Peak oxygen uptake (peak Aerobic fitness refers to the body’s ability to transport oxygen from the environment and utilize it in working muscle. It is well accepted as a health-related and health-determining trait since Blair et al. demonstrated more than 25 years ago that higher aerobic fitness measured during an incremental treadmill exercise test was associated with reduced all-cause mortality in adults . ExercisIncreased attention to the health and fitness of toddlers, children, and adolescents, and to the concept of pediatric origins of adult disease, have brought exercise testing to the forefront as a clinical and research tool; in turn, leading to the imperative of standardizing methodologies. Therapeutic advances have created a growing number of childhood survivors of a wide range of conditions, including preterm birth, congenital heart disease, pulmonary hypertension, cystic fibrosis, sickle cell disease, and cancer. In addition, the same environmental and social factors contributing to the obesity epidemic in otherwise healthy children are at work in children with chronic disease and disability. High levels of physical activity are (or were) the norm in children, and a child’s inability to engage in physical activity is often one of the first indicators of childhood disease. We submit that peakMaximal exercise testing is essential for assessing peakexertion  or the l elusive ,5, rende V˙O2max . However V˙O2max . Some wo V˙O2max , and it  V˙O2max .The attainment of peakl cortex ,11—a motack box” . One must inject a cautionary note when peakut merit . In certut merit ,15. Otheut merit ,16 termsut merit  are purp1) in predicting mortality in CF patients awaiting lung transplantation [1, but also in weight gain and overall enhanced health-related quality of life as well [1 [There is a growing body of literature on the clinical significance of exercise testing and peakpatients , an obsepatients . Indeed,antation . In patiutations , showing as well . As the  of FEV1 . Informa of FEV1 .2 production—is ubiquitous in adults with various types of congenital heart disease, and was the strongest predictor of mortality in non-cyanotic patients [and lungs!There is increasing survival among children with congenital heart defects, prompting the development of specialized clinics for adults with congenital heart disease. In the past decade, reports on exercise testing in this population have proven invaluable for risk stratification . PeakV˙Onotropes ,15,25. Me volume . Thus, iPeakf Fallot , and in f Fallot . The latf Fallot ,30. Whilf Fallot ,32. The Fontan procedure has been lifesaving for many infants with previously lethal forms of congenital heart disease. Measurement of peakpatients ,34. NevePeakl Design . The firldenafil . The finestioned .The use of exercise testing to assess cardiopulmonary fitness in sickle cell anemia (SCA) has been relatively sparse compared to other conditions, despite initial reports in this population over 30 years ago ,39,40. Mnstrated ,42.Peakcontrols . This fidistance ,44. Compwith SCA ,46. Recemolecule . HoweverThere is no agreement as to what constitutes low peak Astrand , as well Astrand ,49, and  Astrand ,51,52. AThe second requirement of the establishment of clinical thresholds is to determine a specific level that adversely impacts the risk of health or disease outcome. Some health-related outcomes have well-accepted clinical cut-points . Based on an increased risk of metabolic syndrome, McMurray et al. suggested that low peakfor boys ; whereasys [V˙O2 . The proys [V˙O2 . One couThe main obstacles to using the physiologic response to exercise as an outcome in clinical trials are: (1) the lack of well-accepted and precise definitions of exercise outcome variables in children; (2) the lack of mechanisms to support the sharing of exercise-derived data among separate institutions; and (3) the lack of commonly-accepted protocols for exercise testing and physical activity assessment in the laboratory. We submit that cardiopulmonary exercise testing and measurement of peakitutions .Much of what is known about exercise physiology has been derived from studies done in adults, yet every pediatrician knows that lessons learned from research in adults do not necessarily apply to children or even to adolescents. There is a growing body of literature defining the state-of-the-art of pediatric exercise medicine, though progress has been slow. This paper advocates the advancement of exercise science in general—and exercise testing in particular—in pediatrics by providing a rationale based on demonstrated prognostic value of data so obtained. Our focus has been on peak oxygen uptake , since peakdulthood ,58,59. I


Supplemental Digital Content is available in the text The vitreous sample has been used for the diagnosis of uveitis and intraocular malignancy for decades. The sample volume is usually limited to 1 mL with current techniques. In the present study, a novel technique for higher amount of vitreous sample acquisition, that is, Binocular Indirect Ophthalmo Microscope-assistant gas-perfused pars plana vitrectomy (BAG-PPV) was invented.For diagnostic purpose, BAG-PPV with 23-ga vitrectomy system was performed on a 54-year-old Chinese male with the symptom of bilateral atypical uveitis. More than 3 mL of vitreous sample per eye was collected without any significant complications. Cytopathology was confirmed on the basis of cell surface markers and released cytokines by flow cytometry analysis and cytokine assays respectively.−, CD10−, cyKi67+, CD71+, FMC7+, CD23−, and kappa light chain single expression for the right eye and a monoclonal B-cell pattern with CD5−, CD10−, cyKi67+, and kappa light chain restriction for the left eye were identified. The cytokine assay revealed high levels of interleukin (IL)-10  and IL10/IL6 ratios for both eyes , while those for the cerebrospinal fluid were low (4.77 pg/mL for the IL10 level and 0.65 for the IL10/IL6 ratio). Based on the results, the patient was diagnosed with primary intraocular lymphoma for bilateral eyes.A monoclonal B-cell population with the pattern of CD5Our results demonstrated that diagnostic vitrectomy with BAG-PPV using the 23-ga vitrectomy system was safe, efficient, and able to provide useful diagnostic information for suspicious intraocular malignancy and other atypical uveitis. However, it is usually difficult to collect high quality of samples using standard methods due to either the small volume or the intensive dilution, which compromises the veracity of the diagnosis.The vitreous specimen obtained with pars plana vitrectomy (PPV) has been used for the diagnosis of intraocular diseases for decades.–4 Most PIOLs are primary vitreoretinal lymphomas with low incidence counting for <1% of intraocular tumors and only 1% of non-Hodgkin lymphoma, plus the highly varieties of clinical manifestations-known as masquerade syndrome, and high progressiveness, so the diagnosis of PIOL is challenging. In this study, we presented a novel method BAG-PPV, the abbreviation for the Binocular Indirect Ophthalmo Microscope (BIOM)-assistant gas-perfused pars plana vitrectomy. With this technique, enough amounts of vitreous sample can be easily collected for further cytokine profiling and flow cytometry analysis, leading to more reliable diagnosis of PIOL, which was previously masqueraded as bilateral uveitis.The most common pathological type of primary intraocular lymphoma (PIOL), one of the highly malignant subtypes of the primary central nervous system lymphoma is the aggressive large B cell-type.22.1Treponema pallidum, human immunodeficiency virus, Mycobacterium tuberculosis, Cytomegalovirus, Epstein–Barr virus, and herpes virus. Serologic markers of autoimmune diseases, including antinuclear antibodies, rheumatoid factor, and anti-double-stranded deoxyribonucleic acid antibodies, were negative. The patient also underwent an enhanced brain magnetic resonance imaging, which revealed multiple gyriform enhancements of bilateral frontal, parietal, temporal, occipital lobes, and corpus callosum, suggesting inflammatory lesions. The cerebrospinal fluid analysis demonstrated a slight increase of protein content (50.40 mg/dL). Flow cytometry analysis and cytokine assay also presented negative results. The ocular examination revealed visual acuity of light perception for both eyes. For the right eye, slit-lamp examination detected cells in the anterior chamber and the keratic precipitates. Vitreous opacity, retinal hemorrhage, yellowish-white retinal, and subretinal infiltrations were also found at the posterior region. For the left eye, the anterior chamber cells were detected, while in the posterior segment, mild vitreous opacity, diffuse nummularis retinal pigment epithelium change, and multifocal yellowish-white butyrous subretinal infiltrations above the optic disc were also found.A 54-year-old Chinese male visited our department with complaint of gradual bilateral vision loss during the past more than 1 year. He had been irregularly treated for uveitis with steroid and cyclosporine A without significant efficacy. All blood tests were negative except for the slight increase of lambda light chain and kappa light chain. Pathogenic examinations were also negative with regard to The patient showed no obvious evidence for infection or autoimmune diseases and had negative response to steroid and immunosuppressive agents, which led us to consider the potential diagnosis of masquerade syndrome. Based on these, BAG-PPV was performed for the purpose of diagnosis and therapy.The patient was fully informed of the potential risks and all possible postoperative consequences before the surgery. The written informed consents were obtained from the patient's family member after the discussion of the procedure. This study was consistent with the Declaration of Helsinki and was approved by the ethics committee of Zhejiang Provincial People's Hospital before applying this surgical procedure clinically.2.2Before surgery, pupils of the patient were sufficiently dilated with tropicamide phenylephrine eye drops . The vitrectomies were performed under retrobulbar anesthesia with injections of 2% lidocaine  and 0.75% bupivacaine . BAG-PPV was performed using 3 suture-less 23-ga sclerotomies in the inferotemporal, superotemporal, and superonasal quadrants 3.5 mm from the limbus. The infusion cannula was inserted into the inferotemporal cannula perfused by aseptic air. The suction pipe of the vitrectomy cutter was connected to a 5-mL aseptic syringe. The noncontact wide-angle viewing system  was used for visualization. First, the core vitrectomy was performed with vacuum aspiration of 280-mm Hg, cutting rate of 6000/min, and 35-mm Hg air pressure , with the assistant pumping the syringe at the same time. The peripheral vitrectomy was required for additional sample only if the volume was not enough. It was noteworthy that the surgeon and the assistant should cut and pump cooperatively so as to maintain suitable intraocular pressure and effective specimen collection Fig. . The und3The BAG-PPV was performed for the bilateral eyes of the patient by the same surgeon (M W) in sequence. Eventually, 3.5 mL  and 3 mL  of vitreous samples were collected. The patient then received intraocular injection of methotrexate. The methotrexate (400 μg) was injected to the affected eyes twice a week during the initial 4 weeks and once a week for the subsequent 8 weeks, and then adjusted to monthly for 9 months.3.1Flow cytometry analysis detected over 3400 cells in sample A. The assay defined an abnormal monoclonal B-cell population with the patterns as positive for CD45, CD19, CD20, CD22, CD71, HLA-DR, FMC7, sIgM, and cyKi67, and negative for CD10, CD5, CD7, CD34, CD117, CD33, CD13, CD34, CD103, CD11c, CD25, CD23, CD27, CD28, CD138, CD30, CD123, CD38, with kappa light chain single expression -10 in both eyes . The fundus of pre- and postoperation were shown Fig. . The vis3.5Up to date, the follow-up period for the patient was already 5 weeks. The patient received bilateral intraocular methotrexate injection at a dose of 400 μg twice a week for the first 4 weeks and once for 1 week. Compared to the previous conditions  and 1.25% (the left eye) of the total cells. These cells had positive staining for CD45, CD19, CD20, CD22, and CyKi67, with kappa light chain single expression or restriction, while CD5 and CD10 were negative. Cytokine assay of these specimens revealed dramatically elevated IL10/IL6 ratios, much higher than 1.0, which led to the definite diagnosis of bilateral PIOL.PIOL has been reported to have a very low rate of occurrence with high malignancy, All the conditions described above might be the reasons leading to the negative result for the cytological examination of the left eye.Unfortunately, we were not thoughtful enough to take the vitreous sample from the right eye for cytological examination. The left eye was at a symptomatic relief period for weeks and then the infiltration above the optic disc increased. Considering the results of flow cytometry analysis from the right eye, we performed the vitrectomy and the cytological examination for the left eye, which turned out to be false-negative partially due to the poor cell viability and small cell quantity in the sample. In addition, the left eye was relatively stable without a solid tumor; vitreous opacity was mild while the vitreous body contained a small amount of lymphocytes and other cells. Of note, the sensitivity of the cytological test is lower than flow cytometry analysis and cytokine assay.In this study, we only demonstrated BAG-PPV work efficiently on the bilateral eyes from 1 patient since the occurrence rate of PIOL is extremely low. Although BAG-PPV with 23-ga vitrectomy system might be a better alternative for current vitreous sample collection in cases of intraocular malignancy, atypical uveitis either infectious or inflammatory, and suspected endophthalmitis, more cases should be collected for further investigations on the safety in a longer postoperative period, which will be beneficial to the accurate diagnosis of intraocular malignancies in the long run.We thank Wanmao Ni, Qi Zhang for assisting in preparation of this manuscript.


Saccharomyces cerevisiae capable of secreting killer toxins, much information has been gained regarding killer toxins and this fact has substantially contributed knowledge on fundamental aspects of cell biology and yeast genetics. The killer phenomenon has been studied in Pichia membranifaciens for several years, during which two toxins have been described. PMKT and PMKT2 are proteins of low molecular mass that bind to primary receptors located in the cell wall structure of sensitive yeast cells, linear (1→6)-β-d-glucans and mannoproteins for PMKT and PMKT2, respectively. Cwp2p also acts as a secondary receptor for PMKT. Killing of sensitive cells by PMKT is characterized by ionic movements across plasma membrane and an acidification of the intracellular pH triggering an activation of the High Osmolarity Glycerol (HOG) pathway. On the contrary, our investigations showed a mechanism of killing in which cells are arrested at an early S-phase by high concentrations of PMKT2. However, we concluded that induced mortality at low PMKT2 doses and also PMKT is indeed of an apoptotic nature. Killer yeasts and their toxins have found potential applications in several fields: in food and beverage production, as biocontrol agents, in yeast bio-typing, and as novel antimycotic agents. Accordingly, several applications have been found for P. membranifaciens killer toxins, ranging from pre- and post-harvest biocontrol of plant pathogens to applications during wine fermentation and ageing .The killer phenomenon is defined as the ability of some yeast to secrete toxins that are lethal to other sensitive yeasts and filamentous fungi. Since the discovery of strains of  Saccharomyces cerevisiae  synthase), join in a step that follows recognition of the primary receptor (chitin) of the toxin but precedes the role of the multi-subunit Elongator, involved in histone acetylation and transcription, the most probable toxin target [In addition, a few receptors for other toxins have been described. The H/KDEL receptor Erd2p mediates K28 toxin binding and uptake in yeast spheroplasts, a viral A/B toxin bearing an HDEL motif at the β-subunit. In vivo toxicity to K28 depends on the existence of Erd2p . In the n target .Pichia toxins, only the membrane receptor for the PMKT toxin has been described. Some unidentified receptors have been hypothesized for other Pichia toxins, in an attempt to explain the binding capability and sensitivity of toxin-treated spheroplasts [d-glucans) and cell wall-localized secondary receptors which are also plasma membrane-associated. PMKT utilizes as the secondary membrane receptor a GPI-anchored protein of the cell wall [cwp2Δ protoplasts are resistant and also unable to bind PMKT, demonstrating once again that Cwp2p is necessary for PMKT toxicity. In addition, it should be indicated that, in its definitive state, Cwp2p is bind to (1→6)-β-d-glucans and this is the most probable way by which PMKT interact closely with Cwp2p, the plasma membrane receptor located in the cell wall. It has been also demonstrated, using a collection of deletion mutants related to GPI (Glycosyl Phosphatidyl Inositol) anchor biosynthesis, that Cwp2p is present in both cellular locations the plasma membrane and cell wall, as usually occur for most GPI-proteins of the yeast cell wall [O-glycosylated and thus, probably bears several phosphodiester groups. At acidic pH values , PMKT bears positive charges and so on could be strongly bound to the negative charges present on Cwp2p [Speaking specifically about roplasts ,116. PMKell wall . This prell wall ,98. The ell wall ,118 and ell wall . The N-ton Cwp2p . Overallon Cwp2p . Until nPichia farinosa KK1 shows its maximum killer activity in the presence of 2.0 M NaCl [Candida nodaensis, presents a strong salt-dependent phenotype [Mrakia frigida 2E00797 and Williopsis saturnus WC91-2 killer toxins [Many targets to the killer toxin action in sensitive cells have been identified and, in consequence, some molecular mechanisms of killing action have been also determined. The first study for determining some aspects of the killing action of PMKT was intended to assess the importance of the addition of salt  in the expression of the killer phenotype . In prel0 M NaCl . Furtherhenotype  and the r toxins ,120. Howd-glucans in the first 2–3 min after toxin addition, and then a lag phase of approximately one hour is necessary to observe reductions in cell viability, probably reflecting the time necessary for the development of the mechanism of action, gradual intoxication and death. The lag phase observed after cell wall binding and previously to cell death, is supposed to involve several sequential interactions and effects: interaction with secondary receptors, ion channel formation in the plasma membrane, alteration of transmembrane gradients and deenergization of cells and eventually, formation of large unspecific plasma membrane pores that permit the passage of other intracellular molecules [+ out of the cells starts (1–1.5 h after exposure to PMKT) and simultaneously, a flow of Na+ began. In the same way as K+, the intake of Na+ is directly related with killing [+ and K+ [PMKT finds its greatest similarity in K1, which share certain biochemical characteristics such as similar isoelectric point and molecular mass. In a similar way to the mode of action in which K1 acts from outside the cell after reaching the cytoplasmic membrane, PMKT exerts its lethal effect by ion channel formation and disruption of cytoplasmic membrane gradients ,122,123.olecules . During  killing . The dis+ and K+ .+ > Na+ > Li+ > Ca2+ > Cl−. Finally, changes in plasma membrane integrity (determined as permeability to propidium iodide) are observed when cellular death was advanced in time, being probably a late consequence of PMKT treatment [In addition, PMKT causes in liposomes a conductance when added at low pH, showing a characteristic channel activity. Furthermore, all the common physiological ions are observed to flow through the pores caused by PMKT in the selectivity sequence: Kreatment .S. cerevisiae to PMKT was explored, 146 genes showed an altered expression in response to PMKT.Yeasts response to harsh physical chemical factors  by activating a wide variety of defenses that include general or specific regulatory pathways, activating multiple MAPK cascades, which transform stimuli into appropriate physiological responses ,127,128.CTT1, HSP12, GPD1, GPP2, TRK2, PDR12, ENA1, SCH9, HAL9, YAP1, XBP1, and STL1  and untreated cells were selected for mRNA isolation and determination of the transcriptional response. The most important functional group with 31 up-regulated genes is the group related to signal transduction, gene expression, transcription and RNA processing, followed by the group of genes related to ionic homeostasis, transport facilitation, carbohydrate metabolism, and cell stress and rescue. Both, the phosphatases and dehydrogenases implicated in the glycerol production pathway are also up-regulated in response to PMKT. In addition, many similarities are found when the group of up-regulated genes in response to the toxin  and thosand STL1 . Further of time ,73.S. cerevisiae, it is known that the response to rapid changes in the osmolarity relies in the activation of the HOG (High Osmolarity Glycerol) pathway, which is manifested by the phosphorylation of Hog1p. Hog1p encodes for the MAPK that is activated by phosphorylation and then translocated to the nucleus to counteract with several environmental stimuli [HOG1Δ mutants are hypersensitive to such environmental stresses, which cause Hog1p activation [HOG1Δ mutants are found to be hypersensitive to PMKT.In  stimuli . HOG1Δ mtivation ,131. Sevtivation , so the tivation . Hog1p ptivation . Hog1p pGPD1 and GPP2, involved in glycerol production, were augmented in the presence of PMKT, agreeing with the glycerol increment observed in response to PMKT, clearly revealing compatible osmolyte accumulation to counteract the toxin  of PMKT and PMKT2 are observed to induce the aforementioned effects on sensitive yeast cells. By contrast, low doses (200 AU/mL) of the toxins lead to a cell death process in S. cerevisiae accompanied by cytological and biochemical indicators of apoptotic cell death . The observed cell death process is characterized by the presence of an initial apoptotic state that rapidly evolves to a necrotic state. The rate at which this process occurs is proportional to the amount of killer toxin used in the study. Low PMKT concentrations (up to 200 AU/mL) lead to a higher number of apoptotic cells, whereas higher concentrations (from 300 to 3000 AU/mL) cause both apoptosis and necrosis. Necrotic cells are usually characterized by a permeabilized plasma membrane , whereas the plasma membrane that have initiated a PCD phenotype is usually intact. As a result of these investigations, it can be determined that cells appear to start the process of death with a PCD response, die as a consequence of apoptosis, and then, as a late consequence, become necrotic [c release, confirming the hypothesis of PCD induction.It has been described how the killing of necrotic . In thesCWP2Δ mutants are resistant to pro-apoptotic, as well as for pro-ionophoric amounts of PMKT, also in agreement with the observations obtained for Kre1p, the plasma membrane secondary receptor involved in the K1 mechanism of action [It has been also demonstrated that the secondary receptors in the sensitive cells have the same function in both experimental conditions. f action ,135, indf action ,135.DCS2, FLR1, GSH1, APH1, VPS13, TSA2, GLR1, ECM4, AAD14 and GTO3, most of which are observed to be induced by different oxidative compounds. Several genes encoding for ROS detoxifying enzymes, such as CTT1, PRX1, TSA1, and SOD1, are also observed to be up-regulated (activations comprised between +16.1 and +11.3), indicating an oxidative stress that can be observed by analyzing the ROS production with dihydroethidium in the presence of PMKT  and phytopatogenic fungi , as well as other killer yeasts [Although killer toxins were originally described and studied for their ecological function in the early 1960s ,136, ther yeasts ,104.W. mrakii var. mrakii HM-1, W. saturnus toxin, P. anomala PaKT and Z. bailii zygocin) have shown activity against pathogenic yeasts, such as Candida albicans, Cryptococcus neoformans, etc., they have been suggested to be potentially useful as potential antifungal agents for infections caused by fungi, their direct application is of limited real importance because of their instability at physiological temperatures and pH values and their antigenicity [Williopsis toxins) could be therapeutically used, but restricted to topical applications such as on superficial skin infections [P. membranifaciens and its killer toxins to control human or animal infections and other clinical diseases is still uncertain.To a large extent, the potential applications that have been proposed for yeast killer toxins have been conditioned by the aforementioned recognized lability of such toxins. The proposed applications are largely restricted to those that provide physical chemical conditions compatible with the stability of killer toxins, that is, those that can be performed at relatively low pH and low temperature values . Althouggenicity ,150,151.P. membranifaciens killer toxins seems to be not really useful at clinical stages, their use for biocontrol purposes has a great interest in agro-food industry. The applications of the biocide activity of P. membranifaciens were initially unveiled by Masih et al. (2001), observing that the mycelium of the B. cinerea treated with P. membranifaciens failed to develop the characteristic grey mould symptoms when it was re-inoculated onto grapevine [P. membranifaciens as a biocontrol agent against B. cinerea, since it was also proved effective in postharvest pears [Since the use of rapevine . This derapevine . Howeverrapevine ,154. Thest pears .B. cinerea spoilage have also been reported. For example, the production of exo-glucanase toxins in W. anomalus was reported by Parafati et al. (2016) [B. cinerea found in this species and in others such as Candida intermedia, Sporidiobolus pararoseus, Starmerella bacilaris and S. cerevisiae [The existence of similar and additional antagonistic mechanisms in other yeasts and their use to control . (2016) , but therevisiae ,161,162.P. membranifaciens has also been used as a biological tool for the control of other crop-fruit-associated fungus pathogens such as Penicillium expansum in postharvest peaches (by rapid colonization and competition for space and nutrients in fruit wounds) [Colletotrichum acutatum causing anthracnose rot in postharvest loquat fruit  [Penicillium roqueforti in storage wheat grain (with no conclusions about the mechanism of action) [Penicillium italicum, Penicillium digitatum and Colletotrichum gloeosporioides causing blue and green mold and anthracnose in citrus fruit (with no conclusions about the mechanism of action) [In a similar context,  wounds)  and in p wounds) ; Colletoucanase) ; Penicil action) ; and Pen action) .P. membranifaciens towards the wine spoilage yeast B. bruxellensis was firstly observed by Yap et al. (2000) [P. membranifaciens CYC 1086 and its killer toxin PMKT2 against B. bruxellensis in simulated winemaking conditions [P. membranifaciens CYC 1086 and PMKT2, killer toxins from Ustilago maydis [Torulaspora delbrueckii (TdKT) [Kluyveromyces wickerhamii (Kwkt), Wickerhamomyces anomalus (Pikt) [Candida pyralidae (CpKT1) [Metschnikowia pulcherrima  [Brettanomyces spoilage. Continuing in the enology context, results from Alonso et al. (2015) and Labbani et al. (2015) conclude that a synergistic effect was operating using PMKT [P. kluyveri) [Zygosaccharomyces or Dekkera/Brettanomyces species. Recently, Velazquez and coworkers described the utility of mixed-inoculation with killer and sensitive yeast strains to speed cell death during second fermentation of sparkling-wines to release of certain compounds from autolyzed yeast cells [The activity of . 2000) , and thi00 , and i (TdKT) , Kluyvers (Pikt)  and Cand (CpKT1)  and the  medium)  have beeing PMKT  and Pkkpst cells .P. membranifaciens shows a broad spectrum of potential applications, sustained by a considerable diversity of mechanisms of action. However, a marked lability of these killer toxins restricts their application to specific environments and matrixes providing adequate chemical physical conditions. These requirements are basically found in food, post-harvest, and fermentative environments, even though the industrial application of killer toxins is far from a reality, since their large-scale production is still expensive. For this reason, the current tendency is only based in applying killer yeasts instead of their toxins. Furthermore, killer resistant strains have been described since the initial studies on killer toxins and, in many surveys, it is possible to found, over a panel of potential sensitive yeast isolates, that some strains are resistant to that potential biocontrol agents [In summary, the killer activity of l agents . In accoP. membranifaciens killer strains to obtain toxins with increased stability at human physiological conditions. Additionally, killer toxins identified as leads for therapeutic uses should also be modulated to reduce their antigenicity [P. membranifaciens killer toxins should be studied in depth, in order to identify key residues causing toxicity and antigenicity, and determine their stability. Finally, the cellular response of the target pathogenic/spoilage microorganism to killer toxins needs to be studied species by species to understand the basis of their susceptibility.For those purposes, regarding toxin stability in both production and application stages, adaptive and directed evolution approaches are of great interest in the development and isolation of phenotypically improved variants. It is possible to evolve populations in laboratory conditions, where it is important that the selection conditions match the industrial parameters as closely as possible . Since tgenicity . For thaP. membranifaciens killer toxins, in the hope of understanding how to work with these killer toxins that have potential biotechnological applications. Elucidation of the molecular mechanisms of their action will be helpful to develop the strategies to fight more and more harmful spoilage yeasts.Additional studies are currently under development to increase the background of knowledge accumulated during the last two decades on the features of


Although imaging of large amounts of accumulated Mn(II) is possible by MRI, quantitative measurement of the biodistribution of manganese, particularly at the trace level, can be challenging. In this study, we produced the positron-emitting radionuclide 52Mn (t1/2 = 5.6 d) by proton bombardment (Ep<15 MeV) of chromium metal, followed by solid-phase isolation by cation-exchange chromatography. An aqueous solution of [52Mn]MnCl2 was nebulized into a closed chamber with openings through which mice inhaled the aerosol, and a separate cohort of mice received intravenous (IV) injections of [52Mn]MnCl2. Ex vivo biodistribution was performed at 1 h and 1 d post-injection/inhalation (p.i.). In both trials, we observed uptake in lungs and thyroid at 1 d p.i. Manganese is known to cross the blood-brain barrier, as confirmed in our studies following IV injection  and following inhalation of aerosol, . Uptake in salivary gland and pancreas were observed at 1 d p.i. (0.5 and 0.8%ID/g), but to a much greater degree from IV injection (6.8 and 10%ID/g). In a separate study, mice received IV injection of an imaging dose of [52Mn]MnCl2, followed by in vivo imaging by positron emission tomography (PET) and ex vivo biodistribution. The results from this study supported many of the results from the biodistribution-only studies. In this work, we have confirmed results in the literature and contributed new results for the biodistribution of inhaled radiomanganese for several organs. Our results could serve as supporting information for environmental and occupational regulations, for designing PET studies utilizing 52Mn, and/or for predicting the biodistribution of manganese-based MR contrast agents.Manganese is essential to life, and humans typically absorb sufficient quantities of this element from a normal healthy diet; however, chronic, elevated ingestion or inhalation of manganese can be neurotoxic, potentially leading to  These images demonstrate a distribution of PET signal in many of the same tissues with high uptake in the current ex vivo biodistribution results, and these images suggest the potential utility of 52Mn for further quantitative PET/CT studies.Comparing the biodistribution of 52Mn in mice, the concentration of 52Mn in most tissues at both 1 h and 1 d timepoints is higher than in the inhalation results. The activity in the gastrointestinal tract was much greater in the inhalation results, suggesting that some of the dose may have been swallowed directly from the aerosol or inhaled, cleared by mucociliary clearance, and then swallowed. Also in our studies, we observed uptake in the pancreas and brain, while 52Mn that entered the stomach or intestines did not appear to be retained, although the one hour amounts were much higher in the inhalation groups likely due to putative swallowing of some of the dose. Retention of 52Mn in the brain, thyroid, and thymus was observed in results from both injection and inhalation. We also found significant uptake of 52Mn at 1 d in the bone (2.6%ID/g) resulting from injection, but not from inhalation.For the comparison of IV injection to inhalation of 52Mn was cleared rapidly from the blood, and the injection data showed 52Mn in the liver and kidney in <1 h. The inhalation data also suggest accumulation in the liver and kidneys, but this accumulation took much longer than one hour. Although manganese appears to accumulate in both liver and kidneys, results from the literature [54Mn]MnCl2 was retained in the lungs to large extent, but was also found in significant amounts in the intestines, liver, kidneys, and in small amounts in the brain. These intratracheal instillation studies also showed that Mn can enter the bloodstream from the lungs, possibly by ion channels, but not by the divalent metal transporter-1 (DMT-1) in that particular tissue MnCl2 [54Mn]MnHPO4 MnCl2  or [54Mnn]MnHPO4  solutionn]MnHPO4  exposed 2, in guinea pigs [52Mn]MnCl2 exhibited higher and faster uptake in salivary gland than in the inhalation study. Our results demonstrate uptake of 52Mn in the brain, thyroid, and pancreas in all trials. Interestingly, uptake from IV administration was higher in brain and pancreas, but lower in thyroid, compared to inhalation results. Importantly, Graves, et al. [β-cells that reduces opening of voltage-dependent calcium channels (VDCCs)—the channels through which Ca(II), Mn(II), and potentially other divalent cations can enter the pancreatic beta cells. In one part of this work, 52Mn(II) in aqueous sodium acetate was administered IV with and without anesthesia by isoflurane, and the uptake in the pancreas at 1 h p.i. under anesthesia was similar to our result (in %ID/g). However, the uptake in the pancreas without anesthesia was ~3x greater than with anesthesia. Since our study only used anesthesia during IV administration of 52Mn(II), and not during inhalation, we expect that in the absence of anesthesia in both routes of administration the difference would be even greater in uptake in the pancreas between IV injection and inhalation.In our studies, relatively high uptake was observed in the lung and thyroid following either route of administration. Although several papers confirmed thyroid uptake of manganese from different administration routes in animal models –83, the nea pigs , manganenea pigs . Interes, et al.  have sho52Mn(II) in radiotracer concentrations. In addition to monitoring brain uptake of 52Mn(II), this study included results for biodistribution from several tissues at 4 and 48 h p.i. Compared to Brunnquell, et al., our biodistribution results were roughly 10x greater for many tissues (in %ID/g), likely due to a roughly 10x lower body mass of the mice in our study compared to rats, along with different timepoints and gender. However, the relative changes from 4 to 48 h p.i. in this study was similar to our results at 1 h and 1 d, including brain, spleen, liver, intestine, heart, lung, and muscle, while results for blood, pancreas, thymus, kidney, and bone demonstrated results that were less similar to our results, based on tissue concentrations relative to other tissues and/or relative changes from 4 to 48 h. Manganese-52 might someday be used as a PET agent for niche clinical research applications, but our biodistribution results in mice can be used, in conjunction with other published results, to inform regulations for safe levels of environmental and occupational exposure to manganese. The primary objective of this work was to contribute to basic research with regards to the in vivo behavior of manganese cations, which are increasingly being studied preclinically for PET agents and MRI contrast agents—with only the possibility for future translation to the clinic.Along with recently renewed interest in manganese for signal in both MRI and PET, Brunnquell, et al.  have pub52Mn via the natCr reaction, chemically isolated this radioisotope of manganese from the chromium metal target material, and reported new data for the biodistribution of manganese in mice following administration by IV injection or inhalation. Manganese-52 was produced by cyclotron bombardment with low-energy protons on chromium metal, and subsequent chemical separation of 52Mn(II) was performed by cation-exchange chromatography and re-dissolved in saline solution for ex vivo biodistribution studies in mice. The doses were administered by IV injection or inhalation, always resulting in uptake of 52Mn in the brain, thyroid, and pancreas. Interestingly, uptake from IV administration was higher in brain and pancreas, but lower in thyroid, compared to inhalation results. While 52Mn might find use as a PET agent in preclinical studies, our biodistribution results in mice may be used to inform regulations for safe levels of environmental and occupational exposure to Mn(II) and the predicted biodistribution of Mn(II) following exposure by IV injection or inhalation.In this work, we have produced “This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or limited, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.”S1 Fig52Mn was observed in the digestive tract, kidneys, and likely the pancreas. At 3 d p.i., 52Mn had cleared from the digestive tract, while it is retained in the kidneys, as well as liver, pancreas, and thyroid gland. Manganese-52 was eluted from the cation-exchange column in 0.067 M ammonium oxalate solution. The ammonium oxalate product was heated to dryness, and then the heat was increased to burn away the ammonium oxalate. Manganese-52 was resuspended by adding water followed by a drop of 6 M hydrochloric acid, then heated to dryness. Evaporation and resuspension in water was repeated and then the 52Mn was finally resuspended in water, resulting in a solution with pH of ~6.5. Mice  were anesthetized by isoflurane (1–2% induction), injected in the tail vein with ~41 μCi of 52Mn in 50 μL total volume, and imaged simultaneously side-by-side at 1 h and 3 d p.i. At the imaging timepoints, anatomic images were obtained by non-contrast CT using an Inveon small animal PET/CT scanner , and PET data were acquired using either the Inveon PET/CT scanner or a microPET Focus 220 scanner . Static PET data were acquired for 30 minutes at the 1-hour timepoint and for 1 hour at the 3-day timepoint. Attenuation maps for each subject were obtained from either the CT scan in the Inveon scanner or by a transmission scan on the Focus 220 scanner. Images were analyzed using Inveon Research Workplace software .At 1 h p.i., (TIF)Click here for additional data file.S1 FileMicrosoft Excel for Mac and contains multiple sheets.This file is a workbook made using (XLSX)Click here for additional data file.S2 FileMicrosoft Excel for Mac and contains multiple sheets.This file is a workbook made using (XLSX)Click here for additional data file.S3 FileMicrosoft Excel for Mac and contains multiple sheets. The workbook includes results from t-test with Welch’s correction at 1 d p.i. only.This file is a workbook made using (XLSX)Click here for additional data file.


Hypovitaminosis D has been identified as a possible new cardiovascular risk factor. However, the results of studies correlating serum vitamin D levels with markers of subclinical atherosclerosis have been conflicting. The aim of this study was to correlate serum levels of 25-hydroxyvitamin D [25(OH)D] with carotid intima-media thickness (C-IMT) and conventional cardiovascular risk factors in Afro-descendants. A cross-sectional analysis was performed on a sample of 382 individuals from a cohort of descendants of African slaves, inhabitants of “Quilombola” communities, with a mean age of 57.79 ±15.3 years, 54.5% of whom were women. Socio-demographic and clinical data were collected and biochemical tests were performed, including serum levels of 25(OH)D by electrochemiluminescence and urinary albumin excretion, evaluated by the albumin/creatinine ratio (ACR) in a spot urine sample. All participants underwent high-resolution ultrasonography for C-IMT measurement. Hypovitaminosis D was defined as serum 25(OH)D levels <30 ng/mL. The mean serum 25(OH)D levels were 50.4±13.5 ng/mL, with a low prevalence of hypovitaminosis D (4.86%). By simple linear correlation, a significant inverse association between 25(OH)D levels and C-IMT  was observed. However, after multiple linear regression analysis, the significance of the association between serum levels of 25(OH)D and C-IMT measurement was lost  and only male gender, age, smoking, systolic blood pressure, glucose and low density lipoprotein (LDL)-cholesterol remained significantly associated with C-IMT. Levels of 25(OH)D were independently and positively associated with HDL-cholesterol and inversely associated with age and ACR. In conclusion, no independent association between 25(OH)D levels and C-IMT was observed in this population. On the other hand, there was an inverse association with albuminuria, a marker of endothelial lesion. Cardiovascular disease (CVD) is the leading cause of death and disability worldwide . AlthougVitamin D, in fact a steroid hormone, has as its primary function the regulation of calcium and phosphorus homeostasis in interaction with the parathyroid glands, kidneys and intestines. Under normal conditions, only about 10% of the vitamin requirement is obtained by food intake, the rest of which is synthesized in the body, starting with the activation of a cutaneous precursor by solar ultraviolet radiation and involving successive hydroxylation processes at the hepatic and renal levels ,5.The role of vitamin D in the regulation of bone metabolism is already well established. Thus, diseases such as rickets and osteomalacia have been classically attributed to hypovitaminosis D . HoweverThe mechanisms by which vitamin D levels may influence cardiovascular risk are still not fully understood and their participation in atherogenesis has been postulated. However, studies attempting to correlate hypovitaminosis D with early signs of atherosclerosis in different populations have reported conflicting findings , and no Thus, in the present study, we sought to correlate serum levels of 25-hydroxyvitamin D [25(OH)D], the stable circulating form of the vitamin, with a marker of subclinical atherosclerosis, the measure of carotid intima-media thickness (C-IMT), and conventional risk factors in Afro-descendant individuals, inhabitants of Quilombola communities in the western coast of the State of Maranhão, Brazil.This cross-sectional study evaluated individuals aged ≥18 years, inhabitants of Quilombola communities in the municipality of Alcântara, west coast of the State of Maranhão, Brazil, that were participants in a prevalence study of chronic kidney disease, the PREVRENAL study , diabetes mellitus (DM), albuminuria and/or reduced estimated glomerular filtration rate (GFR) were referred to a University Hospital for specialized exams and considered eligible for the present study. Thus, out of 416 participants who fulfilled this initial inclusion criterion, 25 were excluded because they presented an overt cardiovascular disease and nine did not attend to perform the complementary exams, so that, at the end, 382 individuals  constituted the sample object of the present research.2 (kg/m2). The waist circumference was measured at the midpoint between the last rib and the iliac crest with the patient in a standing position.Standardized questionnaires were used in the initial evaluation to record information. Data on alcohol consumption (yes/no) and smoking (yes/no) were obtained. Blood pressure was defined as the mean of the last two of three measurements, performed at intervals ≥2 min, using a mercury column sphygmomanometer after sitting for at least 5 min. SAH was defined as the finding of any of the following four criteria: systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg, previous diagnosis by a physician, or use of antihypertensive medication. The diagnosis of DM was performed according to the following criteria: use of hypoglycemic agents, fasting glycemia ≥ 126 mg/dL or an oral glucose tolerance test ≥200 mg/dL. The body mass index (BMI) was calculated using the formula: BMI=weight/height-1/(1.73 m2), based on the definition of chronic kidney disease (CKD) from the National Kidney Foundation  formula, considered low if <60 mL·minundation .The 25(OH)D dosage was performed by means of the electrochemiluminescence fixation assay . A serum level ≥30 ng/mL was considered normal. Values <30 ng/mL were grouped as insufficient 20–29 ng/mL) and deficient (<20 ng/mL) , and reported as mg/g.The carotid artery ultrasound examination was performed with a 7.5 MHz linear transducer in longitudinal section in B mode, by a single experienced examiner, blind to the participants' clinical and laboratory information, in a GE model Vivid 3 device. The C-IMT was measured at the distal wall (farthest from the transducer) of the common carotid artery, 10 mm proximally to its bifurcation, on both sides, according to current recommendations . The meat-test. To analyze the degree of linear correlation between continuous variables, Pearson's correlation coefficient was calculated. Associations presenting a P<0.20 at the bivariate analysis were submitted to multiple linear regression analysis. In the multiple linear regression model, gender  and smoking (absent=0 and present=1) were included as dummy variables. The dichotomization of 25(OH)D serum levels according to the 50th percentile was chosen because of the very low prevalence of hypovitaminosis D, according to the conventional cut-off point. Data were analyzed using SPSS 18.0 for Windows , adopting P<0.05 (5%) as significant.The Kolmogorov Smirnov test was used to evaluate the normality of the continuous variables. Qualitative variables are reported as relative frequency (%) and continuous variables as mean and standard deviation (SD). Thechi-square test was used to compare proportions. For comparison of means, we used the Student’s This research was carried out in accordance with the Brazilian National Health Council Resolution 196/96, active throughout the national territory. The inclusion of the participants was done after signing a Term of Free and Informed Consent that included the information on data confidentiality and its exclusive use in this research. The PREVRENAL project was approved by the Ethics Committee of the university institution involved in the study.The analyzed sample consisted of 382 individuals, with a mean age of 57.79±15.39 years, being almost half with age ≥60 years, with a slight predominance of females. As expected, almost 90% of participants were non-white, and most had low income and low level of schooling. Farmers and fishermen made up half the sample. The expressive prevalence of SAH, DM and albuminuria are justified by the inclusion criteria of study participants. The mean serum levels of 25(OH)D were within the range considered healthy (≥30 ng/mL), with a low prevalence of hypovitaminosis D (4.86%). Slightly more than half of the participants had increased C-IMT and almost half had carotid atheromatous plaques. These and other characteristics of the study population are presented in After multiple linear regression analysis, lower levels of 25(OH)D remained significantly associated with the female gender. In addition, the variables creatinine and HDL-cholesterol were positively associated with 25(OH)D levels, and age, BMI and ACR were inversely associated .As seen in The present study analyzed adult individuals of both genders, Afro-descendants, with cardiovascular risk factors, inhabitants of Quilombola remnant communities, and found no evidence of an independent association between serum levels of 25(OH)D and C-IMT, as well as the presence of carotid atheromatous plaques. On the other hand, an independent inverse association between 25(OH)D levels and urinary albumin excretion, assessed by ACR in an isolated urine sample, was observed.A low prevalence of hypovitaminosis D (<5%), with only 3 cases of deficiency (0.81%), was observed in this population, with a mean serum level of 25(OH)D of 50 ng/mL. This finding differs substantially from what has been described in most studies from around the world, including tropical countries, which show that it affects in general more than half of the individuals . Our finC-IMT is a marker of subclinical atherosclerosis widely validated as an independent predictor of cardiovascular events . The invThe independent inverse association between 25(OH)D levels and the urinary albumin excretion observed in this study should be highlighted. Albuminuria is considered a sensitive marker of endothelial lesion and an independent predictor of cardiovascular events ,25. ThisThe participation of vitamin D deficiency in the pathogenesis of atherosclerosis has been reported in numerous studies, based on the finding that low levels of 25(OH)D correlate with a higher incidence of deaths and cardiovascular events, particularly of atherosclerotic nature ,6,7,30. In addition, there is biological plausibility to justify the antiatherosclerotic role of hypovitaminosis D, which has been explained by several possible mechanisms . The actThus, studies attempting to correlate hypovitaminosis D with subclinical atherosclerosis markers have attracted considerable interest from several researchers in recent years, with variable results. Vitamin D deficiency has been associated with endothelial dysfunction ,10 and hIn vitro studies have shown that the incubation of adipocyte culture with calcitriol promoted an increased expression and activity of lipoprotein lipase, an enzyme related to the reduction of triglyceride levels and increase in HDL-cholesterol (The positive association between 25(OH)D and HDL-cholesterol levels, which is classically linked to lower cardiovascular risk, observed in this study and also by others ,29,33,34The results of the present study, therefore, do not support the hypothesis of the antiatherosclerotic, vasculoprotective role of vitamin D, since the inverse association between levels of 25(OH)D and C-IMT did not persist in the multivariate analysis. However, our study may have had limited statistical power because of the very low prevalence of hypovitaminosis D. With a variation of 25(OH)D levels almost entirely within the normal range, it would be unlikely to find a relevant role of the vitamin in the pathogenesis of atherosclerosis in these individuals. On the other hand, its inverse association with urinary excretion of albumin should be valued and may indicate a role for lower levels of vitamin D, even though currently classified as "normal", at an earlier stage of atherosclerosis in this population. Therefore, based on our findings, the association of hypovitaminosis D with the pathogenesis of atherosclerosis in this population should not be totally discarded.The inconsistency of results in the literature on the association between vitamin D deficiency and early markers of atherosclerosis, coupled with the divergence between observational and intervention studies, have stimulated an intense debate on the causality of hypovitaminosis D in CVD in the last decade ,31,35. MThe present study has some limitations. Its cross-sectional design does not allow establishing a causal relationship in the associations found, as well as the selection of a sample from a specific population does not allow the extrapolation of our results to other populations. In addition, the limited statistical power of the study, due to its relatively small sample size and the low proportion of individuals with hypovitaminosis D observed, should be considered. Moreover, the reproducibility of the C-IMT measurements, performed by a single experienced examiner, was not tested. On the other hand, some strengths should be highlighted. It is a study approaching a population not previously studied, that correlated serum levels of 25(OH)D not only with C-IMT, the main target of this article, but also with albuminuria, in addition to several traditional cardiovascular risk factors/markers. Finally, this seems to be the first Latin American study of this nature.In conclusion, no independent association between serum levels of 25(OH)D and C-IMT was observed in this population. On the other hand, our data suggest an inverse association between C-IMT and albuminuria.


Coronary heart disease (CHD) and abnormal glucose and lipid metabolism are closely associated and generally coexist. The Qi and Yin deficiency syndrome is a common disease pattern encountered in traditional Chinese medicine. We designed a protocol to determine the effectiveness and safety of Zhenyuan capsules for CHD with abnormal glucose and lipid metabolism. This multicenter, randomized, double-blind, parallel-controlled trial was designed in accordance with the CONSORT. We will recruit 200 eligible male patients aged 45–75 years from three participating centers and randomly assign them to treatment and control groups (1 : 1). The primary indicators are glycosylated hemoglobin, fasting blood glucose, 2-hour postprandial blood glucose, and triglyceride levels. The secondary indicators are the Seattle Angina Questionnaire, TCM symptom indicators, ultrasonic cardiography finding, coagulation indicator, and P-selectin level. Measurements will be performed at baseline (T0), the end of the run-in period (T1), and weeks 4 (T2), 8 (T3), and 12 (T4) of the treatment period. Adverse events will be monitored during the trial. This study aims to evaluate the efficacy and safety of Zhenyuan capsules in patients with CHD and abnormal glucose and lipid metabolism. The results will provide critical evidence of the usefulness of the Chinese herbal medicine for CHD with abnormal glucose and lipid metabolism.ChiCTR-TRC-14004639, May 4, 2014. This trial is registered with the Chinese Clinical Trials Registry, with identifier number  Treatment of coronary heart disease (CHD), which poses a significant threat worldwide, has made great progress, including thrombolysis, intervention, and coronary artery bypass grafting. However, according to the COURAGE study, these interventional therapies may not effectively reduce the risk of major adverse cardiac events in patients with stable CHD as compared with conservative treatment . TherefoP = 0.04), microvascular disease by 24% (P = 0.001), myocardial infarction by 15% (P = 0.01), and all-cause mortality by 13% (P = 0.007). Moreover, myocardial infarction or all-cause mortality risk was decreased significantly [In addition, diabetes mellitus as an important independent risk factor of cardiovascular disease has been described in the 1999 American Heart Association statement as “a cardiovascular disease” and regarded as a coronary disease equivalent by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III 2001 . The risWith the publication of the Scandinavian Simvastatin Survival Study, the Cholesterol and Recurrent Events, and other large-scale clinical trials, the relationship between blood lipid levels and cardiovascular atherosclerosis has gradually become clear. Dyslipidemia is closely related to atherosclerosis and is an independent risk factor of CHD. Previous studies showed that lipid lowering can significantly reduce the risk of CHD by 2%, with total cholesterol and low-density lipoprotein cholesterol (LDL-C) both lowered by 1%. Extremely low levels of high-density lipoprotein cholesterol have been shown to increase the risk of CHD by 70% in men and 100% in women. Therefore, controlling abnormalities in serum lipid levels is an important part of the secondary prevention of CHD. The morbidity and mortality of CHD could be significantly reduced by using lipid-lowering drugs, which also could slow down and reverse the progression of atherosclerosis.Furthermore, dyslipidemia, which is associated with diabetes, is the main cause and one of the major risk factors of diabetes-associated macrovascular complications . Owing t Panax ginseng fruit. Previous studies showed multiple components of saponin in GFS, such as ginsenosides Re, Rb1, Rb2, Rc, Rg1, Rg2, and Rd. The structures of these compounds are identified on the basis of physicochemical constants, ultraviolet light, nuclear magnetic resonance, and spectral analysis. The study by Xu et al. [Traditional Chinese medicine (TCM) emphasizes symptomatic treatment and has long-term application in CHD-related diseases. From the view of TCM, patients with CHD and abnormal glucose and lipid metabolism can be divided according to symptoms. A common symptom is the Qi and Yin deficiency syndrome. The effect of Zhenyuan capsules in the treatment of this type of patients has been observed in the clinical setting. The main component of the Zhenyuan capsule is ginseng fruit saponin (GFS), a ginsenoside extract from the mature fruit of theu et al.  showed tTherefore, we designed this randomized, double-blind, parallel-controlled, multicenter, phase IV clinical trial to investigate the efficacy of the Zhenyuan capsule in the treatment of CHD accompanied with glucose and lipid metabolic disorders. The outcome measures are focused on symptom reduction and improvement in physiochemical indexes. We will also determine clinical safety.ChiCTR-TRC-14004639). The multicenter, randomized, double-blind, parallel-controlled, phase IV clinical study follows the recommendations of the CONSORT [This study is registered with the Chinese Clinical Trial Registry . Clinical assessment will be performed at baseline  and at the end of the 2-week run-in period (T1). Participants will then receive the treatment for 12 weeks. In the treatment period, the intervention assessments are performed at weeks 4 (T2), 8 (T3), and 12 (T4). Participants will be recruited from three participating centers, namely, Xiyuan Hospital of China Academy of Chinese Medical Sciences, Guan'anmen Hospital of China Academy of Chinese Medical Sciences, and the Second Affiliated Hospital of Tianjin University of TCM. The leading unit among the three centers is Xiyuan Hospital. In this trial, 200 patients will be recruited and randomly allocated to two groups  coronary angiography or coronary computed tomographic angiography shows that at least one of the main branches of the coronary artery has stenosis of at least 50%; (2) TG levels between 1.70 and 2.25 mmol/L and HbA1c level between 5.7% and 7%; (3) the TCM syndrome differentiation is Qi and Yin deficiency; (4) age between 45 and 75 years at screening; and (5) providing informed consent form.μmol/l) and >2.0 mg/dl (>175 μmol/l) for women; (4) hypohepatia, liver enzyme level of >2 times the normal value; (5) high risks of hemorrhage, hemophilia, and platelet dysfunction, or history of thrombocytopenia, which might influence the use of antiplatelet drugs; (6) active or emerging (<3 months) bleeding, including gastrointestinal bleeding, stool occult blood, or gross hematuria; (7) pregnant or attempting to become pregnant; (8) allergic constitution; (9) joined another trial or received random allocation in another study within the past 3 months; or (10) being unable or unwilling to sign the informed consent form, being noncompliant, or having poor possibility of a follow-up visit.Participants will be excluded if they report any of the following criteria: (1) serious cardiovascular diseases, including severe valvular heart disease, heart valve replacement, refractory heart failure, cardiac function grade IV (NYHA), and cardiogenic shock or serious cardiac insufficiency (EF < 35%); (2) mental disease or malignant tumor; (3) renal insufficiency, serum creatinine level of >2.5 mg/dl for men . Information from dropouts will be collected by phone interviews, such as the reason for the trial discontinuation and the status of the final medication received. Participants who withdraw from the trial will receive appropriate treatment for adverse events.β-blockers, calcium channel antagonists, angiotensin-converting enzyme inhibitors, or angiotensin II receptor antagonists. However, statins are the only Western medicine for lipid treatment. Patients diagnosed as having type 2 diabetes maintain the current Western medicines for hypoglycemic effects. To avoid discussing their medications among themselves, participants are advised to take the drugs at home and required to remain isolated from each other. In addition, the treatment allocation remains blind throughout the study. Participants will not be permitted to use other treatments for angina pectoris in CHD and diabetes, including TCM herbs and Chinese patent medicine. In the run-in period, any TCM medications for improving blood lipid or glucose levels will not be permitted.Patients who meet all the inclusion and none of the exclusion criteria will be randomized into two groups as follows: (1) treatment group, 0.5 g of Zhenyuan capsule, 3 times/day, and (2) control group, 0.5 g of placebo, 3 times/day. According to the Guideline for the Diagnosis and Treatment of Chronic Stable Angina, participants can maintain the conventional drug treatments such as antiplatelet agents, statins, nitrates, anticoagulation drugs, The primary indicators, HbA1c, fasting blood glucose (FBG), 2-hour postprandial blood glucose (2hPBG), and triglyceride (TG) levels, will be tested at T0, T1, T2, T3, and T4. The secondary indicators, ultrasonic cardiography (UCG) finding, coagulation indicator, and P- selectin level, will be assessed at T1 and T4. Cardiac death, nonfatal myocardial infarction, coronary revascularization, Seattle Angina Questionnaire (SAQ) score, and TCM symptom indicators will also be monitored periodically. Adverse events, which include hemorrhage, will be followed up using electrocardiography, routine urinalysis, routine stool test, and liver and kidney function tests  from T1 to T4. Items to be measured and the time window of data collection are shown in Participants are required to honestly report any changes in their symptoms. Any adverse events or unexpected toxic side effects that develop during the trial will be analyzed to determine the cause. These will be recorded and treated appropriately by the intervention administrators. In cases of severe adverse events, the participating patients are asked to take immediate measures to protect their safety and report the events to the Drug Supervision Bureau, the trial applicant, and the ethics committee within 24 hours.Eligible patients are randomly allocated to the treatment or control group in a 1 : 1 ratio. The randomization sequence will be recorded by staff at the sponsor unit. The researchers and outcome assessors participating in this study have no knowledge of the randomization sequence details. In addition, the results are kept in sealed, opaque, and stapled envelopes. They will be unsealed at the end of the outcome assessment. Emergency letters, including random codes and group assignments, are prepared by a statistician. If the blind code is exposed, patients will be excluded accordingly. Any of the following situations could call for a blind breaking and urgent measure: a serious adverse event, serious complication, or a deterioration in the patient's condition.The data of the participants collected according to the Case Report Forms will be imported into the clinical data management system (http://www.xyedc.com/). To keep the data consistency, the supervisor will compare the electronic database with the source documents and correct any identified errors. In addition, computer logic checks will be run after data entry. Manual checks and the Data Coordination Center will also help to identify more complicated and less common errors. All these measures are used to ensure the accuracy and integrity of information.α = 0.05 and a power of 80% (type II error rate of β = 0.2). According to a previous study, the efficacy rate of Zhenyuan capsules is 94% for relieving angina pectoris symptoms and 64% for placebo [n ≈ 25 patients/group.The sample size was determined with a type I error rate of  placebo . The samt-test for continuous variables and the Fisher exact test or χ2 test for categorical variables. In this multicenter trial, the Cochran-Mantel-Haenszel test and the analysis of variance will be used. For diminishing the influence of confounding factors on the curative effect, analysis of covariance and the logistic regression model will be applied. For all the analyses, the difference with a value of P < 0.05 will be statistically significant. All analyses will be conducted using the SAS software version 9.3 .Statistical analyses will be conducted by an independent statistician from Xiyuan Hospital. In the FAS, patients will be dosed with the study drugs at least one time and one clinical observation will be recorded. Participants with good compliance and without any protocol deviations will be included in the per-protocol set (PPS). Subjects with safety data who have completed at least one study visit will be included in the safety analysis. The mean ± SD will be used in continuous variables. The characteristic comparability between the two groups will be evaluated by a two-sample Student Recently, many studies have used the Zhenyuan capsule as treatment for CHD, heart failure , and typThis study first applies the Zhenyuan capsule for blood lipid research. This may reveal new uses for old drugs in TCM. In addition, this is the first study to address the efficacy and safety of the Zhenyuan capsule for CHD with abnormal glucose and lipid metabolism in a double-blind and parallel-controlled trial. We assume that this design, which is in line with the TCM theory, will contribute to more convincing results and robust evidence for the efficacy of study medications.This study has some limitations. Owing to the complexity of the disease, many potential influencing factors may exist. Therefore, to minimize possible errors, participants will have a 2-week run-in period. During this period, any TCM medications for improving blood lipids or glucose will not be permitted. In addition, participants will not be permitted to use other treatments for angina pectoris in CHD and diabetes mellitus, including TCM herbs and Chinese patent medicines at any time during the study.In summary, by rigorous design, the results of this study will provide clinical evidence regarding the efficacy and safety of Zhenyuan capsules for CHD with abnormal glucose and lipid metabolism.


The purpose of this study was to validate a patch-based image denoising method for ultra-low-dose CT images. Neural network with convolutional auto-encoder and pairs of standard-dose CT and ultra-low-dose CT image patches were used for image denoising. The performance of the proposed method was measured by using a chest phantom.Standard-dose and ultra-low-dose CT images of the chest phantom were acquired. The tube currents for standard-dose and ultra-low-dose CT were 300 and 10 mA, respectively. Ultra-low-dose CT images were denoised with our proposed method using neural network, large-scale nonlocal mean, and block-matching and 3D filtering. Five radiologists and three technologists assessed the denoised ultra-low-dose CT images visually and recorded their subjective impressions of streak artifacts, noise other than streak artifacts, visualization of pulmonary vessels, and overall image quality.For the streak artifacts, noise other than streak artifacts, and visualization of pulmonary vessels, the results of our proposed method were statistically better than those of block-matching and 3D filtering . On the other hand, the difference in the overall image quality between our proposed method and block-matching and 3D filtering was not statistically significant . The p-values obtained between our proposed method and large-scale nonlocal mean were all less than 0.05.Neural network with convolutional auto-encoder could be trained using pairs of standard-dose and ultra-low-dose CT image patches. According to the visual assessment by radiologists and technologists, the performance of our proposed method was superior to that of large-scale nonlocal mean and block-matching and 3D filtering. However, this has raised concerns about radiation exposure and the potential cancer risk induced by it Although LDCT has proven useful for screening of lung cancer, the cumulative radiation dose associated with LDCT is a major problem. McCunney et al. showed that the cumulative radiation dose from LDCT screening could exceed that received by nuclear workers or atomic bomb survivors if LDCT lung cancer screening was conducted over a 20–30-year period To overcome this issue, ultra-low-dose CT (ULDCT) has been studied intensively , 5, 6. BOne previous study showed that it was possible to achieve state-of-the-art image-denoising performance with plain multilayer perceptron that maps noisy image patches onto noise-free ones The objectives of the present study were: i) to validate a patch-based, neural-network-trained image-denoising method for ULDCT images; ii) to train the neural network with CAE and pairs of SDCT and ULDCT image patches; and iii) to investigate the performance of our proposed method using a chest phantom. In addition, our proposed method was compared with the method proposed by Chen et al. 2The present study was experimental, and phantom study was performed. No patient information was used in the present study.2.1Helical CT scans of chest phantom  were acquired from lung apices through lung bases using a 16-detector row scanner . During a CT examination session, one set of SDCT images and two sets of ULDCT images were obtained. One set of UDLCT images was used for training, while the other was used for testing. The following CT parameters were used: tube current, 300 mA (SDCT) or 10 mA (ULDCT); tube potential, 120 kV; and gantry rotation time, 0.5 s. Raw CT data were reconstructed into 1.25-mm-thick images using high-frequency kernel. The CT scanner was calibrated regularly.2.2nD = {1x, …, nx} is prepared, where ix is a d-dimensional vector. Then, noise is added to ix , and pairs of original input and noisy input, nT = {, …,  with parameters {W, b}. Here, σ is an activation function, and the sigmoid function was used in our main experiment. Then, the latent representation ih is used to reconstruct the clean output ix’ by reverse mapping ix’ = σ (W’ ih + b’) with parameters {W’, b’}, where the first parameter is constrained as W’ = WT. This constraint means that the same weights are used for encoding the input and decoding the latent representation. The parameters are optimized, minimizing an appropriate cost function between ix and ix’  over the training set nT. If the training is successfully performed, DAE reconstructs the clean output from the noisy input. A schematic illustration of the training DAE is shown in DAE 2.3nT = {, …, }, are given as training set to CAE. For a noisy image y , the latent representation of the k-th feature map is given by kh = σ (y ∗ kW + kb), where ∗ denotes the 2D convolution and kW is the weight of the k-th feature map. The reconstruction x’ is obtained using x’ = c is a bias parameter, H identifies the group of latent feature maps, and W’ identifies the weights. The parameters of CAE are trained, minimizing a cost function. In the present study, the cost function of CAE is the mean squared error (MSE) between ix and ix’  over the training set nT: http://www.python.org/) and chainer (http://www.chainer.org/).Although the DAE ignores the 2D-image structure, CAE preserves the spatial locality of the 2D image 2.4nT = {, …, }, where ix and iy are patches obtained from SDCT and ULDCT images, respectively. Next, for ease of training, the CT value of each pixel in the image patches was converted to results of the following function f(x):Before training CAE, patches of the SDCT and ULDCT images were prepared. First, the SDCT and training ULDCT images were three-dimensionally co-registered using Advanced Normalization Tools x is CT value. After converting the CT value, nT was used for training of CAE. As stated, the number of patches was 100,000 in the training set. A schematic illustration of the preprocessing of CT images and the training of CAE is shown in where 2.5The sizes of the latent representation and the output of CAE were affected by padding and kernel of 2D convolution. To maintain the size of the input, the latent representation, and the output at constant value (28 × 28 pixels), the following value was used: kernel, 3 × 3 pixels; and padding, 1 × 1 pixels. In addition, the following numbers of feature maps were tested: 20, 30, and 40. Typically, CAE is 3-layered neural network. However, a previous study showed that multilayer perceptron with four hidden layers showed better performance than that with two hidden layers The training set was used for training of CAE. Then, the effectiveness of the training was evaluated with the validation set and its MSE. This evaluation was performed for each parameter of CAE, and the best parameters were selected based on the MSE of the validation set.2.6To denoise ULDCT images, ULDCT image was decomposed into overlapping patches, and each image patch was denoised separately 2.7The DAE, LNLM, and BM3D methods were tested for comparison with CAE. As shown in the Results section, because the effectiveness of training for DAE was worse than that for CAE, image denoising was not evaluated for DAE. Only MSE of DAE was compared with that of CAE. To train DAE with image patches, the patches of training and validation set were vectorized. After vectorization, the preprocessing using the function [I] was performed before training DAE.CT images denoised by LNLM and BM3D were compared with those denoised by CAE. For image denoising with LNLM and BM3D, conversion of CT value using the function [I] and inverse transformation of the function [I] were performed as in CAE. For LNLM, the pixel values on denoised image is replaced by the weighted average of pixel values on noisy image located in a neighborhood window. Each weight expresses the similarity in intensity between the central pixel in the window and each neighboring pixel and is given by difference between patch of the central pixel and that of the neighboring pixels , 8. The 2.8The testing ULDCT images of the upper-, middle-, lower-lung fields were denoised with CAE, LNLM, and BM3D. Five radiologists with 1, 4, 8, 8, and 11 years of experience and three technologists visually assessed the denoised CT images in lung window with a level setting of 1,500/−600 HU. They were unaware of the denoising methods or parameters used. The CT images denoised with CAE, LNLM, and BM3D were visually evaluated side-by-side. The readers independently recorded their subjective impression of the following four items: streak artifacts, noise other than streak artifacts, visualization of pulmonary vessels, and the overall image quality. The denoising methods were ranked by the readers for each image and item. The ranks of the three images  were summed, and the sum of the ranks of the three images was recorded as the result of the visual assessment. Because each reader selected rank from 1 to 5 for each slice, the result of visual assessment ranged from 3 to 15.2.9Peak signal to noise ratio (PSNR) and structural similarity index (SSIM) 2.10P-value <0.05 was considered to indicate statistical significance. All analyses were performed by R-3.1.1 (available at http://www.r-project.org/).For each item of visual assessment, the differences in the ranks between various denoising methods were assessed by Mann–Whitney U test. As the readers conducted the visual assessment independently, a statistical test could be performed. 2.11Additionally, our proposed method was extended and compared with the method proposed by Chen et al. •Number of convolution layers was 3. (Number of hidden layers was 2).•Activation function was Rectified linear units (ReLU).•Filter numbers (number of feature maps) in the hidden layers were 64 and 32.•Filter sizes (kernel sizes of convolution) were 9, 3 and 5.•Padding sizes were 4, 1, and 2.The following parameters were used for Chen’s method in the current study, because Chen’s paper does not fully show their parameters of convolutional neural network (padding sizes).The network architecture specified by these parameters of Chen’s method can be viewed as a special case of our method shown in •Numbers of hidden layers were randomly selected from 1, 3, 5, 7, 9, or 11.•Activation function was ReLU.•Filter numbers in the hidden layers were randomly selected from 16, 32, 48, or 80.•Kernel sizes in convolution were randomly selected from 3, 5, 7, 9, or 11.•Padding sizes were calculated from kernel sizes using the following equation: (padding size) = decimal_truncation((kenel size)/2). For example, if kernel size is 7, then padding size is 3.From the viewpoint that Chen’s method can be viewed as a special case of our proposed method of the main experiment, we fully optimized the network architecture of In short, we searched the best parameters of numbers of hidden layers, filter numbers, and kernel sizes in this optimization. The random search was performed 100 times, and the best MSE of validation set and its corresponding parameters were selected. In this additional experiment, the same training set and validation set as the main experiment were used in both our proposed method and Chen’s method. ULDCT images denoised with optimal method in the additional experiment were also evaluated as in the main experiment.3The radiologists and technologists visually evaluated the images of For the additional experiment, MSE of the validation set was as follows: optimal method in the main experiment, 0.478; Chen’s method, 0.365; optimal method in the additional experiment, 0.255. The parameters of optimal architecture in the additional experiment were as follows: number of hidden layers, 7; filter numbers in the hidden layers, 16, 64, 48, 64, 80, 48, 32; kernel sizes, 5, 7, 9, 5, 11, 5, 3, 3; padding sizes, 2, 3, 4, 2, 5, 2, 1, 1. According to these results, MSE of Chen’s method is better than that of optimal method in the main experiment. However, by optimizing network architecture shown in 4The results of the present study show that it was possible to train CAE with pairs of SDCT and ULDCT image patches and to use the trained CAE for patch-based image denoising of ULDCT images. In addition, the results of visual assessment by the radiologists and technologists show that the performance of CAE was better than that of LNLM or BM3D. These results validate the usefulness of image denoising of ULDCT using CAE.Reduction of radiation dose results in an increase of CT image noise, which is an important factor reducing image quality. Because the high contrast between air and other tissues in lungs helps to maintain diagnostic accuracy, radiologists can reliably evaluate chest LDCT images. In contrast, as severe noise and a significant number of artifacts are present in chest ULDCT images, ULDCT hinders the radiologists’ evaluation. To reduce the noise or artifacts in LDCT or ULDCT images, various types of post-processing techniques have shown potential for reduction of CT radiation dose.This paper proposes to denoise ULDCT images using CAE. Previously, non-medical images, such as images of human faces, vegetables, and birds have been denoised with neural network , 21, 22,Although the previous study shows that multilayer perceptron with four hidden layers performed better than that with two hidden layers The advantage of CAE is twofold. First, it can be implemented on any CT scanner without using raw CT data. There have been several studies that have showed the usefulness of iterative reconstruction through raw-data-based techniques  for dose reduction of chest CT , 5, 6. BThe results of visual assessment show that, for noise or artifact reduction, CAE was superior to LNLM or BM3D . Although the difference in the overall image quality between BM3D with sigma = 20 and CAE was not statistically significant , the image quality of CAE tended to be better than that of BM3D. In addition, the image quality of CAE was significantly better than that of LNLM. These results validate the usefulness of CAE for image denoising of ULDCT images.There were several limitations in the present study. First, because the present study was experimental, we could not use clinical LDCT or ULDCT images. It is possible that, if patient data is used, our results will not be reproduced. For further study, we would evaluate CAE with the CT images obtained from clinical patients. Second, the raw-data-based techniques were not evaluated in the present study, as the iterative reconstruction technique was not available in the CT scanner of our institution. Therefore, we could not compare CAE with iterative reconstruction technique. If a CT scanner equipped with the iterative reconstruction technique is available, we will compare CAE with the iterative reconstruction technique.In conclusion, the current study proposed image denoising with CAE, which was trained with the patches of SDCT and ULDCT images. After training of CAE, ULDCT images could be reliably denoised. According to visual assessment by radiologists and technologists, the performance of CAE was superior to that of LNLM and BM3D.Mizuho Nishio: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Chihiro Nagashima, Saori Hirabayashi, Akinori Ohnishi, Kaori Sasaki, Tomoyuki Sagawa, Masayuki Hamada, Tatsuo Yamashita: Performed the experiments.JSPS KAKENHI (Grant Number JP16K19883) and Hyogo Science and Technology Association.This work was supported by The authors declare no conflict of interest.No additional information is available for this paper.


In epidemiologic studies, association between coffee consumption and esophageal cancer risk is inconsistent.The aim of tjis study was to evaluate the effect of coffee on esophageal cancer by combining several similar studies.We conducted a meta-analysis for association of coffee intake and esophageal cancer incidence. Eleven studies, including 457,010 participants and 2628 incident cases, were identified. A relative risk  or odds ratio  of heavy coffee drinkers was calculated, compared with light coffee drinkers or non-drinkers. The analysis was also stratified by cancer types , sex, and geographic region.The summarized OR of having esophageal cancer in heavy coffee drinkers was 0.93 , compared with light coffee drinkers. When stratified by sex, pathologic type of esophageal cancer, and type of epidemiologic study, we did not find any association of coffee consumption and esophageal cancer incidence. However, an inverse association between coffee consumption and incidence of esophageal cancer was found in East Asia participants with OR of 0.64 (95% CI: 0.44–0.83), but not in Euro-America participants .There is a protective role of coffee consumption against esophageal cancer in East Asians, but not in Euro-Americans. As many researchers in the world, we are interested in relationship of coffee consumption and the risk of esophageal cancer incidence because coffee is one of the most widely consumed beverages in the world and incidence and mortality of esophageal cancer are high worldwide. Therefore, we conducted this meta-analysis to examine the association in epidemiologic studies.Esophageal cancer is a major concern in the world, ranking the sixth most common cause of cancer mortality.2The electronic databases, PubMed (1966 to January 2017), Embase (1980 to January 2017), the Science Citation Index (1945 to January 2017), and the Chinese Biomedical Database (1981 to January 2017) were searched for studies of coffee consumption in relation to esophageal cancer risk, which were published in both English or Chinese. We used terms of “coffee,” or “caffeine” combined with medical-subject-heading terms “esophageal neoplasms,” “oesophageal neoplasms,” or “esophagus,” or keywords “esophageal cancer” or “oesophageal cancer” in the paper search. All analyses were based on previous published studies; thus, no ethical approval and patient consent are required.31.Type of study: observational studies: including case-control study and cohort study.2.Exposure factor: coffee consumption.3.Statistical indicators: Relative risk  or odds ratio  of heavy coffee drinkers, compared with light coffee drinkers or nondrinkers, was presented.4We extracted the data for the first author's name, publication year, the country of origin, duration of follow-up, sex, the number of participants (cases and cohort size), measurements of coffee consumption and OR estimates, and their corresponding 95% CIs. When a study provided ORs for both esophageal cancer and invasive esophageal cancer, we used the former because of having more cases. Two reviewers (JZ and BZ) independently inspected the data for eligibility and quality. Disagreements were resolved by the third reviewer (CH).5,9 If P value of heterogeneity was <.10, the random model was used instead of the fixed-effect model for further analysis. Funnel plots with the Begg test and Egger test were visually evaluated to detect a possible publication bias with α = 0.1011. We pooled the ORs for highest versus lowest categories of coffee consumption from each study. We combined data of 11 studies and estimated a summary OR of coffee consumption and esophageal cancer risk. Subgroup analyses were performed by sex, geographic region, pathologic type of esophageal cancer, and type of epidemiologic studies.The Stata version 12.0 software  was used to analyze the OR or RR. Analysis was stratified by cancer types, sex, and geographic region to exclude effect of possible confounders. In the meta-analysis, heterogeneity across the studies was examined using the Q statistic. Statistical significance was set at 0.10 for heterogeneity.66.1In our initial retrieve, 3599 articles were identified, but 3572 articles were excluded for lack of clinical trial (n = 2987), duplication (n = 285), and unrelated statistical indicators (n = 300). After a detailed assessment of full texts, 9 were excluded because of insufficient data and 7 articles were excluded because of being nonobservational studies. Finally, 11 trials were included in this meta-analysis , whereas another 8,16–18,21 studies were conducted in Europe and America (1988 cases). Two studies conducted in Japan and Norway were cohort studies and the others were case–control studies. Only 2 studies,21 provided sex-specific ORs, but 1 study from Taiwan included male participants only. Therefore, the 3 studies were used for male esophageal cancer (1218 cases) and 2 studies for female esophageal cancer (269 cases), respectively.Eleven epidemiologic studies6.3A combined OR of highest coffee consumption was 0.93 (95% CI: 0.73–1.12), compared with lowest coffee consumption , with an exception of the ESCC subgroup (P = .058 for Egger test).There was no evidence of publication bias in the overall trials Fig.  and the 7 The cumulative rate of ESCC is highest among East Asians, and lowest in white subjects. However, ESCC is the dominant type of esophageal carcinomas in Asian countries, especially in China including Hong Kong and Taiwan, Korea, and Japan.,27 The ratio of EAC among esophageal malignancies was as low as 1% to 4% in Korea, Taiwan, and Japan.,28 Therefore, overall incidence of esophageal cancer is much higher in East Asia than that in Europe and America. Coffee consumption may play a more protective role of incidence of esophageal cancer in high-risk area, such as East Asia.In general, our meta-analysis did not find association between coffee consumption and risk of esophageal cancer incidence. However, East Asian who drank coffee every day had a lower incidence of esophageal cancer than those who rarely or never drank coffee, which may be caused by different prevalence of esophageal cancer between Euro-American and East Asian countries. The cumulative rate of EAC is higher in Euro-America than in East Asia. The former is 0.01 to 1.19 and the latter is 0 to 0.15. In our meta-analysis, only 1 study  investigated the association between temperature of coffee drinking and risk of esophageal cancer incidence and concluded that higher temperature of coffee did not increase incidence of esophageal cancer. Owing to little information on relation of temperature of coffee drinking and esophageal cancer, it was difficult to exclude interference from temperature of coffee drinking.A number of experimental and clinical studies have suggested that drinking very hot beverages may be a cause of esophageal cancer. Evidences that tumors were larger sizes of esophageal papillomas rapidly increased when people took hot food with the temperature at 70°C and above and this relationship was confirmed by an experimental study.,17,18,21 included in the present study have investigated the effects of coffee drinking and dose–response relationships and 5 of them did not find dose–response relationships.,15,17,21 In the study conducted by Tavani et al, more coffee consumption daily decreased the risk of getting esophageal cancer (P for trend = .007). We did not do analysis stratified by amount of coffee consumption because of the discrepancy of categorization of coffee consumption in those studies.Six studies Four studies,14,18,20 included in the meta-analysis have observed the association between cigarette smoking and alcohol drinking and risk of incidence of esophageal cancer. In all 4 studies, cigarette smoking and alcohol drinking were found to be substantially associated with an increased risk of esophageal cancer incidence. All studies included in the meta-analysis provided data adjusted for potential interaction factors among age, smoking, and alcohol, which demonstrated a more independent association between coffee consumption and esophageal cancer incidence.Smoking is associated with esophageal cancer and stop smoking may reduce the risk of esophageal cancer. Also there is a strong association between alcohol consumption and esophageal squamous cell carcinoma. Second, almost all original studies have no data on the temperature of the coffee when drinking, which may be a cause of inconsistent data among studies. Finally, although incidence of esophageal cancer is quite high in Africa, we did not find studies conducted among African populations.To minimize the bias, our study referred to many aspects, such as race, sex, age, coffee consumption, and so on. However, there are several disadvantages that should be considered in our meta-analysis. First, the epidemiologic studies did not provide information on the characteristics of the coffee, such as cup size, type of coffee power, and brewing methods, which are responsible for the different concentrations of caffeine and other chemicals in the beverage.In summary, our study suggests that coffee consumption has a protective effect on esophageal cancer risk in East Asian, but not in Euro-Americans. Additional prospective cohort studies should be granted among different populations with carefully control of possible confounders, especially characteristics and temperature of the coffee when drinking.Conceptualization: Bin Zhou.Data curation: Bin Zhou, Juan Zhang.Formal analysis: Bin Zhou, Juan Zhang.Investigation: Juan Zhang.Methodology: Bin Zhou, Juan Zhang.Project administration: Bin Zhou, Juan Zhang.Resources: Juan Zhang.Software: Bin Zhou, Juan Zhang.Supervision: Chuangzheng Hao.Writing – original draft: Bin Zhou, Juan Zhang.Writing – review & editing: Bin Zhou, Juan Zhang, Chuangzheng Hao.