Kidneys have an important role in regulating water volume, blood pressure, secretion of hormones and acid-base and electrolyte balance. Kidney dysfunction derived from acute injury can, under certain conditions, progress to chronic kidney disease. In the late stages of kidney disease, treatment is limited to replacement therapy: Dialysis and transplantation. After renal transplant, grafts suffer from activation of immune cells and generation of oxidant molecules. Anesthetic preconditioning has emerged as a promising strategy to ameliorate ischemia reperfusion injury. This review compiles some significant aspects of renal physiology and discusses current understanding of the effects of anesthetic preconditioning upon renal function and ischemia reperfusion injury, focusing on opioids and its properties ameliorating renal injury. According to the available evidence, opioid preconditioning appears to reduce inflammation and reactive oxygen species generation after ischemia reperfusion. Therefore, opioid preconditioning represents a promising strategy to reduce renal ischemia reperfusion injury and, its application on current clinical practice could be beneficial in events such as acute renal injury and kidney transplantation. The kidneys are involved in several important bodily functions, including blood filtering, resulting in excretion of toxins and metabolic end products, regulating water volume, blood pressure control, secretion of hormones, and acid-base and electrolyte balance . Blood pGlomerular filtration is one of the major purposes of the kidney. The glomerulus filtrates through three specific layers: The fenestrated endothelium, the glomerular basement membrane, and epithelial podocytes with foot processes . MoleculEvaluation of the renal function is vital to determine renal conditions in several pathologies. The glomerular filtration rate (GFR) is used to assess the renal function and indicates the amount of fluid filtered by the kidneys, which is dependent on the hydrostatic and osmotic pressure. GFR is equal to the product of the net filtration pressure, hydraulic permeability, and filtration area. Several equations have been established to obtain the GFR such as Cockroft-Gault and MDRD  equations . KDOQI  5]. Uri. Uri5]. Many different conditions can affect the kidney and its function in a short or long term. The most significant pathologies are acute kidney injury (AKI) and CKD. AKI is defined as a reduction in the kidney function with a decreased GFR . It resu+/K+ ATPase enzyme and depletion of ATP produce uncoupling of the respiratory chain, free radical production, loss of epithelial cell adhesion, and cell death [One of the major causes of AKI is ischemia due to partial or total obstruction of the vessels inflow. . Lack ofll death ,13,14. Ill death . Vasodilll death ,16. Vasoll death ,18,19. TOne of the most critical mechanisms of AKI is IRI . KidneysAKI is an entity that if left untreated, can cause irreversible kidney damage, which can progress into CKD or even end-stage renal disease (ESRD). The postoperative development of AKI occurs in 40% of the cases and is related to increased morbidity and high mortality ,23,24. TSeveral biomarkers besides creatinine, GFR, and urinary output are currently under study as novel diagnostic approaches for AKI. Examples of these are NGAL  and KIM-1 (Kidney Injury Molecule), which are involved in acute responses to injury .2 [CKD is a consequence of several chronic diseases such as diabetes or hypertension . Risk fa2 ,30. This2 . The CKD2 ).Different pathological conditions affecting the glomerulus, vasculature, or tubulointerstitium can result in kidney structural deterioration. Renal fibrosis is the most common pathological factor in advanced kidney diseases and has shown to be the most reliable predictor of CKD progression to ESRD . The conSignaling pathways involved in renal fibrosis are complex and not completely elucidated. Several mechanisms underlying fibrogenesis have debuted, all of them with important characteristics that result in a multifaceted process. Representative molecules that participate in fibrosis are hypoxia-inducible factor 1, transforming growth factor-beta, nuclear factor-kappa b (NF-κb), angiotensin II, reactive oxygen species (ROS), interleukin 6 (IL-6), interleukin 8 and kidney injury molecule 1 .Patients that develop CKD exhibit numerous factors that contribute to the decline of the kidney function and the following clinical symptoms as hypertension, proteinuria, and mineral misbalance. Progression to ESRD is almost inevitable and chronic inflammation appears to be the main trigger. Macrophages and the interstitial fibroblast support the production of cytokines and collagen that modify and accumulate in the extracellular matrix. Finally, pharmacological approaches are minimal and based on controlling symptoms, leaving replacement therapies such as dialysis and transplantation as the best options to patients suffering from ESRD.+/K+ ATPase pump [Kidney transplant is considered one of the most suitable treatments for ESRD patients. However, during the transplant surgery, there is a cessation of renal blood flow with a posterior reestablishment, and therefore, the IRI phenomenon always takes place. Other conditions, such as trauma or vascular and heart surgery also cause IRI. Hypoxia and nutrient deprivation due to ischemia results in an excessive generation of ROS, which cause cell death and inflammatory responses . Biochemase pump . The orgase pump .The IRI mechanism involves the activation of the immune system by neutrophils, macrophages, and dendritic cells in kidneys. Endothelial and tubular epithelial cells also play an essential role in the inflammation induced by IRI ,39. IschKidney injury is worsened by the aggregation of neutrophils to endothelium in tubular capillaries and kidney interstitium. Neutrophil-endothelium adhesion causes neutrophils to activate, release their granules and secrete proteases, which generate ROS. That same activation produces interferon-gamma (INF-γ), interleukin 4 (IL-4), IL-6, interleukin 10 (IL-10), and TNF-α secretion . NeutropPreconditioning refers to the molecular changes that occur at the tissue level, which enable that same tissue to adapt and overcome later adverse events. Preconditioning includes the physiological and molecular adaptations in a changing environment. As a minor event, ischemic preconditioning is an example of how the body uses adverse conditions to improve tissue response for following incidents. Ischemic preconditioning is an adaptive mechanism that takes place in multiple organs like the brain, heart, kidney, liver, and muscle.Brief intervals of ischemia and early reperfusion have been considered as a beneficial therapeutic approach to contain the damage caused by further and more prolonged episodes of ischemia . ProtectPreclinical studies have shown evidence about renal protection conferred by a preceding ischemic event. Classical articles demonstrated that the ischemic preconditioning could reduce IRI improving the renal function, metabolic homeostasis, and preserving cell integrity and tissue morphology. However, they do not leave clarity about the mechanism involved ,51,52. RThe potential clinical use oriented to prevent AKI has already been tested in clinical trials. This strategy is able to reduce the incidence of AKI in patients undergoing a cardiovascular surgical procedure . HoweverThe molecular responses caused by ischemia and reperfusion can be reached pharmacologically with different anesthetic substances ,61. ThisIn summary, tissue exposure to an ischemic condition can enable cells to adapt rather than suffer damage. Therefore, cells under those conditions are able to manage further challenging situations more effectively.A comprehensive understanding of the nature of opioids and its influence on kidney pathophysiology may improve morbidity and mortality in patients undergoing surgical procedures as transplantation, and consequently, has the potential to modify clinical practice. Furthermore, opioids are commonly used to manage pain in CKD and post-transplant patients .Back to the 1800s, the first known opioid (morphine) was isolated from opium . Four naAnalgesia mediated by opioids is induced by binding to μ receptors in GABAergic neurons. These neurons inhibit descendent neurons in the brainstem and produce analgesia. The same effect is also obtained by inhibiting the release of pain mediators such as substance P, nitric oxide, and glutamate . Along wOpioid receptors can also be found in the kidneys, with the δ receptor broadly expressed and, on a smaller scale, the κ type . AlthougMorphine is known as the opioid prototype to which other opioids are compared. Both μ and δ receptors bind morphine to have its effect. These receptors are widely distributed in the human brain, mainly in the amygdala, hypothalamus, thalamus, and several cortical areas. Morphine has two major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide . M6G has been known to have a higher analgesic effect compared to M3G . MorphinUndoubtedly, the analgesic effects of morphine are supported by abundant scientific and clinical evidence. However, the properties on non-nervous tissues such as the kidneys are poorly understood and controversial. Some reports show that morphine has antioxidant properties and is a potent modulator of immune responses ,78. OtheFentanyl was developed in 1960 by Paul Janssen as a synthetic opioid drug for pain treatment and anesthesia. The range of effects consists of analgesia, anxiolysis, euphoria, and drowsiness . It workAlthough much has been studied about the beneficial properties of fentanyl in vivo and in vitro in the heart and cardiomyocytes ,85,86, lThere is considerable research interest in potential methods of renal protection against IRI. Proof from experimental studies shows that anesthetic preconditioning with volatile agents can be achieved and protect the heart, brain, and kidney from IRI . SeveralExperimental renal ischemia and reperfusion in rabbits showed that morphine significantly inhibited superoxide generation by neutrophils, suggesting a potential for reducing the oxidative stress after hypoxia . HabibeyMorphine, fentanyl, and other opioid medications are strong analgesics used frequently in analgesia and pain management, despite reported concerns about drug safety. Long-term administration is commonly associated with the development of side effects including tolerance, dependence, and addiction. Therefore, it is necessary to discriminate between the long-term effects and the effects of single uses in a surgical procedure and preconditioning. Pain management is an essential part of the comprehensive care of patients with CKD at any stage . One of Complex surgical anesthesia schemes in humans make it difficult to dissect the effect of these drugs on the renal function; however, perioperative use showed positive results. This is how the study by Terashi et. al. showed that anesthetic management using remifentanil exerted a renal protective outcome in perioperative adult patients with CKD for at least two weeks after orthopedic surgery . ConversActions of opioid drugs in the kidney have not been well characterized. Nonetheless, most of the information about mechanisms involved in organic protection conferred by opioids come from studies in organs as heart and brain. We will summarize below some of the molecules involved considering the overlapping molecular mechanisms between the kidneys and other organs it is important to mention.i proteins and also can activate the µ receptors, but this has very low expression in cardiomyocytes [Experimental and clinical studies showed that opioids can positively influence the cardiac function and could reduce the size of an infarction resulting from prolonged ischemia . Opioidsmyocytes . The δ rmyocytes ,106. Themyocytes  and invomyocytes . Morphinmyocytes . This remyocytes ,109.Fentanyl, morphine, and remifentanil are frequently used for neurosurgical procedures. Effects on the brain and nervous system undergoing an ischemic event have been documented in experimental models . PrecondEffective and safe anesthesia for successful transplantation depends on an understanding of the influence and interactions with other anesthetics . Here, wPropofol is an intravenous drug widely used in anesthesia as an inducer. It is characterized by its rapid onset of action and the speedy recovery of the patient from its effects. It has been shown to have a protective effect at the cardiac and renal level in reperfusion ischemia models , this efDexmedetomidine is an alpha-adrenergic receptor agonist drug and extensively used as a sedative. Dexmedetomidine protects against myocardial infarction , ischemiIsoflurane is a common volatile anesthetic in the clinic and protects against ischemic brain injury  by supprFinally, barbiturates can also reduce the severity of IRI in cardiac models . The molPotential new uses of old well-known drugs as opioids is an emerging field for kidney research. Anesthetic preconditioning is a promising strategy to reduce renal IRI, and its application on current clinical practice could be beneficial in events such as acute renal failure and kidney transplantation. Current evidence suggests that opioids provide organ protection by decreasing reactive oxygen species and inflammation. However, more experimental evidence is still needed to understand the physiological and molecular mechanisms involved in the protection of the kidney and to translate our current knowledge into clinical settings.


Two artisanal varieties of cheese made in Spain, one made of ewes’ raw milk and the other of goats’ raw milk were selected to evaluate the effect of a high hydrostatic pressure (HHP) treatment at 400 MPa during 10 min at 2 °C on the formation of biogenic amines (BA). These conditions were applied at the beginning of the ripening  and in the case of ewes’ milk cheeses also after 15th days (HHP15). BA formation was greatly influenced by HHP treatments in both types of cheese. HHP1 treatments significantly reduced the amounts of BA after ripening, being tyramine and putrescine the most affected BA in goats’ milk cheeses and tyramine and cadaverine in ewes’ milk cheeses. The BA reduction in the HHP1 samples could be explained by the significant decrease in microbiological counts, especially in the LAB, enteroccocci and enterobacteria groups at the beginning of ripening. The proteolysis in these samples was also affected reducing the amount of free amino acids. Although proteolysis in ewes’ milk cheeses HHP15 was similar than in control samples a reduction of BA was observed probably because the decrease caused on microbial counts. Biogenic amines (BA) are basic nitrogenous compounds formed in different foodstuffs due to the microbial decarboxylation of amino acids. The kind and the amount of BA formed depends on the decarboxylase capability of the bacterial strains present, the availability of substrate amino acids and the physicochemical properties of the matrix . Some arDuring cheese manufacture several factors may contribute to the accumulation of toxic amounts of BA. Good manufacturing practices to minimize the occurrence of BA-producing microorganisms in raw materials, pasteurization of milk or addition of BA-non-producing starter cultures have been suggested as BA risk mitigation options. Although it has been described that milk pasteurization reduces the level of decarboxylase-positive bacteria, later contamination of milk and curd during cheese manufacturing by decarboxylating bacteria and their subsequent growth and metabolic activity during cheese ripening usually results in BA build up ,11,12,13High hydrostatic pressure (HHP) is non-thermal processing method used to extend shelf-life of foods. This induces morphological changes and inhibition of enzymes and genetic mechanisms of microorganism . HHP offThe main objective of this survey was to evaluate HHP processing to reduce BA formation in two varieties of artisanal Spanish cheese made of raw milk from ewe and goat. Treatments were applied at different stages of ripening and the consequences of these treatments on the BA formation and proteolytic activity were evaluated during the ripening.Two types of artisanal ripened cheeses elaborated in Spain were studied in this work, both made with starter culture, enzymatic curd and pressed paste. The first one was produced from goats’ raw milk in the region of Catalonia, northeast of Spain, and the second was made from ewes’ raw milk in Castilla y León, central Spain. Three independent batches of each type of cheese were produced following the usual manufacturing procedures used by the manufacturers.HHP treatments were performed at 400 MPa for 10 min at a temperature of 2 °C using an Alstom HHP equipment  with a 2 L pressure chamber. A mixture of alcohol and water (1:9) was used into the chamber. The pressurization rate and depressurization time were 268 MPa/min and 55 s, respectively. Before processing, goats’ milk cheese samples were separated in two batches: samples not HHP treated (Control samples) and samples HHP treated before the 5th day of ripening (HHP1). In the case of ewes’ milk cheese samples a third batch for samples that were treated after 15 days of ripening (HHP15) was included. In all cases a portion of about 8.0 cm diameter was obtained and shaped to adequate it to the diameter of the cylinder of the HHP equipment and then vacuum packaged. After the HHP treatments, samples were deprived of the plastic bag and kept into a ripening chamber at 14 °C and 88% of relative humidity to continue with the ripening process for 60 days. Analyses of cheeses were performed at the 5th, 15th, 30th, 45th and 60th day of ripening.Lactococcus spp. were made on M-17 agar (Oxoid) supplemented with a bacteriological grade lactose solution  and incubated at 30 °C for 48 h; Lactobacilli were determined on Man Rogose Sharpe agar  and incubated at 30 °C for 48 h; Enterococci were enumerated using KF Streptococcus Agar (Oxoid) supplemented with 2,3,5-trifeniltetrazolium chloride solution 1% (Oxoid) and incubated at 37 °C for 48 h; enumeration of Enterobactericeae was performed on Violet Red Bile Glucose Agar  and counts of Escherichia coli was made on the chromogenic selective media Coli ID  and incubated at 30 °C for 24 h; Staphylococcus aureus counts were determined on Bair Parker Agar supplemented with rabbit plasma fibrinogen  and incubated at 37 °C for 24–48 h.Ten grams of each sample were homogenized in 90 mL of sterile Buffered Peptone Water  with a paddle blender . Counts of WSN) fraction was obtained and determined by the Dumas combustion method [WSN fraction was expressed as a percentage of Total Nitrogen (TN), described as the Ripening Index (RI), according to the formula:Water Soluble Extracts (WSE) of cheese were prepared according to the method described by Kuchroo and Fox . From thn method . The nitl-Leucine per g of cheese.The measurement of the amino group content was determined on WSE by the Trinitrobenzensulphonic Acid method (TNBS) according to the procedure described by Hernández-Herrero et al. . ResultsThe RP-HPLC method described by Eerola et al.  and modip < 0.05. Comparisons of mean values of proteolysis indexes were performed using the Student-Newman-Keuls test with the significance level set at p < 0.05. All tests were performed with the SPSS for windows (v.15.01) program .Analysis of variance (ANOVA) was performed on all data from each batch and treatment of goats’ and ewes’ milk cheeses at different ripening stages. Comparisons of mean values of physicochemical, microbiological and BA were followed by Duncan test with significance level set on 10 CFU/g, probably because strains of Lactococcus lactis were added as the starter culture, while lactobacilli counts were significantly lower at the beginning of the ripening, although their counts rose as the ripening progressed, achieving similar counts to lactococci. Enterococci counts remained steady at a level about 6 log10 CFU/g at the beginning of the process, showing a slight decrease around the 15th and the 30th day in the goats’ and ewes’ cheeses, respectively. S. aureus, enterobacteria and E. coli counts decreased during the ripening until becoming undetectable in most of the samples. These results are in agreement with those reported by other authors in other varieties of cured cheeses [In control samples (non HHP-treated) of both types of cheese, Lactoccocci was the main microbial group at the beginning of the ripening, showing counts above 8 log cheeses ,28,29,3010 CFU/g and 1.5 log10 CFU/g in the goats’ and ewes’ milk cheeses, respectively. In the case of lactobacilli mean reductions were 3.41 log10 CFU/g and 1.03 log10 CFU/g. However, these counts recovered along the ripening process and no statistical differences were observed at the end of the work with respect to control samples. Ewes’ milk cheeses HHP15 showed a reduction of about 1.91 log10 CFU/g and 1.33 log10 CFU/g for lactoccocci and lactobacilli counts, respectively. In that case the subsequent recovery of these counts was not so clear and they did not achieve the same counts than control samples. Novella-Rodríguez et al. [10 CFU/g in goats’ milk cheeses as a consequence of HHP, although a subsequent increase was found during ripening. Rynne et al. [10 CFU/g in starter and non-starter LAB in cheddar cheese treated at 400 MPa on the first day of ripening. In ovine milk ripened cheeses treated on day 1 and 15 at 300 MPa and 400 MPa proved to cause similar reductions although recovery of LAB counts was observed only in samples treated 1st day. HHP treatments also affected significantly (p < 0.05) the counts of enteroccocci in both type of cheeses in either HHP1 and HHP15 samples, being unable to recover the initial counts in any case. Arqués et al. [10 CFU/g in enteroccocci counts when a treatment of 400 MPa for 10 min at 10 °C was applied on the 2nd day of ripening to “La Serena” cheeses, remaining constant during the rest of the ripening. Although high counts of enteroccoci have been associated with the unhygienic processing of cheese, their presence is also considered important for the development of the typical aroma and flavour of traditional Mediterranean cheeses. Their counts may range from 104 to 106 CFU/g in curds and 105 to 107 CFU/g in ripened cheeses [S. aureus HHP1 treatments also caused significant reductions in both kind of cheeses and HHP15 in ewes’ milk cheeses, becoming undetectable in most cases at the last day of the work. Nevertheless, the initial counts were already quite low. Similar results were reported in “La Serena” cheeses treated at 400 MPa on the 2nd day of ripening [S. aureus has been described as one of the most HHP resistant non-sporulated bacteria. López-Pedemonte et al. [10 CFU/g.HHP treatments reduced significantly the Lactic Acid Bacteria (LAB) counts. In HHP1 treated samples, lactoccocci counts decreased 2.2 logz et al.  also obse et al.  reporteds et al.  reportedripening  and in Cripening . S. auree et al.  describe10 CFU/g of enterobacteria in ewes’ milk cheeses after a 400 MPa treatment applied the 1st and the 15th day of ripening. Initial counts of E. coli were significantly lower than enterobacteria. O’Reilly et al. [E. coli to HHP in cheeses with reductions above 6 log10 CFU/g after HHP treatments at 400 MPa.HHP treatments showed to be very effective reducing Gram-negative bacteria except for HHP1 in goats’ milk cheese samples, where a slight growth was noted on the 15th day probably due to a possible recovery of the sub lethal injured cells after the HHP treatment. However, cheese ripening conditions, with low pH, increasing salt concentration and presence of LAB, made difficult this recovery to consolidate and no positive counts were detected later at the 30th and 45th days of ripening. Juan et al.  also desy et al. , Capellay et al.  and De Ly et al.  have preThe proteolytic activity parameters measured in the goats’ and ewes’ cheeses are presented in Significant differences between control and HHP1 samples in TNBS were observed from the 45th to the 60th days of ripening in goats’ milk cheeses, while the differences in FAA content were mainly noted from day 30 to 60. In both parameters control samples showed the highest values. The ratio of RI displayed a different trend where an increment of around two times was observed during the first 15 days period in control and HHP-treated samples but after this point the proteolysis rate became slower. In the ewes' milk cheeses, an increment on the three-proteolysis index evaluated was observed during the ripening period . In the WSN is produced mainly by the rennet and to a lesser extent by plasmin or cellular proteinases, whereas starter peptidases are primarily responsible for the formation of small peptides and free amino acids [RI values indicated that proteolysis was more intense during the first 15 days of ripening in the goats’ and ewes’ cheeses in control and HHP treated samples. This proteolytic activity was probably caused by milk and rennet proteinases, being not so clear the role of microbial proteinases. On fact, no acids . Messensno acids  observedno acids  in a stuno acids  found inWSN/TN values than control samples, at the end of the ripening but higher than those obtained in samples with HHP-treatment on the 1st day. In contrast, some works reported after application of HHP treatments not differences on proteolysis indexes during ripening in Gouda [On ewes’ milk cheeses HHP15 samples showed slightly higher values on the three-proteolysis index evaluated than control samples during the ripening period, although no significant differences were observed at the end of the ripening, reflecting that this treatment did not significantly affect the proteolysis process. However, the HHP application during the initial stages of the ripening in ovine and caprine milk cheeses led to a decrease of the proteolysis rate. Similar results were obtained by Juan et al.  who in pin Gouda  and chedin Gouda  or an inin Gouda ,42. Starin Gouda , being oin Gouda . HHP-indin Gouda  and possin Gouda  indicatein Gouda  pointed in Gouda  and Calzin Gouda  noticed in Gouda ,42 suggeEnterococcus spp. and LAB isolated from cheese samples [TY and PU were the main BA formed in untreated goats’ milk cheeses, showing concentrations of about 492 and 476 mg/kg, respectively, at the end of the ripening. Whereas in ewes’ milk control samples the predominant BA were TY and CA with 277 and 106 mg/kg, respectively. Several authors reported, in variable ranges, TY (88.6–445 mg/kg), HIS (not detected–697 mg/kg), PU 74.15–446.5 mg/kg) and CA (44–269.77 mg/kg) as the most abundant BA in goats’ and ewes’ milk ripened cheeses .5 mg/kg ,55,56. T samples ,59,60,61Enterococcus spp. also have shown this capability “in vitro” [Lactococcus lactis, are also able to form PU via the agmatine deiminase [PU amounts in control goats’ milk cheeses were almost the same than TY but HHP1-treatments caused a decrease on the first 15 days, remaining almost stable throughout the rest of the ripening. PU levels were about 83% lower in the HHP1 samples than in control samples at the 60th day. The pressure application in ewes’ milk cheeses also affected the PU amounts formed, showing that HHP1 treatments limited the production of this diamine around 93% compared with control cheeses at the end of the ripening. HHP15 showed to be less efficient reducing the formation of PU. With respect to CA, untreated and HHP-treated goats’ milk cheeses displayed amounts below 100 mg/kg without appreciate significant differences between them. In control ewes’ milk cheeses this diamine increased mainly during the first 15 days, while in HHPI and HHP15 cheeses remained without significant changes throughout the ripening. The amounts of TR and PHE increased during the ripening in control goats’ milk cheese samples without significant differences in relation to HHP1-treated samples, while low amounts were detected in ewes’ cheeses remaining practically constant during ripening and without showing significant differences between treatments. Formation of PU and CA is usually associated with Gram negative bacteria, although some strains of n vitro” . Some LAeiminase . The appeiminase .Enterobacteriaceae strains able to decarboxylate histidine in diverse foodstuffs [In goats’ raw milk control cheeses the concentration of HIS was very low at the beginning of ripening but increased later reaching its maximum after 45 days (18 mg/kg). No significant changes were observed until the 60th day. In HHP1-treated samples HIS showed a similar behaviour but in this case, differences were found after the 45th day displaying levels 68% lower than control samples at the 60th day of ripening. HHP1 ewes’ milk cheese samples showed a reduction of 92% of HIS when compared with control samples. This treatment was more efficient than the HHP15. Diverse authors reported low HIS amounts in cheese (below 100 mg/kg), relating the production of this BA with some LAB ,56,63,64odstuffs ,67,68,69odstuffs ,59,70.In cheeses made from raw goat milk, low levels of polyamines were found at the beginning of ripening, increasing their concentration very slightly during ripening until the 60th day . HoweverThe effectiveness of HHP treatments depends on different factors, such as the type of cheese, the stage of ripening, the HHP processing conditions applied, the kind and number of microorganisms present. In this work, the use of HHP applied at the initial phases of ripening affected significantly the microorganisms responsible of forming BA, and, in consequence, reduced its content, especially of TY and PU in goats’ milk cheeses and TY and CA in ewes’ milk cheeses, assuring the safety of this product for the most BA sensitive consumers. As previously mentioned, HIS and TY are the BA that most frequently have been related with food-borne outbreaks, being suggested threshold values in cheese between 200–400 mg/kg of HIS ,72,73 an


The incidence of breast cancer and immediate breast reconstruction is on the rise particularly in the US and Western Europe. Over the last decade, implant based breast reconstructions have gained popularity. The prepectoral breast reconstruction has emerged as a novel technique, minimally invasive, preserves the chest wall anatomy while restoring body image. However, implant rippling appears to be an adverse effect associated with this technique. We have described a new grading system for rippling following prepectoral implant breast reconstruction and discussed its management. We then evaluated the new grading system in our practice. We looked at the first 50 consecutive patients who underwent prepectoral implant based breast reconstruction. In our experience, 45 patients (90%) had grade 1, 3 patients (6%) had grade 2, 1 patient (2%) had grade 3 and 1 patient (2%) had grade 4 rippling. The observed rippling was seen more often in patients with low BMI<20 and in those who had poor subcutaneous fat preoperatively (pinch test<2 cm). Prepectoral implant based breast reconstruction adds a whole new dimension to breast reconstruction. However rippling can be an undesired adverse effect associated with this technique and patients need to be informed. The current technique of creating a new breast in the pre-pectoral plane usually involves ex-vivo coverage of the breast implant with a mesh and subsequent attachment to the chest wall, preserving both the pectoralis major and serratus anterior muscles. As the breast remains in its anatomical plane animation deformity is prevented,2 postoperative pain is reported to be lower, and shoulder function is not impaired.3,4Implant-based breast reconstructions account for 40-60% of all breast reconstructions performed in the UK and approximately 75% in the United States.5 We aim to provide a new grading system for rippling with prepectoral implant breast reconstruction in order to guide its management. The prepectoral mesh forms an internal bra with the mesh implant wrap, which in turn is secured to the chest wall. Biological meshes integrate through collagen remodelling, which ultimately integrate with host tissue becoming vascularised.6 The collagen matrix in biological grafts aids in remodelling and new collagen deposition.7 Synthetic meshes create a scaffold and promote fibrous tissue growth to cover for the implant. Integration occurs via a fibroblastic reaction alongside a mild inflammatory response.8Implant rippling, however, remains a large concern.8Over time, and under the weight of the implant, the upper part of the prepectoral reconstruction atrophies. This, coupled with thinning of the collagen in the skin, can result in visible implant rippling. Indeed, subcutaneous cover influences the degree of rippling and is often less prevalent in those with a high body mass index (BMI).We have tabulated the degree of rippling  to aid w9Patients were selected for this new procedure according to the Association of Breast Surgery and the British Association of Plastic, Reconstructive and Aesthetic Surgeons’ guidelines for ADM-assisted implant-based breast reconstruction. Inclusion criteria includes a body mass index (BMI) of <35 kg/m2, no previous radiotherapy, an estimated mastectomy weight of <500 g and a good subcutaneous layer (pinch test>1 cm). The technique of prepectoral implant based breast reconstruction has been described by the author previously.9,10 All patients in this series had fixed volume silicone implant with an average volume of 360 mL (range: 150-540 mL). Basic demographics of our series are noted in We looked at the incidence of rippling in our centre. All consecutive patients had prepectoral implant-based breast reconstruction using a pre-shaped Braxon® mesh: a porcine derived acellular dermal matrix (ADM) that is 0.6 mm thick.We looked at the first 50 consecutive patients who underwent prepectoral implant based breast reconstruction. In our experience, 45 patients  had grade 1, 3 patients  had grade 2, 1 patient  had grade 3 and 1 patient  had grade 4 rippling. The observed rippling was seen more often in patients with low BMI<20 and in those who low subcutaneous fat preoperatively (pinch test<2 cm). All patients with rippling were offered correction of which only 10% underwent treatment. Two patients with grade 2, One patient with grade 3 underwent lipomodelling, while the patient with grade 4 underwent exchange to a larger implant and lipomodelling.11,12 As such, there was a paradigm shift towards submuscular implant based breast reconstruction. However, over the recent years, prepectoral breast reconstruction has regained its popularity due to the problems associated with submuscular implant based reconstruction, that largely being animation deformity.3Breast cancer appears to be the most frequent cancer among women with an increase in the number of patients having mastectomy with immediate implant based reconstruction each year. Historically, subcutaneous implant based reconstruction was associated with poor cosmetic outcome including rippling and visible implant contours.12 However, the same plane gives rise to rippling, an unwanted side effect that can be observed over time. The current literature reports the incidence of rippling to lie between 0-35%  fat injections.14 Lipomodelling is often used to correct rippling and can be carried out in stages with fat harvested and stored but injected over a period of time. Along with lipomodelling, a number of adjunct procedures can be employed: if saline implants were initially used then converting to gel based implants may provide some improvement.15To avoid rippling in the patients with low BMI, Salibian 15,16 Combination of ADM and lipomodelling  restores the thickness of mastectomy skin flaps resulting in improvement in the aesthetic results.17 Finally, a capsulorrhaphy can be performed with exchange of implants along with fat injections. Based on our experience, we have developed a grading system of the implant rippling and its management depending on the grade (The addition of another ADM to the thinned out flap can provide thickness to the upper pole making the implant less visible reduce rippling.he grade .Patients with grade 2 and grade 3 rippling were corrected with lipomodelling. The fat is grafted from the patient’s abdomen, thigh, or knee areas. Patients with grade 4 rippling benefit from higher volume lipomodelling with or without exchange of implant. Further follow up of the patients after the fat injections is important with re-evaluation and possible repeat of lipomodelling procedure, if necessary, in 3-6 months. This should be discussed with the patient before the first lipomodelling session to manage the patient’s cosmetic expectations. 18 However, formal evaluation of this is required. Prepectoral or muscle sparing implant-based breast reconstruction is a minimally invasive method of breast reconstruction. This new technique brings a new choice in implant-based breast reconstruction with preservation of normal anatomy. However, rippling is an adverse effect associated with this technique and patients should be well informed.Indeed, further longitudinal evaluation is required to validate our results. In our series we mainly used lipomodelling while other adjuncts such as change of implants, tightening of excess skin can be offered to correct rippling. We also postulate that using an expander inflated with air may cause less tissue atrophy as it is lighter and results in less stretching of the skin.The authors declare no conflict of interest.


N-[2--hexyl]]-nicotinamide hydrochloride (3e) as the most promising compound with inhibitory potencies against EeAChE and EqBuChE in the low nanomolar level 67 and 153 nM, respectively. Moreover, 3e compound is non-hepatotoxic, able to inhibit amyloid beta aggregation, and shows a mix-type of cholinesterase’s inhibition. The mixed type of inhibition of the compound was confirmed by molecular modeling. Then, yeast three-hybrid (Y3H) technology was used to confirm the known ligand-receptor interactions. New derivatives do not show antioxidant activity (confirmed by the use of two different tests). A pKa assay method was developed to identify the basic physicochemical properties of 3e compound. A LogP assay confirmed that 3e compound fulfills Lipinsky’s rule of fiveHere we report the two-step synthesis of 8 new cyclopentaquinoline derivatives as modifications of the tetrahydroacridine structure. Next, the biological assessment of each of them was performed. Based on the obtained results we identified 6-chloro- Alzheimer’s disease (AD) is the most common form of dementia and chronic, neurodegenerative disease. AD attacks the brain leading to impaired memory, thinking and behavior, mainly among the elderly. AD has been proven to be a multifactorial disease associated with several aspects including low levels of acetylcholine (ACh), formation of β-amyloid (Aβ), hyperphosphorylated tau aggregates, oxidative stress and so on. Genetic studies have shown that dysfunction of Aβ or tau is sufficient to cause dementia. Ongoing molecular research is expected to lead to a true understanding of the disease’s pathogenesis. The neuropathological alterations described above suggest that the target at these factors could create the possible and effective treatment of AD ,2.N-Methyl-d-aspartate (NMDA) receptor antagonist. Unfortunately, none of the currently available strategies can completely prevent neurodegeneration and cure AD  was tested for bait autoactivation. All negative and positive controls described in the manual of Y2H Matchmaker Gold system were performed.The Y3H method was applied to evaluate if the synthesized hybrid ligand induces in vivo the interactions between AChE and A4, BACE1A, MAO B and MAPT. Plasmids suitable for Y3H screening were prepared by Gene Universal Inc. . The company performed cDNA synthesis, molecular cloning of inserts in a proper frame as the NdeI/BamHI fragments into the pGBKT7 (ACHE) or pGADT7  vectors. Gene Universal Inc. performed also the yeast codon optimization to facilitate the high expression of human recombinant proteins in The small scale mating procedure (5mL) based on Matchmaker Gold Y2H system manual recommendations was applied to exclude the putative protein–protein interactions between bait (AChE) and four preys . Obtained cell suspension was transferred on DDO agar plates to initially screen for putative protein–protein interactions. DDO agar plates were supplemented with Aureobasidin A (200 ng/mL) and X-α-Gal (40 µg/mL) (DDO/X/A).Finally, a small scale mating procedure (10 mL) was performed. The mating mixture contained the hybrid ligands at the concentration of 10 µM to promote the putative ligand-mediated protein interactions. The products of mating were placed on DDO/X/A agar plates containing 10 µM of hybrid ligand. The blue colony was transferred to the more stringent QDO agar plates containing Aureobasidin A (200 ng/mL), X-α-Gal (40 µg/mL) (QDO/X/A) and hybrid ligand (10 µM) to confirm the presence of interactions. The obtained blue colony was spread on QDOX/A agar plates without the hybrid ligand to confirm that the interaction depends on the hybrid ligand. The strength of hybrid-ligand induced interactions was quantitatively evaluated by yeast β-galactosidase assay kit .ADMET  prediction was performed to estimate the risk and effectiveness of use our compound as a medicine. Calculated parameters were confronted with the values defined by the Lipinski’s “Rule of Five”. Our experimental parameters like logP and pKa (Base) were used as basic parameters to predictions. The ADMET analysis was done with the help of ACD/Percepta Version 14.0.0 .3a–3h were evaluated by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free-radical scavenging assay according to the method of Blois (1958) with slight modifications [50 values of test compounds were determined. Results are expressed as the mean ± SD of at least three different experiments performed in triplicate.DPPH Assay. The antioxidant activities of compounds ications . Briefly•+ Assay. The ABTS•+ solution was prepared by reaction of 5 mL of a 7 mM aqueous ABTS solution with 88 µL of a 140 mM potassium persulfate. After storage in the dark for 16 h, the solution was diluted until the absorbance value of 0.70 ± 0.05 at 734 nm was reached. Solutions of each tested compounds were prepared in PBS (pH = 7.4). For each compound and concentration, measurements were made in triplicate with blank solutions using a microplate reader Synergy H1 (Biotek) [ABTS(Biotek) .2 before the initiation of the assay.The SH-SY5Y (human neuroblastoma) (European Collection of Cell Culture) was chosen to determine the potential neuroprotective properties of novel compounds. SH-SY5Y were grown in Ham’s F12:EMEM (1:1) (Sigma Aldrich) containing 15% Foetal Bovine Serum , 2mM Glutamine (Sigma Aldrich), 100 units/mL penicillin and 100 mg/mL streptomycin  and 1% non-essential amino acids . Cells were grown in an incubator at 37 °C with 5% CO3 cells per well and cultured for 24 h in the incubator . After 24 h of cells incubation, medium was removed and cells were exposed to the 100 µL of the compound solutions over a range of concentrations ranging from 0.0001 to 10 µM or nothing but culture medium (control). After 24 h, the medium was removed and 50 µL of the MTT solution was added. Plates were incubated for additional 2 h in the incubator . Then, the MTT solution was removed and 100 µL of DMSO was added. Plates were incubated for 10 min at room temperature and 5 µL of Sorensen Buffer was added. The plate was swayed and the absorbance was measured in the microplate reader  at a wavelength of 570 nm. The cell viability was expressed as a percentage of the control values (blank). The experiments were done in triplicate [Cells were seeded in 96-well plates at the density of 5 × 10iplicate .3e against oxidative stress were determined in three different experiments. Hydrogen peroxide (H2O2) was used to generate exogenous free radicals in the first experiment. SH-SY5Y were incubated with compounds in the range of concentrations (from 10 µM to 0.01 µM) for 24 h before the addition of H2O2 (100 µM). Then, H2O2 was added and the incubation in the presence of compounds as conducted for the next 24 h. In the second and third experiments, mix of rotenone (30 µM) with oligomycin A (10 µM) (R/O) was used to cause mitochondrial reactive oxygen species. A second experiment involved “pre-incubation”. SH-SY5Y were incubated with 3e before the addition of the rotenone and oligomycin A mixture for 24 h. Next, the mixture was added, and cells were incubated with 3e for additional 24 h. The last experiment, “co-incubation”, involved adding the cells at the same time as the mixture of rotenone with oligomycin A and 3e. Incubation took 24 h. Trolox was used as a positive control. Each experiment was done three times in quadruplicates and cell death was tested by the MTT assay. Data were shown as the percentage of the reduction of MTT in regard to non-incubated cells [The neuroprotective properties of ed cells ,23.The 3-dimentional structure of synthesized compounds were drawn in Corina on-line (Molecular Networks and Altamira)  and subs3e is a dual AChE and BuChE inhibitor and a promising compound for further development. This compound is a good EeAChEI and EqBuChEI with IC50values . 3e compound shows a mixed-type of inhibition confirmed by in vitro study and molecular modeling. What is very important is that 3e derivative are non-hepatotoxic, show low antioxidant activity, and have the ability to inhibit Aβ aggregation. The results of ADMET prediction and LogP assay, which confirmed the fulfillment of Lipinsky’s rule of five, shows a good pharmacokinetics and future potential use in medicine. Moreover, we conducted an innovative study—a yeast three-hybrid technology (Y3H) which is an extension of the two-hybrid system (Y2H). In the Y2H, the interaction between two hybrid proteins activates the expression of reporter genes facilitating the yeast cells to grow on particular selective media [To sum up, a novel series of eight cyclopentaquinoline derivatives were designed, synthesized, and evaluated as potential multifunctional compounds for the AD therapy. In vitro studies concerned their ability to inhibit AChE and BuChE. The results demonstrated that compound ve media ,43. In tve media ,45,46,47


Some polyphenols are known to improve the symptoms of diabetes. In the present study, we investigated the effects of a polyphenol-rich extract of maple syrup (MSx) on a diabetic mouse model.Ay mice were fed a normal or 0.05% MSx-supplemented diet for 42 days. Body weight, food intake, serum biochemical parameters, and fecal total bile acid were measured. Gene expression of liver and epididymal white adipose tissue (WAT) and cecal microbiota were analyzed. Data were analyzed with an unpaired two-tailed Student’s t test or Welch’s t test according to the results of the F test.KK-Serum low-density lipoprotein cholesterol levels were significantly reduced in mice that consumed MSx. Hepatic genes related to fatty acid degradation and cholesterol catabolism were upregulated in mice that consumed MSx. In contrast, the expression of genes related to lipid metabolism in WAT was unaffected by the intake of MSx. There were no significant differences between the two groups in terms of total bile acid level in the feces and the relative abundance of bacteria in the cecum.Our results primarily indicate that MSx can help alleviate one of the symptoms of dyslipidemia. Ay mice, a model for type 2 diabetes accompanied by obesity, and acacia polyphenols improved dyslipidemia and insulin resistance in KK-Ay mice [The increasing prevalence of diabetes is a global problem . Diabete-Ay mice , 4.Acer saccharum, which contains a large number of polyphenols [Maple syrup is a sweetener made from the sap of the sugar maple tree, yphenols . A butanyphenols . Inhibityphenols . In our yphenols . Maple syphenols .Ay mice and fed them an ethanol extract of maple syrup (MSx) containing 15.02% polyphenols as gallic acid equivalents for 42 days. Then, serum biochemical parameters related to nutrient metabolism, gene expression profiles of liver and epididymal white adipose tissue (WAT), and composition of intestinal bacteria were evaluated to determine the effects of MSx on nutrient metabolism, with the results demonstrating that MSx alters lipid metabolism.Based on the above reports, we speculated that maple syrup, particularly its polyphenol-enriched extract, could alleviate chronic hyperglycemia in individuals with diabetes. In the present study, we used KK-MSx prepared from Canadian maple syrup (Canada No. 2/Amber) by SiliCycle Inc.  was purchased by the Federation of Quebec Maple Syrup Producers  . BrieflyAy mice 4 weeks of age were purchased from CLEA Japan . The mice were individually housed in a room maintained at a temperature ranging from 21 to 23 °C with 50–70% relative humidity and a 12:12-h light/dark cycle. Normal and 0.05% MSx-supplemented diets were prepared based on the AIN-93G formula by Oriental Yeast Co.  (Table n = 8 each) with approximately equal mean body weights. Mice were fed the normal or MSx diet for 42 days and were allowed free access to the diet and ultrapure water during this period. Body weight and food intake were measured every 2 days. Feces were collected on days 36–38 of the testing period and were stored at − 80 °C until use. After 16 h of food deprivation, mice were sacrificed under sodium pentobarbital anesthesia. Blood was collected from the heart chamber and centrifuged at 830×g for 10 min for serum isolation. The liver was treated with RNAlater . Epididymal WAT and cecum contents were immediately frozen in liquid nitrogen. All samples were stored at − 80 °C until use.Male KK-Eight serum biochemical parameters, including glucose, glycated albumin, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein cholesterol (HDL), triglycerides, nonesterified fatty acids (NEFA), and total ketone bodies, were measured on a 7180 Clinical Analyzer  by Oriental Yeast Co. Serum insulin and tumor necrosis factor (TNF)-α levels were measured with a Mouse Insulin ELISA Kit  and a Mouse TNF-α Quantikine ELISA Kit , respectively. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula:HOMA-IR = insulin (ng/ml) × 26 μIU/ml × glucose (mg/dl).Total cholesterols were isolated as total lipids from the liver according to the Folch method  and its t test or Welch’s t test according to the results of the F test. Differences were considered significant at P < 0.05.Data were analyzed with the unpaired two-tailed Student’s Total RNA was isolated from the liver and WAT with TRIzol Reagent (Thermo Fisher Scientific Inc.) and was purified with an RNeasy Mini Kit and RNase-Free DNase Set . Total RNA concentration was measured on a spectrophotometer. RNA integrity was evaluated with an Agilent RNA 6000 Nano Kit and on an Agilent 2100 Bioanalyzer  and RNA Integrity Number was confirmed greater than 8.0. cRNA was prepared from purified 100 ng total RNA with the GeneChip 3′ IVT PLUS Reagent Kit and hybridized to a GeneChip Mouse Genome 430 2.0 Array (Thermo Fisher Scientific Inc.). The array was stained using a GeneChip Hybridization, Wash and Stain Kit and a GeneChip Fluidics Station 450 (Thermo Fisher Scientific Inc.). The fluorescence signals of the probes were scanned with a GeneChip Scanner 3000 7G (Thermo Fisher Scientific Inc.) and converted to an intensity value with Affymetrix GeneChip Command Console software (Thermo Fisher Scientific Inc.).p value [p value using the Benjamini and Hochberg method [The intensity values of probe sets were normalized by the distribution-free weighted method in the case of liver and by the quantile normalization factor analysis for robust microarray summarization method in the case of WAT , 13. Datp value . FDR wasg method . GO termg method . DEGs ang method . Ensemblg method .http://primer3.ut.ee/) and these sequences are shown in Additional file ctin, beta (Actb) whose expression showed the smallest differences between the two groups among 5 housekeeping genes. PCR reactions were performed with 3 technical replicates for each gene. DNA contamination was not detected.Total RNA 1 μg) isolated from the liver as described above was reverse transcribed with a SuperScript IV VILO Master Mix (Thermo Fisher Scientific Inc.). The synthesized cDNA (1 ng) was amplified in a 10-μL reaction volume with a PowerUp SYBR Green Master Mix (Thermo Fisher Scientific Inc.) on a CFX Connect Real-Time PCR Detection System with CFX Maestro 1.1 software  under the following conditions: 50 °C for 2 min, 95 °C for 2 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Primers were designed with Primer3web v.4.1.0  using a modified terminal restriction fragment length polymorphism (T-RFLP) method . BrieflyAy mice were fed a normal diet with or without 0.05% MSx for 42 days. Feces were collected on days 36–38. After fasting, mice were sacrificed, and serum samples were obtained. Values for physical and biochemical parameters are shown in Table To examine the effect of MSx on diabetes, KK-To examine changes in cholesterol metabolism, we performed a hepatic transcriptome analysis using the DNA microarray technique. There were 272 and 265 DEGs that were up- and downregulated, respectively, in the MSx group compared to the expression levels in the control group (FDR < 0.001). A total of 537 DEGs were annotated with GO terms to categorize their function. DEGs were shown to be related to lipid metabolism  and the immune system  (Table 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 (Hmgcs2), encoding the rate-limiting enzyme in the ketogenesis pathway, was upregulated. These results suggest that fatty acid degradation is accelerated by the intake of MSx. All DEGs mapped to the cholesterol catabolic pathway were upregulated in the MSx group. Moreover, cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1), encoding the rate-limiting enzyme, was included among the DEGs mapped to the pathway. This result suggests that cholesterol catabolism was stimulated by the intake of MSx.To examine the effect of MSx on lipid metabolism, we mapped DEGs annotated with GO terms related to lipid metabolism in four KEGG pathways as follows: mmu00071 fatty acid metabolism, mmu00100 steroid biosynthesis, mmu00120 primary bile acid biosynthesis, and mmu00072 synthesis and degradation of ketone bodies. We integrated the KEGG pathways into a single metabolic pathway , Hmgcs2, and cytochrome P450, family 8, subfamily b, polypeptide 1 (Cyp8b1) showed significant differences between the two groups  and serum amyloid A 1/ 2/ 4 (Saa1/ 2/ 4) are synthesized in the liver in response to inflammatory cytokines [Inflammation leads to the aggravation of diabetes symptoms by activating the immune system . To examytokines . These DThe hepatic gene expression profile suggested that lipid catabolism increased with the intake of MSx. A gene expression analysis of WAT was performed to examine the effect of MSx on lipid metabolism in the tissue. There were 241 and 298 DEGs that were up- and downregulated, respectively, in the MSx group compared to the control group (FDR < 0.02). A total of 539 DEGs were annotated with GO terms. DEGs related to lipid metabolism were not distinctly presented (Table The gut microbiota is reported to affect host lipid metabolism . Therefop = 0.23). Thus, MSx intake may cause the use of acetyl-CoA for ketogenesis rather than cholesterol synthesis, resulting in a decrease in serum LDL cholesterol levels. The enhanced ketone body production in diabetic patients is caused by the promoted utilization of fatty acids as an energy substrate due to the impairment of glucose utilization. However, there were no significant differences between the two groups with respect to the levels of serum glycated albumin, glucose, or insulin. This result indicates that MSx intake does not aggravate diabetic symptoms. Detailed research is needed to understand the mechanism of the underlying effect.According to the results of this study, dietary intake of MSx alters lipid metabolism Fig. . The serAy mice. The results of qRT-PCR analysis were also affected by an individual difference, especially C1 in the control group. The effects of MSx intake on lipid metabolism of KK-Ay mice will be accurately revealed by increasing sample size.Various parameters  in this study showed the large variance, which may be derived from an individual difference of KK-Ay mice, the catabolism of lipoproteins such as VLDL and LDL is suppressed due to insulin resistance, resulting in the elevation of these lipoprotein cholesterol levels in blood [In the context of diabetes, including in KK-in blood , which iUsing gene expression analysis of the liver and WAT, this study showed the possibility that MSx intake may affect the immune system. However, the results demonstrating the effect of MSx intake at the protein level could not be shown. Thus, further study is required to understand the effect of MSx intake on the immune system.The present study examined the effect of MSx intake in obese diabetic mice. The intake of MSx does not influence chronic hyperglycemia, but it reduces the LDL cholesterol level. Hepatic gene expression analysis suggested that the intake of MSx promotes cholesterol catabolism, although further studies are required to identify the detailed mechanism underlying the reduction in LDL cholesterol levels. Our findings provide evidence that MSx contributes to alleviating one of the symptoms of dyslipidemia.Additional file 1. Primer information.Additional file 2. GO terms and annotated DEGs in the liver. Up and down represent DEGs that were up- and downregulated, respectively, in mice fed MSx compared to the levels in controls. Fold change was calculated from the expression value of each probe normalized by the Affymetrix Micro Array Suite 5.0 method.Additional file 3. DEGs related to the immune system in the liver. Down represents DEGs that were downregulated in mice fed MSx compared to the levels in controls.Additional file 4. GO terms and annotated DEGs in WAT. Up and down represent DEGs that were up- and downregulated, respectively, in mice fed MSx compared to the levels in controls. Fold change was calculated from the expression value of each probe normalized by the Affymetrix Micro Array Suite 5.0 method.


A minority of patients have a late-onset form of disease that presents from late-childhood to adulthood and has a slowly progressive course with prolonged survival.The GM2 gangliosidoses (GM2), Tay-Sachs and Sandhoff diseases, are rare, autosomal recessive genetic disorders caused by mutations in the lysosomal enzyme β-hexosaminidase A Little research has been published documenting patient experiences with late-onset Tay-Sachs and Sandhoff diseases and how the disease impacts their daily lives and functioning. This study explored the most frequent symptoms and functional impacts experienced by patients with late-onset GM2 gangliosidosis through interviews with patients and caregivers.A qualitative research study design was employed, using three focus groups and 18 one-on-one interviews with patients who were recruited at the National Tay-Sachs and Allied Diseases Annual Family Conference. Transcripts were generated from the discussions, and patient quotes were analyzed using a content analysis approach. Concepts were aggregated into symptom and functional impacts, and the frequency of mention in the focus groups and individual interviews was calculated.n = 19, 95%), “clumsy” gait , fatigue ] and impacts  disclosed by patients and caregivers were similar to those previously reported in the literature. However, less frequently described symptoms such as gastrointestinal issues  and coughing fits  have been expanded upon. This study evaluated the immediate impact of these symptoms on the patients’ lives to highlight the burden of these symptoms and the functional limitations on daily living activities, independence, and emotional well-being. The findings were used to develop a conceptual disease model that could serve as a foundation for patient-centered outcomes in clinical trials and provide insights to the medical community that may benefit patient care.Many of the frequently described symptoms [muscle weakness (This study contributes to the current understanding of symptoms associated with late-onset GM2 gangliosidosis, and further identifies the many consequences and impacts of the disease. These symptoms and impacts could be measured in clinical trials to examine the effects of novel treatments from the patient perspective. HEXA) or β subunit (HEXB), respectively, of the two β-hexosaminidase isoenzymes, hexosaminidase A (αβ) and hexosaminidase B (ββ). Tay-Sachs disease is caused by a deficiency of β-hexosaminidase A activity, whereas Sandhoff disease results from a combined deficiency of β-hexosaminidase A and B activities. The reduced ability of patients’ cells to catabolize GM2 ganglioside leads to progressive lysosomal accumulation of GM2 and related glycolipids, hence their classification as GM2 gangliosidoses  and just kind of like give up. Some people just say ‘Huh? Huh?’ … He’ll order a lager and they give him a water and he’ll get annoyed” .Embarrassment was centered on symptoms, such as muscle weakness, gastrointestinal issues, and coughing, as well as impacts such as falling and going out. One patient was embarrassed by how muscle weakness prevented him/her from being able to stand up to use the bathroom, stating “that’s really bad for my psyche and ego … I wear “Depends” and I have my help clean it up at night” (Interview #4). One patient mentioned that the frequent coughing was embarrassing as it caused a lot of unwanted concern from others, particularly when “going out to eat and then you eat and you’re either…you start that cough and people think you’re constantly choking” (Interview #13).A number of patients were worried about disease progression and worsening symptoms. After a bad fall that resulted in a broken patella, one patient was worried, stating “I was scared I would never walk again” (Interview #14). Though some patients were worried about falling, another patient specified that his/her fear was not of falling, but of not being able to get up again. Lastly, the fear of the future and how quickly the disease might progress was a tangible concern, and a patient stated that “I’ve noticed over the last probably six months that I’m definitely starting to progress…not quickly, but much quicker than I have in the previous four to five years” (Interview #16). What was even more concerning for this patient was that it would be many years before he/she would be able to collect a disability check, and “I don’t know if I’m going to be able to make it that far” (Interview #16).Patients were acutely aware of the effect that late-onset GM2 gangliosidosis has on other people. They felt that their disease is burdensome to others and that these feelings made it difficult for them to proactively engage with other people. Difficulty communicating was reported in 11 interviews (55%) and affected communication with family, friends, and strangers. Despite patients’ best efforts to communicate, “some people won’t take the time to try and listen” (Interview #6).Due to the actual and perceived social hurdles, patients and caregivers felt that their disease negatively impacted relationships as “friends, family, everything, his whole life was affected by this” (Interview #6). Engaging in romantic relationships was difficult, particularly for one patient who “used to be engaged and it never worked out because she couldn’t handle my condition” (Interview #4). Another patient mentioned that having the disease was very challenging for his/her self-esteem to handle having “such a difficult diagnosis, looking for a partner. Most women, they’ll run for the hills when they see that” (Interview #11). One patient felt socially isolated due to limited mobility, stating “I can’t dance at weddings. When people go to weddings and they are getting up to do the Hora or whatever, I am not and I am not too happy about that. My girl got married two years ago and I was sitting down. My husband danced, but I could not dance” (Patient Focus Group #1).Most patients were unable to work in the career of their choice due to the physical limitations of the job, communication obstacles, or financial constraints. Six patients (30%) reported that the disease affected their productivity, as muscle weakness affected jobs that ranged from physical labor –“I just could not handle the climbing up and down in the truck all day long” (Interview #15) to fine motor skills – “He had problems with the weakness and typing in the computer” (Interview #14). A caregiver stated that due to the patient’s worsening speech difficulties, “she ultimately lost her job. … So rather than having a career in marketing, she’s a cashier at a supermarket. …. Her career is no longer an option for her” (Interview #8). Lastly, patients had few opportunities available as the job needed to meet certain requirements, such as “I have to get a job that I could do in my wheelchair. And I have to get a job that’s close enough to home for me … That dictates where I can work” (Interview #1).n = 10, 50%). Being unable to work caused financial difficulties and many patients needed to live with their parents for symptom and financial support. As one patient was unable to work a full-time salaried job and was on disability, her caregiver stated that “The disability only allows her to make a certain amount of money a month, which limits whatever kind of job she can get. … She could no longer afford to support herself” (Interview #8). Due to their disease symptoms, patients often had to purchase specialty items, such as an electric chair to navigate stairs, and others made changes to their homes such as “put[ting] a foam floor on the top of a packed … floor, so when she falls, it’s not as bad” (Caregiver Focus Group). When a patient mentioned in a focus group that “I will have to sell my house and move to a one level eventually” (Patient Focus Group #1), others also echoed the need to move to a more accommodating residence. Since some patients required non-medical services, such as a trainer or nursing service, these were not covered by health insurance and required out-of-pocket payment. Patients were acutely aware of the amount of money they earned in order to remain eligible for Medicaid, which was necessary as “prescriptions are over $1,000 a month, so I can’t be without Medicaid” (Interview #1).Patients and caregivers were significantly impacted financially due to disease management or securing their future financial stability. Patients expressed an overwhelming concern about being eligible for or staying on disability (n = 3) and speech and walking (n = 2 each). For treatment, four patients each felt that preventing disease progression and being able to walk would be the most important treatment benefits. A meaningful improvement in symptoms would be reducing muscle weakness (n = 4), improving the ability to communicate, improving clarity of speech, and not needing to use a wheelchair (n = 2 each). Overall perspectives are presented in Tables Patients were asked to consider what the most bothersome symptoms of late-onset GM2 gangliosidosis were and which were the most important to treat. Due to time constraints, not all patients were asked these questions. Five patients felt that the most bothersome symptom was muscle weakness, followed by lack of independence . Caregivers noted that many patients experienced frustration in their inability to self-care, help their caregivers around the household, and move independently. One caregiver stated that, “I think the biggest problem [he] had was stopping driving. That was his last vestige of independence. It didn’t matter that he was in a wheelchair” (Caregiver Focus Group). With the progression of disease over time, caregivers expressed that patients felt increasingly isolated as a result of their reduced ability to express themselves or actively engage with others in social situations.The aspects of late-onset GM2 gangliosidosis perceived by caregivers to be most burdensome for patients were the lack of independence, impact on self-esteem, and muscle weakness, confirming patient reports that muscle weakness was among their most bothersome symptoms. Caregivers noted that the lack of independence and impact on self-esteem were often linked. For one caregiver, the patient who he/she cared for “feels he is a burden. It doesn’t matter what I say or do. I can’t seem to help him overcome that lacking that he has” (Caregiver Focus Group).A conceptual model Fig.  was deveThis study used a semi-structured interview method that consisted of focus groups and one-on-one interviews to elicit patient perspectives (complemented by caregivers’ views) on the symptoms and functional limitations experienced by patients with late-onset GM2 gangliosidosis. A conceptual model was developed to capture the relationship between symptoms, functional limitations, and other impacts of the disease. As no instruments exist that specifically measure the impact of late-onset GM2 gangliosidosis on the patient experience, this conceptual model may be used to develop new disease-specific clinical outcome assessments (COAs) and inform clinical trial and registry designs.7,8 In both late-onset Tay-Sachs and Sandhoff diseases, patients described their balance and frequent falling as being “clumsy” (Patient Focus Group #2). Development of a broad-based ataxic gait that made walking difficult is well-documented, and this study captured various adaptations by patients, including assistive devices, specialty adaptations to the home, and changes to gait to assist with mobility.Patients mentioned experiencing symptoms of muscle weakness and muscle tremors, as well as altered speech , all of which are cited as being more apparent in the first decade of disease manifestation, indicating underlying neurologic problems.Some of the symptoms of late-onset GM2 gangliosidosis disclosed by patients and caregivers in this study are similar to those reported elsewhere, particularly problems with balance and walking, muscle weakness, and cognitive function , 10. HowThe main strength of this study is the patient-centered approach, which focused on generating a representative view from patients and caregivers. Considering the rarity of the disease, a relatively large sample size of 20 patients was attained. The qualitative study design involved both focus group and individual interviews, which minimized any potential limitations that may exist with either method of data collection alone. Data saturation was achieved with the patient interviews, and the caregiver group corroborated the most salient concepts. The consistent input from participants supports the likelihood that the perspectives documented here are representative of patients with late-onset GM2 gangliosidosis. However, it is possible that since the population of this study was drawn from people who attend a disease-specific patient advocacy conference, they may differ from other patients with late-onset GM2 gangliosidosis. Nevertheless, the opportunity to use national patient/family meetings to collect qualitative data from a broad spectrum of patients and caregivers is an important one and represents a patient-centered approach that recognizes patients as the experts of their conditions.The limitations of this study are those that are common to similar studies in a population of patients with a rare disease, especially one that has limited peer-reviewed literature. The retrospective nature of the research, the self-reporting by parents and other family members, and the presence of disease-related features makes it difficult to exclude recall bias and ascertainment bias. As this study was not designed to evaluate subgroups such as gender or age, there may be different experiences of the disease across populations. Additionally, there were only four patients with late-onset Sandhoff disease who participated in the study, and thus the findings may not fully reflect the experiences of all patients with late-onset Sandhoff disease. However, the four patients with late-onset Sandhoff disease who participated in this study largely reported similar experiences to what was reported by patients with late-onset Tay-Sachs disease. Clinically, late-onset Tay-Sachs and Sandhoff diseases are considered as one entity.This research has demonstrated that patients experience many consequences of the symptoms of their disease, some of which have a direct impact , while other, more indirect impacts, extend over a longer term . The symptoms with the most impact present viable opportunities for evaluation as possible primary or secondary endpoints in late-onset GM2 gangliosidosis clinical trials. Patients were most frustrated by their muscle weakness, lack of independence, altered speech, and lack of/limited mobility, and many expressed a desire to improve these symptoms. In more advanced states of the disease, speech becomes more burdensome for patients as communication becomes more difficult. The conceptual model will be validated in a future study and can provide insight to the medical community. In addition, a repeated measures approach could be useful in order to better understand the trajectory of the condition and how patients are impacted by and cope with it over time.There is a clear need for additional support and solutions for patients that would enable more independence and management of their own lives. Research into ways to enable patients to move more independently, especially in moderate to advanced disease stages, as well as being able to communicate more effectively, would strongly improve patients’ quality of life by enabling them to be mobile, better understood, and independent.This study revealed that patients with late-onset GM2 gangliosidosis experience difficulties with walking, balance, and communication that limit many aspects of their lives, from relationships to career choices. The findings and development of this preliminary conceptual model may prove useful to the recognition or development of instruments that will optimally measure relevant clinical outcomes in clinical trials, registries, or other studies. In the long term, these findings are an important contribution to the current understanding of patients’ and caregivers’ experiences with late-onset GM2 gangliosidosis and may lead to the introduction of interventions designed to better support patients.Sanofi Genzyme collaborated with the National Tay-Sachs and Allied Diseases (NTSAD) patient association in the United States (US) to identify potentially eligible patients and to conduct the interviews. Adult patients with late-onset GM2 gangliosidosis and their caregivers were recruited through their attendance at the NTSAD Annual Family Conference in 2015 and/or 2018. No other screening was conducted to determine eligibility for participation.The protocol, informed consent form, and relevant supporting information were submitted for review and approved by Schulman IRB and Advarra IRB. The study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and consistent with Good Clinical Practice and applicable regulatory requirements.Informed consent was obtained in writing from interested patients and caregivers prior to participation in the focus groups and individual interviews. To preserve patient confidentiality, all transcripts and audio recordings were de-identified and no personal identifying information was collected.A qualitative research design using semi-structured focus group and individual interviews with patients and caregivers was adopted to elicit multiple perspectives on each topic . In 2015Each focus group comprised between five and seven participants. To ensure homogeneity between focus group participants, the GM2 gangliosidosis patient groups differed by age distribution, time since diagnosis, and living situation: one group with older and more advanced patients (Patient Focus Group #1), and the other one (Patient Focus Group #2) with primarily younger patients diagnosed within the previous 5 years and living alone or with parents. The sessions were audio recorded with participant permission to capture the discussion verbatim in the transcripts in order to be analyzed a posteriori. The patient focus groups were designed to improve the understanding of the signs, symptoms, and burden of disease by obtaining the views directly from the perspective of patients with late-onset GM2 gangliosidosis. To further confirm the symptoms and impacts that were experienced by patients, a single focus group interview was held with seven caregivers of some of these patients. This focus group was designed to expand upon the concepts spontaneously elicited by patients and obtain information on concepts that were not fully discussed with patients.In 2018, 18 interviews were conducted with patients with late-onset GM2 gangliosidosis, nine of whom had also participated in the 2015 focus group interviews. All of the interviews were conducted one-on-one, with the exception of five patients who were interviewed with their caregivers assisting in communicating the full breadth of their experience. The interviews lasted approximately 75 min and the goal was to confirm the most salient symptoms and impacts for patients with GM2 gangliosidosis.Data were pooled across the 2015 and 2018 interview cohorts to describe the symptoms and functional limitations of GM2 gangliosidosis. While this included data from the nine patients who participated in both the focus group and individual interviews, their 2018 data was retained in the analysis because the one-on-one interviews provided a more in-depth assessment of their experiences. There also was the possibility that patient experiences may have changed in the three-year period between the interviews. The initial analytic step involved repeatedly listening to recordings and reading transcripts while taking notes, and comparing the recordings and transcripts with the minutes made during the focus group sessions. Meaningful information was organized into themes that described and categorized the possible observations. De-identified interview transcripts were reviewed and analyzed in ATLAS.ti 8 software using content analysis techniques to extract meaningful themes and concepts. The frequency with which themes and concepts were mentioned was the primary method for establishing the salience of their relevance to GM2 patients. The transcript from each focus group was treated as a single patient in analysis, as it was not possible to distinguish the identities of individual speakers from the focus group transcripts.Concept saturation was assessed to ensure that the number of interviews was sufficient to confirm that all of the most relevant concepts related to the late-onset GM2 gangliosidosis patient experience were identified.


Drosophila recapitulates many previous findings about gene duplication in this lineage, showing that new functions often emerge rapidly and asymmetrically in younger duplicate gene copies, and that functional divergence is driven by strong natural selection. Hence, CLOUD represents a major advancement in classifying retention mechanisms and predicting evolutionary parameters of duplicate genes, thereby highlighting the utility of incorporating sophisticated statistical learning techniques to address long-standing questions about evolution after gene duplication.Learning about the roles that duplicate genes play in the origins of novel phenotypes requires an understanding of how their functions evolve. A previous method for achieving this goal, CDROM, employs gene expression distances as proxies for functional divergence and then classifies the evolutionary mechanisms retaining duplicate genes from comparisons of these distances in a decision tree framework. However, CDROM does not account for stochastic shifts in gene expression or leverage advances in contemporary statistical learning for performing classification, nor is it capable of predicting the parameters driving duplicate gene evolution. Thus, here we develop CLOUD, a multi-layer neural network built on a model of gene expression evolution that can both classify duplicate gene retention mechanisms and predict their underlying evolutionary parameters. We show that not only is the CLOUD classifier substantially more powerful and accurate than CDROM, but that it also yields accurate parameter predictions, enabling a better understanding of the specific forces driving the evolution and long-term retention of duplicate genes. Further, application of the CLOUD classifier and predictor to empirical data from  Gene duplication is a mutational process that creates copies of existing genes. Experimental studies in several diverse species have revealed that duplication occurs faster than all other types of spontaneous mutation , thus seYet, the evolutionary path leading from gene duplication to functional innovation remains unclear. According to traditional evolutionary models , gene duOn the other hand, genomic studies from the past two decades show that the duplication process itself can often generate a child copy that is distinct from its parent copy . A key eDue to their vastly different evolutionary forces and functional outcomes, differentiating among duplicate gene retention mechanisms is critical to understanding how gene duplication drives phenotypic innovation. Accordingly, many studies have tackled this problem through applications of comparative  and modeDrosophila , making it applicable to a wide range of single- and multi-cellular biological systems.Though OU processes have been used to model expression evolution of single-copy genes , they haosophila  recapituDrosophila. First, we introduce our OU framework for modeling expression evolution after gene duplication, which forms the basis of the CLOUD classifier and predictor. Next, we formally define the multilayer neural network architecture implemented by CLOUD for both classification and prediction tasks. We then employ simulations to evaluate the relative classification powers and accuracies of CDROM and CLOUD across a wide range of parameters, as well as in more targeted regions of the parameter space. We also use these simulations to probe its accuracy in predicting parameters driving gene expression evolution after duplication, specifically its ability to estimate optimal gene expression, selection strength, and phenotypic drift for each of the classified retention mechanisms. Last, we apply CLOUD to empirical data from Drosophila . We assumed two hidden layers when training and testing CLOUD, as this resulted in the best cross-validation performance . Our training set consisted of 50,000 observations, of which 10,000 were simulated under each class. We trained CLOUD on these data, and explored evolutionary parameters drawn on a logarithmic scale across many orders of magnitude. Specifically, we independently drew the five parameters er CDROM  to the sAnalysis of the resulting classifications reveals that CLOUD generally has substantially higher power A and accα and α) or random phenotypic drift is weak  and phenotypic drift is strong  to make predictions for each of the five parameters (α), which are more precise than those of phenotypic drift  or phenotypic drift is weak  or phenotypic drift becomes stronger (larger α and α and α) with weak phenotypic drift  with strong phenotypic drift . As for the trained CLOUD classifier and predictor, we assumed m = 6 tissues, and explored all possible distinct scenarios in which k tissues shared one mechanism and m − k tissues shared a different mechanism. For each setting, we evaluated 1,000 independent replicate test data sets, with tissue model parameters drawn from the same wide distributions as in the training data set.We showed that under ideal settings, CLOUD is a superior classifier to CDROM, and is also adept at predicting underlying evolutionary parameters. Thus, we next explored the performance of the trained CLOUD classifier and predictor on test data generated under scenarios that violated model assumptions of the training data. In particular, we considered test data in which the duplicate gene retention mechanism was non-uniform across the simulated tissues. Specifically, we evaluated scenarios in which Comparisons among these diverse scenarios illustrates that the classification performance of CLOUD is dependent on a combination of the difference between flexibilities in model parameters of the two retention mechanisms and the numbers of tissues sharing each retention mechanism  online. α for the setting of three “Conserved” and “Subfunctionalized” tissues  compared to those classified as conserved or subfunctionalized  estimates were shifted toward higher values for retention mechanisms in which there were acquisitions of new functions  relative to those in which ancestral functions were preserved  L2-norm regularization when γ  =  0, L1-norm regularization that incorporates feature selection when γ = 1, and both types of regularization when To prevent overfitting, we include an elastic net-style regularization penalty term  on the wi is from class k, and 0 otherwise. Hence, all output values are zero except for that corresponding to class k, which has a value of one. Based on this output, we employ the loss function used in In the classification problem, for training observation deviance ℒ(y^(i),kth parameter value from simulated replicate i. Based on this output, we employ the loss function used in In the prediction problem, 4 observations from each of the five classes, for a total of N = 50,000 training observations. We assumed that tissues were independent, and that there were a total of m = 6 tissues as in an empirical data from Drosophila  and the second is only present in one species (child). Quantile-normalized gene expression abundances for carcass, female head, ovary, male head, testis, and accessory gland tissues in D. melanogaster and D. pseudoobscura were obtained from the Dryad data set associated with https://doi.org/10.5061/dryad.742564m. In that study, paired-end RNA-sequencing reads were downloaded from modENCODE (https://www.modencode.com, and aligned to reference transcriptomes of each species with Bowtie 2 (We applied CDROM and CLOUD to empirical data consisting of py genes  and theipy genes . In part species , was dowodENCODE  at httpsBowtie 2 . AbundanBowtie 2  were calBowtie 2 , quantilN = 50,000 observations as described in subsection Training the neural network on data simulated from OU processes above. Gene expression data from single-copy genes in Drosophila (L = 2 hidden layers, and through five-fold cross-validation (Because CLOUD requires estimates of th MACSE , which ath MACSE  for eachth MACSE . The emposophila  were uselidation , estimatMolecular Biology and Evolution online.www.flybase.org, modENCODE at https://www.modencode.com, and Dryad at https://doi.org/10.5061/dryad.742564m.The data underlying this article were derived from sources in the public domain: FlyBase at https://msaa267_Supplementary_DataClick here for additional data file.


Loligo vulgaris among two closely related and less valuable species. The assay, coupled to rapid genomic DNA extraction, is suitable for large-scale screenings and on-site applications due to its easy procedures, with fast (30 min) and visual readout.A colorimetric assay, exploiting the combination of loop-mediated isothermal amplification (LAMP) with DNA barcoding, was developed to address the authentication of some cephalopod species, a relevant group in the context of seafood traceability, due to the intensive processing from the fishing sites to the shelf. The discriminating strategy relies on accurate design of species-specific LAMP primers within the conventional 5’ end of the mitochondrial COI DNA barcode region and allows for the identification of  Loligo vulgaris and its discrimination from two genetically similar and often mislabeled species, Loligo reynaudii and Loligo forbesii [Food adulteration has become a concern of great interest worldwide. This issue is strictly connected to the phenomenon of trade globalization and overexploitation of biodiversity, which are causing profound changes in the whole food supply chain and market systems. Seafood is among the most adulterated food categories . Its lonforbesii . This onLoligo spp. tissues were obtained from a previous study assessing the mislabeling rate of cephalopod seafood items in the Italian market using a DNA barcoding approach [Loligo vulgaris, L. forbesii, L. reynaudii, and closely related species , were downloaded from GenBank and aligned using MUSCLE online [approach . COI seqE online  to perfoTo perform LAMP experiments, we homogenized squid tissues (≤5 mg) with a sterilized plastic pestle, and DNA was extracted using the InstaGene Matrix , following the manufacturer’s instructions. With this protocol, DNA was rapidly extracted (approximately 15 min) and required no further purification steps prior to perform LAMP experiments.4, 1.4 mM of each deoxyribonuclotide triphosphate (dNTP) , 0.32 U/µL Bst 2.0 WarmStart DNA polymerase , and 0.06 mM Xylenol Orange . The colorimetric reaction was performed at 63 °C and the result was visualized after 30 min.Colorimetric LAMP reactions were performed in a Bio-Rad T100 Thermal Cycler. Specifically, 4 µL of genomic DNA (≈25 ng/µL) was used as a template and mixed to 21 µL of reaction mix, containing 0.2 µM of forward outer primer (F3) and backward outer primer (B3), 1.6 µM of forward inner primer (FIP) and backward inner primer (BIP), 1.2 µM of forward loop primer (LF) and backward loop primer (LB) , 0.6 M betaine , 1x LAMP reaction buffer , 6 mM MgSO4, 1.4 mM of each dNTP , 0.32 U/µL Bst 2.0 WarmStart DNA polymerase , and 0.2x SYBR Green . The reaction was performed at 63 °C for 60 min.Fluorimetric LAMP reactions were performed in an Applied Biosystems StepOne Real-Time PCR System. Specifically, 4 µL of genomic DNA (≈25 ng/µL) were used as a template and mixed to 21 µL of reaction mix, containing 0.2 µM of F3/B3, 1.6 µM of FIP/BIP, 1.2 µM of LF/LB , 0.6 M betaine , 1x LAMP reaction buffer , 6 mM MgSOColorimetric LAMP products were electrophoresed on a 1% agarose gel to assess the amplification reactions. In detail, 500 mg of ultra-pure agarose  was dissolved in 50 mL of 1x tris-borate-EDTA buffer (TBE)  using a microwave oven. A total of 5 µL of SYBR Safe DNA gel stain  was added to the solution, which then was poured into the plate with a spacer for gel polymerization. Each product of reaction was analyzed by mixing 5 µL of LAMP products to 1 µL of gel loading dye. A 0.1–10 Kb DNA ladder  was used as a molecular weight marker. The electrophoresis was performed at 80 V, and the result of the run was visualized in a Bio-Rad Gel Doc XR system.Loligo vulgaris products is representative of an important issue in the field of seafood since the global demand for cephalopods has shown a positive trend in the last decades, with the main importers and consumers being Italy, Spain, and Japan . Given this growing market importance, the correct identification and labelling of traded cephalopods is of primary importance for food safety issues due to the possible occurrence of direct or indirect toxicity phenomena in different species [The species substitution of labelled  species ,35.The schematic procedure of the entire method is displayed in Loligo tissue was used as starting material prior to homogenization with a sterilized plastic pestle. Then, without any purification steps, a few microliters of the extracted target were directly added to the reaction tube, containing all reagents for the amplification reaction. The six primers were designed to be highly specific to the authentic species (unlike the polymorphic variants), and annealed to the selected regions in the presence of the target, triggering the amplification process. The colorimetric result is directly visualized by the naked-eye, due to a color change from purple to pink/orange when amplicons are produced. Remarkably, the overall colorimetric method, including the DNA extraction, the amplification reaction, and the colorimetric readout of the result, requires no instrumentation, apart from a simple heating block.A rapid DNA extraction from the squid tissue was performed using the InstaGene Matrix  following the manufacturer’s instructions. Less than 5 mg of L. vulgaris, and allow its discrimination from the two closely related species. In detail, all the available DNA barcodes of each Loligo species and close relatives (The key point of the entire strategy relies on the accurate design of species-specific LAMP primers, which target the mitochondrial COI gene of elatives  were alielatives  were desL. vulgaris, L. reynaudii, and L. forbesii specimens, using 100 ng/µL of each crude DNA extract. The isothermal amplification process was performed in a simple heating block, and the colorimetric result was rapidly and easily visible within 30 min (The one-step colorimetric LAMP was applied to various genomic DNA samples, rapidly extracted from the tissues of n 30 min A.L. vulgaris, as shown by the typical profile of LAMP products, unlike the closely related species that were not amplified (As is shown, the colorimetric assay was able to correctly identify the target species, which exhibited a clear purple-to-orange color change, whilst the other species maintained the same initial color as the control sample. The reaction was further controlled by agarose gel electrophoresis, confirming that the amplification occurred specifically for mplified B. In addmplified  by usingThus, the positive control reaction was tested on each sample under the same conditions. The visual inspection of the results showed that all samples turned pink/orange , demonstBesides on-site assessments, this reaction may also represent an interesting laboratory-based methodology due to its simplicity and rapidity. Actually, when coupled to a real-time fluorescence reader, using a classical fluorescent intercalating reporter instead of a colorimetric dye, the assay exhibited high discrimination efficiency and ultra-rapidity, being capable of providing clear authentication results in only 15 min .L. vulgaris, L. reynaudii, and L. forbesii specimens by fluorimetric LAMP, observing fast amplification after 15 min of reaction only in the case of the target species L. vulgaris. Hence, the optimized design of the LAMP primers, combined with the DNA barcoding approach, enabled us to address the authentication of L. vulgaris. Notably, both the colorimetric and the fluorimetric LAMP were also proven effective when associated with a rapid DNA extraction procedure.Specifically, we analyzed three genomic DNA extracts from In this work, we reported the development of a fast, on-site assay for the authentication of squid species, addressing the specific discrimination of one species from two genetically similar and less valuable ones. By coupling the LAMP technique with DNA barcoding and rapid DNA extraction procedures for a one-step readout, we showed the possibility of achieving fast, naked-eye readout of the species discrimination. Such features make these methodologies suitable for large-scale screenings and on-site industrial quality controls in several fields.