13.5 mm, Lamp length: 160 mm). After polymerization, the specimens were stored in distilled water at 37°C and 100% humidity before performing the flexural strength test.Thirty rectangular prism-shaped specimens (2mm × 2 mm × 25 mm) of each composite were prepared  with ad  minutes  into a lEach group of composites  was divided into three subgroups  of ten specimens each: the first subgroup (A) was used as control and were tested immediately after storage in distilled water for 24 hours; the specimens of the second subgroup (B) were immersed in 50 ml of acidic drink  for 1 week; the specimens of the last subgroup (C) were immersed in 50 ml of acidic drink  for 1 month. All specimens were immersed at 37°C and 100% humidity environment. A single specimen has been immersed for each of the 50 ml aliquots. The acidic drink was changed once a week for all the time . The pH Each sample was placed in an appropriate aluminum framework . The spaAfter specimen failure  the flexσ) and the elastic modulus (E) have been calculated [After collecting the data, the flexural strength . Descriptive statistics were calculated . Normality of the distributions was assessed with Kolmogorov and Smirnov test. Nonparametric analysis of variance  was applied to determine whether there were significant differences among the various groups. Mann–Whitney post hoc test was applied. Significance for all statistical tests was predetermined at P<0.05.Mean and standard deviations of the various groups are illustrated in When evaluating the samples of Subgroups B (after 1-week Coca Cola immersion) and C (after 1-month Coca Cola immersion), Filtek Supreme XTE showed a significant decrease in flexural strength if compared with distilled water immersion (Subgroup A) (P<0.05). All the other composites tested showed no significant differences in flexural strength values among the three different storage conditions (P>0.05), except for Filtek Supreme XTE (Group 1) that showed significantly higher values after storage in distilled water than after 30 days of acidic drink storage (P<0.05).Mean and standard deviations of the various groups are illustrated in When evaluating the samples of Subgroups B (after 1-week Coca Cola immersion) and C (after 1-month Coca Cola immersion) similar results were reported, except for Filtek Supreme XTE (Group 1) that showed no significant differences (P>0.05) with Ceram.X Universal (Group 6).Each of the composites tested showed no significant differences in elastic modulus values among the three different storage conditions (P>0.05), except for Filtek Supreme XTE (Group 1) that showed significantly higher values after storage in distilled water than after both one week and 30 days of acidic drink storage (P<0.05).Durability of restorations in the oral cavity is highly affected by the resistance to dissolution or disintegration caused by foods, drinks, and the acidity produced by bacteria . PostpraIn the present report, the flexural strength and elastic modulus of different composite materials have been tested. Previous studies evaluated flexural strength and elastic modulus of various dental materials. Many reports have been presented about fiber-reinforced composites used as endodontic posts , nets 2, and lonThe persistence of an acidic environment can cause a loss of mechanical properties of composites, glass-ionomer cements, and polyacid modified composites , 12. In In the present report, the most affected composite is the nanofilled composite , which is also the composite that has reached the highest value of flexural strength under physiologic solution storage. Furthermore, value of flexural strength of nanofilled composite (Group 1) after one month of Coca-Cola reached the average value of all the other composites. Regarding other composites, the effect of the acidic beverage does not affect significantly the flexural strength. Therefore, nanofilled composites present higher flexural strength than all other materials tested without immersion in acidic drink solution. On the other hand, in acidic environment no difference was reported among all materials tested.It is also possible to notice that Admira Fusion (Group 3) has the lowest value of flexural strength. This is a nanohybrid Ormocer based composite, and it is a different type of composite compared to all of the other composites tested. Ormocer is a composite technology that literally means ORganically MOdified CERamics . These mFlexural strength is a clinically relevant property for restorative materials, as it simulates composite use in high-stress bearing areas , 36. MorFurthermore, the data collected in the present investigation suggest that the percentage of the filler in the composites presumably does not affect flexural strength. This is in agreement with previous reports evaluating physical properties and depth of cure of composites , 39. FloIn the present report, also elastic modulus has been tested. Higher values were reported with nanofilled composite  and lowest values were reported with microfilled composite , flowable microfilled hybrid composite , and flowable bulk fill . As Filtek Supreme XTE (Group 1) showed the highest value of elastic modulus, this material can be considered the stiffest composite tested. The importance of the elastic modulus is mainly related to the choice of the right composite for a specific clinical situation: in fact, flowable composites are usually used in restorations of V class because their higher elasticity can absorb at best the chewing force in this specific region of the tooth. On the other hand, in occlusal restoration, where the material resistance has to be maximized, stiffer composites are more fit to the scope , 40. PreRecent research showed a significant increase in elastic modulus of various composite materials, with the increase of filler content . In factThe results of the present report, showed the following:(i) The various composite materials showed different flexural strengths and elastic moduli. Higher values were reported with nanofilled composite (Filtek Supreme XTE).(ii) Immersion in acidic drink lowered flexural strength and elastic modulus of nanofilled composite (Filtek Supreme XTE). Other materials were not affected by acidic drink immersion.