According to guidelines, the histological diagnosis of severe alcoholic steatohepatitis (ASH) can require liver biopsy if a specific treatment is needed. The blood test AshTest  has been initially validated for the non-invasive diagnosis of ASH in a large population of heavy drinkers. The aim was to validate the AshTest accuracy in the specific context of use of patients with suspected severe ASH, in order to reduce the need for transjugular biopsy before deciding treatment.The reference was liver biopsy, performed using the transjugular route, classified according to its histological severity as none, minimal, moderate or severe. Biopsies were assessed by the same experienced pathologist, blinded to simultaneous AshTest results.A total of 123 patients with severe clinical ASH  were included, all had cirrhosis and 80% had EASL histological definition of ASH. 95% of patients received prednisolone; and the 2-year mortality was 63%. The high AshTest performance was confirmed both for the binary outcome [AUROC = 0.803 (95%CI 0.684–0.881)] significantly higher than the AST/ALT AUROC , and for the severity of ASH-score system by the Obuchowski measures for , as well as for the diagnosis and severity of ballooning, PMN and Mallory bodies. According to attributability of discordances, AshTest had a 2–7% risk of 2 grades misclassification.These results confirmed the diagnostic performance of AshTest in cirrhotic patients with severe clinical ASH, in the specific context of use of corticosteroid treatment. AshTest is an appropriate non-invasive alternative to transjugular liver biopsy. In patients with suspected alcoholic liver disease, recent guidelines recognized that the precise indications of liver biopsy are not well established in routine practice due to significant morbidity/mortality related to liver biopsy. There is however a consensus that transjugular liver biopsy "should be considered" for the diagnosis of severe alcoholic steatohepatitis (ASH) that is amenable to specific therapy such as corticosteroids, both in the EASL and AASLD guidelines,,2 These In 2006 we published the first accuracy validation study of a non-invasive biomarker called AshTest . Two hundThe specific aim of the present study, in comparison to the first validation, was to focus on the performance of AshTest in the real context of use, which is a patient with suspected severe clinical and histological ASH who requires specific treatment such as corticosteroid. The following performances of AshTest as a surrogate have been assessed: be in the diagnosis of mild, moderate and severe histological steatohepatitis, in the binary diagnosis of histological ASH according to different definitions, and the correlation with ASH score.AshTest was prospectively assessed in consecutive patients admitted to the Hepatology department’s intensive care unit at "Groupe Hospitalier Pitié Salpêtrière" for suspected severe ASH, clinically defined in a patient with severe liver dysfunction in the context of excessive alcohol consumption and the exclusion of other causes of acute and chronic liver disease, ,2 such aThis non-interventional study was exempt from IRB review after institutional IRB review . All data were analyzed anonymously. All clinical investigation were conducted according to the principles expressed in the Declaration of Helsinki.We ask to all patients of our unit to sign an inform consent for this type of utilization of blood sample. All co-authors had access to the study data and had reviewed and approved the final manuscript.Liver biopsies were fixed, paraffin-embedded and stained with hematoxylin–eosin–safran and Masson’s trichrome or picrosirius red for collagen. A single, experienced pathologist (FC), unaware of the patient characteristics, including biomarker results, analyzed the histological features using a specific scoring system, at x200 and x400 magnification.Scoring procedures that focus on the main "independent" elementary lesions, as proposed for chronic viral hepatitis  or non-aHistological ASH was defined as the presence of steatosis, ballooning and PMN (EASL definition), In sensiThe non-binary primary endpoint , was calculated in the identical fashion as the NAS score. The sum of the three elementary lesion grades , resulting in a 4-grade severity score: H0, no ASH; H1, minimal ASH (score 1–2); H2, moderate ASH (score 3–5); and H3, severe ASH (score 6–9). In sensitivity analyses, the severity of histological ASH, also given by the pathologist in four grades in his conclusion, was also used to evaluate the test performance.S1 File). with the serum AST activity and specific algorithms adjusted for age and gender. AshTest scores range from 0 to 1.00, with higher scores indicating a greater probability of significant lesions. FibroTest and SteatoTest  were determined using published recommended pre-analytical and analytical procedures.,11GGT, ALT, AST and BILI were measured by a Hitachi 917 analyzer and Roche Diagnostics reagents , A2M, APOA1, and HAPTO were measured using a Modular analyzer . All coefficient of variation assays were lower than 10%.Discordance between biopsy and biomarker tests for the prediction of histological ASH was analyzed according to their respective risk factors of failure.,11 Risk According the obvious risk of commercial bias we performed several analyses of association between risk of false positive/negative and specific conditions of severe ASH, which could interact with AshTest components. We compare the value of AshTest and its components between patients with or without sepsis at admission to identify if infected patients had an inflammatory profile with increasing haptoglobin  or A2M  in comparison without sepsis.High risk factors of biopsy failure were specimen length and fragmentation: less than or equal to 15 mm long, or between 15 and 19 mm but with more than 10 fragments. Failure was attributable to biopsy when there was no high risk of AshTest failure and if the biopsy specimen was at high risk of failure.Due to the risk of commercial bias another analysis was made as the worse scenario for AshTest and AST/ALT, assuming that biopsy was a perfect reference without any false positive/negative.After fasting overnight, patients were placed in the supine position, and the wedged and free hepatic venous pressure gradients [HVPG] were measured by two experienced operators using an 8F catheter  inserted into the right hepatic vein. Two similar consecutive values of the HVPG were used. PHT  was defined as an HVPG ≥5 mmHg, and severe PHT as an HVPG ≥12 mmHg..,16.Between 2004 and 2013, of 157 eligible patients, 123 were included and 34 not included. In 31 patients AshTest was not performed due to forgotten prescription or too small blood sample. Among the 123 included patients, AshTesT and biopsy were obtained the same day except for one case obtained 3 days apart. .Table 1) According to the definition, the prevalence of histological ASH varied from 78% , 80% for the EASL definition , to 91% for at least one activity lesion . All patients had cirrhosis both observed at histology and presumed by FibroTest ; one FibroTest was not interpretable which can be considered as a failure in intention to diagnose.There were no significant differences in the main patient characteristics.), and alpha2-macroglobulin: 0.89 g/L (0.12) and 1.84 g/L (0.74) among the 23 patients with sepsis vs 0.73 (0.28) and 1.99 (0.70) in 100 patients without sepsis at baseline (P = 0.28 and P = 0.29) respectively. Only the MELD prognostic index was associated with sepsis, as well as a dramatic decrease in apoA1 as previously observed.. AshTest AUROCs varied according to the binary endpoint from 0.777 (0.651–0.861) for presence for 3 lesions, to 0.815 (0.639–0.910) for presence of any single activity lesion.S3 Table) The main differences were observed for non-ASH vs. minimal ASH and for moderate vs. severe ASH.(Table 3) Using pathologist conclusion as endpoint, AshTest performances were similar than using EASL endpoint.(S4 Table) AshTest had also higher performance for histological ASH scores than MaddreyDF and MELD score. vs. 0.833 (0.023), P = 0.01].(LD score.(S5 TableS6 Table) Two or three grades discordance was attributed to failure of biopsy  in five (4%) cases (including the only "three-grade discordances"); the lengths ranged from 10 to 14 mm, and there were 2 to 12 fragments. Discordance was attributable to AshTest  in three cases, as the biopsy was 20 mm or longer, and the ApoA1 was not decreased, as is usually observed in histological ASH. In the remaining cases, the cause of discordance could not be determined, though cardiac insufficiency, cirrhosis and sinusoid dilatation, as well as minimal PMN and ballooning were suspected. Univariate comparisons between the 54 concordant cases and the 69 discordant cases identified the associated factors as the clinical non-severity of ASH (P<0.0001), the number of fragments (P = 0.02) and the inferior vena cava pressure (P = 0.03). In multivariate analysis the only factor associated with discordance was the clinical non-severity of ASH (P<0.001).(S7 Table)Concordance between the histological scores and AshTest scores was significant (P<0.0001) and was rated as moderate to fair . Discordance was minimal, and a difference of only one grade was found in 60/123 (49%) patients, and of 2 grades or more in only 9/123 (7.3%) patients., 4.1% (5/123) for biopsy and unknown in the remaining case (0.8%).Finally, due to the severity of liver disease all these patients were considered for treatment by corticosteroids. Based on AshTest only, or based on biopsy only, 109 (89%) and 88 (72%) patients would have been treated respectively.(S7 TableS8 Table). There was no significant association between AshTest and gradient, VCI and OD pressure.(S9 Table).In this population that includes only severe end-stage liver diseases, only the Maddrey discriminant function had a significant prognostic value in multivariate analysis [Risk ratio = 1.01 (95%CI 1.006–1.020) P = 0.0005]. AshTest, AST/ALT, MELD, AHHS and hemodynamic measurements had no prognostic value., which is now prescribed and available in more than 50 countries. FibroTest also enables confirmation of the fibrosis stage with 100% positive predictive value in the present study for cirrhosis, including in the case of cardiovascular insufficiency.The recognized advantages of transjugular liver biopsy are its status as the gold standard; its direct assessment of elementary lesions; the simultaneous assessment of the HVPG, which was previously validated as a prognostic index in cirrhotic patients; and its potential ability to diagnose associated causes, such as nodular hyperplasia or heart failure.,25,26S8 Table). Biopsy was useful in only one patient, showing mixed liver lesions including sinusoidal dilatation, cirrhosis, minimal PMN and minimal ballooning. The pathologist diagnosed cardiac-related liver disease without obvious histological ASH. This patient probably had a mixed cause of cirrhosis, heart failure with an elevated gradient (23 mmHg) together with a high (19 mmHg) inferior vena cava pressure (IVC), and minimal histological ASH. Interestingly, we observed that 31 out of 97 patients (32%) had IVC pressures greater than 15 mmHg, suggesting frequent undiagnosed right heart failure in these patients. These possible relationships between clinical or histological ASH and alcoholic cardiovascular disease should be explored.The utility of HVPG in our population was unclear, as its prognostic value was not significant and was lower than that of the Maddrey DF .In the severe clinical ASH context of use, the other major biopsy limitations were the small specimen length, as well as the large number of fragments. Indeed, the mean number of fragments was significantly higher in discordant versus concordant patients 11 vs. 8). The analyses of severe discordance (2 grades or more) suggested that low quality specimens were associated with 5 false negatives of biopsy and 3 false negatives of AshTest. Therefore, and as observed in NAFLD  and chro. The anaInterobserver variability is another cause of false positives or false negatives. Even in 392 drinkers without severe histological ASH, we observed moderate or fair concordance (binary outcome) between two observers using intercostal biopsy specimens, which were of better quality than those from the transjugular route: only 17% were fragmented and 88% were longer than 10 mm.[In a recent prognostic study of severe histological ASH, similar limitations of biopsy were observed, including a relatively limited number of subjects despite the international multicenter design , small specimen length (median 6 mm) and substantial interobserver variability (non-weighted kappa concordance coefficient ranging from 0.46 to 0.65) compared to the usual coefficient of variation of blood tests lower than 10%.The cost of transjugular biopsy is estimated at £1,500 (€1900), and requires an overnight stay and possible transportation costs.The cost of AshTest/Ash-FibroSure varied according to countries and health care systems, from 30 euros to 487 dollars, but 50 Euros for FibroMax  is the competitive median price of patented blood liver tests.The four advantages of AshTest compared with biopsy are its absence of adverse events, its rapid assessment, its lower variability and lower cost. All components of AshTest can be measured on a single analyzer, and the algorithms, including the security algorithm directly integrated in the biochemistry routine process, enable the results to be obtained in less than 2 hours in our department.Table 2). This overall greater performance was mainly due to the ability to discriminate between moderate and minimal histological ASH (S3 Table). The advantage of the AST/ALT ratio is its cost and availability. Another advantage of the AshTest versus the AST/ALT ratio is the possibility of simultaneously assessing the stage of fibrosis using FibroTest, and the steatosis grade using SteatoTest in the FibroMax combination using the same sample. A combination of white blood cell and platelet counts had significant performance for the binary diagnosis of histological ASH, in one study.[We acknowledge that there is no recognized reference blood test for ASH, and that AST/ALT ratio is considered to be reflection of alcoholic liver disease in general and not specific for diagnosis of alcoholic hepatitis. However AST/ALT ratio was the only "routine" biochemical test discussed in the diagnostic paragraph of EASL guidelines. One origne study. However ne study.In our first AshTest validation, we recognized that the small number of patients with severe histological ASH (53 out of 225 patients and 299 controls), was a limitation.[We focused on the diagnosis of histological ASH and not on its prognostic performance, as the AshTest was developed from its inception for diagnosis. We acknowledge that the presence of megamitochondria was not prospectively predetermined with the appropriate recommendations, such as the high magnitude power field (600 vs400). Since alPatients were consecutive suspected severe ASH, clinically defined, and all of them had cirrhosis, which could be also viewed as a limitation. The first validation validated AshTest in a population with a broad spectrum of clinical ASH. In this second validation the population was patients with cirrhosis and severe ASH, which is the target of an unmet need: non-invasive biomarker as treatment such as corticosteroid are discussed and they needed transjugular biopsy. Patients with cirrhosis represented 82% of patients with suspected ASH in of a recent multicenter study with the higher odds ratio for short-term survival.Finally according to the limitation of biopsy it seems fair to simplify the definition the ASH (necro-inflammatory activity) from the non-activity features of ALD , as it had been achieved for elementary histological features of chronic viral hepatitis 21 years ago with the METAVIR scoring system, and receThese results confirm the performance of AshTest in 123 new patients with suspected severe clinical ASH and within the specific context of corticosteroid treatment use. AshTest has limitations, including a 2–7% risk of two grades misclassification, but is an appropriate non-invasive alternative to transjugular liver biopsy, allowing an estimate of histological activity grades. It could be included in updated algorithms for the diagnosis and treatment of histological and clinical ASH.,29S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableOver all and stage by stage performances.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableDetails versus Maddrey and MELD scores.(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.


Leishmania infantum, the dog acts as the main reservoir for the disease. Non-invasive sampling for Leishmania detection is pivotal for rapid and affordable diagnosis. Recently, the use of conjunctival swab (CS) has been evaluated as a non-invasive sampling technique for quantitative real-time PCR (qPCR). However, few investigations have been made on the applicability of CS qPCR in particular cases such as dogs with borderline IFAT titres, suspected disease relapse with comorbidity and therapy monitoring. The aims of this study were i) to confirm the efficacy of CS, comparing these samples to buffy coat (BC) samples, as effective non-invasive samples for Leishmania quantitative detection by qPCR and ii) to verify the usefulness of qPCR compared to conventional laboratory and clinical parameters to assist in therapeutic decision making regarding dogs with complex clinical cases.In leishmaniasis caused by Eighty dogs were divided into 4 groups based on their IFAT titres and clinical histories. Two qPCR assays were performed both on CS raw lysates and on purified DNA from BC samples. The assays were then compared. Z tests for difference of proportion, with Bonferroni correction, were carried out to evaluate the qPCR results. Logistic regression with backward stepwise elimination was performed to detect the subset of haematochemical variables significantly associated with PCR positivity.Leishmania kDNA load in CS and body condition scores or IFAT titres were calculated in dogs with new leishmaniasis diagnoses. The Leishmania kDNA load in CS correlated moderately with IFAT titres (R = 0.59) but a very weak correlation (R = 0.37) with body condition score (BCS) was found.The qPCR performed on CS samples showed better sensitivity (87%) and specificity (96%) than assays carried out using BC samples, regardless of the primers used. The haematochemical parameters haemoglobin and globulins were found to be significantly associated with qPCR positivity. Pearson correlations between Leishmania detection in dogs was confirmed, revealing the usefulness of raw lysates for quantitative purposes. Moreover, the qPCR was found to be particularly useful in cases lacking a clear clinical diagnosis, where the haematochemical values cannot be predictive.The applicability of CS for The online version of this article (doi:10.1186/s13071-014-0460-3) contains supplementary material, which is available to authorized users. Leishmania species. The parasite, primarily transmitted by phlebotomine sand flies, cause 0.2–0.4 million cases of visceral leishmaniasis (VL) and 0.7–1.2 million cases of cutaneous leishmaniasis (CL) per year. Currently, 12 million people are affected by the disease in 98 countries [Leishmaniasis is a parasitosis caused by at least 15 Leishmania infantum is the aetiological agent of zoonotic leishmaniasis, which is endemic in the Mediterranean Basin, Central America, South America and parts of Asia [L. infantum infection and is the main reservoir for this parasite. In light of this fact, the canine leishmaniasis (CanL) is acknowledged as a very important problem in veterinary medicine [Trypanosoma cruzi, Trypanosoma caninum, Leishmania braziliensis, and Ehrlichia canis [Leishmania minicircles has been widely used as a specific target because of its very high number of target sequences [et al. [Leishmania parasite in different ocular tissues of infected dogs. More recently, Lombardo et al. [et al. [Leishmania detection in CanL using whole blood or buffy coat is not without difficulties [Specifically,  of Asia . The dogmedicine ,4 and humedicine ,6. Althomedicine ,8. The imedicine . Neverthmedicine . Moreoveia canis . In addiia canis , may be ia canis  and theria canis ,15, in bequences -19. Moreequences ,21. Unfoequences ,23. More [et al.  characteo et al.  verified [et al.  showed c [et al. . Contexticulties . It showiculties -29, and iculties . On the iculties . MoreoveThe study was approved by the Ethical Committee for Animal Experiments of the University of Urbino (CESA) on July 31st 2012. The study’s title was “Diagnosi biomolecolare della leishmaniosi attraverso l’uso di campioni clinici non invasivi e loro utilizzo per il monitoraggio terapeutico” (Prot. CESA 2/2012).L. infantum is present as a parasite. A total of 80 dogs were enrolled . The dogs either lived in kennels or with their owners. Two of the dogs were of unknown breed, 24 were mongrels and 54 were pure breeds. The median age of 51 dogs was 6 years (range: 2 months −15 years); the remaining 29 dogs were adults of unknown age. The dogs were examined by veterinary practitioners for the presence of signs attributable to Leishmania infection and scored for clinical signs from 0 to 14. A single point was attributed for each of the following 14 clinical signs: alopecia, dermatitis, skin ulceration, ocular lesions, dysorexia, weight loss, asthenia, vomiting and/or diarrhoea, epistaxis, hyperthermia, lymphadenopathy, lameness, onychogryposis and other . Dogs with a body condition score (BCS) of 0 were defined as asymptomatic; those with a score of 1–2, paucisymptomatic; those with a score of 3–6, oligosymptomatic; and animals with a score of 7–14, polisymptomatic.The study was carried out in the Marches region , where In light of the IFAT results (see below) and considering their clinical history, the dogs were divided into 4 groups: A) IFAT negative dogs (n = 24), composed of healthy dogs  and also symptomatic dogs suspected of having leishmaniasis by veterinary practitioners ; B) suspected for leishmaniasis ; C) dogs monitored after successful therapy , composed of dogs that were diagnosed with leishmaniasis before the beginning of this study and brought to the veterinary clinic for a re-evaluation, in some cases because of the reappearance of symptoms ; D) newly positive dogs . A further subgroup (group E) comprised dogs monitored during therapy . This group included dogs with qPCR results available both at the time of diagnosis and during therapy .Canine peripheral blood  and conjunctival swabs (CS) were provided by the “Santa Teresa” Veterinary Clinic . Serum for biochemical assays and protein electrophoresis was obtained after the centrifugation of peripheral blood collected in a tube with serum-granules clotting activator at 1,900 × g for 5 min. The separated serum was stored at −20°C until it was analyzed. The whole blood (1 ml) for haematological assays was collected in tubes with EDTA and immediately analyzed. The buffy coat samples (100 to 320 μl) were obtained after centrifugation of residual peripheral blood at 210 × g for 10 min. Conjunctival swabs were collected from the right and left conjunctivas using sterile cotton swabs. The swabs were rubbed with a rotational movement back and forth three times in the lower conjunctival sac.et al. [IFAT was performed on serum samples with an in-house assay validated and provided by the Institute of Experimental Preventive Veterinary Medicine  of Sicily (IZS) . In accoet al. , IFAT tiProtein and albumin were quantified using an automated spectrophotometer  and Giesse Diagnostics commercial kits , based on the biuret and bromocresol green methods, respectively. Protein fractions were measured in 25 μl of serum by electrophoresis on cellulose acetate membranes  and buffer  in a semi-automated electrophoresis analyzer  according to the manufacturer’s instructions. The haematological assays where performed using the coulter system Genius Vet haematology analyzer .The DNA was extracted from buffy coat and conjunctival samples as previously described . BrieflyL. infantum kDNA minicircle. The standard curve was established using serial dilutions of Chelex-purified L. (L.) infantum MHOM/TN/80/IPT1 DNA, ranging from 100 to 0.001 parasites equivalent.Two qPCR assays were performed using two different primer pairs  as previously reported . Both prLeishmania parasites to the number of canine cells. The primers B2Mcanis_F (5′-GTCCCACAGATCCCCCAAAG-3′) and B2Mcanis_R (5′- CTGGTGGATGGAACCCTGAC-3′) were used under the same conditions of qPCR1 and qPCR2. The B2M standard curve was constructed with serial dilutions of the B2M PCR product quantified using Qubit fluorometer (Life Technologies). In addition to the parasite quantification, a ΔCt value between Leishmania and B2M amplification curves (ΔCt Leish-B2M) was evaluated as an additional parameter to monitor parasite load changes in the same animal during the course of the illness or treatment. In fact, increases or decreases in ΔCt Leish-B2M correlate with decreases or increases in parasite loads, respectively.In CS samples, for quantitative purposes and to exclude false negative results due to low DNA extraction efficiency and/or the presence of PCR inhibitors, the canine beta-2-microglobulin (B2M) gene was used as a reference (qPCR3). In fact, quantification of the B2M gene was used to estimate the number of cells withdrawn in CS samples and consequently to normalize the Descriptive statistics were performed with quantitative variables; PCR quantifications were log10 transformed in order to meet assumption of normal distribution. Analysis of variance (ANOVA) was used to test the associations between dog groups, clinical chemistry and haematological values. Z tests for proportions were performed in order to compare specificity and sensitivity values. The area under the receiver operating characteristic curve (AUC) allows an evaluation of the overall performance of a test or classification, i.e. its diagnostic accuracy; reported effect size intervals for AUC are: small effect sizes (0.528–0.556); medium effect sizes (0.584–0.638); large effect sizes (0.714–0.760); very large effect sizes (above 0.760) . A good Leishmania therapy . Therefore, the qPCR assays were used to detect and quantify the Leishmania parasites in CS and BC samples.Clinical parameters at the time of sampling for molecular assays for all the dog groups in our study are shown in Table Leishmania parasites was assessed in the dogs assigned to all groups by qPCR both from conjunctival swabs (CS qPCR) and buffy coat (BC qPCR). The qPCRs were initially performed with MaryF-MaryR primers (qPCR1); the positive results were subsequently confirmed using MLF-MLR primers (qPCR2). The amount of CS raw lysates to be used as a template in qPCR without any inhibitory effects was established as 1 μl per 25 μl reaction tube, maintaining the previously reported sensitivity (1×10−3 par/reaction tube) [The presence of on tube)   of the qPCR negative CS samples were tested for B2M amplification. In order to quantify the B2M copy number, a calibration curve was constructed using serial dilution of a purified PCR product , but the correlation with BCS was very weak (R = 0.37), according to previous literature data [Pearson correlations between the quantitative load of ure data .Leishmania DNA and to evaluate the usage of these parameters as a first line diagnostic approach in suspected cases with borderline IFAT titres 1:40–1:80 (i.e. group B), and previously infected dogs (i.e. group C).The clinical parameters shown in Table Logistic regression with backward stepwise elimination showed that only globulins  and haemoglobin  were significantly associated with group membership. Nagelkerke R square was 0.39, showing a modest but significant goodness of fit. Logistic regression specificity and sensitivity values were 0.704 and 0.739, respectively. If both globulins and haemoglobin parameters are above and below the normality range, respectively, the probability to belong to the infected group is 80%, whereas when both clinical parameters are in normality range, the probability to belong to the infected group is 20%.Leishmania kDNA and the canine B2M gene. The qPCR efficiencies were calculated using serial dilutions of two CS lysates  spiked with known amounts of L. infantum MHOM/TN/80/IPT1 DNA. The ΔCt between B2M (qPCR3) and Leishmania kDNA (qPCR1 and qPCR2) were plotted against dilutions of CS lysates spiked with L. infantum DNA. The slope of the trend lines was < 0.1, indicating comparable PCR efficiencies [Leishmania kDNA and canine B2M gene could be used to evaluate the trend of therapy or illness progression in the same animal.The efficiency of the qPCR assays used in this study were calculated to evaluate the possibility of making a direct comparison between the Ct values of ciencies   diagnosed with leishmaniasis, entered a therapeutic protocol and were monitored for parasite load in CS samples during therapy. Three dogs resulted qPCR negative in left and right CS samples after initiation of therapy; one dog (number 67) resulted qPCR negative in the left CS, and presented a parasite reduction in the right CS. Notably, in this sample the decrease in parasite load was verifiable by the increase in the ΔCt Leish-B2M value Table .Table 3The development of non-invasive sampling techniques is a worthy goal in human and veterinary medicine. In addition, in veterinary medicine, even more than in human medicine, there is a need for rapid sampling, limiting the use of patient sedation. Conjunctival swabs appear to be a very useful non-invasive sampling method although the technique may have drawbacks regarding sensitivity  and quanLeishmania in dogs is a much debated issue [Leishmania DNA positivity without symptomatology may reflect seasonal contact with the infected vector and does not necessarily involve the appearance of specific symptoms also in the presence of borderline IFAT titres [The present study assessed the diagnostic value of CS qPCR in dogs with borderline IFAT titres, suspected disease relapse or the presence of comorbidity, comparing the CS qPCR results with BC qPCR and haematochemical analyses. The use of BC samples to detect ed issue -37. Our T titres ,38. BC qT titres .−3 par/reaction tube), at a low cost and without inhibitory effects when 1 μl per reaction tube was used. We found a CS-qPCR sensitivity and specificity of 87% and 96%, respectively, confirming and improving on the results obtained by Gramiccia et al. [In order to obtain quantitative data from CS samples, the parasite load was normalized using the B2M gene and the results were expressed per 50,000 canine cells. The normalization can overcome the problem due to the intrinsic variability of the swab sampling and would be helpful to compare data from different laboratories also because of the non-standardized way of collecting conjunctive cells using swabs. This procedure allowed us to quantify the CS parasite load and to monitor the same animal during therapy; however, it increased the cost of analysis and required more time. This disadvantage was partially reduced by using SYBR Green and CS raw lysate as the source of the DNA template. In fact, the use of CS raw lysate, instead of DNA extracted with the phenol-chloroform method ,26,39 ora et al. . These da et al. ,26 althoInterestingly, among the clinical and haematological parameters considered, those most associated with qPCR positive results were globulins and haemoglobin. This finding seems to suggest that these parameters in particular should be considered during first-line laboratory tests in the case of suspected CanL or evaluation of disease relapse. However, even considering these parameters, relevant uncertainty remained, supporting the need of a wider use of qPCR.et al. [Leishmania DNA is rapidly degraded following parasite death; hence, its identification in conjunctival tissue could be indicative of recent extraepidermical dissemination that, in presence of clinical signs or, as in this case, haematological signs, could require treatment or close monitoring. Another complicated case study, in terms of therapeutic decisions, was dog 40 because of the presence of comorbidity. This dog was in therapy for hypothyroidism when Leishmania was detected. Subsequently, Dirofilaria spp., Trichuris spp. and Toxocara spp. were also diagnosed by optical microscopy. There were no significant alterations in biochemical and haematological parameters but the BCS was 8. Regarding the qPCR positive results, although indicating low parasite load, the veterinary practitioner started the animal on a canine Leishmania therapy with allopurinol alone and specific therapy for the other parasitosis mentioned above. An evident improvement in symptoms was reported four months later.The present study also investigated the use of CS qPCR in borderline and disease relapse cases with complex diagnosis, including comorbidity. In group B (borderline cases) only 2 dogs 27 and 40) out of 17 tested positive with CS qPCR. Dog 27 showed a very low parasite load in the left CS . The BCS was 1, IFAT titre was 1:40 and serum electrophoresis and biochemical analysis were normal; however the haemochrome showed a moderate anaemia compatible with leishmaniasis. Prina  out of 1Furthermore, eight dogs  out of 16 in group C (monitored after therapy) tested positive in at least one of the BC and CS samples analyzed. It is noteworthy that only dog 42 had important alterations in its biochemical values; 6 out of 6 with available data had alterations in their electrophoretic patterns  and relative normalization.On the whole, the CS qPCR results obtained in dogs suggest more extensive investigation of the use of this kind of samples in human cases also.


Wolfram syndrome (WS) is an autosomal recessive neurodegenerative disorder characterized by diabetes mellitus (DM), optic atrophy (OA), central diabetes insipidus (CDI) and deafness (D).WFS1 gene, a nuclear gene localized on chromosome 4. Since the discovery of the association between WFS1 gene and Wolfram syndrome, more than 150 mutations have been identified in WS patients.The phenotype of the disease has been associated with several mutations in the We previously described the first case of perinatal onset of Wolfram syndrome newborn carrying a segmental uniparental heterodysomy affecting the short arm of chromosome 4 responsible for a significant reduction in wolframin expression.WFS1 gene in foetal and neonatal neurodevelopment.Here we review and discuss the pathophysiological mechanisms that we believe responsible for the perinatal onset of Wolfram syndrome as these data strongly suggest a role for We described a male patient of 30 weeks’ gestation with intrauterine growth restriction and poly-hydramnios.During the first days of life, the patient showed a 19% weight loss associated with polyuria and hypernatremia. The presence of persistent hypernatremia (serum sodium 150 mEq/L), high plasma osmolarity (322 mOsm/L) and low urine osmolarity (190 mOsm/l) with a Uosm/Posm ratio < 1 were consistent with CDI. The diagnosis of CDI was confirmed by the desmopressin test and the brain magnetic resonance imaging (MRI) at 34 weeks of age, that showed the lack of posterior pituitary hyperintense signal. In addition, a bilateral asymmetrical optic nerve hypoplasia associated with right orbital bone hypoplasia was observed, suggesting the diagnosis of WF.During the five years follow-up the patient did not developed glucose intolerance or diabetes mellitus. By the end of the second year of life, primary non-autoimmune central hypothyroidism and mild neurodevelopment retardation were diagnosed.The analysis of our case, in the light of the most recent literature, suggests a possible role for WFS1 gene in the development of certain brain structures during the fetal period.Wolfram syndrome should be considered in the differential diagnosis of the rare cases of congenital central diabetes insipidus developed in the neonatal period. Wolfram syndrome WS1) MIM #222300), first described by Wolfram and Wagener in 1938 [ . A wide spectrum of abnormalities of the endocrine glands, the central nervous system (CNS) and the urogenital tract has been described, comprising hypothyroidism, hypogonadism and ACTH deficiency, anosmia, ataxia, seizures, nystagmus, gaze palsies, dysarthria, dysphagia, psychiatric disorders, cognitive deficits, hypo or areflexia and neurogenic bladder, central sleep apnea, neurogenic upper airway collapse, myoclonus, Parinaud’s syndrome [The diagnosis of Wolfram syndrome is based on the presence of the two minimal diagnostic criteria: early onset DM (<15 years) and bilateral progressive OA. DM is usually the first symptom to present at a median age of 6 years, followed by the onset of OA at a median age of 11 years. The incidence of central DI varies considerably, ranging from 48% to 78%, as generally it does not appear until the 2WFS1, a nuclear gene mapped on chromosome 4p16.1, encoding for the endoglycosidase membrane glycoprotein named wolframin. Wolframin plays a protective function against endoplasmic reticulum (ER) stress and it is involved in calcium storage, redox regulation, steroid synthesis, and cell death [The disease phenotype has been associated to mutations in ll death -12. Acqull death -16.WFS1 gene and Wolfram syndrome, more than 150 mutations have been identified in WS patients. However, no evident genotype-phenotype correlations were found [Since the discovery of the association between re found .We recently described the first case of Wolfram syndrome perinatal onset, presenting with congenital OA and CDI, in a growth-restricted male infant, born at 30 weeks gestation,. A detailed description of the underlying genetic defect, a segmental uniparental heterodysomy affecting the short arm of chromosome 4 responsible for the significant reduction in wolframin expression, have been previously published .WFS1 role in fetal and neonatal neurodevelopment.Here we review the pathophysiological mechanisms possibly responsible for the perinatal onset of Wolfram syndrome according to the recent published evidence of We described a male patient, born to a 38 years old mother at 30 weeks’ gestation by emergency Cesarean section, performed because of pre-eclampsia and IUGR associated with poly-hydramnios, detected by 26 weeks. The pregnancy was complicated by maternal type 2 DM, requiring subcutaneous insulin, Antenatal serologies  were protective. Cytogenetic investigation of the fetus by villocentesis confirmed a normal 46,XY karyotype. Paternal medical history was unremarkable. Parents were non-consanguineous Caucasians.Apgar scores were 6 at 1 minute and 7 at 5 minutes of life. Physical parameters were as follows: weight 1190 g (<10° centile), length 36 cm (<3° centile) and head circumference 26 cm (<10° centile). Physical examination was normal except for a moderate respiratory distress, During the first hours of life, the respiratory status worsened, evolving towards a third-degree hyaline-membrane disease that required surfactant administration, mechanical ventilation for a 24 hour period and non-invasive ventilation for 10 days.Throughout the first 3 days of life, the patient experienced a significant weight loss (as high as 19% of the birth weight), associated with polyuria (4.7-5.5 ml/kg/h) and hypernatremia (serum sodium 152–159 mEq/l), despite adequate fluid intake. At first, hypernatremia was interpreted as a consequence of the excessive weight loss and treated with large amounts of daily intravenous fluids (180–200 ml/kg/d). The clinical course was complicated by one episode of Serratia Marcescens septic shock during the second week of life and disseminated intravascular coagulation requiring plasma administration, platelet transfusion and aggressive antibiotic therapy.CDI was subsequently suspected as a result of persistent hypernatremia (serum sodium 150 mEq/L), high plasma osmolarity (322 mOsm/L) and low urine osmolarity (190 mOsm/l) with a Uosm/Posm ratio < 1 associated with failure to thrive during the first four weeks of life. The diagnosis of CDI was confirmed by the desmopressin test, showing a normal urine concentrating capability after nasal desmopressin (DDAVP) administration with rapid raise in urine osmolarity and reduced plasma osmolarity and the brain MRI at 34 weeks postmestrual age demonstrating the lack of posterior pituitary hyperintense signal. Bilateral asymmetrical optic nerve hypoplasia associated with right orbital bone hypoplasia was also observed, suggesting the diagnosis of WF . No otheOphthalmologic examination revealed eyeball and right optic disc hypoplasia, associated with exotropia. Visual evoked potential confirmed the pathological findings of the right optic pathway, while the electroretinogram resulted normal. Audiological examinations were normal. Borderline TSH values with normal thyroid function were recorded during hospitalization, but no treatment was required until the end of the first year of life, when levo-thyroxine substitutive therapy was started because of primary non-autoimmune central hypothyroidism with a small thyroid gland. During the five years follow-up mild psychomotor and mental retardation were diagnosed. No glucose intolerance or diabetes mellitus have been detected so far.After the catch-up growth, length and head circumference has stabilized around the 50° centile and, after the first year of life, body weight was above the 97° centile.Water and electrolyte balance is maintained with standard doses of nasal desmopressin.No progression of the bilateral asymmetrical OA was noted at the 1-year follow-up MRI. However, new pathological signs   were notWFS1 that we hypothesized could remove splicing regulatory motifs located in intron 1. Direct sequencing analysis of WFS1 cDNA showed a mixed sequencing pattern including the wild-type transcript sequence and two splice variants. The first splice variant was a previously described 4-bp deletion in exon 2, while the second one exhibited the complete skipping of exon 2 and, probably, the loss of the transcription start site. RT-PCR confirmed a normal mRNA expression compared to young controls, while Western blot analysis showed a significantly decreased Wolframin expression [Genetic investigation of the patient and his relatives revealed a segmental paternal heterodisomy of the chromosome 4 , involving at least part of the promoter and the first exon of pression . InformeIn this report we deepened from the clinical point of view the case of an atypical Wolfram patient, presenting during fetal and neonatal life. As a matter of fact, we discovered DI and OA during the examination performed after the delivery, that occurred long before the term of pregnancy, albeit the antenatal origin of the CDI was suggested by poly-hydramnios, polyuria and rapid body-weight loss occurred since first days of postnatal life. In addition, we speculate that the OA and the hypoplasia of the eyeball were caused by a pathological process originating during fetal life.WFS1 is a key element of the ER stress activated signalling cascade, also known as unfolded protein response (UPR). In particular, WFS1 regulates ATF6A activity, one of the ER stress-sensor molecules that induces UPR target genes and prevents cells from apoptosis caused by the imbalance between productive folding of secretory proteins and degradation of misfolded proteins [The Wolfram syndrome is caused by mutations of the protein Wolframin, essential for the regulation of intracellular calcium homeostasis, the cell cycle progression and the retrieval of homeostasis under conditions such as ER stress -12. Indeproteins ,13  and pathological  processes, causing the production of toxins, cytokines and mutant proteins expression ,18,19.Patients affected by Wolfram syndrome exhibit a dysregulated ER stress signaling. The uncontrolled ATF6α up-regulation determines apoptosis of sensitive cell types, in particular pancreatic β cells and neural cells ,13.Recent insight arising from animal studies led to the conclusion that Wolframin may also be essential for the processing of the arginine vasopressin (AVP)-precursor and for the survival of the supraoptic (SO) and the supraventricular hypothalamic (PV) nuclei neurons . GabreelInvestigations in our patient did not allow neither to determine the mechanisms underlying the development of early onset CDI, nor to correlate his rare and previously undescribed mutation to his unique phenotype . The mosWFS1 expression has been fully characterized in the murine CNS. Wolframin presence has been confirmed in several CNS structures, comprising the amigdala, the hippocampal region, the hypothalamic magnocellular neurosecretory system, the brainstem nuclei, the globus pallidus, and the posterior caudate putamen [ putamen ,27. Hils putamen .WFS1, but even more widely the white and gray matter. However, we can not exclude that the radiological findings as well as the mild psychomotor and mental retardation may be a consequence of prematurity and co-morbidities; In fact, disabilities affect roughly 30% of patients born between 29 and 32 weeks of gestational age and septic shock is an established cause of white matter damage [The neuroimaging evolution of our patient at 1 year of life showed new pathological features such as reduced corpus callosus, moderate ventricular enlargement, asymmetric hippocampus and altered occipital peri-ventricular white matter signal. This suggests a neurodegenerative process affecting brain structures, known to be damaged by the loss of The bilateral asymmetrical OA associated with the eyeball hypoplasia and the right orbital bone hypoplasia were evident by the first month of life, suggesting an alteration in the development rather than an involution of optic nerves.WFS1 mutations may affect their ability to provide nutrients to optic axons when they suffer from ER dysfunction [In control mice, Wolframin is expressed in neurons and glial cells of the optic nerve, the SC, the SCN and the visual cortex, while in the retina it was expressed in all neuron subtypes . In humafunction .In our patient, the presence of optic nerve hypoplasia with orbital bone hypoplasia long before term corrected age allowed new speculations about the underlying pathogenetic mechanisms, supported by recently published work. The vast majority of the critical processes involved in the retinal development occur between 24 weeks gestation and 3 to 4 months of postnatal life, while the axonal development of the optic tract occurs between 20–30 weeks of gestation . A recenHeterozygous missense mutations in WFS1 have been linked to autosomal dominant low frequency neurosensory hearing impairment and psychiatric disorders . Eight fWFS1 plays a role in the development of neural structures, such as the optic nerve and the neuro-hypophyseal tract. After a careful examination of the patient and the extensive review of the literature, we speculate that this early and severe OA cannot be entirely explained by a progressive degenerative mechanism, although further studies are needed in order to identify all cerebral structures expressing Wolframin during embryogenesis and fully elucidate its role in the neurodevelopment.The analysis of our case reinforces the intriguing hypothesis that the Written informed consent was obtained from both parents for the publication of this Case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.No ethical approval was required for this case-report.


Recent studies have suggested a possible association between outdoor or indoor temperature and obesity. We aimed to examine whether ambient temperature is associated with the prevalence of obesity or abdominal obesity in the Korean population.2 and a waist circumference (WC) ≥ 90 cm (men) or 85 cm (women) were considered to represent obesity and abdominal obesity, respectively.Data on anthropometric, socio-demographic, laboratory and lifestyle factors were retrieved from National Health Insurance System data obtained in 2009–2010. Thirty years (1981 to 2010) of meteorological parameters for 71 observation areas were acquired from the Korea Meteorological Administration. Included in this analysis were 124,354 individuals. A body mass index (BMI) ≥ 25 kg/mP = 0.0065). WC was positively correlated with MAT  and negatively correlated with the number of days with mean temperature < 0°C . After adjusting for age, sex, smoking status, alcohol consumption, exercise, income, residential area and altitude, the odds ratios (95% CI) for obesity and abdominal obesity in the highest quintile MAT group were 1.045  and 1.082 , respectively, compared with the lower four quintiles of the MAT group. Similarly, subjects in the area of the lowest quintile of DMT0 had significantly higher odds of abdominal obesity compared with the higher four quintile groups of DMT0.The mean annual temperature (MAT) ranged from 6.6°C to 16.6°C, and BMI was positively correlated with MAT (r = 0.0078, This study finds an association between ambient temperature and prevalence of obesity in the Korean population when controlling for several confounding factors. Adaptive thermogenesis might be a possible explanation for this phenomenon. Compareor women . For insor women , 8.Most people in the developed world live and work in a relatively narrow temperature range of comfortable ambient temperatures known as the thermoneutral zone, which is approximately 23.0°C for humans , 10. LitIn this study, we aimed to examine whether ambient temperature is associated with the prevalence of obesity or abdominal obesity using nationally representative data of the Korean population.The National Health Insurance System (NHIS) contains comprehensive health-related information for approximately 50 million Koreans and is composed of an eligibility database , a medical treatment database , a health examination database  and a medical care institution database . The source population for this system is the Health Insurance Review and Assessment (HIRA) service. Under the NHIS, Koreans are entitled to medical coverage as either an employee or a member of a community , 18. HeaA customized database from NHIS that includes approximately 2.2% subjects of the Korean population was used in this study. Subjects were selected by stratified random sampling to represent the total population. Of the 235,148 participants who underwent health examinations in the year 2009 and 2010, those with missing variables  and those living in the area of missing meteorological data  were excluded. Ultimately, the study population consisted of 124,354 subjects. This study was approved by the Institutional Review Board of The Catholic University of Korea (No. KC15EISI0431). Anonymized and de-identified information was used for analyses, and therefore informed consent was not obtained.The data on mean annual temperature (MAT), the number of days with mean temperature (DMT) < –5°C (DMT–5), < 0°C (DMT0), ≥ 5°C (DMT5) and ≥ 25°C (DMT25), altitude (height of observation area above mean sea level) and information on the observation area were extracted from the Climatological Normals of Korea published by the Korea Meteorological Administration in 2011. These data were obtained for 73 observation stations that are located throughout South Korea and have latitudes ranging from 33° 14’ to 38° 15’. The statistics are based on a 30-year period (1981 to 2010). In this study, data from 71 observation areas were merged with the NHIS data  .2, respectively [Anthropometric measurements were performed while the subjects were wearing light clothing. BMI was calculated as the subject’s weight in kilograms divided by the square of the subject’s height in meters. Obesity and normal weight were defined as BMI ≥ 25 and < 25 kg/mectively . Waist cectively , 21. Bloectively . Hospitat-tests, the Mann-Whitney test or chi-squared tests. The cutoff value of the highest quintiles of MAT was 14.1°C. The correlation between BMI, waist circumference and meteorological parameters were tested using regression analysis, and the Spearman correlation coefficient was calculated. The mean values of meteorological parameters in the 71 observation areas were separately plotted, and the correlations with BMI and waist circumference were analyzed accounting for sampling weights of each area. The risks of obesity and abdominal obesity according to the MAT quintile groups were tested using the lower four quintile MAT groups as a reference group. To account for possible confounding factors, age and sex were adjusted in Model 1 and alcohol consumption, smoking, exercise, income, residential area and altitude were further adjusted in Model 2. The risks of obesity and abdominal obesity according to the DMT0 quintile groups were tested using the highest four quintile DMT0 groups as reference groups after adjusting for the abovementioned variables. A P value < 0.05 was considered significant. The statistical analyses were performed using SAS version 9.3 .The data are expressed as the means ± standard deviations (SDs), median (25th–75th percentiles) or as percentages. The characteristics of the study subjects according to MAT quintile groups (lower four quintiles vs highest quintile) were compared using The MAT ranged from 6.6°C to 16.6°C. The study subjects were categorized according to the MAT quintile groups . CompareP = 0.0065) and DMT5 . Waist circumference was positively correlated with MAT  and DMT5  and negatively correlated with DMT–5  and DMT0 . However, no significant correlation was observed between BMI or waist circumference and altitude  and 0.275 (P < 0.0001), respectively (P < 0.0001) and waist circumference  were negatively correlated with DMT0  did not attenuate this association and demonstrated an odds ratio (95% CI) of 1.045  for obesity. Similarly, the odds ratios (95% CI) for abdominal obesity were 1.080  and 1.082  in Models 1 and 2, respectively .Compared to subjects in the higher four quintiles of DMT0, those in the lowest quintile group demonstrated a 1.062 times higher odds of abdominal obesity after adjusting for age and sex. Further adjustment for alcohol consumption, smoking, exercise, income, residential area and altitude demonstrated an odds ratio (95% CI) of 1.063 . However, subjects in the lowest quintile of DMT0 did not exhibit a significantly higher odds for obesity compared to those in the higher four quintile groups .Using nationally representative data of the Korean population, we demonstrated significant associations between obesity or abdominal obesity and ambient temperature. BMI and waist circumference were positively correlated with MAT and negatively correlated with DMT0. The odds for obesity and abdominal obesity were 1.045 and 1.082 times higher in subjects living in the highest quintile MAT group compared to those living in the lower four quintile MAT groups. Similarly, subjects in the area of the lowest quintile of DMT0 had significantly higher odds of abdominal obesity compared with the higher four quintile groups of DMT0. These results suggest that high ambient temperature might be associated with an approximate 5% increased odds of obesity in the Korean population.Previous studies regarding an association between ambient temperature and BMI are scarce. In Tibetans, BMI, waist circumference and waist-to-height ratio were lower among subjects living at higher altitude compared to those living at lower altitude, and this finding was explained by a possible catabolic effect of low temperature and low oxygen in higher altitude . Valdés The discrepancy surrounding ambient temperature and obesity might be a consequence of the different ranges in mean temperatures, different altitudes of observed areas, different ethnic groups, and the heterogeneous adjustment for confounding variables in previous studies. However, although some reports do not include a significant number of locations with extreme ambient temperatures , 15, theIn contrast to previous studies that included high elevation areas , 14, ourSeveral lines of evidence have suggested a close link between ambient temperature and obesity or energy expenditure in humans. In the range of 15 to 28°C which encompasses normal indoor temperature, energy expenditure was negatively associated with the thermal environment . In addiThere are several limitations to our study. As this was a cross-sectional study, we were unable to determine the cause-effect relationship between ambient temperature and the prevalence of obesity. In addition, because our database only contained the subject’s current area of residency, people who had moved to or from different areas could not be excluded. Therefore, we could not rule out a potential reverse causation between obesity and ambient temperature of residential regions. Moreover, as our study was conducted only among Korean subjects, further studies in different countries and among different ethnic groups as well as a wider range of temperatures are needed to confirm our data. Subjects in the highest quintile of the MAT group had a significantly higher age and lower income level compared to those in the lower four quintiles of the MAT group. These two factors alone might have influenced the prevalence of obesity. However, the highest quintile of the MAT group demonstrated independent odds for obesity or abdominal obesity after statistical adjustment for age and income level. Moreover, different latitudes of observational areas might have influenced the prevalence of obesity, considering Bergmann’s rule which states populations of larger size are found in colder environments and those of smaller size are found in warmer regions. However, Foster et al.  suggesteIn conclusion, this study shows a significant association between ambient temperature and prevalence of obesity among the Korean population when controlled for several confounding factors. Cold-induced thermogenesis may be a possible explanation for this phenomenon and should therefore be the subject of further study.


Wukong elements in the maize genome. The Wukong family shows a distinct pattern of tandem arrangement in the maize genome suggesting a high rate of unequal crossing over. Estimation of insertion times implies a burst of retrotransposition activity of the Wukong family after the allotetraploidization of maize. Using next-generation sequencing data, we detected 87 new Wukong insertions in parents of the maize NAM population relative to the B73 reference genome and found abundant insertion polymorphism of Wukong elements in 75 re-sequenced maize lines, including teosinte, landraces, and improved lines. These results suggest that Wukong elements possessed a persistent retrotransposition activity throughout maize evolution. Moreover, the phylogenetic relationships among 76 maize inbreds and their relatives based on insertion polymorphisms of Wukong elements should provide us with reliable molecular markers for biodiversity and genetics studies.TRIMs , which are characterized by their small size, have been discovered in all investigated vascular plants and even in animals. Here, we identified a highly conservative TRIM family referred to as  The NGS data of 75 genomes of maize and its wild relatives from the Maize Hapmap2 project  and BSSS53  were downloaded from NCBI GenBank.The maize B73 reference genome sequences (RefGen_V2) were downloaded from maizesequence .The nucleotide sequences of TSDs and their 5-bp flanking sequences at both sides served as the query to identify their positions in the genome. A total of 118 elements with intact TSD, including 90 complete TRIM elements and 28 solo-LTRs, were used to generate the sequence logo with Weblogo 3  using MEGA v5.0 with the Kimura-2 parameter model. Finally, the insertion times (T) of the intact Wukong elements were measured with the equation T = K/2r, where r measures an average nucleotide substitution rate, which has been set to 1.3×10−8 substitutions per site per year, as proposed previously . PCR reaction was conducted in 20 μl volume solution, including 50 ng of genomic DNA, 10 pmol of each primer, 1× PCR buffer, 250 μM dNTPs, and 1.0 unit Ex Taq DNA polymerase. The cycling conditions were 95° for 2 min, 35 cycles of 95° for 15 sec, 56° for 30 sec, and 72° for 40 sec, followed by a final extension at 72° for 5 min. PCR products were analyzed by agarose gel electrophoresis stained with ethidium bromide (EB).Genomic DNAs from B73 and 25 parents of NAM population were extracted from 2-wk seedlings using standard cetyltrimethyl ammonium bromide (CTAB) methods, and three Table S1).Total RNA of 2-wk seedlings and pollen of B73 were isolated with TRIzol reagent  according to the instructions of the manufacturer and then treated with RNase-free DNaseI  to remove contaminating genomic DNA. Go Script Reverse Transcription System  was used to prepare cDNA from treated total RNA. Finally, PCR reaction with cDNA as templates was performed as stated previously. The β-actin gene was used as a control to correct the concentration of cDNAs among different samples . The majority of the intact and solo elements have 5-bp target site duplications (TSD) (Table S2). In addition, the majority of the elements shared up to 90% identity of their LTRs, internal domains, and even overall sequences, which suggested a series of recent retrotransposition events of Wukong elements.To investigate the abundance of Wukong elements were investigated by analyzing the 5-bp long TSDs and their 5-bp flanking sequences of a total of 122 Wukong elements including 71 intact and 28 solo elements. However, there appeared to be no bias of insertion sites, suggesting a rather random distribution of elements in the maize genome . Other genomic regions frequently contained a variable number of elements even within the same chromosome . For instance, the B3r10 element contained an insertion of 689-bp DNA transposon of the hAT family, whereas B8r8 had a 5.5-kb copia-like LTR retrotransposon insertion  have two identical LTRs, suggesting that the Wukong element family could still be active recently. Comparison of orthologous regions of the two progenitors of maize and rice genomes showed an uneven expansion in genic regions with Ty1/copia-like elements rather than Ty3/gypsy-like elements after allotetraploidization, which could also explain the distribution of Wukong elements in the maize genome . Furthermore, out of 87 elements, 22 (25%) were present in multiple lines, 13 (15%) were in two lines, and 52 (60%) were line-specific (Table S4), suggesting that Wukong elements maintained their activity at least during the process of crop improvement.The nested association mapping (NAM) population contains 25 diverse lines (25DL) across today’s maize germplasm (Wukong elements detected by NGS reads were selected to be subjected to PCR amplification and sequencing. One element (R78) detected from five NAM parents were also validated by PCR and sequencing. The results showed that all these sites contain Wukong insertions in the corresponding lines, whereas B73 has none of them . A total of approximately 20,000 reads containing the sequences immediately flanking Wukong insertion sites in the genome were identified from 781 Gb of resequencing data. For each examined site there are, on average, three short reads supporting the presence or absence of Wukong elements. On average, 82% (5038/6156) of putative insertion sites could be predicted with current genome coverage of the population. However, it is noted that the teosinte group has the lowest proportion of detectable sites with an average of 75% in comparison to modern lines and landrace groups with an average of 84% and 85%, respectively (Table S5).Given the recent activity of Wukong elements, 65  were predicted in all three germplasm groups, including 30  insertions present in all 76 accessions, suggesting that their retrotransposition occurred before maize domestication; 11  were detected only in the landrace and modern lines group, indicating that they were presumably activated during domestication; four  were unique to modern lines, showing that their insertion presumably happened during crop improvement. In addition, given that the 25 parents of the NAM population were classified into two subgroups, tropical subgroup and temperate subgroup, we found that four Wukong elements  were specifically present in the tropical subgroup, suggesting that these Wukong elements had a unique amplification in tropical improved lines relative to the others. These implied that Wukong elements not only possessed a persistent retrotransposition activity throughout maize evolution but also could be used as molecular markers for biodiversity and genetics studies.Of 80 Wukong sites, including two insertions absent in B73 and one B73 insertion, for PCR amplification to confirm the presence or absence in the parents of the NAM population . A neighbor-joining phylogenetic tree was then constructed based on the presence or absence matrix formed by allelic variations. The 75 accessions can be grouped into three clusters  was set at three, the three groups corresponded with the data from the phylogenetic analysis, lending further support to our phylogenetic analysis.After excluding 29 elements that are present in all investigated strains, a total of 51 s I–III; . Specifis I–III; . These rWukong elements are nonautonomous retrotransposons and retrotransposition needs to be activated in trans by other autonomous elements. To identify such an element with pairing specificity, Wukong sequences were subjected to a Blast analysis of the sequenced B73 maize genome. We could identify a copia-like LTR retrotransposon on chromosome 5 , which could have given rise to Wukong elements through internal deletions because they share more than 90% identity in their LTRs and the internal domain of Wukong elements   . Although the majority of them possess TSDs, others do not (Table S2). To some extent, our results support the notion that elements with multiple LTRs probably resulted from recombination between two adjacent intact elements  were far lower than those of the PbCassandra elements in pear . On the contrary, the ratio of the elements with multiple LTRs to the total number of Wukong elements (8%) was slightly higher than for PbCassandra (5%)  . Like otWukong members, which were identified from the parents of maize NAM population (Table S5), and many of them were unique to a particular germplasm group, subgroup, or even individual lines. Moreover, these inbred lines not only could be distinguished not only from each other but also could be clustered together with members from the same group or subgroup in their phylogenetic relationships based on insertion polymorphism of TRIMs .Considering the high abundance and dispersion, combined with the known ancestral states, TRIMs have been demonstrated to be an ideal molecular marker for investigating evolutionary relationship and genetic diversity among species. In the tribe of the Brassiceae, four TRIMs members have been used as clade markers to infer the taxonomic lineage based on insertion site polymorphisms (of TRIMs . These rWukong insertions was very similar to that inferred from a large number of SNPs (Wukong insertion is a simple, cost-efficient approach for classifying the maize germplasm that only requires PCR amplification with locus-specific primers. It is doubtless that Wukong is a promising marker for maize diversity and evolution studies.The phylogenetic tree generated by polymorphic Wukong element family. Because TRIMs have no coding capacities, they have to achieve their retrotransposition through a transacting autonomous element. Despite the evidence for recent transposition events, identification of the controlling element of TRIMs in plant genomes has been rather elusive (Sanzang element as a potential controlling element for the Wukong element family. In support of this proposal, we offer the presence of a 12-bp motif residing in LTRs of both elements (Sanzang in maize pollen would be consistent with this role (It appears that TRIM elements have expanded in size over a relative long period of time in plant genomes . This is elusive . In maizelements . Specifihis role . Therefo


The present study compared the effects of three rice cultivars on postprandial glycemic control and appetite. A single-blind, randomized, crossover clinical trial was performed with 18 healthy subjects, nine males and nine females. Three treatments were administered at three separate study visits: commercially available conventional white rice (short grain), specialty high amylose white rice 1 (Dixiebelle), and specialty high amylose white rice 2 (Rondo). Postprandial capillary blood glucose, venous blood glucose and insulin measurements, and appetite visual analog scale (VAS) surveys were done over the course of two hours. The capillary blood glucose concentrations were significantly lower for Rondo compared to short grain rice at 30 min, and for Dixiebelle and Rondo compared to short grain rice at 45, 60, and 120 min. Capillary blood glucose area under the curve (AUC) was significantly lower for Dixiebelle and Rondo compared to short grain rice. Subjects were significantly more hungry at 30 min after Dixiebelle intake than Rondo intake, but there were no other significant effects in appetite ratings. The present study determined that intake of high amylose rice with resistant starch (RS) can attenuate postprandial blood glucose and insulin response in comparison to short grain rice. Rice consumption in the United States has increased in recent decades from 27% of adults reporting consumption of >1/4 oz of rice per day (NHANES 1999–2004) . With trin vitro digestibility among the cultivars.Rice is typically considered a highly digestible source of carbohydrate, but rate of digestion and the resulting glycemic response varies among rice cultivars and preparation techniques ,5. AmyloResistant starch, a type of dietary fiber, is defined as all types of starch and starch degradation products that resist digestion and absorption in the small intestine when consumed, and enter the large intestine ,8. ResisThe objective of this randomized, single-blind, crossover clinical trial study was to investigate the effects of white (milled) rice of two cultivars having high RS on postprandial blood glucose, insulin, and appetite in healthy adults, compared to a control short-grain conventional white rice. Palatability of the rice cultivars was also investigated. It was hypothesized that high RS rice consumption would result in lower blood glucose and insulin concentrations and would be more satiating, compared to the control rice.2, a habitual breakfast eater, habitually eats rice, able to fast for 12 h, and available in the morning on weekdays for study visits. Exclusion criteria included diagnosis of diabetes mellitus (type I or II), hyper- or hypoglycemia, hyperinsulinemia, history of gastrointestinal disease, and history of disordered eating. A subject was also excluded if she was pregnant or lactating, he or she was taking any medications that controlled blood glucose or insulin, appetite, or weight, or if the subject had any known food allergies. The necessary sample size to detect difference in serum insulin response is 18 subjects based on a standard power calculation (α = 0.05 and β = 0.80). Twenty six subjects were enrolled in the study and 18 subjects  completed all of the study visits  and registered through clinicaltrials.gov (registration #NCT01685879). To recruit subjects, fliers were placed around the University of Hawaii at Manoa and Queen’s Medical Center. Subjects were screened by telephone or email for initial eligibility. After passing the initial screening, subjects attended a screening visit to complete a health history questionnaire to confirm eligibility and receive study materials. Inclusion criteria included general “good health”, 18–40 years old, non-vegetarian, non-smoking, BMI < 30 kg/my visits .The high amylose rice cultivars containing high RS, Dixiebelle and Rondo, were obtained from the USDA Dale Bumpers National Rice Research Center in Stuttgart, Arkansas. The amylose content of the selected rice cultivars was previously reported Dixiebelle (30.3% dsb) and Rondo (29.0% dsb) . The conSubjects completed a total of three study visits, and treatment order was randomly assigned at the time of enrollment. Subjects were blinded to the treatment identities. Study visits were held on weekday mornings. The day prior to the study visit, subjects kept a 1-day food record to document habitual diet. The subjects were instructed verbally on how to keep a food record by study staff, and the food record also included instructions and an example food record. Subjects began fasting at midnight (except for water), and were instructed to drink plenty of water the night before and the morning of the visit to assist with venous blood draws. Subjects arrived fasted for a minimum of 8 h.Each visit lasted approximately 2.5 h. A timeline of a typical study visit is shown in During the study visit, subjects read, used an electronic mobile device, or watched television. If necessary, a heating pad was used in between venous blood draws to aid with the draws. Subjects received refreshments and compensation after each study visit.et al. determined appetite VAS scores to be reproducible and valid for appetite research studies [Appetite was evaluated using a 100 mm VAS. After reading a question, subjects marked where they felt their answer belonged on a 100 mm line, using it as a scale (0 mm to 100 mm). Questions were: How hungry do you feel? Response options: Not hungry at all (0 mm) to I have never been more hungry (100 mm); How satisfied do you feel? Response options: I am completely empty (0 mm) to I cannot eat another bite (100 mm); How full do you feel? Response options: Not full at all (0 mm) to Totally full (100 mm); How much do you think you can eat? Response options: Nothing at all (0 mm) to A lot (100 mm). Flint At time 15 min, palatability of the treatment was assessed by five characteristics, from bad (0 mm) to good (100 mm). These characteristics were visual appeal, smell, taste, texture, and overall pleasantness of the treatment given.Blood glucose concentrations at 0, 15, 30, 45, 60, 90, and 120 min were measured by finger stick with a One Touch Ultra glucose meter . The blood samples from the venous blood draws at 0, 60, and 120 min were analyzed by Diagnostic Lab Services for blood glucose and insulin concentration. Capillary blood glucose area under the curves (AUC) were calculated using the trapezoid rule .The Nutrition Data System for Research (NDSR) 2012  was used to determine total nutrient and energy intakes for each food record. If a food was not listed in the NDSR program, the best match for the food was selected.p < 0.05.Data were analyzed with SAS statistical software . Results are presented as mean ± standard error of the mean. Treatment effects on biochemical parameters, appetite ratings, and dietary intake were determined using “PROC MIXED” procedure for analysis of variance to control for subject variation. Dietary intake and visit order were also included as a predictors of biochemical parameters and appetite ratings to determine if prior intake affected the study visits. Treatment groups were compared using LSMeans. Significant differences were determined at The RS content of one portion (1¼ cup US) of rice was as follows: short grain white rice, 1.40 g RS; Dixiebelle rice 2.25 g RS; Rondo rice, 2.10 g RS.The demographic information on the study subjects is presented in Dietary intake 24 h prior to study visits among treatments was not significantly different .The mean capillary blood glucose concentration at 30 min was significantly higher for the short grain rice than Rondo rice . At 45, The mean venous insulin concentration at 60 min was significantly higher for the short grain rice than both Dixiebelle and Rondo . At 120 Although treatments were not significantly different for most of the satiety measurements, only at 30 min, were subjects significantly more hungry after Dixiebelle intake than Rondo intake . Before The taste, texture, and pleasantness of the short grain rice were all significantly preferred over both Dixiebelle and Rondo . There w2) was 8.05 g RS, about 6 g more than the present study [et al. (2013) found that a 4.4 g RS dose in a serving of rice had no effects on postprandial blood glucose response in healthy adults  [Solid foods containing RS have shown therapeutic effects on postprandial blood glucose response and insulin in healthy individuals. Slowly digestible starch content may also reduce postprandial glucose response. The high RS rice samples, when matched for volume, significantly attenuated postprandial blood glucose response with an RS dose amount of 2.25 g (Dixiebelle) and 2.10 g (Rondo), compared to the short grain rice (1.4 g). In a previous study, the RS dose per serving of rice that significantly improved postprandial blood glucose response in healthy adults  study, the RS dose value of 8.05 g in a serving of rice significantly decreased insulin concentrations after intake starting at 45 min, then at 60, 90 and 120 min [The present study also found that insulin response was significantly lower at 60 min with an RS dose of 2.25 g (Dixiebelle) and 2.10 g (Rondo), and at 120 min with the RS dose of 2.10 g (Dixiebelle), compared to the short grain rice. In the Li  120 min . The add 120 min  suggestsp for linear trend < 0.001), the range of intakes differed tremendously between Asian cohorts and Western cohorts. For example, the upper threshold for lowest quintile or quartile in the Chinese and Japanese cohorts reviewed by Hu et al. exceeded the lower threshold for the highest quintile or quartile in the Western cohorts [et al.’s review collected dietary intake using a food frequency questionnaire that did not differentiate the starch digestibility of the white rice samples used. As shown in the present study, white rice yields differing physiological effects, depending on the carbohydrate identity.Prospective studies have noted a correlation between rice intake and increased risk of type 2 diabetes in multiple cohorts (as reviewed by ). Althou cohorts . Prospecet al. (2011) found a serving of bread (7.7 g RS) had no significant effects on insulin response in healthy adults  [et al. (2005) study, subjects were Japanese, but ethnicity is not stated for the subjects in the Larsen et al. (1996) study [For other solid foods , a range from 6.5–15.6 g RS dose per serving significantly lowered postprandial blood glucose response and insulin response from 30 min to 2 h in healthy adults after intake ,18,19,20 stated) . However6) study ,22. Howe6) study . The rolThe RS dose in rice of the present study is much lower than the RS dose in the studies previously discussed. The inconsistency across food forms suggests that RS dose, alone, may not be relevant, and that food source and/or the proportions of digestible starch, slowly digestible starch, and RS may be more important.et al. (2009) found that among muffins with different fiber types , the RS muffin (8 g RS) was the most satiating in healthy adults  [Studies on RS in solid foods and its effects on appetite are limited. Willis  stated) . However stated) .et al. (2013) found no significant differences in appetite between short grain rice and the rice containing 4.4 g RS in healthy adults [Few studies have investigated the effect of rice, without any added ingredients or meal items, on appetite. Chiu et al. 20 found noet al. (2010) compared the starch characteristics of 16 rice cultivars grown in the southern United States, at five different growing locations [The high RS rice cultivars were chosen for this study based on potential for expanded commercial production. Patindol There are some limitations to this study. First, only three measurements of venous blood glucose and insulin concentrations were taken, thus AUC for venous blood glucose and insulin was not calculated. The study visit protocol was modified to have only three venous blood draws  for subject comfort and study visit ease. Also, the study subjects were healthy individuals, with no issues in carbohydrate metabolism. The influence of slowly digestible starch content on the observed glycemic response is unknown, and while the present finding suggest a beneficial role in mediating blood glucose concentrations, further work is necessary to confirm this effect in individuals with pre-diabetes or diabetes.The present study demonstrated that Dixiebelle rice and Rondo rice with higher levels of RS are capable of attenuating postprandial blood glucose and insulin response compared to short grain rice with lower RS in healthy individuals. The serving size of these rice samples was practical and can be easily incorporated into the diet. Future work should include an investigation on the RS dose in rice needed to have effects on postprandial glucose and insulin response in pre-diabetic and diabetic individuals.


Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide. Most cancer cells display high rates of glycolysis with production of lactic acid, which is then exported to the microenvironment by monocarboxylate transporters (MCTs). The main aim of this study was to evaluate the significance of MCT expression in a comprehensive series of primary CRC cases, lymph node and hepatic metastasis.Expressions of MCT1, MCT4, CD147 and GLUT1 were studied in human samples of CRC, lymph node and hepatic metastasis, by immunohistochemistry.All proteins were overexpressed in primary CRC, lymph node and hepatic metastasis, when compared with non-neoplastic tissue, with exception of MCT1 in lymph node and hepatic metastasis. MCT1 and MCT4 expressions were associated with CD147 and GLUT1 in primary CRC. These markers were associated with clinical pathological features, reflecting the putative role of these metabolism-related proteins in the CRC setting.These findings provide additional evidence for the pivotal role of MCTs in CRC maintenance and progression, and support the use of MCTs as biomarkers and potential therapeutic targets in primary and metastatic CRC. Colorectal cancer (CRC) is the third most common cancer in men and the second in women, being one of the most prevalent diseases of the occidental world .Altered metabolism in cancer cells was recently recognized as a hallmark of cancer . Most caThe MCT family comprises 14 members; however, only the first four (MCT1-4) were identified as mediating the proton-coupled transport of monocarboxylic acids across the plasma membrane –24. It iData on the role of MCTs in CRC is somewhat contradictory. Koukoukaris et al.  describeThe aim of the present study was to evaluate the role of MCTs in CRC, by assessing the immunohistochemical expression of the MCT isoforms 1, 4, CD147 and the glycolytic metabolic marker GLUT1, and correlate their expressions with clinicopathological parameters in a comprehensive CRC series, including primary tumours and both lymph node and hepatic metastasis. Our results provide additional evidence of MCTs role in primary CRC and CRC metastasis, supporting their use as biomarkers and potential therapeutic targets in primary and metastatic CRC.Tissue samples and data from 487 patients treated in Hospital de Braga, Portugal, between 1st January of 2005 and 1st January of 2010 with CRC diagnosis were collected prospectively. Tumour localization was recorded and classified as colon and rectum . The histological type of CRC was classified by an experienced pathologist and tumour staging was graded according to the TNM classification, sixth edition . Tissue Additionally, an independent series of 45 patients with histological diagnosis of CRC hepatic metastasis operated between 1st January of 2003 and 1st January of 2011 was retrieved from the files of Hospital de Braga and data were retrospectively collected.CRC samples and CRC lymph node metastasis were included into tissue microarrays (TMAs). Prior to TMA construction, haematoxylin and eosin sections were reviewed to select representative areas of the tumour. Normal-adjacent tissue was also included in the TMAs for primary tumours. Each case was represented in the TMA by at least two cores of 0.6 mm.The study protocol was approved by the Ethics Committee of Hospital de Braga. The data of CRC and lymph node metastasis series were collected prospectively, patients were informed and signed a written consensus for collecting data and samples collection.Protein expression in primary CRC samples, lymph nodes and hepatic metastasis was evaluated by immunohistochemistry, as previously described . DetaileImmunohistochemical evaluation was performed as previously described .Briefly, sections were scored semi-quantitatively for immunoreaction extension (score 0–3) and intensity (score 0–3). Immunoreaction final score was defined as the sum of both parameters, and grouped as negative (0–2) and positive (≥3). Both cytoplasm and plasma membrane staining were assessed, but for statistical analysis only membrane staining was considered. Evaluation of protein expressions was performed by blind analysis by two observers and discordant cases were discussed in a double-head microscope in order to define the final score.Mutation analysis of BRAF (exon 15) and KRAS (codons 12 and 13) hotspot mutations, was performed by PCR, using primers and methods previously described , 45, folMicrosatellite Instability (MSI) was determined using a multiplex PCR of five quasimonomorphic mononucleotide repeat markers was end-labeled with a fluorescent dye , as described . PCR wasχ2) test and Fisher’s exact test (when n < 5).All data were analyzed using the Statistical Package for the Social Sciences, version 19.0 . Comparisons were examined for statistical significance using Pearson’s chi-square  and their 95 % confidence intervals in univariate and multivariate analysis. For multivariate analysis, variables that reached a To infer about the importance of the proteins MCT1, MCT4, CD147 and GLUT1 in the progression of CRC, their expression was evaluated by immunohistochemistry in 487 samples of CRC, 210 samples of CRC lymph node metastasis and 45 samples of hepatic metastasis. Representative images of MCT1, MCT4, CD147 and GLUT1 positive staining in CRC normal adjacent (NA) epithelium, primary tumour, lymph node and hepatic metastasis are presented in Fig. p < 0.001, Fig. p < 0.001, for both), with a decrease of MCT1 expression in CRC primary tumour to lymph node and hepatic metastasis  and a decrease of MCT4 expression in CRC primary tumour to hepatic metastasis (p = 0.0001). Compared to the MCTs expressions, the percentage of CD147 and GLUT1 positivity reactions were lower in CRC primary tumour; however, there was an increase in their expression from CRC primary tumour to lymph node  and hepatic metastasis   Fig. . In the p < 0.001, for all proteins, data not shown), namely MCT4 and GLUT1 with no expression and MCT1 and CD147 with 64.4 and 30 %, respectively, at NA hepatic tissue.We also matched the expression of these metabolism-related proteins in CRC hepatic metastasis with NA hepatic tissue, and we observed that these proteins presented a low expression in the liver tissue . On the other hand, MCT4 expression in lymph node metastasis seems to be independent of its expression in CRC primary tumour. Interestingly, primary CRC with negative MCT1 and MCT4 expressions can originate lymph node metastasis with positive expression for both markers. Detailed information is depicted in Table Since CRC primary tumours and lymph node metastasis belong to the same group of patients, we could compare the expression of the proteins in the two types of samples. We observed that MCT1, CD147 and GLUT1 positivity in CRC primary tumour samples associates with MCT1, CD147 and GLUT1 positivity in their respective lymph node metastasis (p = 0.003) in CRC primary tumour samples and with GLUT1 in CRC hepatic metastasis (p = 0.002) (Table p = 0.001) in CRC primary tumour and with CD147 expression (p = 0.050)  and hepatic metastasis samples    Table .Table 8Ap = 0.012)  Fig. , while n12) Fig. . The pre12) Fig. . No signMCTs play an essential role in the maintenance of cancer glycolytic metabolism. On one hand, they perform the efflux of lactate and, on the other hand, they help in the regulation of the cell pH, by co-transporting a proton , 17, 18.Our group has previously analyzed the immunoexpression of MCT isoforms 1, 2 and 4 in a series of 126 cases of CRC. Expression of all MCT isoforms in tumour cells was significantly increased, with a significant gain in membrane expression for MCT1 and MCT4 and loss for MCT2 in tumour cells, when compared to adjacent normal epithelium . In the Our results showed that most proteins studied  were overexpressed at the plasma membrane of CRC cells and CRC lymph node and hepatic metastasis when compared with CRC NA tissue, with exception of MCT1 in CRC lymph node and hepatic metastasis. Here we showed that in CRC samples, MCTs were the most frequently expressed proteins followed by CD147 and GLUT1. The MCT results are in concordance to our previous study, in which we showed upregulation of MCT1 and MCT4 in the tumour samples, compared to NA tissue . We founMost CRC cells, as many other solid tumours, rely mostly on glycolysis to meet their energetic demands . Thus, tTo the best of our knowledge, this is the first report that compares the expression of these proteins in CRC primary tumour with the respective lymph node metastasis,. MCT1, CD147 and GLUT1 positivity were positively associated in CRC and lymph node metastasis, although the expression of MCT1 was less pronounced in the metastasis than the primary tumour, which suggests that metabolic profile of the lymph node metastasis may be different from the primary tumour. For MCT4, the maintenance of membrane expression in lymph node metastasis, suggests the predominance of glycolytic metabolism, but more studies are necessary to demonstrate this hypothesis. In studies performed in breast cancer, MCT expression is reduced in lymph node metastasis compared to primary tumour .Lymph node metastasis are initially independent of vascularization, relying on the stroma to provide the required nutrients , 55. It Regarding the association between the proteins under study in primary CRC and clinicopathological data, we found that MCT1 positivity was associated with older patients; CD147 was associated with both larger tumours and more advanced tumour stage. Our results are supported by previous observations showing CD147 might enhance CRC growth, thus being associated with poor clinical prognosis –58. GLUTn = 16 and n = 1, respectively), which may have compromised statistical power.Regarding the association between the protein expression in lymph node metastasis and the same clinicopathological data, MCT4 positivity was associated with colon tumours and more advanced tumour stage and CD147 with tumour differentiation. MCTs and CD147 work synergistically, increasing invasiveness and metastatic potential trough microenvironment acidification and extracelular matrix destruction, via metalloproteinase induction –63. StudData on associations between protein expression in hepatic metastasis with the clinicopathological revealed that MCT1 expression was associated with primary tumour localization in colon. Association with left colon is a poor prognosis factor since CRC located in the left colon is associated with worse prognosis .Analyzing the CRC survival curves, we observed that MCT1 plasma membrane expression was associated with better patient survival in stage IV, however this association was not confirmed by multivariate analysis. MCT1 plays a pivotal role in colon epithelial cell metabolism, being critical for the metabolic communication between cells and for the transport of short chain fatty acids (SCFA), including lactate , 66, 67.No significant differences were found in primary tumour, CRC lymph node and hepatic metastasis survival curves for the different proteins.Overall, our findings support the role of MCT1, MCT4, CD147 and GLUT1 in CRC maintenance and progression. Moreover, since we found upregulation of these molecules either in primary tumours or metastasis, our results also support their exploitation as molecular targets in CRC treatment.CEA, Carcinoembryonic antigen; CRC, Colorectal cancer; MCTs, Monocarboxylate transporters; MSI, Microsatellite Instability; MSI-H, High MSI; MSI-L, Low MSI; MSS, Microsatellite stable; NA, Normal adjacent epithelium; SCFA, Short chain fatty acids; SMCTs, Sodium-coupled monocarboxylate co-transporters; TMAs, Tissue microarrays