Oral lichen planus (OLP) is a chronic inflammatory disease of unknown etiology with significant impact on patients' quality of life. Malignant transformation into oral squamous cell carcinoma (OSCC) is considered as one of the most serious complications of the disease; nevertheless, controversy still persists. Various factors seem to be involved in the progression of malignant transformation; however, the mechanism of this process is not fully understood yet. Molecular alterations detected in OLP samples might represent useful biomarkers for predicting and monitoring the malignant progression. In this review, we discuss various studies which highlight different molecules as ominous predictors of OLP malignant transformation. Oral lichen planus (OLP) is a chronic inflammatory T cell-mediated disease, clinically manifested as white, lacy plaques, located mainly on the buccal mucosa and tongue , 2. SeveAccording to the World Health Organization, OLP is categorized into the group of potentially malignant disorders , 4, whilOSCC accounts for 90% of malignant tumors of the head and neck region. It is most commonly diagnosed in 60–70-year-old individuals. It is a multifactorial disease; tumor development is based on both genetic and environmental factors , 6. VariThe first case of malignant transformation of OLP was reported in 1910 . LandiniCurrent research shows that the malignant process is based on increased proliferation of the basal-layer cells under the influence of mediators released from the inflammatory infiltrate that activate different pathways and can lead to tumor development . RecentlWe performed a nonsystematic review using Pubmed and Google Scholar databases. We selected articles written in English based on relevance and our experience in the presented topic; we did not use conventional inclusion and exclusion criteria.We identified a great number of markers of oral lichen planus malignant transformation and classified them into the following categories: apoptosis-related biomarkers, cell cycle regulators, tissue remodeling factors, inflammation-related factors, galectins, and intercellular adhesion proteins.Alterations of apoptosis pathways proved to be involved in general in the onset of malignant processes , 24. Twoα and FasL, respectively ..58].Katayama et al. have revealed that overexpression of MDM2 as an effect of SUMO-1 overexpression may function as a marker of tumor development and aggressiveness even in OSCC's early stages. In this light, SUMO-1 in conjunction with MDM2 might be employed not only as an indicator for tumor occurrence but also as a possible target for future pharmacological therapy .However, another study that has analyzed the expression of proteins p53, MDM2, and SUMO-1 in 4 diseases localized on the oral mucosa  compared to normal mucosa proved p53 and MDM2 overexpression in OLP, establishing hence a promalignant transformation environment. As for the expression of SUMO-1 in OLP, it was found to be similar in both normal mucosa and inflammatory fibrous hyperplasia, implying that alterations of SUMO-1 develop at later stages of carcinogenesis, as an important overexpression of this protein was found in oral epithelial dysplasia and established OSCC .BCL-2 is an inhibitor of apoptosis, whereas BAX, also a member of BCL-2 family, participates in the activation of intrinsic apoptotic pathway, having an opposite effect to BCL-2. Elevated expression of BCL-2 promotes a high survival rate of malignant cells that predisposes to an increased risk of developing new mutations , 36, 61.An increased expression of BCL-2 was found in the lymphocytic infiltrate which is characteristically encountered in OLP lesions, on one hand. On the other hand, the expression of BAX was elevated in epithelial basement keratin in OLP group compared to that in the control group, which included healthy subjects. These observations suggest a link between alteration of apoptosis and carcinogenesis . The samMCL-1 is an antiapoptotic protein pertaining to the BCL-2 family, it binds proapoptotic protein BAK in normal healthy cells. BAK is therefore sequestered until various cytotoxic signals activate a combination of BH3-only proteins that can displace BAK from this bondage, such as NOXA. Consequently, BAK can form oligomers that will organize as channels in mitochondria, leading to cytochrome c exiting into the cytosol and, subsequently, to caspase activation and apoptosis execution  66]..66].Shin et al. suggested that MCL-1 could be a novel biomarker for the malignant potential of OLP. However, the study included a small number of samples . Expression of MCL-1 was increased in OLP lesions and in the 2 tested cell lines, compared to that in the normal mucosa. The study highlighted the decrease of MCL-1 expression in cancer lines after treatment with sorafenib and mithramycin A, along with a decrease in cell replication, suggesting that the decline of MCL-1 expression could influence the process of malignant transformation .In many neoplasms, it was observed that MCL-1 overexpression is associated with cell resistance to apoptosis. This phenomenon is based on the interaction between MCL-1 and proapoptotic members of the BCL-2 family . In another study on OSCC, Shin et al. evaluated the role of mithramycin and it was found that it inhibits MCL-1 expression. Using this chemotherapeutic agent, good therapeutic results were obtained, therefore supporting once more the role of MCL-1 in carcinogenesis .Survivin is a member of the inhibitor of apoptosis (IAP) gene family. It seems that survivin plays an important role in carcinogenesis, being a molecule that modulates apoptosis and inhibits cell division . MultiplA recent study investigated the role of survivin in OLP. Survivin expression with moderate intensity was observed in the basal layer of OLP lesions. Its expression in normal tissues was modest or absent, while a higher expression of survivin was identified in OSCC samples, suggestive for its role in carcinogenesis .Deregulators of cell cycle controllers have also been studied as a possible important process involved in the malignant transformation of OLP. Most studies focus on the role of p16, B-cell-specific-Moloney murine leukemia virus integration site 1 (BMI1), and Ki67.Cell cycle is governed by the action of cyclin-dependent kinases (CDKs) and their main inhibitors p16, p21, and p27, which are important tumor suppressors . CDKs asα and an increased p16 expression [As aforementioned, p16 protein is involved in the antitumor response, promotes tumor suppression, and acts on the cell cycle . Increaspression . Montebupression . Moreovepression . Howeverpression –82.Goel et al. have revealed an increased expression of cytoplasmic p16 and CDK4 in OLP patients compared to that in normal mucosa. However, compared to OSCC, cytoplasmic expression of p16 and CDK4 were lower in OLP. Comparing nonerosive and erosive OLP, only the overexpression of cytoplasmic CDK4 was observed in erosive OLP samples. Cytoplasmic expression of p16 and CDK4 might be a predictor of the OLP malignant progression . In addiBMI1, a polycomb-group protein and a stem cell factor, is involved in cell cycle and in cell proliferation and plays a role in the self-renewal of stem cells. Increased expression of BMI1 has been determined in many tumors  and seemBMI1 was proposed by Ma et al. as a marker for identifying oral lesions at high risk of progressing into OSCC . After tOther research has investigated the possible links between BMI1 and p16 functions, as previously discussed in this section. Thus, Huber et al. studied 252 samples of oral and oropharyngeal SCC. They found an increased BMI1 expression along with a decreased p16 expression in SCC cells. Moreover, they obtained correlations of these markers with survival and recurrence rates. Thus elevated BMI1 expression and reduced p16 expression correlate with a poor prognosis and a high rate of relapse. It seems that BMI1 has an inhibitory effect on p16 .Another study by Kang et al. brings other important information to our attention regarding BMI1 involvement. The authors blocked endogenous BMI1 in cultures of normal keratinocytes and SCC keratinocytes and found that cell replication was affected suggesting its role in cancer cells proliferation. In addition, the study offers information about the link between BMI1 and p16. Differences in p16 expression in cells with and without BMI1 suppression were not observed and it was suggested that BMI1 may act through p16-independent pathways to stimulate the malignant proliferation process. Moreover, BMI1 overexpression was observed in pre-neoplastic oral lesions, showing various degrees of dysplasia, an important clue that suggests that BMI1 is expressed early in the process of carcinogenesis .It is commonly known that Ki67 is involved in the active phases of cell cycle and is considered an ubiquitary marker of cell proliferation. Ki67 is being expressed starting with S phase of the cell cycle and it reaches a maximum when mitosis occurs . Zargaraβ) and interleukin 8 (IL-8), macrophages, neutrophils, and fibroblasts release various MMPs. MMPs participate in the process of malignant transformation through mechanisms such as stimulation of different growth factors or inhibition of natural killer (NK) cell function. Additionally, MMPs regulate the bioavailability of vascular endothelial growth factor receptor (VEGFR) and consequently promote angiogenesis [Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in inflammatory and malignant processes , 90–93. ogenesis , 95.Giannelli et al. conducted the first study that revealed the role of MMPs in the pathogenesis of OLP and hypothesized that these molecules may be involved in the destruction process of the basement membrane through an imbalance between the level of MMPs and their inhibitors .Other research that included patients with oral leukoplakia showed a positive correlation between MMP-9, VEGFR2, and the degree of epithelial dysplasia .β1, a modulator of the MMPs activity. The study suggested that MMPs, especially MMP-9, might be a predictor of OLP lesions' malignant transformation. This research showed that atrophic OLP apparently has a higher risk of malignant progression than nonatrophic OLP [Chen et al. investigated the expression of MMPs  in normal mucosa, nonatrophic OLP, atrophic OLP, and OSCC, and they observed a progressive increase of their expression. The same progressive increase was observed for tissue inhibitor of metalloproteinases (TIMP) 2, an inhibitor of MMP-2, and TGF-phic OLP . In addiphic OLP . Immunohphic OLP , 100. Agphic OLP .The physiological inhibitors of MMPs are TIMPs. Shrestha et al. have studied the complex MMP-2/TIMP 2 in OSCC lesions and revealed that the activity of MMP-2 might be a marker associated with a low survival rate. The two markers correlated with the stage of the disease and the existence of metastases . In addiα, TGF-β, or IL-6 has a direct effect on the cell growth and survival rate [Inflammatory cells release a variety of molecules that may be involved in cell proliferation and angiogenesis. Studies have shown that in the course of a chronic inflammatory process, cytokines can participate in malignant cell transformation, contributing to an increase in mutation rate –107. Cytval rate .α, IL-1, IL-6, and IL-8 (NF-κB-dependent cytokines) in the saliva of 13 OLP patients with different degrees of dysplasia, 13 OSCC patients and 13 control subjects. TNF-α levels were elevated in OLP patients with moderate or severe dysplasia, similar to those with OSCC, while IL-6 and IL-8 levels were lower in OSCC. IL-1a level in moderate dysplasia was similar to that identified in OSCC samples, whereas in severe dysplasia, it was lower [α and IL-6 as markers with prognostic significance [δ) is decreased in advanced cases of leukoplakia and OSCC [It seems that inflammatory processes may play a role in the progression of OLP lesions into OSCC . Rhodus as lower . Similarificance . Lisa Chificance . Anotherand OSCC .Like in other types of cancer, the proinflammatory cytokine types prevail upon the anti-inflammatory ones sustaining thus the tumorigenesis potential of inflammation.As previously discussed, the presence of inflammation, especially chronic inflammation, is a condition contributing to the occurrence of carcinogenesis. Inflammatory stimuli lead to the activation of numerous signaling pathways, including cyclooxygenase (COX) expression . COX hasChangkong reported an increased COX-2 expression in OLP lesions and the correlation between COX-2 expression and disease severity . The stuGalectins comprise a family of endogenous carbohydrate-binding proteins with affinity for b-galactosides . GalectiMuniz et al. emphasized the role of galectins in the differentiation of OSCC and premalignant lesions. They studied the expression of galectins 1, 3, and 9 in OSCC, OLP, and oral leukoplakia in comparison with a normal histopathological profile. They have noticed a higher expression of galectin 9 in OSCC samples compared to that in samples of oral premalignant conditions and samples with normal histopathological aspect. The results regarding the expression of galectin 1 and 3 were heterogeneous in the studied groups .Ding et al. proposed galectin 1 as a predictor for progression of oral leucoplakia lesions into OSCC, revealing the overexpression of this protein in biopsies taken from patients with OSCC and oral leukoplakia . GalectiTransmembrane cadherins are proteins involved in intercellular adhesion and cellular differentiation. Reduction in their expression is associated with loss of characteristics of epithelial cells, and therefore, cadherins are involved in tumor differentiation, lymph nodes invasion, and occurrence of metastases. Altered E-cadherin expression was reported in large, poorly differentiated and metastatic tumors, E-cadherin being known as a molecule which inhibits tumor progression , 133. ReDu and Li have revealed abnormal positive expression of E-cadherin among OLP patients. Thus, 51.9% of the OLP patients had an abnormal positive expression of E-cadherin compared to only 4.8% of healthy controls. Based on the results of various studies in which E-cadherin expression was associated with the development of malignancies, it has been suggested that E-cadherin may be a marker of malignant transformation of OLP . Srideviα, IL-6, and COX-2). Although many researchers have suggested various biomarkers that may be useful in the detection of malignant progression, further studies are needed in order to establish the role of these biomarkers in current medical practice.OLP is considered by many researchers as a premalignant lesion; therefore, patients should be monitored in order to identify ominous signs of malignant transformation into OSCC in the very early stages. It is of paramount importance to understand the pathogenesis of OLP and establish what determines its progression to OSCC, as well as to monitor it adequately. Up to now, numerous biomarkers show promising results, including modulators of apoptosis , cell cycle regulators , tissue remodeling factors (MMPs), and inflammation-related factors (TNF-


Then, only four regions  were included in the simulated HRM (High-resolution Melting) analysis as the results from previous analysis showed that rpoC and trnL may not be suitable to be used to identify Zingiberaceae species in HRM analysis based on both the percentage of nucleotide variation and GC content. Simulated HRM analysis was performed to test the feasibility of Bar-HRM. We found that ITS2 is the most effective region to be used for identification of the studied species and thus was used in laboratory HRM analysis. All seven tested Zingiberaceae plants were then able to be distinguished using the ITS2 primers in laboratory HRM. Most importantly the melting curves gained from fresh and dried tissue overlapped, which is a crucial outcome for the applicability of the analysis. The method could be used in an authentication test for dried products. In the authentication test, only one of seven store-sold Zingiberaceae products that were tested contained the species listed on their labels, while we found substitution/contamination of the tested purchased products in the rest.It is long believed that some spices may help protect against certain chronic conditions. Spices are usually parts of plants that have been powdered into small pieces. Have you ever wondered what the curry powder in your dish is made of? The aim of this work was to develop an appropriate DNA-based method for assessment of spice identity. Selecting the best marker for species recognition in the Zingiberaceae family. Six DNA regions were investigated  Zingiber officinale, Curcuma longa, Curcuma zedoaria, Kaempferia parviflora, Boesenbergia rotunda, Zingiber montanum, Alpinia galangal and Amomum uliginosum [Spices are primarily used for flavouring, colouring or preserving food, and they usually come from seeds, fruit, roots, bark, berries, and buds of plants. In addition to culinary use, spices are sometimes used in medicine, cosmetics or perfume productions and in religious rituals. Modern science has now shown that many of them do indeed carry remarkable health benefits. There has been great expansion of spicy products in the Thailand market as the number of products increased rapidly, particularly spices in the ginger or Zingiberaceae family, which includes several important spice producers. There are many plants in the Zingiberaceae family that have been deemed as economic crops that can generate national income, such as iginosum . The extmatK [rbcL [Molecular identification through DNA barcoding is an efficient technique for the identification of plant species. A considerable amount of literature has actually been published, and it shows that DNA barcoding can be used to distinguish closely related medicinal plants –4. The imatK ,9,10, antK [rbcL . DeveloptK [rbcL –15. ThisBar-HRM has been used in a number of comparable applications. Bar-HRM is revolutionising the way we can trace plant species in herbal, agricultural and food products; however, the approach faces challenges that can hinder our ability to produce robust results. Recently, a comprehensive evaluation of the use of Bar-HRM in plant species identification addressed the choice of markers used, which could lead to success in different plant groups . DifferematK, trnH-psbA spacer region, rbcL, rpoC and trnL retrieved from GenBank, which included sequences from the entire family of Zingiberaceae   . With th2 genus) .matK, trnH-psbA spacer region, rbcL, rpoC and trnL were extracted from GenBank using the keyword ‘Zingiberaceae + (region title)’ at the end of September 2015. Some of the sequences retrieved from public databases, including GenBank, were found to be low quality with no known associated herbarium vouchers. For this reason, all of the sequences were subjected to critical evaluation and any low-quality sequences were removed. Criteria used to filter the sequences were as follows: (1) sequences were not ‘unverified’ without a species name, (2) sequences contained <3% ambiguous base ‘N’, and (3) a maximum of 3 samples (sequences) were included from a species. After processing, multiple alignments were made from the selected sequences using MEGA6 [Sequences of ITS, ng MEGA6 , and theAlpinia galangal, Amomum uliginosum, Boesenbergia rotunda, Curcuma longa, Kaempferia parviflora, Zingiber montanum and Zingiber officinale, were investigated. Dried and fresh plant tissues for DNA extraction were kindly provided by the Queen Sirikit Botanic Garden (QSBG) and the faculty of pharmacy, Chiang Mai University, respectively . Species included in the assay were as follows: 1) Amomum krervanh 2) Boesenbergia rotunda 3) Curcuma longa 4) Curcuma zedoaria 5) Kaempferia parviflora 6) Zingiber montanum 7) Zingiber montanum and 8) Zingiber zerumbet  that was capable of distinguishing the seven selected species of Zingiberaceae, PCR amplification using real-time PCR and DNA was performed using a Rotor-Gene Q 5plex HRM system  in a total volume of 20 μl on a plate. The reaction mixture for the real-time PCR and HRM analysis contained 10 μl of MeltDoctor HRM Master Mix , 1.2 μl of 10 mM forward primer, 1.2 μl of 10 mM reverse primer, 1 μl of 30 ng DNA and 6.6 μl of ddH2O. The primer pair was derived from the ITS2 sequence data retrieved from an online database (GenBank) (Forward 5’- CGCCTGCTTGGGCGTCATGGC -3’ and Reverse 5’- GGGCCTCGCCTGACTTGGGGCC -3’). Fluorescence dye was used to monitor both the accumulation of the amplified product and the high-resolution melting process in order to derive the Tm value during PCR.To acquire the characteristic melting temperature  was used to analyse the TZingiber officinale (P1.0-P1.6), Curcuma longa (P2.0-P2.6), Kaempferia parviflora (P3.0-P3.6), Boesenbergia rotunda (P4.0-P4.6), Zingiber montanum (P5.0-P5.6), and four of Alpinia galangal , two of Amomum uliginosum (P7.0-P7.1), were included in the analyses  analysis. Seven commercial products of each species, analyses .matK, 425 of rbcL, 60 of rpoC, 647 of trnH-psbA spacer region, 436 of trnL and 930 of ITS were retrieved, of which 242, 323, 60, 87, 102 and 252 sequences of each barcoding region were considered as being useful for further analysis , followed by trnH-psbA, trnL, rpoC, rbcL and ITS, with 29.5%, 33.1%, 38.9%, 44.0% and 55.8%, respectively. Based on the results above, it was estimated that the ITS would be a suitable barcoding marker for the HRM analyses for the target species. ITS has also been used for plant species identification with excellent results [The average %GC content of all barcoding region amplicons was calculated, and thus, the predicted possible variation in the melting curves for the different markers was used. The  content 9.3%, fol results ,20.SM to define the discriminatory power via prediction of fluorescent high-resolution DNA melting curves of PCR products. Eight species  were included in the simulated HRM analysis as the results from previous analysis showed that rpoC and trnL may not be suitable to be used to identify Zingiberaceae plants in HRM analysis based on both the percentage of nucleotide variation and the GC content. However, only seven sequences were used in the rbcL assay, as there is no sequence of B. rotunda that could be retrieved from the database (GenBank). Although we expected eight different melting curves from the eight tested species, as seen from trnH-psbA showed the highest number of distinctive curves (seven), which made them the most promising regions for our study. Thus, we chose only ITS and trnH-psbA for further analyses.The DNA-fragments or sequences of selected Zinbearaceae species were analysed using a web-based application called uMelt species  were chotrnH-psbA, ITS1 and ITS2. To indicate the most suitable pair, HRM analysis was performed in triplicate on each of the three selected taxa  to establish the Tm and melting profiles for each primer set. The DNA of each tested sample was isolated in triplicate and used in the HRM analysis. The melting profiles of all studied amplicons are illustrated in m values and melting curves, all three primer pairs, trnH-psbA  [trnH-psbA for the next analysis. When considering the differences in Tm values, melting profile and nucleotides variation (data not shown) of both ITS1 and ITS2, we only chose ITS2 for further experiments.Although the three tested primer pairs were efficient enough to differentiate the three selected species, the e pairs) . As amplWe used ITS2 primers in the HRM analysis in order to confirm its ability to differentiate the seven selected Zingiberaceae plants before using it to identify the species origin in spice products. The melting profiles of ITS2 amplicons from all tested species are shown in C. longa and Z. montanum products (P2.0 and P5.0) presented similar melting curves to the reference species and thus implied that the products contained the indicated species. In addition, only one store (of seven) in this study, sold all tested spices that were identified as the same species indicated by the seller or on the labels .The ITS2 primer pair was then used in HRM analysis for authentication to see whether they were consistent with the species indicated by the sellers. The tested spice products were purchased from various stores or producers in northern Thailand. HRM analysis was performed in triplicate on each of the tested products to establish the melting profiles. The shapes of the melting curves were analysed using a Rotor-Gene Q Series Software (version 2.3.1) to indicate plant species contained in the products. The melting profiles of all product amplicons are illustrated in Spices were the main focus of our study as they were not only used for medicinal purposes but also serve as aromatherapy products, natural pest repellents, skin soothers, and even weapons (pepper spray). Several studies have shown that substitution of plant species occurs in herbal medicines. This could pose a serious problem in quality control in production, especially for those with high consumption and economic value. Only 41.46% of the tested spices here were found to be consistent with the species indicated by the sellers or labels. Whether intentional or not, substitution, contamination and adulteration of species should not happen. As most of the spices are sold in powdered form, it is hard to tell whether a product is actually the indicated species based on visual inspection. In addition, the Bar-HRM was previously reported to be able to discriminate an herbal mixture at various concentrations (1%–50%) . Thus, tSeveral scientific research reports have noted that spices have health benefits, such as boosting the immune system, and they may help protect against certain chronic conditions, such as cancer, diabetes, and heart disease. Many traditional remedies have hinged on the use of spices as well, so they have always been in high demand. Before selling, most spices are powdered into small pieces, so it is difficult to identify the species by using morphological approaches. For detecting substitution and adulteration in spice products using molecular techniques, the Bar-HRM, has a number of advantages: (1) the HRM analysis method is highly sensitive for detecting a 1%–0.1% presence of an adulterated sample; (2) it is a high throughput technique that is capable of analysing multiple samples at the same time; and (3) no post-PCR processes are needed, and thus, cross-contamination can be avoided.S1 Table(DOCX)Click here for additional data file.


We measured the fasting blood glucose, insulin, hemoglobinbA1c, homeostasis model assessment for insulin resistance (HOMA-IR), carboxymethyl lysine, total antioxidant capacity, and malondialdehyde levels at the beginning and the end of the study. Thirty-nine patients (20 in the intervention group and 19 in the control group) completed the study. After an eight-week intervention, changes in the level of fasting blood glucose, insulin, hemoglobinbA1c, HOMA-IR, carboxymethyl lysine, total antioxidant capacity, and malondialdehyde were not significant in either group, nor were any significant differences between groups observed in these glycemic and inflammatory indicators at the end of the intervention. Our study revealed that cinnamon supplementation had no significant effects on glycemic and inflammatory indicators in patients with type 2 diabetes.The aim of the current study was to determine the effect of a daily intake of three grams of cinnamon over eight weeks on glycemic indicators, advanced glycation end products, and antioxidant status in patients with type 2 diabetes. In a double-blind, randomized, placebo controlled clinical trial study, 44 patients with type 2 diabetes, aged 57 ± 8 years, were randomly assigned to take either a three g/day cinnamon supplement ( Diabetes is one of the most common multifactorial public health diseases and a leading cause of death worldwide . AccordiManagement of diabetes to prevent its complications and increase life expectancy and quality of life of diabetic patients becomes possible with appropriate intervention and modifications in lifestyle, including changes in dietary pattern, regular physical activity, and anti-diabetic medications ,8. RecenCinnamomum zeylanicum Blume), as one of the common spices, contains various antioxidant compounds that are routinely used in a traditional system of medicine to treat chronic diseases such as cardiovascular diseases and diabetes in various regions of the world [Cinnamon  are key participants in the development and progression of type 2 diabetes and its complications . On the Considering the limited studies available on the effects of cinnamon on managing and controlling type 2 diabetes, in the current study, we have investigated the effect of cinnamon supplementation on glycemic indices, AGEs, and antioxidant status in patients with type 2 diabetes. To do this, we performed a double-blind, randomized, placebo controlled clinical trial to assess the effect of three grams per day of cinnamon supplementation for eight weeks on FPG, insulin, HbA1c, HOMA-IR, carboxymethyl lysine, total antioxidant capacity, and MDA in 44 patients with type 2 diabetes.This is a double-blind, randomized, placebo controlled clinical trial conducted in Tehran province between 2016 and 2017 for which 44 patients, aged 25–70 years with type 2 diabetes, were randomly enrolled in two groups, the placebo and intervention. We used the formula for parallel clinical trials, considering type I error (α) of 0.05 and type II error (β) of 0.20 (power = 80%) to calculate the sample size of 20 for each group. However, assuming an estimated 10% dropout rate, there were 22 patients in each group  to compensate for the probable loss to follow-up.2, fasting blood glucose <180 mg/dL, 2 h blood glucose <250 mg/dL, glycemic control with metformin therapy, women without pregnancy and lactation, not having allergy to cinnamon, and free of some chronic diseases . Exclusion criteria were noncooperation of patients during the study, pregnancy and lactation, detection of any side effects while taking cinnamon supplements, changes in the routine management of diabetes, drug abuse, alcohol and anti-inflammatory drug consumption.Inclusion criteria were: history of diabetes <8 years, body mass index (BMI) ranged 18.5–30 kg/mThe current study was conducted according to the principles of the Declaration of Helsinki, and the study protocol was approved by the Ethics Committee of the Shahid Beheshti University of Medical Sciences, Tehran, Iran (No.: IR.SBMU.NNFTRI.REC1394.36). We explained the study protocol and risks of experimental procedures carefully to all participants, after which their written informed consent was obtained. The study was registered at the Iranian registry of clinical trials, registration number IRCT2016061128392N1.n = 22) or placebo (n = 22) for 8 weeks. Supplements and placebos were packaged in the same form and investigators and subjects were blinded to group assignment and to capsule content until the end of the analysis  guidelines in the microbiology laboratory. Then, in the traditional medicine pharmacy, cinnamon sticks were finely ground. Cinnamon as treatment and also microcrystalline cellulose as placebo placed in capsules were not distinguishable from each other in color, smell, taste, shape, and size. Finally, the capsules were packaged and labeled equally. Each capsule contained either 1000 mg of cinnamon or microcrystalline cellulose. In the beginning and again at the fourth week of the study, the participants were given cinnamon supplements or placebos that were sufficient for use over four weeks and they were asked to consume one 1000 mg capsule of cinnamon or placebo after each main meal  for eight weeks. In addition, patients were asked not to change their routine physical activity or normal dietary pattern and to continue their medications (metformin therapy) during the intervention.We contacted the patients weekly to ensure that participants would act in compliance with the protocol of study, and remind them to take their supplements/placebos daily; they were also asked to return the original tablet bottles for capsule counts and assess compliance, so that the all unused capsules could be checked by counting the unconsumed capsules at each visit (every four weeks). The patients were interviewed by trained interviewers using pretested questionnaires for collection of socio-demographic and other information, including data on age, sex, education levels, marital status, employment status, duration of diseases, medication use and smoking at the beginning and end of the study. Weight was measured and recorded without shoes and wearing light clothing, using a digital scale with an accuracy of up to 100 g. Height was measured in a standing position without shoes, using a stadiometer with a minimum measurement of 5 mm. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared.A 10 mL venous blood sample was taken after 12 h of overnight fasting in a sitting position according to the standard protocol and centrifuged within 30–45 min of collection at baseline and after the eight-week intervention. All blood analyses were performed on the day of blood collection at the laboratory of the Endocrinology and Metabolism center, Shahid Beheshti University of Medical Sciences.Two mL of whole blood was added to a vial containing ethylenediaminetetraacetic acid (EDTA) anticoagulant for analyzing HbA1c level, immediately, and blood samples were centrifuged at room temperature for 10 min in a clinical centrifuge at 3000 rpm for plasma separation. Plasma samples were stored in a freezer at −70 °C for later analyses in micro tubes with a mL capacity. FPG was measured by the enzymatic colorimetric method with glucose oxidase, using commercial kits . The blood level of HbA1c was measured by high performance Ion Exchange Chromatography (IEC) method . Fasting plasma levels of insulin were analyzed using the auto-analyzer with 2 micU/mL sensitivity, by a commercially available enzyme-linked immunosorbent assay (ELISA) kit . In addition, a commercial kit with 0.1 mmol sensitivity  was used for measuring of total antioxidant capacity. Plasma level of carboxymethyl lysine was measured ELISA method . We also determined the MDA concentration (μmol/L) by chromatography assay. Finally, the homeostasis model assessment for insulin resistance (HOMA-IR) was conducted according to their suggested formulas as follows ,25:HOMp-values < 0.05 were considered statistically significant. To assess the normality of the variables, Kolmogorov–Smirnov analysis was used. The characteristics of the patients at baseline and at the end of the study are expressed as the mean ± SD for normally distributed contentious variables, median (25–75 inter-quartile range) for skewed continuous variables, and percentages for categorical variables. To compare quantitative variables between intervention and placebo groups, we used Student’s t-test and Mann–Whitney test for means and medians, respectively. In addition, a Pearson Chi-Square test was applied for comparison of qualitative variables between the two groups. Paired t-test was used to compare variables before and after the intervention within each group. Statistical analyses were conducted using the Statistical Package for Social Sciences  and 2, respectively. Forty-four patients were recruited in this study and during the intervention. Five patients were excluded from the study due to non-cooperation or travel and withdrawal of two patients (case group) and three patients (control group). Therefore, thirty-nine patients with type 2 diabetes, 15 men and 24 women completed the study. The mean ± SD age and body mass index of the participants were 57.6 ± 8.7 years and 27.7 ± 4.5 kg/mp = 0.83). In addition, mean ± SD body mass index was 26.4 ± 3.0 kg/m2 for the intervention group and 29.0 ± 5.5 kg/m2 for the placebo group.According to A comparison of glycemic indices and inflammatory factors before and after of cinnamon consumption are presented in p > 0.05).In addition, after eight weeks of intervention, there were no significant intra-group differences based on FPG, fasting levels of insulin, HbA1c, carboxymethyl lysine, total antioxidant capacity, and MDA in patients of either group  region that assessed the effects of cinnamon intake on glycemic indicators along with inflammatory factors, and AGEs in diabetic patients. Limitations of the current study include the short follow-up period (eight weeks) and dose of the cinnamon supplementation (three grams per day). In addition, the intake of high doses of cinnamon supplementation  or a lonIn conclusion, findings of this study revealed that an 8-week intervention of three grams of cinnamon supplement per day had no beneficial effects on FPG, insulin levels, HbA1c, HOMA-IR, carboxymethyl lysine, total antioxidant capacity, and MDA levels. Further studies are needed to better evaluate the impact of cinnamon intake on glycemic markers and indicators of oxidative stress or inflammation in diabetic patients.


Racial disparities in the incidence of major cancers may be attributed to differences in the prevalence of established, modifiable risk factors such as obesity, smoking, physical activity and diet.Data from a prospective cohort of 566,398 adults aged 50–71 years, 19,677 African-American and 450,623 Whites, was analyzed. Baseline data on cancer-related risk factors such as smoking, alcohol, physical activity and dietary patterns were used to create an individual adherence score. Differences in adherence by race, gender and geographic region were assessed using descriptive statistics, and Cox proportional hazards models were used to determine the association between adherence and cancer incidence.Only 1.5% of study participants were adherent to all five cancer-related risk factor guidelines, with marked race-, gender- and regional differences in adherence overall. Compared with participants who were fully adherent to all five cancer risk factor criteria, those adherent to one or less had a 76% increased risk of any cancer incidence , 38% increased risk of breast cancer , and doubled the risk of colorectal cancer . However, risk of prostate cancer was lower among participants adherent to one or less compared with those who were fully adherent . The proportion of cancer incident cases attributable to low adherence was higher among African-Americans compared with Whites for all cancers (21% vs. 19%), and highest for colorectal cancer (25%) regardless of race.Racial differences in the proportion of cancer incidence attributable to low adherence suggests unique opportunities for targeted cancer prevention strategies that may help eliminate racial disparities in cancer burden among older US adults.The online version of this article (doi:10.1186/s12885-017-3557-1) contains supplementary material, which is available to authorized users. Colorectal, prostate and breast cancer are three of the four most common cancers among adults in the U.S. Combined, they are estimated to account for over 560,000 new cases and 115,000 deaths due to cancer in 2016  cancers,In this prospective cohort of African-American and White older adults, we examined adherence to body weight, physical activity, alcohol, smoking and nutrition guidelines by race, gender and region, and estimated the proportion of overall, breast, prostate and colorectal cancer incidence attributable to poor adherence. Understanding the contribution of these modifiable risk factors to cancer incidence may be useful for public health interventions focused on cancer prevention and inform strategies to eliminate racial and/or geographic disparities in cancer risk.n = 49,318), proxy respondents , death record data only (n = 4255) or who had missing data on behavioral risk factors  and race (9566) were excluded from analysis. The final analysis included a total of 470,000 adults; 19,677 African-American and 450,623 Whites with no prior history of any cancer. With a sample size of 19,677 for African-Americans, we were well powered with Type 1 error of 0.05 and Type II error of 80% to detect effect sizes as low as 1.1 and adherence levels as low as 20%.Data for this study was obtained from the prospective National Institutes of Health-American Association of Retired Persons (NIH-AARP) Diet and Health Study. The cohort consists of 566,398 adults AARP members aged 50–71 years recruited in 1995–1996   and the We assessed adherence to each cancer prevention guideline overall  and for each risk factor separately. We compared baseline characteristics and adherence by race and gender using chi-square tests and ANOVA as appropriate. We also examined differences in adherence by geographic region, categorized as: Northeast, Mid-West, South, and West. We conducted Cox proportional hazards models to determine the association between adherence and cancer incidence, and reported the results from Cox models as hazard ratios (HR) and 95% confidence intervals. We examined Kaplan-Meier survival cures and found no evidence of violations of the proportional hazards assumption. All statistical models were stratified by race, and adjusted for baseline characteristics such as age, marital status, education, health status, and gender . Trend tests were performed by assessing the linear relationship between adherence and cancer incidence. Censoring occurred at the time of first primary cancer diagnosis, loss to follow up or the end of incidence follow-up period, whichever occurred first. The attributable risk (AR) due to adherence was calculated from models based on individual’s region, race, background covariates, and adherence value using the appropriate model, and the counter-factual estimate for that individual assuming the highest rate of adherence. The proportions of individuals categorized as affected (i.e. for which the random number did not exceed the risk estimate) for both situations  were divided to form a risk ratio (RR), and AR calculated using the formula (RR-1)/RR. Confidence intervals for the AR were generated from bootstrapped resamples of 1000 draws of random numbers from a uniform distribution and compared to the estimates, and this was repeated for the counterfactual estimates to provide a measure of the precision of AR estimates. All analyses were conducted using SAS 9.4 and R statistical package.The majority of NIH-AARP participants were between ages 65 to 69 years 32%), and most participants were male (60%), married (69%) and 39% had at least a college degree , 22% did not meet the criteria at all, and 43% were overweight. Alcohol Use: Adherence to guidelines regarding alcohol was high, with over 98% of participants meeting the criteria i.e. consuming 7 or less alcoholic drinks per week for females and 14 or less alcoholic drinks per week for males. Smoking: Less than 40% of participants were adherent to guidelines regarding smoking i.e. never smokers, while 52% were partially adherent meaning that they were former but not current smokers. Nutrition: Only 26% of study participants were adherent to nutrition guidelines, and 36.5% were totally non-adherent i.e. did not consume at least 5 servings of fruits and vegetables per day. Physical Activity: Only 23% of study participants were adherent to physical activity guidelines i.e. at least 210 min of moderate physical activity per week.Only 1.5% of study participants were adherent to all five cancer-related risk factor guidelines, with marked race-, gender- and regional differences in adherence overall Fig. . ObesityIncreasing adherence to cancer prevention guidelines was associated with progressively reduced risk of any cancer incidence Table , Fig. 2.The proportion of cancer incidence attributable to low adherence was higher among African-Americans compared with Whites for all cancers (21% vs. 19%), and highest for colorectal cancer (25%) regardless of race. Racial difference in the attributable fraction was observed for breast and prostate cancer: 16% of breast cancer incidence was attributable to low adherence for African-American and less than 8% for Whites. Notably, 18% of prostate cancer incidence was prevented due to low adherence overall; 12% for African-American and 18% for Whites Fig. .Fig. 3AtIn one of the largest prospective cohort studies of older adults in the US, we observed racial, gender and regional differences in the level of adherence to AICR/WCRF cancer-related risk factor guidelines. At baseline, adherence was overwhelmingly low, with less than 2% of older adults adherent to all five criteria; less than 1% of African-American and 1.5% of Whites met all five criteria for body weight, physical activity, smoking, alcohol and diet. Adherence was highest in the West for obesity and physical activity, and in the North East for alcohol use, smoking and nutrition. Cancer risk overall increased significantly with reduced adherence to the cancer-related risk factor guidelines; adherence to one or fewer criteria (relative to five) increased the risk of all cancers by 76%, breast cancer by 38%, and colorectal cancer by 100%, however lower adherence was associated with a 21% reduced risk of prostate cancer. Although the magnitude of the associations was similar between African-American and Whites, the only statistically significant association for African-Americans was for the risk of any cancer and not for specific cancers. Overall, lower adherence was associated with increased cancer risk consistently across regions, except for colorectal cancer where there was a higher but non-significant association in the Mid-West. About 20% of all cancers, 10% of breast and 24% of colorectal cancers are attributable to low adherence, however among White women, only 8% of breast cancer incidence was attributable to low adherence, compared with 18% for African-American women, and close to 20% of prostate cancer cases were actually prevented by low adherence.Several studies have examined the influence of cancer-related risk factors in general, and adherence to cancer prevention guidelines, on the risk of developing cancer and have observed similar results to ours –22. HoweThe biological mechanisms linking modifiable lifestyle factors and cancer development have been well established, including in a comprehensive review by . Excess Genetic and epigenetic alterations in cancer-related genes, influenced by lifestyle factors, have also been shown to influence cancer tumorigenesis . NeverthDespite convincing evidence regarding the negative influence of obesity, smoking, and low physical activity on health outcomes in general, and cancer risk specifically, we observed that in 1995–1996 only about a third of older US adults met each of the modifiable lifestyle risk factors  . These eWe observed an inverse association between adherence and prostate cancer risk. This is similar to findings from other studies , 44, 45,In conclusion, for the major cancer types observed among US adults, lack of adherence to lifestyle related cancer risk factor guidelines significantly increased cancer risk, with up to 25% of new cancer cases attributable to low adherence. A larger proportion of breast cancer incidence in African-American women compared with Whites was attributable to examined lifestyle related risk factors, suggesting that there may be unique opportunities for targeted clinical and public health strategies to reduce the burden of breast cancer among older African-American adults.


Chinese women residing in Hong Kong for the past 7 years were recruited by media advertisements . The control participants were recruited by convenience sampling from health workshops held in clinics and communities of 15 districts of Hong Kong. After completing test–retest reliability, all participants were asked to complete diet pattern questionnaires about their food preferences and dietary patterns. The Student’s unpaired hot, neutral, and cold. The participants with breast cancer exhibited a stronger preference for hot food than the control group . A higher frequency of breast cancer was associated with a higher frequency of dining out for breakfast  and lunch . The participants with no history of breast cancer consumed CM supplements and Guangdong soups  more frequently than those with breast cancer.Three major CM dietary patterns were identified: neutral  dietary pattern, and consumed CM supplements and Guangdong soups more frequently.Non-breast cancer participants adopted a The online version of this article (doi:10.1186/s13020-017-0138-9) contains supplementary material, which is available to authorized users. Breast cancer is the most common and one of the most fatal cancers in women worldwide. The number of breast cancer cases among women in Hong Kong increased from 2273 in 2004 to more than 2870 in 2009, and average incidence rate per 100,000 women between 2004 and 2009 was 386.7 . Howeverhot or cold features) may increase the risk of related diseases, such as cancers [The dietary patterns of women in Hong Kong have been changing with western lifestyle in recent years , 7. The  cancers . Althoug cancers –11, the cold, cool, hot, and warm) can be used to classify diets as hot, neutral, or cold; these types of food have different functions in the body. Under this classification, beef, shrimp, chicken, and even ice cream are hot foods  , who were compared with healthy women (controls). The cases were recruited by advertisements in the local media, such as in newspapers, in booklets issued by the University of Hong Kong, on posters, and on the radio. Convenience sampling was used to recruit the control group from health workshops in clinics and communities in 15 Hong Kong districts. All participants had to fulfill the following conditions: (1) female, (2) Chinese women residing in Hong Kong, (3) 28–60 years of age, (4) living in Hong Kong for the past 7 years, (5) able to understand the questionnaire items in Chinese, and (6) provision of an informed consent. Controls were required to fulfill the following conditions: (1) no breast cancer or other gynecologic diseases; (2) no metabolic, nutritional, or other severe diseases; and (3) no serious disease after doctor consultation in the past 3 months. Case inclusion criteria were: (1) new primary breast cancer patients, and (2) a diagnosis of primary breast cancer through biopsy and/or discharge report. Participants were excluded from the study if they had (1) other prior cancer diagnosis, (2) treatments with psychotropic drugs, (3) any medical conditions that limited their physical activity, and (4) not completed the questionnaire.hot foods and an expected odds ratio (OR) of 2.32 according to the formula of OR was found. For the pilot study, we invited 37 people from the oncology outpatient clinic and the general outpatient clinic in the School of Chinese Medicine, University of Hong Kong, to complete the questionnaire: 20 were breast cancer cases and 17 were not. After the pilot study, we analyzed the responses and calculated the consumption of 12 kinds of hot foods including beef, mutton, shrimp, soft drinks, and milk. Of the breast cancer cases, 45.8% consumed hot foods regularly, whereas only 26.7% of the controls did so. We chose 10% as the estimated prevalence of hot food consumption for control group and calculated the estimated prevalence of hot food consumption for breast cancer group as 0.2 the expected OR of 2.32 to estimate the sample size of our research. The estimated sample size was calculated as about 207 participants in each group. Taking into account the nullified questionnaires, 202 participants were eventually included out of the total 207 participants recruited in each group.We conducted a pilot study with 37 participants whose total diet comprised mainly A self-administered FFQ was used to collect the dietary intake information. This questionnaire was adapted from the Fred Hutchinson Cancer Research Center Food Questionnaire, which is based on Western dietary patterns , 14. Twocold, neutral, and hot foods, classified according to CM). A total score for each subscale was obtained by summing the relevant item scores. Higher scores indicated higher consumption frequencies [The questionnaire consists of two sections. The first section assessed sociodemographic information, including age, working status, education level, weight, height, and marital status. The second section contained the CM FFQ items, which measured consumption of very common foods. The FFQ consists of 74 items scored on a 5-point scale, ranging from 1  to 5 (consumed very often). It comprises nine subscales of food consumption frequency for CM-defined foods, and a general preference score for three kinds of food . The results produced a Every potential participant was interviewed by a trained research doctor at the clinic center of the School of Chinese Medicine, University of Hong Kong. Participants completed a self-administered questionnaire and had a face-to-face consultation for the same research doctor to check the questionnaire.Three important steps were taken throughout the process to ensure the quality of investigation. First, to ensure consistency of the survey across sites and over time, the same interviewer used the same procedures and standards to investigate all women. Second, each questionnaire was checked by the same researcher carefully and those questionnaires with missing data on foods or dietary patterns were excluded. Third, to prevent input error, we used a double data entry method by different researchers to enter all data into SPSS.cold, neutral, and hot properties and have different nutritional constituents, each with their own cold, neutral, and hot properties. These food properties are based on the functions the foods perform after consumption. All vegetables, meat, fruits, and grains are classified according to one of these three properties, as summarized in Additional file hot, neutral, or cold was determined by the average score for all the foods listed. For example, if the overall FFQ score for neutral food was the highest, then the overall preference score for food was considered neutral. The hot and cold preference scores were calculated in the same manner.According to CM theory, different foods possess different dim sum/tea drinking) and Western-style food consumption  . Demographic information such as age, marital status, current height and weight (to obtain body mass index), and education level was also collected.Additionally, participants were asked to indicate the frequency of (1) eating out for breakfast, lunch, and dinner ; (2) CM intake, CM supplement intake, and Chinese soup consumption ; and (3) Chinese-style food  was used for data analysis. Descriptive statistics were calculated; continuous variables were presented as mean ± standard deviation, and categorical variables were presented as frequencies. The mean difference between continuous variables was tested using Student’s Regarding the validity and reliability assessment of the questionnaire, we examined test–retest reliability by inviting participants in the pilot study to complete the questionnaire a second time. We compared both test results using ANOVA, which produced a value of 0.915, showing high test–retest reliability. All the pilot study participants were interviewed about the interpretability of the questionnaire, and the interpretability was reported to be good.The case and control groups were similar in terms of body mass index, occupation, education, marital status, age at first menstrual cycle, and family history of cancer Table .Table 1SP < 0.001): participants in the control group were 2.4 years younger. However, when age was categorized according to five different age groups, there was no significant difference between the case and control groups (P = 0.061). Because there was a marginally significant difference in age between the two groups, we considered age a confounder in multivariate analysis of variance (MANOVA).When age was treated as a continuous variable, there was a significant difference between the case and control groups (P < 0.001). We analyzed the between-group differences in food preferences of hot, neutral, and cold using logistic regression with age as a variable. The consumption of hot foods such as roast meat, shrimp, shellfish, soft drinks, popcorn/fries, and candies was significantly different between groups. Participants with a strong preference for roast meat, shrimp, shellfish, and popcorn/fries had 6-, 9.2-, 13.3-, and 7.6-times higher risk, respectively, of having breast cancer compared with participants with an average preference for these foods. Women who preferred soft drinks had a 4.9-times higher risk of having breast cancer compared with women who had an average preference. Higher preferences for nuts and freshwater fish  and tofu and squid (cold) showed a lower OR for breast cancer  and lunch  was positively associated with the risk of breast cancer. In general, participants who dined out for breakfast and lunch (at least four times per week) had at least twice the risk of having breast cancer compared with participants who dined out 0–1 time per week   dietary pattern, along with consumption of CM supplements and Guangdong soups.Non-breast cancer participants adopted a healthy and balanced (Additional file 1. Classification and characteristics of foods according to TCM theory.Additional file 2. Reference food questionnaire.Additional file 3. Minimum standards of reporting checklist.Additional file 4. Questionnaire-N-dietary.Additional file 5. IRB.


To evaluate the effectiveness and safety of first-generation protease inhibitors for the treatment of genotype 1 hepatitis C virus-infected patients at Brazilian reference centers.This multicenter cross-sectional study included hepatitis C virus genotype 1 monoinfected patients treated with Peg-interferon, ribavirin, and either boceprevir (n=158) or telaprevir (n=557) between July 2013 and April 2014 at 15 reference centers in Brazil. Demographic, clinical, virological, and adverse events data were collected during treatment and follow-up.p=0.190). Serious adverse events occurred in 44.2% of patients, and six deaths (0.8%) were recorded. Cirrhotic patients had lower sustained viral response rates than non-cirrhotic patients  and a higher incidence of serious adverse events . Multivariate analysis revealed that sustained viral response was associated with the absence of cirrhosis, viral recurrence after previous treatment, pretreatment platelet count greater than 100,000/mm3, and achievement of a rapid viral response. Female gender, age>65 years, diagnosis of cirrhosis, and abnormal hemoglobin levels/platelet counts prior to treatment were associated with serious adverse events.Of the 715 patients, 59% had cirrhosis and 67.1% were treatment-experienced. Based on intention-to-treat analysis, the overall sustained viral response was 56.6%, with similar effectiveness in both groups (51.9% for boceprevir and 58% for telaprevir, Although serious adverse events rates were higher in this infected population, sustained viral response rates were similar to those reported for other patient cohorts. Infection with hepatitis C virus (HCV) is one of the leading causes of chronic liver disease worldwide In addition to protease inhibitors, other classes of direct-acting antiviral agents have gradually been introduced into clinical practice, including NS5A and NS5B inhibitors Between 2013 and 2015 in Brazil, triple therapy with BOC and TVR was the standard treatment for patients infected with HCV genotype 1 This multicenter cross-sectional study included patients chronically monoinfected with HCV genotype 1 (treatment-naïve or previously treated with Peg-IFN and RBV) who were treated with Peg-IFN (α2a or α2b), RBV and either BOC or TVR according to the guidelines of the Brazilian Ministry of Health All patients who started treatment in these centers between July 2013 and April 2014 were eligible for this study. The exclusion criteria were individuals younger than 18 years of age and individuals co-infected with human immunodeficiency virus.This study was approved by the Ethics Committee for the Analysis of Research Projects (Comissão de Ética para Análise de Projetos de Pesquisa–CAPPesq) of the Clinics Hospital of the Medical School of the University of São Paulo  under protocol no. 11,934.3; and 3- albumin levels <3.5 g/dL. Regarding hepatic fibrosis staging, liver biopsy results obtained using the Metavir Cooperative Study Group Data were collected using a standardized questionnaire. The variables selected for analysis were grouped into the following different categories: 1) variables related to the patient: age, gender, body mass index, and comorbidities; 2) variables related to HCV infection before the present treatment: genotype 1 subtype, presence of extrahepatic manifestations, presence of previous hepatic decompensation, the last value recorded in medical records for hemoglobin levels, platelet counts, albumin levels, HCV viral load, and staging of liver fibrosis; and 3) variables related to the treatment of hepatitis C: history of previous treatment as well as the type of viral response observed and the occurrence of SAEs during the current treatment. The variables analyzed in our study were standardized according to specific considerations. For the laboratory test conducted prior to treatment, the following results were considered abnormal: 1- hemoglobin levels <12 g/dL for female patients and <13 g/dL for male patients; 2- platelet count <100,000 per mm10 in HCV RNA levels at week 12 of treatment but with detectable HCV RNA at week 24; and null responders - reduction under 2 log10 in HCV RNA levels at week 12 of treatment. In addition, those who did not have a defined viral response to previous treatment were considered non-responders.Regarding the type of response observed after previous treatment with Peg-IFN and RBV, non-responders were considered those patients with any of the following types of viral responses as a result of previous treatments: partial responders - reduction of at least 2 logSAEs were defined as events that resulted in death, represented a threat to life, required hospitalization or prolongation of an existing hospitalization, resulted in persistent or significant disability, or promoted congenital malformation or anomaly, as defined in previous studies The decision to initiate treatment and the choice between BOC or TVR and Peg-IFN-α2a (180 mg) or Peg-IFN-α2b (1.5 μg/kg) was entirely at the discretion of the attending physician of each participating service, according to the guidelines of the Brazilian Ministry of Health and the product manufacturers HCV RNA levels were measured at weeks 4 and 12 of treatment, at the end of therapy and at weeks 12 and/or 24 after the end of therapy. We performed real-time polymerase chain reaction (PCR) with a detection limit of 12 IU/mL  The rapid viral response (RVR) was defined as undetectable HCV RNA (<12 IU/mL) at week 8 for those treated with BOC and week 4 for those treated with TVR (we considered week 8 to be when “lead in” was performed).Effectiveness was evaluated using the intention-to-treat principle. The primary outcome of the study was SVR, which was defined as undetectable levels of HCV RNA (<12 IU/mL) at least 12 weeks after discontinuation of treatment.The safety of triple therapy using BOC and TVR was assessed by determining the occurrence of SAEs during treatment.t-test or the Mann-Whitney U-test was applied for continuous variables as required.A descriptive analysis of the baseline demographic and clinical characteristics was performed. The results are presented as frequency tables for qualitative variables; central tendency and dispersion measures were estimated for quantitative variables. Pearson’s chi-square test was used to compare the patients according to the treatment provided. Fisher’s exact test was used for situations in which this test was not appropriate. Student’s The prevalence of treatment effectiveness, rate of SAE occurrence, and respective 95% confidence intervals (95% CIs) were calculated. The factors associated with the effectiveness of treatment and SAE occurrence were determined in patients with chronic hepatitis C by performing a bivariate analysis and determining the prevalence ratios (PRs) and respective 95% CIs.p-values smaller than 0.20 as determined by performing bivariate analysis were selected for multivariate analysis. The Poisson regression model with robust variance was used in the bivariate and multivariable analyses. PRs and their 95% CIs were estimated for each of the variables in each final model. A significance level of 5% was adopted.Variables with A total of 715 adult patients with chronic HCV infection were included in this study. The majority of patients were male (56.1%), with a mean age of 54.1±10.1 years; 267 individuals (37.3%) had HCV subtype 1a, and 315 (44.1%) had subtype 1b. Among the studied patients, 422 (59%) had liver cirrhosis, and 67.1% were treatment-experienced. In total, 557 patients (77.9%) received treatment with Peg-IFN, RBV, and TVR, and 158 (22.1%) patients received treatment with Peg-IFN, RBV, and BOC.vs. TVR). Patients treated with BOC reported a higher prevalence of extrahepatic manifestations before treatment and a more frequent history of previous hepatic decompensation than those treated with TVR . However, there was a predominance of treatment-naïve patients in the group treated with TVR compared to the number in the group treated with BOC . Regarding the laboratory tests conducted before treatment, abnormal hemoglobin levels  and platelet counts <100,000/mm3 in the BOC group were significantly more frequent in the BOC group than in the TVR group .vs. 58.0%; p=0.190). However, the SVR rate was higher in patients without cirrhosis than in patients with cirrhosis . Taking into account a history of previous treatment, SVR rates were 59.1% for treatment-naïve patients, 74.3% for relapsers, and 43.2% for non-responders  (p<0.001) , the overall percentage of patients with SVR was 56.6% . The effectiveness of treatment between the groups that received BOC or TVR was similar (51.9 p<0.001) . Of the p<0.001), history of previous treatment in non-responders , being treatment-naïve  vs. being a relapser, a platelet count <100,000/mm3 prior to treatment , and the presence of RVR .The results obtained for the 2 groups of patients were then combined to identify factors associated with treatment effectiveness. The results of the univariate and multivariate analyses are shown in vs. 34.8%, p<0.001). Premature discontinuation of treatment in cases of SAE was observed in 121 (16.9%) patients, and the most common SAEs were hepatic decompensation (n=28), anemia (n=23), and infection (n=20). Furthermore, six deaths (0.8%) were observed among the treated patients. Most of these cases were associated with infections, including spontaneous bacterial peritonitis (n=1), septic arthritis (n=1), and skin sepsis (n=1). Other causes of death included hepatorenal syndrome (n=1), pulmonary embolism (n=1), and cerebral aneurysm (n=1).Of the 715 patients, 316  had at least one SAE episode. A total of 581 SAE episodes were observed. The SAE rate was significantly higher among patients with cirrhosis than among patients without cirrhosis (50.7 vs. TVR). Patients treated with TVR had a higher frequency of degree 3 or 4 anemia than patients treated with BOC and an increased need for blood transfusion .p<0.001), age >65 years , the presence of liver cirrhosis , and abnormal hemoglobin levels or platelet counts before treatment   .3 before therapy, and the presence of RVR. Regarding treatment safety, approximately 44.2% of study patients had SAEs. Multivariate analysis indicated that the factors associated with the occurrence of SAEs were the presence of liver cirrhosis, female gender, age >65 years, and abnormal hemoglobin levels or platelet counts before treatment.Based on our results, 56.6% of patients with chronic hepatitis C treated with therapies involving the use of BOC or TVR achieved SVR. Multivariate analysis indicated that the factors associated with the achievement of SVR were the absence of cirrhosis, a history of relapse after previous treatment with Peg-IFN and RBV, a platelet count >100,000/mmComparison of our results with those observed in other large real-life cohorts involving the use of triple therapy with BOC or TVR suggested the rate of SVR was similar to that observed in previous studies, in which this rate ranged between 44% and 58% The presence of liver cirrhosis has often been associated with a lower likelihood of SVR in the treatment of chronic hepatitis C, regardless of the treatment used With respect to the SVR rates observed in our study for non-responders to previous treatments with Peg-IFN and RBV, our results corroborate those obtained in registration studies with BOC RVR was the best predictor of treatment success (SVR) in our cohort  and in other clinical studies Regarding treatment safety, we observed a high prevalence of SAEs in our study group (44.2%); this rate was higher than that reported in phase 3 studies for BOC The most important factors associated with the occurrence of SAEs were the presence of liver cirrhosis, female gender, age >65 years, and abnormal hemoglobin levels or platelet counts before treatment. All of these factors have been extensively described in studies similar to ours Despite the high prevalence of SAEs in our study, the rate of discontinuation of therapy due to adverse events was low 16.9%) compared to rates observed in real-life studies % compareWe believe that this study describes the largest cohort of patients infected with HCV genotype 1 and treated with BOC or TVR in combination with Peg-IFN and RBV in a real-life context in Latin America. We sought to include patients from different Brazilian service centers and with different characteristics to obtain a representative sample of patients during this phase of treatment for hepatitis C in Brazil. However, this study has some limitations. The first limitation is its cross-sectional, retrospective, observational, and multicentric nature. In this type of study, the leading center researchers involved are fully responsible for the choice of treatment offered and for providing the data. In this respect, the selection of patients for treatment and the choice between different treatments is at the discretion of the local investigator. This type of study may lead to selection bias for the patients included. Moreover, in this study, it was not possible to determine how many patients with chronic HCV infection and treatment indication were initially considered eligible for treatment or refused treatment. We cannot exclude the possibility that patients selected for treatment who had a higher likelihood of achieving SVR were included in our analysis. However, we believe that by following the standards described in the Brazilian protocol at the time Another potential bias in our study is the possibility of missing data, as only the data recorded in the medical records of the patients were included in the study. This has also been the case in other retrospective studies. In terms of monitoring of adherence to treatment, it was not possible to access this information due to the retrospective nature of this study. However, it is important to note that in our study, only variables that did not exceed 15% patient loss were included for analytical purposes. Conversely, to evaluate the effectiveness of treatment in clinical practice, real-life studies offer the advantage of eliminating the potential bias of a change in behavior and conduct that might occur during the execution of a prospective study According to data from the Brazilian Ministry of Health, approximately 7,800 treatments with BOC or TVR were performed at the national level between 2012 and 2016 In conclusion, although most of our patients achieved SVR after treatment, the rate of SAEs was very high in this population. Patients with cirrhosis had a lower risk of achieving SVR and an increased risk of developing SAEs. Therefore, despite the beneficial outcome and cure of a significant number of patients, the risk of the development of SAEs, including severe clinical complications and death, particularly in those with advanced liver disease, should be taken into consideration. In addition, the use of therapies such as BOC or TVR has been proscribed, and safer, more effective direct-acting drugs should be considered. Our results confirm the need to reformulate the guidelines for the treatment of chronic hepatitis C in Brazil, which recently incorporated safer and more effective drugs for the treatment of this condition.Callefi LA and Mendes-Correa MC were responsible for the study design, data collection and analysis and manuscript preparation. Villela-Nogueira CA contributed to data collection and participated in data analysis and manuscript revision. Tenore SB, Carnaúba-Júnior D, Coelho HS, Pinto PT, Nabuco LC, Pessoa MG, Ferraz ML, Ferreira PR, Martinelli AL, Chachá SG, Ferreira AS, Bisio AP, Brandão-Mello CE, Álvares-Da-Silva MR, Reuter T, Ivantes CA and Perez RM were responsible for data collection, participated in the data analysis and critically revised the manuscript. All authors read and approved the final version of the manuscript.


Renal cell carcinoma (RCC) is the most prevalent and lethal malignancy of the kidney. Despite all the efforts made, no tissue biomarker is currently used in the clinical management of patients with kidney cancer. A search for possible biomarkers in urine for clear cell renal cell carcinoma (ccRCC) has been conducted. Non-targeted metabolomic analyses were performed on paired samples of surgically removed renal cancer and normal tissue, as well as on urine samples. Extracts were analyzed by liquid chromatography/high-resolution mass spectrometry (LC-HRMS). Hydroxybutyrylcarnitine, decanoylcarnitine, propanoylcarnitine, carnitine, dodecanoylcarnitine, and norepinephrine sulfate were found in much higher concentrations in both cancer tissues  and in urine of cancer patients (compared with control urine). In contrast, riboflavin and acetylaspartylglutamate (NAAG) were present at significantly higher concentrations both in normal kidney tissue as well as in urine samples of healthy persons. This preliminary study resulted in the identification of several compounds that may be considered potential clear cell renal carcinoma biomarkers.The online version of this article (10.1007/s00216-018-1059-x) contains supplementary material, which is available to authorized users. Biomarkers provide a powerful approach to understanding diseases with applications in epidemiology, clinical trials, screening, diagnosis, and prognosis. Defined as alterations in the constituents of tissues or body fluids, they often offer the means for classification of a disease and can extend our knowledge about the underlying pathogenesis of disease. Theoretically, efficient biomarkers can also reflect the entire spectrum of disease from the earliest manifestation to the terminal stage. The development of cancer therapies is increasingly dependent on the understanding of tumor biology, and biomarkers are becoming essential tools in the field of medicine.Renal cell carcinoma (RCC) is the most prevalent and lethal malignancy of the kidney, accounting for nearly 90% of all renal tumors and representing 2–3% of all adult malignant tumors , 2. RCC RCC is now thought to be a morphologically and genetically heterogeneous disease that can be classified into several different subtypes, such as clear cell RCC (ccRCC), papillary RCC, and chromophobe RCC , 5. CleaA favorable prognosis  can be achieved by radical nephrectomy with nephron-sparing surgery when kidney cancer is detected and treated at an early stage . UnfortuRecent studies have renewed interest in the alterations in cellular metabolism associated with a range of diseases, including cancers, and it is now widely accepted that metabolomics can be a powerful tool not only for disease detection, diagnosis, as well as treatment guidance and assessment but also for the elucidation of the molecular processes behind the disease states , 2.1H nuclear magnetic resonance (NMR) + in positive ion mode). The monoisotopic mass of the corresponding neutral was obtained from that of the parent ion by subtracting the proton mass. The alignment procedure results in a list of “features,” each associated with a monoisotopic mass , a retention time, and a total ion abundance. The calculated mass values for the neutral compounds, M, were used throughout the manuscript, instead of m/z for the MH+ ions. Unless the identification of a parent ion in a group of peaks as MH+ is erroneous, each feature will correspond to an actual compound. Alignment of detected peaks was performed separately for the set of samples extracted into THF and into water, respectively.In the alignment procedure, peaks obtained in three different UHPLC-HRMS experiments (triplicate injections) are determined to be formed from the same compound, based on their appearance at nearly the same retention time and A major objective of this metabolomic study is to identify (putative) compounds that are over- or under-expressed in renal cancer as opposed to normal renal tissue. For features, the terms “over-abundant” and “under-abundant” were used, while “over-expressed” and “under-expressed” were used for (putatively) identified metabolites. Detailed LC-MS data discussed in this work is available in the Electronic supplementary material  metabolite database  using a Multivariate statistical analyses were performed using Metaboanalyst 3.0 .Examples of total ion chromatograms for cancer and normal tissue extracts are shown in the ESM Fig. . SimilarA total of 948 features were detected in the set. The relative similarities and differences between the metabolomes of the different samples were studied using statistical methods. Principal component analysis (PCA) did not yield a clear separation between cancer and normal tissue. Therefore, supervised statistics, i.e., partial least square discriminant analysis (PLS-DA), was applied as it enhances the separation between groups by rotating the PCA components.Figure It is seen that it is mainly C2 that separates cancer from normal tissues, while C1 is associated mainly with those interpatient differences that are not strongly dependent on the development of renal cancer. In the present sample set, C1 mainly separates out patients 2 and 3 from patients 4 and 5. It is seen that C2 achieves a clear separation between normal and cancerous renal tissue. .It is noted that the respective cancer tissues have, for all four patients, a somewhat more negative value for C1. Based on this observation, it would seem that the metabolome differentially associated with the development of renal cancer has a pattern in common with interpatient differences for normal tissue. Expressed differently, the normal tissue metabolisms of some patients are, in some respects, more “cancer like” than those of other patients.As seen in Fig. In conclusion, it is demonstrated that with PLS-DA of metabolomic data, it is possible to discriminate between cancerous and normal renal tissue . “While 1, 2, 12–16, 6, 10, 13, 16, 18, 23, 24, and 26–28, respectively. A listing of compounds with standard errors is in Tables Of the 948 features detected in the tissue samples, the abundancies for a large majority were not significantly different in cancer versus normal tissue. Using a minimum fold change of 2, seven features were found to have a higher average abundance in the cancer tissue and nine compounds a higher average abundance in the normal tissue. These are listed in Table 14, 15). In particular, acetylcarnitine (14) had a very high abundance in cancer tissue, while the abundance in normal renal tissue was two orders of magnitude lower. Decanoylcarnitine (15), was also over-expressed in the cancer tissue, though at much lower abundance. Similar observations have been made previously [14–28). Five of these (16–20) were over-expressed in cancer tissue, though they did not fulfill minimum fold change requirements. Acetylcarnitine, is an acetic acid ester of carnitine, which for example is used by the body to transport fatty acids into the matrices of mammalian mitochondria where fatty acid metabolism occurs [Two features that were over-abundant in cancer tissue were putatively identified as carnitines  were high both in tissue and urine samples. Cancer-to-control ratios were found to be moderately high being 1.6 and 1.5 for tissue and urine cancer/control pairs, respectively; it should be noted that tissue result is similar to the one shown by Ganti and co-workers [18) and hydroxypropionylcarnitine (17) were found in tissue with 2.3 and 3.1 cancer-to-control ratios, respectively. What is interesting, first compound—hydroxybutyrylcarnitine was also found to be even in higher abundance in cancer urine with a ratio of 5.7; result similar to the one shown by Ganti et al. who showed also higher cancer patients’ concentration of this compound (ca. four times) in urine comparison. Two other carnitines were found to show similar pattern of ratios—4,8-dimethylnonanoylcarnitine (21) and 2-dodecenoylcarnitine (20) for which cancer-to-control ratios grow from 1.0 to 7.5 in case of 21 and from 1.4 to 4.1 for 20 when switching from tissue to urine results. Two other carnitines—hexanoylcarnitine (22) and 3-methylglutarylcarnitine (23) present higher abundances in normal tissue samples, and only 23 was detected in urine with 3.4 cancer-to-control ratio.The abundances of carnitine (-workers . Much mo24), heptanoylcarnitine (25), 2,6-dimethylheptanoylcarnitine (26), pimelylcarnitine (27), and dodecanedioylcarnitine (28) were detected exclusively in urine, all of them were in higher abundances in cancer patient urine with ratios of 3.1, 1.8, 4.1, 6.7, and 7.2 for compounds 24–28, respectively. It should be noted that 26 was also found by Ganti et al. with a cancer-to-control ratio of 2.0.Other carnitines found exclusively in urine samples such as butenylcarnitine .As mentioned above, 13 features were found to have a higher abundance in normal tissue, as compared wwith cancer tissue, features 11 and 12. Feature 11, but not 12, was present also in urine but without significant difference between RCC and control patient groups. Feature 11 is putatively identified as N-(3-oxooctanoyl)homoserine, and feature 12 as alanyl-α-thiophenylglycine or S-(phenylacetothiohydroximoyl)-cysteine. The latter compound is involved in the biosynthesis of glucosinolates from phenylalanine.The biggest fold changes, > 50, for normal versus cancer tissue were observed for features 13 (NAAG), this was the case also for 9, which has the putative identification of riboflavin. Both will be discussed in more detail below.Two features that were over-abundant in normal tissue, were also significantly more abundant in normal versus cancer urine. Apart from 6, 10, and 23. This pattern can be expected for compounds that are not retained or consumed, by cancer tissue, but excreted. Three of the four features have putative identifications based on mass alone but without additional evidence they remain highly uncertain.For several of the features that were over-abundant in normal tissue, it was observed that the abundances in the urine samples were reversed, i.e., higher in cancer than in normal urine, albeit with a smaller fold change. This was the case for features 7, 5, 8, 12, 15, and 75. Feature 8 was 15-fold over-abundant in normal as compared with cancer tissue. Assignment to a compound was difficult as metabolite data base searches yielded 18 isomers  is shown to be an excellent candidate for a tissue-based biomarker in both water and THF extracts; unfortunately, it was not found in urine samples (vide infra).Additional analysis based on THF-extracts of metabolites was performed. Selected results of this analysis are presented in Table 2, 4, 6, 10, 15, 16, 18–21, 23–28) and 44 to be under-abundant  in cancer urine, when compared with control urine. The abundance data is presented as bar charts in Figs. Among the 3368 features detected in the set of urine samples, 16 were determined to be over-abundant  over-abundant in cancer urine have a dominant common pattern. All except features 24–28 were detected also in tissue. Seven of the features were either not detected at all, or detected with an insignificant abundance, in urine from non-cancer patients. Third, the hits in the MMDB and HMDB were few, and none were obvious candidates for known human metabolites. While other scenarios are possible, this pattern is consistent with the features being due to drugs given to the cancer patients or to their metabolites. Such compounds are indeed expected to be present in both types of tissue and in cancer urine, but not in control urine. Because a wide range of synthetic compounds are possible, any putative identifications would be very unreliable and not included in Table The abundance pattern for the 11 features . This feature was only detected in normal tissue and not in cancer tissue. While NAAG is normally present in urine of healthy subjects, it was here found to be less abundant in urine from the cancer patients. NAAG is one of the three most prevalent dipeptide neurotransmitters in the mammalian nervous system. The simplest explanation for the absence of NAAG in renal cancer may be denervation of the tumor, something that is commonly observed and has been reported to enhance cancer metastasis [Feature tastasis . Howevertastasis , 56, andNAAG in urine does not seem to previously have been considered for cancer detection. However, the observation that the decrease in NAAG in the urine of cancer patients is substantial, suggests that the concentration of NAAG is urine may be used as an indicator of RCC size or activity.9 is also observed to be over-abundant in both cancer tissue and urine. This feature was putatively assigned to riboflavin. This compound is one of eight B-complex vitamins, and it plays a key role in maintaining human health. The significance of riboflavin was discussed in many publications [Feature ications , 58.2, 4, 15, 16, 18–20) are, as mentioned above, strong candidates for potential kidney cancer biomarkers. Feature 15 is putatively identified with decanoylcarnitine, which is a carnitine ester with decanoic acid. This compound is present in blood plasma in cases of fatty acid oxidation defects (FAOD), such as long-chain 3-hydroxylacyl-CoA dehydrogenase (LCHAD) deficiency, carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, and medium-chain acyl-coenzyme A dehydrogenase deficiency [Features that are over-abundant in both cancer tissue and cancer urine . Thus, it is a very good candidate for a new kidney cancer biomarker. Acylcarnitines, which are intermediates in the key energy metabolic pathways of fatty acid β-oxidation and amino acid catabolism, were found previously at higher concentrations in tumor tissues , 24 and 2, Fig. 2, Fig. Norepinephrine sulfate (NE sulfate) showed significant increases both in cancer tissue and urine samples from patients with kidney cancer , Fig. 2.Liquid chromatography/high-resolution mass spectrometry analysis of extracts from cancer and healthy tissue regions allowed the identification of up- and downregulated compounds that could potentially serve as renal cancer biomarkers, ccRCC. Similar analyses of urine from cancer patients and from a healthy control group yielded additional putative biomarkers. The putative identifications of compounds were based on exact mass and on data base hits on important human metabolites, known to be relevant for cancer. Cross-comparison of two sets of results allowed the identification of four kidney cancer biomarkers that are either over- or under-expressed in both cancer tissue and urine from cancer patients. Hydroxybutyrylcarnitine, decanoylcarnitine, propanoylcarnitine, carnitine, dodecanoylcarnitine, and norepinephrine sulfate were found in distinctly higher concentrations in both cancer tissues and in urine of cancer patients compared with controls. In contrast, feature assigned to riboflavin and NAAG were present at significantly higher concentrations both in normal kidney tissue as compared with renal cancer tissue and in urine samples of healthy persons than in urine from the cancer patients. All eight mentioned compounds may be considered potential clear cell renal carcinoma biomarkers. Preliminary research presented in this work will be followed in the future with larger-scale study based on higher amount of patients.ESM 1(PDF 590 kb)


Salmonella and multidrug-resistant Escherichia coli O157:H7 strains. Bacteriophage phiE142 belongs to the Myoviridae family due to the presence of long non-flexible tail and icosahedral head. The genome is composed of 121,442 bp and contains 194 ORFs, and 2 tRNAs. Furthermore, the phiE142 genome does not contain any genes coding for food-borne allergens, antibiotics resistance, virulence factors, or associated with lysogenic conversion. The bacteriophage phiE142 is characterized by broad host range and compelling genetic attributes making them potential candidates as a biocontrol agent.The emergence of antibiotic-resistant foodborne bacteria is a global health problem that requires immediate attention. Bacteriophages are a promising biotechnological alternative approach against bacterial pathogens. However, a detailed analysis of phage genomes is essential to assess the safety of the phages prior to their use as biocontrol agents. Therefore, here we report the complete genome sequence of bacteriophage phiE142, which is able to lyse The online version of this article (doi:10.1186/s40793-016-0211-5) contains supplementary material, which is available to authorized users. Bacteria cause the majorities of foodborne illnesses; Escherichia coli and Salmonella are among the most common foodborne pathogens that affect millions of people annually [E. coli and Salmonella strains makes more difficult its control [Foodborne diseases are an important cause of morbidity and mortality worldwide, therefore are a serious public health problem . Bacteriannually . Further control . Hence, In this context, bacteriophages have several potential applications in the food industry; these killing-bacteria viruses are alternatives to conventional antimicrobials method for the control of pathogenic bacteria and have great potential in the improvement of food safety –6. BacteThe primary aim of our research group is increase knowledge of phage biodiversity and contribute to the understanding of different types of phages in several regions of Sinaloa, an important agricultural region in Northwestern Mexico. Recently, a new bacteriophage, designated as phiE142, one of phages isolated, exhibits a high potential as a biocontrol agent . HoweverE. coli strain EC-48 , was also isolated from the same geographical region two years before the isolation of the phage [E. coli EC-48 lawn; the plaques were already visible after four to six hours of incubation time at 37 °C.The bacteriophage phiE142 was previously isolated in Food and Environmental Microbiology Laboratory at the Research Center for Food and Development from animal feces samples collected on a farm in Northwestern Mexico. An he phage . Phage pSalmonella strains and 33 E. coli strains  and classified into its appropriate viral morphotype according to Ackermann’s classification . The anaEnterobacteriaceae were clustered in the same group . Considering the close relationship among these phages, it is likely that phiE142 also belongs to this genus. This result confirms the findings obtained by electron microscopy.The sequence of DNA polymerase has become a commonly-used marker for constructing phylogenetic analysis, therefore the phylogenetic tree was performed based of DNA polymerase deduced amino acid sequences. According to the phylogenetic tree, the phage phiE142 and others eight phages that infect the bacterial family up Figs. . All of E. coli and Salmonella strains isolated from environmental sources in Northwest Mexico. The analysis of more genomes of bacteriophages is necessary to increase our understanding of the genetic diversity of bacteriophages, phage biology, basic molecular mechanisms, and provide a deeper insight into the relationship of phages with their hosts. Furthermore, analysis of phage genomes may reveal novel antimicrobial peptides and enzymes with bactericidal activity. In addition, the genome well understood is an essential requisite to ensure the safety of the phages prior to their use as biocontrol agents. Therefore, the genome project was deposited in the Genomes On Line Database (GOLD). The genome sequence of bacteriophage phiE142 was deposited in GenBank under accession number KU255730. The summary of genome project is available in the Table The bacteriophage phiE142 is one of the first genome to be completely sequenced publicly available for a phage infecting E. coli strain EC-48 were mixed with 3 ml TSB with 0.4% agarose, spread on TSA plates, and incubated overnight at 37 °C. After, phage was subsequently collected by adding 6 ml of SM buffer  to the surface of each plate and the soft agar was scraped off the surface of the agar plates. Cell debris was removed by subsequent centrifugation at 5,500 × g for 10 min, the supernatant was filtered with 0.22 μm syringe filters, and phage particles were precipitated by centrifugation at 40,000 × g at 4 ° C for 2 h. The phage pellet was suspended in SM buffer and stored at 4°C. Bacteriophage DNA was isolated by the method of proteinase K and phenol–chloroform as previously described [g for 15 min at 4 °C, the pellet was washed twice with 70% ethanol, centrifuged at the same conditions. Finally, the dried DNA pellet was suspended in nuclease-free water. Concentration of phage DNA was estimated with a NanoDrop spectrophotometer  and also the quality of extracted DNA was also tested visually with electrophoresis on a 1% agarose.Standard double-layer agar plate method was used to obtain high-titer stocks of the phage phiE142 , with sode novo assembly was conducted with Velvet , resulting in one final contig with coverage from approximately 10,000-fold. Additional manual functional annotation and genome map was performed using Geneious software.High-throughput DNA Sequencing of phage genomic DNA was performed using HiSeq 2000 technology (Illumina) to produce 100 bp paired-end reads, library construction and sequencing were performed according to the manufacturer’s instructions. In total, about 18 million pair reads of 100 bases in length were obtained with a quality filter threshold of Q30. The reads were analyzed and quality checked using FastQC and Geneious software package R8  was used to trim raw reads with a low quality score. The E value of 10−4. Predicted protein sequences were analyzed against InterProScan [Open reading frames (ORFs) were identified using Glimmer 3.02 , GeneMarrProScan , Pfam [1rProScan  and TMHMrProScan  for consrProScan  and tRNArProScan . The orirProScan . MoreoverProScan  and the rProScan . AdditiorProScan . The lifrProScan . Whole grProScan .E. coli (about 50% GC). Genome analysis of the phage revealed 194 putative open reading frames (94.4% of the genome consists of a coding region), with 26 oriented in a forward orientation and 168 in a reverse orientation, and two tRNA genes were identified. Based on BLAST results, functions were assigned to 95 of the genes; most of the annotated genes (98 genes) were hypothetical proteins, probably due to the enormous diversity of bacteriophages and the insufficient database information about the functional genes of phage. Only one gene product is hypothetical novel proteins  with coliphage vB_EcoM_PhAPEC2, which belong to the The phiE142 genome is functionally organized into four modules containing gene clusters for virion morphogenesis, DNA replication/regulation, DNA packaging, and host cell lysis. This modular organization of the genome is typical of bacteriophages.Thirty-one ORFs were found to encode proteins involved in the morphogenesis of virions. These include the ORFs 1–3, 170, 172, 175–185, and 187–194, which are proposed to be genes encoding the components of the tail fiber and baseplate. Databases homology searches suggested that ORFs encoding capsid protein are 46, 139, 142, and 174. Additionally, the proteins encoded by ORFs 185 and 186 are most similar in its amino acid sequence to neck protein.Overall, a total of 46 ORFs are associated with processing of the viral DNA. Our analysis of the phage genomes reveals several genes potentially involved in nucleotide metabolism, including ORFs 14–15, 38–39, 47, 64, 70, 96, 100–101, 125, and 171. In addition, genes that encode proteins involved in replication and transcription of its own DNA were identified in ORFs 5, 7, 12–13, 18, 20–21, 24–25, 28–29, 32, 34–35, 37, 49, 56, 59, 61, 66, 71, 73–76, 78, 81, 86, 102, 106, 130, 132, 141, 144, and 173.Two ORFs exhibit similarity to a gene involved in the host cell lysis, including endolysin and holin. The protein encoded by ORF 143 displays a high degree of identity with the endolysin. This ORF contained one glycohydrolase domain , which indicates that this protein is probably an enzyme that degrades peptidoglycan. While the putative protein of ORF 4 was identified as a holin protein. Unusually, this ORF is not located adjacent to the endolysin ORF, in most genomes bacteriophages, the holin ORF is adjacent or overlaps a ORF encoding an endolysin. The deduced holin encoded by phiE142 phage has one putative transmembrane domain, and thus resembles class III holins.The phage lifestyle prediction result of PHACTS indicated that the phiE142 is a virulent phage, consistent with the results of genomic analysis, which revealed the absence of genes associated with the establishment and maintenance of lysogenic cycle.The DNA packaging module includes ORF 60, which encode the putative portal protein. However, it was not possible to identify the terminase subunits.Myoviridae family and the Tevenvirinae subfamily. Interestingly, in silico analyses of phiE142 genome did not exhibit homology to known virulence-associated genes, genes involved in lysogeny nor to antibiotic resistance genes or potential immunoreactive allergens. These results indicate that phage phiE142 exhibits genetics properties suitable for evaluation as a biocontrol agent.Our data suggest that phiE142 is a member of T4-like virus genus of the