Quercetin is a bioactive compound that is widely used in botanical medicine and traditional Chinese medicine due to its potent antioxidant activity. In recent years, antioxidant activities of quercetin have been studied extensively, including its effects on glutathione (GSH), enzymatic activity, signal transduction pathways, and reactive oxygen species (ROS) caused by environmental and toxicological factors. Chemical studies on quercetin have mainly focused on the antioxidant activity of its metal ion complexes and complex ions. In this review, we highlight the recent advances in the antioxidant activities, chemical research, and medicinal application of quercetin.  Quercetin a,b is a The antioxidant activity of quercetin is mainly manifested through its effect on glutathione (GSH), enzymatic activity, signal transduction pathways, and reactive oxygen species (ROS) caused by environmental and toxicological factors. Quercetin shows a strong antioxidant activity by maintaining oxidative balance.2- and transforms it into H2O2. This enzyme further catalyzes the decomposition of H2O2 to the non-toxic H2O. This reaction requires GSH as a hydrogen donor. Animal and cell studies found that quercetin induces GSH synthesis [2O2 is converted to H2O and GSH is oxidized to GSSG (oxidized glutathione disulfide). GSH reductase catalyzes the reduction of GSSG in the liver and red blood cells (by providing H) to form GSH. Thus, the dynamic balance of GSH is produced, which may cause the inhibition of GSH levels in low doses. This inhibition of quercetin on GSH at the level of 0.5% is reported in the literature [Quercetin increases the body’s antioxidant capacity by regulating levels of GSH. This is because, once oxygen free radicals are generated in the body, superoxide dismutase (SOD) quickly captures Oynthesis ,14. It wynthesis . When quterature .The –OH groups on the side phenyl ring of quercetin are bound to important amino acid residues at the active site of two enzymes. In this way, it has a stronger inhibitory effect against key enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which are associated with oxidative properties . QuercetIt was reported that pretreatment with quercetin significantly enhanced the expression levels of endogenous antioxidant enzymes such as Cu/Zn SOD, Mn SOD, catalase (CAT), and GSH peroxidase in the hippocampal CA1 pyramidal neurons of animals suffering from ischemic injury. This indicates that it strongly protects the hippocampal area CA1 pyramidal neurons from I/R injury. Thus, quercetin may be a potential neuroprotective agent for transient ischemia . In addition, as one of the most metabolically active tissues of the body, bone undergoes a continuous and complex remodeling process throughout life. In particular, osteoblasts, which are derived from osteoprogenitor cells produced by self-renewing, pluripotent stem cells, play a critical role in this cycle. The primary function of osteoblasts is to generate a new bone matrix and, together with osteocytes, support the bone structure itself. Damage to osteoblasts can therefore result in several dysfunctions. Smokers have low bone mass and stability. It was reported that the application of quercetin can promote fracture healing in smokers by removing free radicals and upregulating the expression of heme-oxygenase- (HO-) 1 and superoxide-dismutase- (SOD-) 1, which protects primary human osteoblasts exposed to cigarette smoke .Quercetin has also been shown to prevent heart damage by clearing oxygen-free radicals caused by lipopolysaccharide (LPS)-induced endotoxemia. LPS induces histopathological and biochemical damages to the myocardium in endotoxemia model. In a rat model experiment, rats treated with LPS showed a significant increase in the malondialdehyde (MDA) level in tissues and a decrease in SOD and CAT activity in heart tissues. In contrast, quercetin treatment increased the levels of SOD and CAT and reduced the level of MDA after LPS induction, suggesting that quercetin enhanced the antioxidant defense system .Quercetin has several effects on various signal transduction pathways, such as activating, inhibiting, upregulating, or downregulating many molecules of the body. In this way, quercetin can improve the antioxidant state of the body and repair injury such as spinal cord injury, atherosclerosis, and lead or cadmium toxicity. By influencing signal transduction pathways, quercetin modulates the enzymes or antioxidant substances that enhance antioxidant properties, thereby preventing disease development. Studies have shown that the protective mechanism of quercetin against acute spinal cord injury is mediated by its inhibitory effect on the p38MAPK/iNOS signaling pathway, the downregulation of MDA levels, and the upregulation of SOD activity to promote antioxidant activity . In psorSeveral enzymes regulate signaling pathways to enhance antioxidant activities, such as oxidative stress protection. Quercetin reversed the oxLDL-induced decrease in AMPK activation and oxLDL-induced increase in NADPH oxidase expression, thereby maintaining AKT/eNOS function and suppressing NF-κB signal transduction to combat atherosclerosis . MoreoveOxidative damage is mostly caused by ROS. Quercetin can remove ROS, thereby resisting oxidative damage such as respiratory damage, ultraviolet radiation b (UVB) skin lesions caused by radiation, and oxidative damage induced by paraquat, as well as sperm change associated with ROS and oxidative damage of gastric epithelial cells. Respiratory damage is caused by exposure to fine particulate matter (PM2.5) in the environment, leading to the cellular activity of 16HBE cells, increased production of ROS, and the inhibition of the expression of mitochondrial genes. It has been recognized that quercetin may stimulate 16HBE cells to repair oxidative damage after exposure to PM2.5 by regulating ROS production and anti-inflammatory processes .Human skin is the body’s largest organ that can resist all kinds of environmental damage. However, UVB induces a transient increase in ROS and an imbalance of endogenous antioxidant systems, thereby increasing the level of free radicals and inflammation, which affect cellular processes. Studies have shown that quercetin prevents UVB-induced radiation damage by removing ROS and strengthening the cell membrane and mitochondrion against ROS-induced damage. In addition, it also inhibits cell membrane mobility and mitochondrial membrane depolarization. Therefore, the consumption of quercetin also inhibits this imbalance and is used to prevent UVB-induced skin damage ,46.2O2 can be suppressed by quercetin treatment due to its protective effects on gastric epithelial GES-1 cells. In these cells, quercetin prevents oxidative damage and inhibits the production of ROS during acute gastric mucosal injury in mice [Quercetin prevents oxidative damage induced by paraquat by reducing the ROS levels and increasing the total GSH levels. Other studies have shown that quercetin can alleviate oxidative stress by modulating the expression of antioxidant-related genes in A549 cells . Gastric in mice . MoreoveOn the other hand, quercetin inhibits oxidative stress, thereby preventing antioxidant damage. Oxidative stress is caused by the imbalance between oxidants and antioxidants in the body, and it tends to be oxidized. Once oxidized, it results in neutrophil inflammatory infiltration, high protease secretion, and other oxidative intermediates. Quercetin inhibits oxidative stress by regulating the balance between oxidant and antioxidant effects. In various experimental studies, quercetin suppressed radiation-induced brain damage in rats, oxidative damage in rats induced by acrylamide, nerve damage in retinas of diabetic rats, as well as neurodegenerative diseases and oxidative stress induced by cadmium fluoride. By modulating the antioxidant levels, quercetin protects the brain, nerves, or other cells in the body from damage caused by oxidation ,52,53,54Ionizing radiation induces various types of damage by increasing free radical formation or increasing cell damage and cell death due to ROS. Quercetin effectively protects cells from genetic toxicity and damage induced by radiation by scavenging free radicals and increasing endogenous antioxidant levels. Bioflavonoids act as reducing agents for hydrogen or electronic agents, which inhibit or reduce free radical toxicity and enhance antioxidant properties in the body, thereby providing protection against radiation ,56,57.Quercetin exerts antioxidant and hepatoprotective effects against acute liver injury in mice induced by tertiary butyl hydrogen peroxide. This is attributed to its strong antioxidant effect and free radical scavenging effect. It inhibits lipid peroxidation and increases antioxidant activity and thus can be an effective treatment for oxidative liver injury . QuercetQuercetin removes free radicals and strengthens antioxidant defense systems in the body. Thus, quercetin can suppress oxidative stress including the production of ROS induced by nicotine to treat diseases such as tobacco addiction .Due to the poor water solubility and low bioavailability (5.3%) of quercetin, several studies have been performed to modify its structure to increase its water solubility and bioavailability, and thus enhance its antioxidant activity .The modification process of quercetin is generally divided into two types—namely, the derivation of quercetin or recombination with other active groups. The former changes the structure of quercetin and improves its solubility through derivation, while the latter produces a synergistic effect based on the properties of active groups and quercetin, such as metal complexes of quercetin. Moreover, the bioactivity and pharmacological action of quercetin are significantly enhanced after forming complexes with some metal or complex ions. Therefore, many researchers have attempted to improve the antioxidant activity of quercetin using the complex formation method.Combining quercetin with metal ions improves the reducibility of flavonoids by enabling them to be oxidized by free radicals more easily compared to unmatched flavonoids. Therefore, when complexed with metal ions, quercetin shows excellent antioxidant activity. The scavenging capacity of quercetin combined with vanadium , copper 2O2 and does not effectively reduce the amount of ROS and MDA in ARPE-19 cells. However, a quercetin–phospholipid complex significantly increased the activity of these enzymes and significantly reduced ROS and MDA levels. These datasets show that the antioxidant activity of quercetin–phospholipid complexes is higher compared to that of free quercetin. The poor water solubility of quercetin limits its use. Therefore, a quercetin–phospholipid complex was developed to improve its water solubility, enhance its absorption through the gastrointestinal tract, and increase its bioavailability [A study reported that quercetin does not enhance the activity of SOD, CAT, and GSH-PX in ARPE-19 cells treated with Hlability ,74. The Some complex ionic complexes, such as glucan–quercetin conjugate , calciumGiven the clinical importance of oxidative damage, antioxidants are expected to treat some diseases. Therefore, quercetin can be exploited in medicinal field due to its strong antioxidant properties. This basic principle of antioxidant activity of quercetin is shown in Quercetin has been found to influence malignant tumors such as tumors of epithelial tissue and malignant tumors of interlobular tissue. The term "cancer" generally refers to all malignant tumors, including cancer and sarcoma.Quercetin has been used in cancer prevention and to prevent the spread of various cancers, such as lung, prostate, liver, breast, colon, and cervical cancers. Its anticancer properties are mediated by various mechanisms involving cell signaling pathways and enzymatic activities that inhibit carcinogens. Here, we discuss the treatment or prevention aspect of quercetin for cancer.High levels of ROS induce oxidative stress, which in turn causes the over-activation of signal transduction pathways and promotes cell proliferation, as well as survival and metabolic adaptation to the tumor microenvironment. In this way, ROS promotes tumorigenesis. Quercetin regulates both internal and external pathways of ROS-mediated protein kinase C (PKC) signaling. PKC is a key regulator of cell growth and differentiation in mammalian cells and its activation partially depends on ROS signaling. PKC inhibits cell proliferation and survival and induces apoptosis in cancer cells. Quercetin prevents cancer development by upregulating p53, which is the most common inactivated tumor suppressor. It also increases the expression of BAX, a downstream target of p53 and a key pro-apoptotic gene in HepG2 cells ,83.2O2, and antioxidant GSH level were measured in rats. The result showed that rats treated with carcinogen and testosterone had higher levels of LPO and H2O2 and lower levels of GSH compared to quercetin-treated rats. This implies that quercetin may be used to target signaling molecules in prostate cancer, which is the second highest cause of cancer-related deaths in men [In addition, quercetin prevents cancer by modulating oxidative stress markers and antioxidant enzymes. In a previous study, histology and oxidative stress markers such as lipid peroxidation (LPO), Hs in men . Other ss in men . Moreoves in men .2+ overload-induced myocardial cell injury. Therefore, quercetin can efficiently prevent injury induced by oxidative stress [Based on its antioxidant activity, quercetin has a therapeutic effect on cardiovascular diseases. For instance, coronary heart disease may lead to acute myocardial infarction (AMI). A recent study showed that oxidative stress is an important factor involved in the development of AMI. Quercetin significantly decreases MDA content, increases SOD and CAT activity, and regulates anti-inflammatory and anti-apoptosis processes to effectively protect against myocardium injury . In addie stress .Depression is a common mental disease that negatively affects human physical and mental health. Chronic stress is connected with depression and anxiety. Several studies have shown that quercetin treatment significantly lowers oxidative and inflammatory stress and prevents neural damage by regulating oxidative stress markers such as TBARS and nitric oxide, antioxidants such as total thiol and catalase, and pro-inflammatory cytokines in the hippocampus . ElsewheQuercetin has the potential to treat diseases caused by pathogenic factors. Such diseases are often caused by an imbalance between oxidation and antioxidation processes. Quercetin protects the body from urotoxicity induced by cyclophosphamide (CYP) via the inhibition of oxidative stress and the restoration of pro-inflammatory/anti-inflammatory cytokine balance. Compared with normal group, CYP treatment showed a marked reduction in bladder levels of catalase, SOD, and IL-10. However, quercetin prevented urotoxicity by reversing the changes in these biochemical markers and histopathology . By inhiNumerous studies have shown that quercetin has the potential to treat other diseases, such as necrotizing enterocolitis (NEC), diabetes, and lung injury. In NEC, quercetin modulates the total antioxidant status by regulating serum levels of MDA and GSH . In typeQuercetin is a typical flavonoid that is abundant in fruits and vegetables. Its application in the medicinal field has shown potential to improve human health due to its antioxidant activity in vivo. Some studies show that quercetin can be used as a nutraceutical to offer protection against various diseases. It is effective in the treatment and prevention of human diseases since it influences glutathione, enzymes, signal transduction pathways, and ROS production. However, its application in the pharmaceutical field is limited by its low absorption into the body based on its poor solubility, low bioavailability, poor permeability, and instability. When it forms complexes with metal ions or complex ions, its bioavailability and antioxidant effect are enhanced and strengthened. In addition to quercetin complexes, newer preparations of quercetin have emerged in recent years, including nanoparticles loaded with quercetin ,106,107,Therefore, strategies that improve the solubility and bioavailability of quercetin will potentiate its properties such as its antioxidant and antimicrobial activities. This will contribute to the full exploitation of quercetin as a rich natural drug resource for medicinal uses.


Exposure to toxic metals such as mercury has been proposed to be a risk factor for the development of breast cancer since some metals can promote genetic mutations and epigenetic changes. We sought to find what toxic metals are present in normal breast tissue and in the tumours of women who had mastectomies for invasive ductal breast carcinoma.Formalin-fixed paraffin-embedded blocks from mastectomies for breast carcinoma were examined from 50 women aged 34–69 years. Paraffin blocks selected for elemental analysis were from breast tissue not involved by carcinoma and from the carcinoma itself. Seven micrometer-thick sections were stained with autometallography to demonstrate the presence of mercury, and subjected to laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to confirm the presence of mercury and to detect other toxic metals.Autometallography-detected mercury was seen in intraductal secretions and some luminal epithelial cells of normal breast lobules in 26 (55%) of the 47 samples where lobules were present, and in 10 (23%) of carcinomas from the 44 samples where carcinoma was present. In eight samples ductal carcinoma in situ was present and one of these contained mercury. LA-ICP-MS confirmed the presence of mercury in samples that stained with autometallography, and detected lead, iron, nickel, aluminium, chromium and cadmium in some samples.Mercury was present in normal breast lobules in more than half of mastectomy samples that contained an invasive carcinoma, and in a smaller proportion of carcinomas and ductal carcinomas in situ. Other toxic metals that may interact synergistically with mercury could be detected in some samples. These findings do not provide direct evidence that toxic metals such as mercury play a role in the pathogenesis of breast cancer, but suggest that future molecular biological investigations on the role of toxic metals in breast cancer are warranted. Breast cancer is the most common cancer, and the leading cause of cancer death, in women. Many of the risk factors for breast cancer are associated with estrogens, such as early menarche, late menopause, and obesity . Other rBRCA1 that play a part in DNA repair are associated with breast cancer, suggesting that these environmental toxins and gene mutations may have injury to DNA repair in common [Toxic metals such as mercury could promote carcinogenesis directly through gene mutations –32, epign common .Atmospheric mercury is the toxic metal most strongly associated with breast cancer . ExposurThe findings of epidemiological studies on links between toxic metals and breast cancer have varied and are often contradictory, so other methods to assess toxicant exposures have been attempted, such as measuring metal levels within breast cancer tissue, but these too have yielded diverse results –44. ThesThis study was approved by the Human Research Committee, Sydney Local Health District , in accordance with the Declaration of Helsinki as revised in 2000. The institutional review board waived the need for written informed consent from individuals studied since this was a de-identified retrospective study of archived paraffin-embedded surgical samples. Patient records were searched for age at time of surgery, month and year of surgery (between January 2010 and December 2014), microscopic diagnosis of tumour and non-tumour tissue, tumour grade, presence of ductal carcinoma in situ, and estrogen, progesterone, and human epidermal growth factor receptor status of the carcinoma.Hematoxylin and eosin-stained microscopic slides from the Royal Prince Alfred Hospital tissue repository were examined to find two paraffin tissue blocks that contained (1) normal breast tissue and (2) carcinoma, from 50 women aged between 34–69 years who had mastectomies for invasive ductal breast carcinoma. Paraffin blocks were sectioned at 7 μm with a Feather S35 stainless steel disposable microtome blade and deparaffinised before AMG and LA-ICP-MS analyses. Nottingham tumours grades  were available for all samples. The carcinoma estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status had been measured according to the College of American Pathologists Template for Reporting Results of Biomarker Testing of Specimens from Patients with Carcinoma of the Breast.Breast terminal duct-lobular units are focal collections of intralobular terminal ducts and acini within relatively dense fibrous tissue . The smaSections were stained for inorganic mercury bound to sulphide or selenide using silver nitrate AMG, which represents the presence of mercury as black grains . AMG is In addition to mercury, AMG can detect inorganic silver and bismuth ,46. TherMercury in breast secretions was identified by black AMG staining within the lumens of lobules and terminal ducts Figs –4. ContrAbout one-fifth of lumens with mercury-containing secretions had fine granular mercury staining within the cytoplasm of luminal epithelial cells. This mercury often surrounded the epithelial cell nucleus Figs –4. FaintThe ages of the patients, the density of lobules, the proportion of lobules, carcinomas and ductal carcinomas in situ (DCIS) with AMG staining, and the carcinoma biomarkers are shown in The relation of mercury in normal breast lobules to age and tumour characteristics are shown in Mercury was seen in carcinomas in 10 (23%) of the 44 samples where carcinoma was present. The mercury was usually within the lumen of carcinoma ducts. Mercury could be found within the carcinoma cells surrounding some of these lumens .In eight samples, foci of DCIS were present . In seveLA-ICP-MS was carried out on seven samples containing normal breast tissue  and on five carcinomas .Mercury was detected in all ten AMG-positive lobule, carcinoma, and DCIS samples, but not in the three AMG-negative lobule samples or in the AMG-negative carcinoma sample Figs –8. MercuCadmium was seen in a few lobules. Chromium was seen occasionally in lobules and in blood vessels. Iron was seen often in lobules and blood vessels, and in carcinomas. Lead was found occasionally in stroma or lobules, and often in carcinomas. Nickel was present in a few lobules, blood vessels and in carcinomas. Aluminium was localised in some lobules, and in some carcinomas.Non-mercury metals were detected on LA-ICP-MS in some normal breast lobules, some normal stroma and some carcinomas Figs –8. Metalaluminium, bismuth, cadmium, chromium, gold, lead, nickel and silver was seen in some samples, possibly due to contamination from elements introduced during formalin fixation and processing of tissue for paraffin embedding; of these elements, only chromium is present in the stainless steel blades used for microtomy, so these elements were not introduced at the section cutting stage. To eliminate from consideration potentially-contaminating elements, only elements localised to breast lobules, tumour cells, stroma or blood vessels were considered to be biologically significant. Bismuth, gold and silver were not localised to any of the breast compartments  are normally found in many capillaries, but in only a few lobules, in the human breast . It is lThe luminal epithelial progenitor cell is the proposed cell of origin for most breast cancers –53. DuriMercury persisted in luminal secretions in some of our post-menopausal breast samples, and the proximity of these secretions to the luminal epithelial cells would ensure continual exposure of these cells to mercury . On the Human exposure to environmental sources of mercury, for example from seafood consumption or dental amalgam fillings, is common . FurtherMercury within carcinomas was usually found in only a small proportion of tumour cells, probably in relation to mercury transporters such a breast cancer resistance protein being expressed in these cells . MercuryNon-mercury metals in our breast samples that have previously been implicated in the pathogenesis of breast cancer were aluminium ,65–67, cPrevious workers comparing exposure to metallic air pollutants to breast tumour estrogen and progesterone receptor status have reported either that arsenic and cadmium exposure increase ER/PR-negative cancer risk , that me++ cation from the mercurous, mercuric, and vapor forms of mercury is the proximate toxic form of the metal in tissues, and methylmercury is slowly converted to inorganic mercury in the body [This study has several limitations. (1) Autometallography demonstrates inorganic mercury bound to sulphides and selenides, but not organic mercury. However, the Hgthe body , so inorthe body ,60 and cthe body ,72. The the body ; howeverthe body  to compaIn conclusion, a large proportion of breast samples taken at mastectomy for carcinoma had normal breast lobules that contained mercury, as well as other toxic metals. A smaller proportion of breast carcinomas contained mercury and other toxic metals. Although our results do not provide an unequivocal link between mercury in normal breast lobules and breast cancer, it would seem prudent to limit exposure to mercury as much as possible by restricting the intake of larger predatory fish and considering alternatives to mercury-containing amalgam fillings , and to S1 Table(DOCX)Click here for additional data file.S1 FigBreast samples showing the absence of localised staining for Ag, Au and Bi. Phosphorus (P) staining indicates cellular density. Selected regions stained with autometallography (AMG) are indicated in the dashed boxes. Element distribution = A: absent, N: non-localising. Scale = counts per second .(TIF)Click here for additional data file.


An increasing number of studies have investigated the clinical epidemiology and outcomes of ventilator-associated pneumonia (VAP) in intensive care units. However, these findings have not been clearly defined in broad subgroups of mechanically ventilated adults. Hence, this protocol for a systematic review and meta-analysis is designed to better understand the clinical and epidemiological features of VAP in these patient populations by establishing its overall prognosis of and risk factors for morbidity and mortality and to determine the differences in clinical and economic outcomes between VAP and non-VAP patients.X2 and I2 statistics. In addition, sensitivity and subgroup analyses will be performed to ensure consistency of pooled results. The review protocol described herein is in accordance with the PRISMA-P standards.This present review will systematically search available full-text articles without date and language restrictions and indexed in PubMed, CENTRAL, CINAHL, Web of Science, and EMBASE databases. In addition, reference lists and citations of retrieved articles and relevant medical and nursing journals will be manually reviewed. Supplementary search in other databases involving trials, reviews, and grey literatures, including conference proceedings, theses, and dissertations, will be performed. Study investigators will be contacted to clarify missing or unpublished data. All prognostic studies meeting the pre-defined eligibility criteria will be included. The study selection, risk of bias assessment, data extraction, and grading of the quality of evidence will be carried out in duplicate, involving independent evaluation by two investigators with consensus or a third-party adjudication. The degree of inter-rater agreement will be calculated using the kappa statistic. For meta-analysis, dichotomous and continuous outcome measures will be pooled using odds ratios and standardized mean differences with 95% confidence intervals, respectively. The Mantel-Haenszel or inverse variance methods with random effects model will be used as a guide for analysis. The heterogeneity of each outcome measure will be assessed using both The investigation of the epidemiological profiles, prognostic factors, and outcomes associated with VAP is critical for the identification of high-risk groups of mechanically ventilated patients and evaluation of possible clinical endpoints. This may provide substantial links for improved VAP prevention practices targeting modifiable risk factors. Implications for future research directions are discussed.PROSPERO CRD42017048158The online version of this article (10.1186/s13643-019-1080-y) contains supplementary material, which is available to authorized users. The present review will not consider clinical criteria with radiologic findings as primary bases for VAP diagnosis due to its inherent clinical limitations and issues on sensitivity and specificity [Critically ill patients will be followed up for the development of initial episode of microbiologically confirmed VAP until extubation for more than 48 h, ICU discharge, or death. This event of interest will be regarded as the primary outcome measure. Secondary outcomes include duration of MV, ICU/hospital LOS, microbiological findings, antibiotic and hospital costs, and VAP mortality . Studies with clinically defined VAP outcome measures will be excluded. Clinically defined VAP studies may be regarded if study investigators only used a conventional diagnostic method in any of the following: clinical criteria  will be excluded.This present review will consider peer-reviewed articles written in languages other than English where translation is possible with the resources of our respective institution.There is no maximum length of follow-up or retrospective analysis that will be imposed for this review. However, to fulfill the criteria for VAP, case patients must be mechanically ventilated for ≥ 48 h at the time of enrollment; hence, studies not meeting this MV cutoff value will be excluded. This time window is crucial in distinguishing primary from secondary infections or in categorizing pneumonia as community-acquired or ventilator-associated. The durations of endotracheal intubation/MV prior to VAP development (time to VAP) and ICU/hospital LOS prior to death (time to death) will be determined.To identify potential search terms and retrieve the best set of results possible, a pre-determined MEDLINE search strategy was constructed using the PICO  equivalents: patients, exposures of interests, comparison, outcomes, and study design The Cochrane Central Register of Controlled Trials (CENTRAL) (via The Cochrane Library)host)Cumulative Index to Nursing and Allied Health Literature (CINAHL) Excerpta Medica dataBASE (EMBASE)The present review will exhaust all available full-text articles (without date and language restrictions) indexed in the following major scientific databases:The pre-determined MEDLINE search strategy will be adopted in searching relevant articles in all other databases. Modifications to indexing terms  for other databases will be applied as necessary.All reference lists of the included original articles and some relevant reviews will be manually screened to reduce the possibility of excluding important citations.® database to capture relevant citations that were possibly missed during the initial search. The PI (JMG) has chosen Scopus® because it is considered as the largest abstract and citation database of peer-reviewed literature, including scientific medical and nursing journals, books, and conference proceedings.The present review will perform citation tracking using the ScopusTo further find additional articles, a select number of medical and nursing journals focusing on infection control and intensive-critical care will be electronically handsearched from date of inception to present issue  and review (PROSPERO) registers will be carried out to identify ongoing or recently completed trials and systematic reviews, respectively. In addition, relevant studies in grey literatures, including conference proceedings and studies indexed in OpenGrey/ProQuest dissertations and theses databases, will be checked. Study investigators or corresponding authors will be contacted to clarify missing or unpublished data.An additional search in supplemented databases using trial  to independently cross-validate the abovementioned procedures following a search strategy decision algorithm  and subsequently by six trained RAs . Each RA will be assigned to specific databases including other resources  tool will be utilized  with consensus for each included study. A third person (AB) will be consulted if disagreements cannot be resolved. A total of three independent external reviewers  will be part of this process. Each will be given assigned studies by the PI (JMG) to reach the target review timeline. To illustrate the decisions for each risk of bias domain in every included study, a risk of bias summary 3 × 3 table will be created and this will be used for kappa calculations.General characteristics of the study;Sociodemographic and clinical characteristics of subjects;Outcome definition (VAP diagnostic criteria);Onset and incidence or prevalence of VAP among exposed and non-exposed subjects;Numbers of VAP-associated microorganisms;Risk factors associated with VAP and clinical outcomes of morbidity and mortality;VAP clinical outcomes including patient mortality, duration of MV, and ICU/hospital LOS; andAntibiotic treatment and hospitalization costs.For each included study, data from both published and unpublished reports will be independently extracted in duplicate by the PI (JMG) and one data extractor (RAF or RE) using a structured, pilot-tested, author-made data extraction form  will enter four important data items , check all the totals matching the published study (step 5), check the calculated effect size (step 6), and assess the validity of entered data (step 7) to ensure completeness and accuracy. For data with continuous outcome measures including pooled data set, similar procedures will be applied as appropriate. These will be validated by one RA (RAF or RE). Any disputes will be resolved by discussion.For data management, the PI (JMG) developed a platform using MicrosoftList of studies awaiting assignment.In handling missing or unpublished data, the study investigators will be consulted if applicable. A request to obtain missing or incomplete data from study investigators will be forwarded. All requests will be documented and included in the cases − crude mortalitycontrols/crude mortalitycontrols [All patient clinical information, including the outcomes of VAP-related morbidity and mortality, will be pooled using frequencies, percentages or proportions, and means (± SDs). Descriptive epidemiology will be employed for incidence density, prevalence rate, and proportional crude mortality. To estimate the number of patients with VAP, all new and pre-existing cases in a given period of time will be included and divided to the total population during the same time period. To estimate the proportional mortality attributable to VAP, relative risk increase (RRI) will be calculated using the following formula: crude mortalitycontrols . Chi-squcontrols : poor, sd) with 95% CI will be calculated for continuous outcomes to compare the duration of MV, ICU/hospital LOS, and antibiotic and hospitalization costs between VAP and non-VAP patients. Pooled estimates with 95% CI for each outcome measure will be calculated using random effects model. Forest plots will be constructed to illustrate a summary of findings from individual prognostic studies. Such analyses are essential to accurately estimate the overall association between the abovementioned variables. A statistically significant association will be considered if p value is < .05. A summary of the research questions and sample planned data analyses is presented in Additional file To combine all relevant studies investigating VAP epidemiological outcomes, a meta-analysis will be performed using Review Manager software  for Mac OS X, version 5.3. In this analysis, study weights for dichotomous and continuous outcomes will be generated using Mantel-Haenszel (M-H) and inverse variance (IV) methods, respectively. Due to the inclusion of case-control studies with dichotomous outcomes, odds ratio (OR) with 95% confidence interval (CI) will be calculated to identify risk factors imposed on VAP development and clinical outcomes of morbidity and mortality. Odds ratio will also be used to investigate VAP attributable mortality among patients with VAP. In addition, standardized mean difference  are more likely to acquire healthcare-associated infections (including VAP) following hospital admission compared to other age groups . An a priori subgroup analysis by age (in years) is planned for the following categories: ≤ 30, 30 to 60, 60 to 75, and ≥ 75.Type of illness severity. The severity of illness is defined as the extent of patient’s physiologic decompensation or organ system derangement measured by ICU severity scoring systems at admission, as patients with higher severity of illness index have higher risk of developing poor clinical outcomes . An a priori subgroup analysis by type of illness severity is planned for the following categories (based on previously defined cutoff points): according to acute physiology and chronic health evaluation (APACHE) II scores, < 20, 20 to 29, and ≥ 30 [Type of country by development status. This may refer to a country’s economic or trade status, as resources play a critical role on the incidence and risk factors for VAP development. The concept of resource inadequacy is one of the reasons why VAP rates can be substantially different in other settings. For instance, in developing countries, a larger number of VAP incidence are reported with varying risk factors involved than developed countries. An a priori subgroup analysis by country classification is planned for the following categories (based on previously defined status): developing, in transition, and developed .Type of patient population. This may refer to medical, surgical, trauma, and mixed ICU patients, as trauma patients might have greater need of mechanical ventilation compared to other ICU populations, therefore increasing their degree of exposure to VAP potential factors. An a priori subgroup analysis by type of patient population is planned for the following categories: medical (including coronary), surgical, cardiac surgical , neurosurgical, trauma surgical, trauma , burn, neurological, oncological, and mixed ICU patients .Type of disease onset. Onset (in days) may refer to the initial manifestations of VAP as examined by the physician intensivist or other outcome assessors. The pathogenesis and risk factors associated with VAP are dependent on the disease onsets. For this review, a definite cutoff period will be provided to categorize VAP onset based on the mean cumulative reports of primary studies, as VAP onset definitions may differ between studies. An a priori subgroup analysis by type of onset is planned for the following categories: early-onset and late-onset VAP.Candida as a pathogen in our immunocompromised population. An a priori subgroup analysis by type of causative microorganisms is planned for the following categories : high-risk and low-risk microorganisms.Type of causative microorganisms. This may refer to the pathologic agent causing VAP, as the attributable risk of VAP appears to vary with infecting pathogens, considering microbial virulence. This review will not consider Method of diagnosis. This refers to the different methods of VAP diagnosis, considering clinical criteria and radiological findings with microbiological confirmation  obtained via bronchoscopic and non-bronchoscopic investigations. This review will take necessary precaution when interpreting subgroup analysis for studies reporting unspecific microbiological confirmation. An a priori subgroup analysis by method of diagnosis is planned according to the following categories: blinded outcome assessment with adjudication, unblinded outcome assessment with adjudication, blinded outcome assessment without adjudication, and unblinded outcome assessment without adjudication.Presence and type of immune suppression. This may refer to patients who are immunocompromised, as patients with human immunodeficiency virus (HIV) infection or acquired immune deficiency syndrome (AIDS), oncologic conditions, or history of immunosuppressant drug use have increased risks of developing VAP and clinical failure  following institution of invasive mechanical ventilation. An a priori subgroup analysis by presence (or absence) and type of immune suppression is planned for the following categories: according to presence (or absence), immunocompromised and non-immunocompromised; according to type, deliberately induced (by immunosuppressive medications) and non-deliberately induced immunosuppression .Presence and type of initial antimicrobial therapy. This may refer to patients who received empirical antimicrobial treatment(s). The term “empirical” is defined as treatment given prior to obtaining susceptibility results of microbial analysis. An a priori subgroup analysis by presence (or absence) and type of initial antimicrobial therapy is planned for the following categories: according to presence (or absence), with initial antimicrobial therapy and without initial antimicrobial therapy; according to type, appropriate and inappropriate empiric antibiotics.Presence and type of VAP protocol. This may refer to studies reporting the presence or absence of a VAP prevention protocol, as the number of VAP prevention strategies  that are routinely available may influence VAP occurrence. An a priori subgroup analysis by presence (or absence) and type of VAP protocol is planned for the following categories: according to presence (or absence), units with VAP prevention strategies and units without VAP prevention strategies; according to types, recommended VAP bundles and modified VAP bundles.If adequate data pertaining to patient age, type of illness severity, type of country (by development status), type of study population, type of onset, type of causative microorganisms, methods of diagnosis, and presences (as opposed to absences) and types of immune suppression, prior or initial antimicrobial therapy, VAP protocol are reported from the included studies, the review investigators plan to carry out a priori subgroup analyses between studies to explain inconsistency between important subgroups. The following are definitions of subgroup analyses with corresponding categories, considering the study hypotheses and pre-specified direction of effect:The abovementioned subgroup analyses will be formally applied to all outcome measures even if the variability appears to be explained by chance. If studies yield large inconsistent results due to serious or unexplained heterogeneity, the review investigators plan to rate down the quality of evidence for each outcome measure using the recommended approach .Furthermore, this present review anticipates that all these proposed a priori subgroup analyses might not be adequately reported in the included studies. Hence, additional caution will be regarded when exploring and interpreting the review results, as underpowered subgroup analysis, in full awareness of the review investigators, may lead to an incorrect conclusion.A sensitivity analysis will be performed in studies with low risk of bias to ensure the consistency of pooled results concerning the association between variables. This approach involves the omission of outliers or reports with a higher risk of bias, indicating less rigorous designs. These may include studies with methodological and statistical issues , non-equivalent controls , inconsistent VAP definition or questionable diagnostic criteria for VAP, and studies that did not report/assess the presence lung infection at admission. Pooled results with 95% CIs will again be analyzed to test for the robustness of association. The conclusion would be regarded as stable if the results do not change substantially, indicating a higher degree of certainty. The comparison of original risk estimates with post-assessment results will be displayed in a summary table.If there are at least ten relevant studies with robust designs, a visual asymmetry assessment using funnel plot will be generated to detect publication bias .The present review will use the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach  to assesVentilator-associated pneumonia is a complex and multifactorial clinical condition associated with high morbidity and mortality and has a staggering impact on healthcare costs. This premise is largely recognized in the international body of literature  with litThe relevance of this present review highlights the urgent need to identify medium- and high-risk critically ill patients targeting important risk factors for modification and stratified management. The review results may be used as a springboard for tool development  and clinical protocol design, which raises a number of critical issues for research, policy, and clinical practice considerations. Moreover, the review results are critical to shed light on other potential predictors of VAP morbidity and mortality outcomes, warranting further investigation.This present review, of course, has inherent strengths and limitations. Strengths include an exhaustive search of evidence, published or unpublished, without date and language restrictions in five major scientific databases and other sources; a comprehensive risk analysis plan with a justified number of a priori subgroup analyses (including a priori hypotheses with a pre-specified direction); and the utilization of the GRADE approach to rate the quality of evidence. Limitations relate to the anticipated variability in critically ill populations, diagnostic methods used, and study designs reported from included studies; paucity of data regarding the clinical epidemiology and outcomes of VAP in some countries; and the lack of subgroup and adjusted analyses of prognostic factors within-study comparisons, as no individual patient data will be obtained.With this initiative effort to provide a substantial link for the successful implementation of the VAP prevention practices in critical care settings, the key players such as healthcare practitioners, researchers, policymakers, and hospital administrators are hoped to make productive use of the review results, which is soon to be disseminated following this protocol.Additional file 1:Preferred Reporting Items for Systematic review and Meta-Analysis for Protocols (PRISMA-P) 2015 guidelines. (DOCX 44 kb)Additional file 2:Supplementary tables. (DOCX 132 kb)Additional file 3:Flow diagrams. (DOCX 402 kb)Additional file 4:Data forms. (DOCX 161 kb)


The most fundamental process for the perpetuation of a species is the transfer of information from parent to offspring. Although genomic DNA contributes to the majority of the inheritance, it is now clear that epigenetic information −information beyond the underlying DNA sequence − is also passed on to future generations. However, the mechanism and extent of such inheritance are not well-understood. Here, I review some of the concepts, evidence, and mechanisms of intergenerational epigenetic inheritance via sperm small RNAs. Recent studies provide evidence that mature sperm are highly abundant in small non-coding RNAs. These RNAs are modulated by paternal environmental conditions and potentially delivered to the zygote at fertilization, where they can regulate early embryonic development. Intriguingly, sperm small RNA payload undergoes dramatic changes during testicular and post-testicular maturation, making the mature sperm epigenome highly unique and distinct from testicular germ cells. I explore the mechanism of sperm small RNA remodeling during post-testicular maturation in the epididymis, and the potential role of this reprograming in intergenerational epigenetic inheritance. The possibility that our life experiences can influence phenotypes in our descendants has tremendous implications for basic biology and public health and policy . Indeed,Challenging these preexisting notions about inheritance, many recent studies in mammals provide evidence that paternal exposure to different environmental stressors, such as diet, psychological stress, toxicants, etc., can influence offspring phenotypes, and sperm epigenome plays a pivotal role in the transmission of such phenotypes. Here, I review recent studies on intergenerational inheritance via male germline, discuss the potential role of small RNAs as epigenetic information carriers in sperm, and explore the mechanisms of such inheritance. Intergenerational inheritance involves transmission of parental environmental effects via exposure of the developing embryo and/or germ cells to the environmental insult (transmission from parent to offspring or grandoffspring). For instance, if a female (F0) pregnant with male offspring is exposed, both its embryo (F1) and germ cells of the embryo (F2) are directly exposed. On the other hand, transgenerational inheritance affects generations that were not directly exposed to the environmental insult (F3 and onwards) . I primaMultiple studies in mammals over the past decade have demonstrated that parents pass on information about their environment to their children. In humans, undernutrition in parents has been linked to metabolic diseases in children, with epidemiological studies of the Dutch Hunger Winter of 1944–1945  providinA large number of laboratory studies in rodents have linked paternal treatment regimes with changes in offspring phenotype, such as dietary alterations , psycholIn rats, paternal consumption of high-fat diet leads to glucose intolerance in F1 female offspring . Similarin utero environment programs offspring for a similar environment after birth, and thus, links poor fetal and infant growth with increased risk of metabolic diseases in adults born in an unmatched ex utero environment  and foster mothers, where the only contribution from parents is their gametes, allows a direct test of whether paternal environmental information is passed via sperm. A few recent studies reported the effects of paternal exposure  on offspring generated via IVF, similar to those seen in offspring produced using natural mating, suggesting that paternal environmental information is indeed transmitted by sperm , which is one of the most well-characterized epigenetic information carriers. For instance, cytosine methylation is required for germline epigenetic inheritance of imprinted genes — genes that are expressed from only the maternal or paternal alleles and thus, retain the “memory” of the gender of germline they developed from C. elegans  pos elegans . It is i elegans .Small RNAs can be distinguished based on their biogenesis and biological function, which in turn is often determined by their size and specific sequence characteristics . miRNAs O methylation  clade of Argonaute proteins , are highylation . Importahylation . Mechanihylation , and poshylation .Tetrahymena thermophila, RNase T2 family endonucleases process tRNAs (RNase5) cleaves tRNAs (tRNA-fragments (tRFs) have only recently been studied in depth , and aress tRNAs , whereases tRNAs . tRFs ares tRNAs . For inses tRNAs . Treatmees tRNAs . Functioes tRNAs  and tranes tRNAs , ribosomes tRNAs , targetees tRNAs , regulates tRNAs , and reges tRNAs .in situ hybridization, quantitative real-time PCR, microarrays, and RNA high-throughput sequencing, studies report that sperm RNAs are localized in specific compartments. Small RNAs such as miRNAs and tRFs have been sequenced in detergent-treated-sperm heads  to induce early life stress. The MSUS mice showed depressive-life behavior, and such behavior was transmitted to their offspring. Injection of RNAs purified from sperm of MSUS mice into one-cell control embryos lead to the transmission of a subset of the behavioral and metabolic trait of MSUS mice to their offspring. This study provided the first direct evidence that paternal environmental information is transmitted by sperm RNAs, and identified a specific set of miRNAs that change in MSUS sperm in response to stress . AnotherOverall, multiple recent studies suggest that various environmental exposures direct distinct small RNA changes in sperm —diet mainly affects tRFs, while psychological stress influences miRNAs. Whether such differences arise from variation in the methods used for sperm small RNA profiling by different groups or distinct environmental insults are signaled to different classes of small RNAs is not clear. Irrespective, these studies provide strong evidence that small RNAs are environmentally-responsive epigenetic molecules in sperm. Moreover, a recent study reported that both small and long RNAs influence paternal exposure phenotypes of early life trauma in offspring, with long and small RNAs regulating different behavioral traits . AlthougWhether one epigenetic mark is more potent at responding to a certain environmental insult is still not clear. Depending on the type of environmental exposure and the developmental timing of exposure, one epigenetic mark might be more responsive compared to the other. For instance, sperm of mice fed a low protein diet from birth to weaning displayed changes in DNA methylation at ribosomal DNA (rDNA) , howeverThe ability of environmental conditions, such as diet, to influence phenotypes in future generations requires that environmental exposure induces changes in the epigenome of gametes. The nature and mechanism of shaping and altering the epigenome of mammalian gametes are not well-understood. One possibility is that somatic cells communicate information to the gametes and, thus, to progeny. Although the Weismann barrier has long been thought to be a problem for such communication , over thPost-testicular sperm maturation takes place in the epididymis, a long, convoluted tubular organ where sperm enter after exiting the testis. Intriguingly, recent studies report that sperm small RNA payload undergoes dramatic changes during epididymal transit. Deep sequencing of small RNAs from sperm at various stages of development revealed that while testicular sperm populations such as spermatocytes, round spermatids, and mature testicular spermatozoa are highly enriched in piRNAs , mature During the process of spermatogenesis, spermatozoa shed most of their cytoplasm as residual body  and in tin vitro reconstitution studies revealed that epididymosomes can deliver small RNAs to relatively “immature” testicular spermatozoa , as has been seen in other mammalian cells . FinallyThe functional significance of epididymis specific small RNA remodeling during post-testicular maturation is not well-understood. Recent studies report that small RNA shipment from the epididymis to sperm is essential for embryo implantation . EmbryosAn understanding of how environmental conditions influence epigenetic marks in the gametes is essential to elucidate how paternal environment affects phenotypes in offspring. As discussed above, numerous studies provide evidence that paternal environmental conditions, such as altered diet or stress, influence small RNA levels in sperm, however, the mechanistic understanding of how any of the environmental exposures noted above cause specific changes in sperm small RNA levels is largely lacking. Broadly, paternal environment can affect sperm small RNA levels by (1) regulating the transcription of small RNA precursors, (2) regulating small RNA processing, (3) modulating small RNA decay/stability, (4) influencing sorting of small RNAs into epididymosomes, and (5) affecting delivery of epididymosomal cargo to sperm.N-2-methylguanosine (m2G) (5s2U) and formylcytidine (f5C) (S-adenosyl methionine (SAM), resulting in global changes in levels of specific tRNA modifications and alterations in tRNA processing and/or stability   and micene (f5C) . Not onlne (f5C) , they pone (f5C) . The modne (f5C) , dietarytability . HoweverCommunication between somatic cells and germ cells is not only crucial for the maintenance of germ cell integrity, as suggested in model organisms , it alsoHow do changes in sperm small RNAs in response to various environmental conditions result in the transmission of paternally acquired phenotypes in offspring? Mature sperm deliver their RNAs to the oocyte at the time of fertilization, and hence, potentially regulate early embryonic gene expression to affect offspring phenotypes. Indeed, recent studies provide evidence that sperm small RNAs play important, and in some cases, essential role during early development. Microinjection of a synthetic 5′ fragment of tRNA-GlyGCC (tRFGlyGCC) or an inhibitor of tRFGlyGCC −among the most abundant small RNAs in sperm, which is up-regulated in low protein sperm− in control zygotes revealed that tRFGlyGCC represses transcription of a specific set of MERVL retroviral element-driven genes in early embryos . While tWhile these studies examined the functions of specific tRFs, another group examined the broad functions of 30–40 nts sperm small RNAs (comprising chiefly of tRFs). Microinjection of 30–40 nts small RNAs purified from sperm of mice fed high-fat diet in control zygotes resulted in altered expression of multiple genes involved in metabolic regulation in eight-cell embryos and blastocysts, suggesting that a cascade of gene expression changes in preimplantation embryos potentially leads to metabolic phenotypes in adult pancreatic islet cells . ImportaFirst, the RNAs that change in response to environmental conditions are not particularly abundant in sperm. For instance, the most abundant tRFs in sperm are estimated to be at ∼100–100,000 molecules per sperm  or pre-loading in some RNA–protein effector complexes. Furthermore, epididymosomes adhering to the surface of the sperm might play important regulatory roles in delivering additional RNAs to the female reproductive tract or the oocyte , are some common pathways targeted by distinct classes of small RNAs? Finally, what is the mechanism of transgenerational inheritance? Since mammals lack a known RNA-dependent RNA polymerase, sperm small RNAs potentially influence early embryonic chromatin and/or DNA methylation states to transmit epigenetic information to the future generations. With recent advances in the field of single-cell omics, a more in-depth examination of sperm and embryonic epigenome will help shed light on these and other outstanding questions, and elucidate the mechanism of transgenerational inheritance of acquired traits.The author confirms being the sole contributor of this work and has approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As neurons are one of the most highly polarized cells in our body, they require sophisticated cellular mechanisms to maintain protein homeostasis in their subcellular compartments such as axons and dendrites. When neuronal protein homeostasis is disturbed due to genetic mutations or deletions, this often results in degeneration of neurons leading to devastating outcome such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and fragile X syndrome (FXS). Ribonucleoprotein (RNP) complexes are macromolecular complexes composed of RNA binding proteins (RBPs) and their target RNAs. RBPs contain RNA binding domains and bind to RNA molecules via specific sequence motifs. RNP complexes have various functions in gene expression including messenger RNA (mRNA) trafficking, RNA processing and silencing. In neurons, RBPs deliver specific sets of mRNAs to subcellular compartments such as axons and dendrites to be locally translated. Mutations or deletions in genes coding for RNPs have been reported as causes for neurological disorders such as SMA, ALS, and FXS. As RBPs determine axonal or dendritic mRNA repertoires as well as proteomes by trafficking selective mRNAs and regulating local protein synthesis, they play a crucial role for neuronal function. In this review, we summarize the role of well-known RBPs, SMN, TDP-43, FUS, and FMRP, and review their function for local protein synthesis in neurons. Furthermore, we discuss their pathological contribution to the neurological disorders. Dysfunctional RNA processing in neuronal tissue is often observed in neurodegenerative diseases and plays a crucial role in neuronal pathology. This implies the essential part of ribonucleoprotein (RNP) complexes, that play an important role in RNA metabolism. RNP complexes contain RNA molecules with specific sequences/motifs bound to RNA-binding proteins (RBP). RNPs regulate eukaryotic gene expression in various cellular mechanisms including formation of ribosomes, spliceosomes and RNA-induced silencing complexes (RISCs) , and fragile X syndrome (FXS). Protein synthesis is an essential cellular process for function, development and survival. In this process, a coordinated interplay between proteins and RNA is crucial to produce functional proteins. Mutations in genes involved in RNA metabolism lead to various diseases. Interestingly, many of these mutations are associated with neurodegenerative, neurodevelopmental and neuromuscular disorders. Because neurons are post-mitotic, highly polarized and larger in size compared to other types of cells, less severe mutations in genes involved in RNA metabolism may cause dysfunction in neurons, but other types of cells might tolerate these mutations. Dysfunction of RNA-related processes including tRNA synthesis, ribosome biogenesis, sequestration and mislocalization of mRNAs has been reported as pathological mechanisms in neurological disorders  to neuronal projections is often located in the 3′-untranslated region (UTR) of the mRNA  and tRNP granule  proteins can be synthesized in mature axons SMN1 gene encoding the full length SMN protein. Humans possess a duplication of the SMN1 gene, SMN2, which produces a transcript lacking exon 7 due to a single point mutation located in exon 7  is mis-spliced in motor neurons of SMA mice, and notably it is important for organizing acetylcholine receptors at the NMJ  has been developed to restore SMN protein levels. Briefly, ASO binds to an intronic silencer in intron 7 and thereby it enhances the inclusion of exon 7 of SMN2 , annexin 2 (Anxa2), and neuritin/cpg15 , TDP-43, FUS and a hexanucleotide expansion repeat in Chromosome 9 Open Reading Frame 72 (C9orf72)  and around 1% sALS  via RAN translation. These dipeptide proteins form aggregates in neurons and cause cellular toxicity , which is characterized by insoluble inclusions containing TDP-43 aggregates  is a DNA/RNA-binding protein and mutations in FUS are implicated in ALS (about 4% of fALS and <1% of sALS) and FTLD  in FUS  Da Cruz. Due to Interestingly, it has been shown that motor and cognitive deficits in ALS are caused by increase of mutant FUS in axonal compartments, that lead to reduced intra-axonal translation and therefore to synaptic dysfunction   is a genetic disorder with intellectual disability caused by loss/deficiency of fragile X mental retardation protein, FMRP. FMRP is a polyribosome-associated RNA binding protein, which is encoded by the fragile X mental retardation 1 gene  and one RGG domain, which can bind to poly-riboguanylic acid . This process is important to NGF induced axonal outgrowth , and Mmp-9  (Schaeffer et al., in vivo studies are required, this data unveiled a new cellular mechanism underlying neuronal activity induced FMRP-mediated protein synthesis at the synaptic site. Taken together, FMRP regulates local mRNA translation in neurons via multiple layers of cellular mechanisms.The cellular mechanism of dendritic local translation has been intensively studied as well as the role of FMRP in postsynaptic protein synthesis. FMRP participates in dendritic local translation in various steps . First, Here, we have reviewed the current knowledge about the role of RNA binding proteins in local protein synthesis in subcellular compartments of neurons. While there are many more RNA binding proteins in neurons and their roles are equally important and interesting, we have only covered the most well-known four proteins, SMN, TDP-43, FUS, and FMRP. Importantly, dysregulation of these proteins caused by mutations and deletions is often associated with human neurological disorders. As neurons are highly polarized, often large in size, and their function is transient, neuronal protein homeostasis needs to be regulated timely and spacially. Neurons solve this issue by quick production of proteins at local sites when new proteins are required. For this process, RNA binding proteins traffic mRNAs and store them in mRNP granules in synaptic compartments, and upon stimulus, mRNA can be released to produce new proteins. As this process is important for neuronal function, dysfunction of RNA binding proteins often cause neurological disorders, in the worst case, leading to organismal death. While many underyling cellular mechanisms have been characterized, the molecular functions and biophysical properties of RNA binding proteins are still not fully understood. Furthermore, it will be important to understand how dysfunctional RNA binding proteins and impaired local translation contribute to pathology of human diseases.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TERT gene in most normal human somatic cells, whereas activated only in small subsets of cells, including stem cells, activated lymphocytes, and other highly proliferative cells. In contrast, telomerase activation via TERT induction is widespread in human malignant cells, which is a prerequisite for malignant transformation. It is well established that TERT/telomerase extends telomere length, thereby conferring sustained proliferation capacity to both normal and cancerous cells. The recent evidence has also accumulated that TERT/telomerase may participate in the physiological process and oncogenesis independently of its telomere-lengthening function. For instance, TERT is shown to interact with chromatin remodeling factors and to regulate DNA methylation, through which multiple cellular functions are attained. In the present review article, we summarize the non-canonical functions of TERT with a special emphasis on its cross-talk with epigenetics: How TERT contributes to epigenetic alterations in physiological processes and cancer, and how the aberrant epigenetics in turn facilitate TERT expression and function, eventually promoting cancer either initiation or progression or both. Finally, we briefly discuss clinical implications of the TERT-related methylation.Telomerase, an RNA-dependent DNA polymerase with telomerase reverse transcriptase (TERT) as the catalytic component, is silent due to the tight repression of the  TERT gene is stringently repressed in most human somatic cells, which consequently results in telomerase silencing ..71].TERT transcription and expression. It is well characterized that the TERT harbors a single promoter embedded in a CpG island (−1800 to +2300 relative to ATG), but the TERT promoter is unmethylated in normal human cells, whereas methylated in malignant cells [TERT gene is transcriptionally de-repressed. TERT induction in turn further facilitates the THOR hypermethylation, promoting cancer progression  impede reliable utility of the direct TERT expression or telomerase activity assay as routine cancer diagnostic or disease monitoring tools. The hypermethylated TERT promoter has been shown unique to human cancer, as described above ,72, and In addition, the TERT promoter hypermethylation has been implicated in cancer prognostication. The association between the TERT promoter hypermethylation and poor outcomes or progression was reported in brain tumors, adrenocortical carcinoma, and other cancers ,80. The From the therapeutic point of view, targeting the interaction between TERT and epigenetics may contribute to the development of novel anti-cancer strategies. TERT inhibition results in the disruption of the TERT-DNMT3B and TERT-β-catenin loop signals, thereby exerting detrimental effects on cancer cells. On the other hand, telomerase inhibition can be achieved by targeting DNMT3B or β-catenin or both, or re-activating PTEN and other TSGs.TERT gene de-repression/telomerase reactivation is essential to malignant transformation of human cells. Telomere lengthening is the canonical function of TERT or telomerase; however, the evidence has also accumulated that TERT displays much broader activities independently of its telomere elongation. The cross-talking between TERT and epigenetics leads to the interaction of TERT with key signaling pathways, including DNMT3B, PI3K-AKT, and WNT/β-catenin. These interactions are of importance for physiological and aging processes. In cancer, these cross-talks form positive feedback loops to amplify oncogenic signaling outputs. The disruption of these loops or networks by targeting TERT may be a novel anti-cancer strategy and are expected to be detrimental to cancer cells. Therefore, these mechanistic understandings of the TERT contribution to various cancer hallmarks should be helpful to the rational development of TERT-based tools for clinical diagnosis and management of cancer patients.Human beings have acquired a robust cancer resistance strategy by repressing telomerase and maintaining shorter telomere over a long evolution period, however, such a cancer protective mechanism is a trade-off for aging/degenerations. Appropriate TERT/telomerase expression is required for stem cell biology, tissue homeostasis, and physiological aging, while the aberrant


Mastomys natalensis rodents and other rodent species. Due to a high fatality rate, lack of treatment options and difficulties with prevention and control, LASV is one of the high-priority pathogens included in the WHO R&D Blueprint. The WHO LASV vaccine strategy relies on availability of effective diagnostic tests. Current diagnostics for LASV include in-house and commercial (primarily research-only) laboratory-based serological and nucleic acid amplification tests. There are two commercially available (for research use only) rapid diagnostic tests (RDTs), and a number of multiplex panels for differential detection of LASV infection from other endemic diseases with similar symptoms have been evaluated. However, a number of diagnostic gaps remain. Lineage detection is a challenge due to the genomic diversity of LASV, as pan-lineage sensitivity for both molecular and immunological detection is necessary for surveillance and outbreak response. While pan-lineage ELISA and RDTs are commercially available (for research use only), validation and external quality assessment (EQA) is needed to confirm detection sensitivity for all known or relevant strains. Variable sensitivity of LASV PCR tests also highlights the need for improved validation and EQA. Given that LASV outbreaks typically occur in low-resource settings, more options for point-of-care testing would be valuable. These requirements should be taken into account in target product profiles for improved LASV diagnostics.Lassa fever virus (LASV) causes acute viral haemorrhagic fever with symptoms similar to those seen with Ebola virus infections. LASV is endemic to West Africa and is transmitted through contact with excretions of infected  Diagnostics are key to effective prevention and control of Lassa fever virus (LASV), a WHO R&D Blueprint priority pathogen that causes acute viral haemorrhagic fever.Current diagnostics include laboratory-based serological and nucleic acid amplification tests as well as rapid diagnostic tests.Challenges to LASV diagnostics include commercial availability of clinically validated pan-lineage tests, few options for point-of-care testing, differentiation from other agents that cause similar symptoms and a need for improvements to test validation, regulation and external quality assessment; target product profiles for LASV diagnostics should be refined to take into account these needs.Lassa fever virus (LASV) causes acute viral haemorrhagic fever (VHF) and is endemic to West Africa. Every year, approximately 100 000–300 000 people contract LASV, and 5000 people die from the infection.Arenaviridae family. First identified in 1969 in Nigeria,LASV is a single-stranded RNA virus of the Mastomys natalensis multimammate rodents are the most common rodent across the African continent, found predominantly in rural areas and human dwellings.M. natalensis populations.Hylomyscus pamfi and Mastomys erythroleucus have been shown to host LASV.M. natalensis rodents readily colonise human areas where food is stored, contributing a significant risk for spillover, especially in communities with poor sanitation or crowded living conditions.Human-to-human transmission is less common, but LASV can be spread through direct contact with bodily secretions of persons infected with Lassa fever, presenting a higher risk for healthcare and humanitarian personnel,Prevention of Lassa fever relies on promoting good community hygiene to reduce the potential for human-rodent contact. Measures to discourage rodents include storing grain and other food in rodent-proof containers, good hand and food hygiene, disposing of garbage away from the home, maintaining clean households and trapping rodents or employing cats as a natural deterrent.Healthcare settings should employ standard infection prevention and control precautions when caring for patients.Early detection is critical for LASV containment, and a strong surveillance system is necessary to support interventions in endemic or ‘hot spot’ areas for LASV and other VHF.Early detection of LASV infection is difficult as the clinical course is highly variable, with symptoms ranging from 2 to 21 days postinfection. Lassa fever symptoms can mimic other endemic diseases such as malaria, typhoid fever and other VHFs.Around 80% of people who become infected with LASV are asymptomatic or have mild symptoms of gradual fever, weakness and malaise, which often go unreported.In non-fatal cases, the fever subsides and the patient’s condition improves over 1–3 weeks, although renal damageMembers of the arenavirus family are composed of an ambisense RNA genome and a nucleoprotein (NP), surrounded by a lipid envelope and a glycoprotein. The LASV genome consists of two single-stranded RNA segments: the small segment  encodes the NP and the glycoprotein precursor (GPC); the large segment  encodes the RNA-dependent RNA polymerase (LP) and the matrix protein (Z).Once thought to be genetically stable, considerable genomic variation has been detected for geographically distant LASV strains, with phenotypic differences even among closely related isolates. LASV phylogeny is far more genetically diverse than Ebola virus in that <75% sequence is conserved for LASV compared with >97% for Ebola.The recommended course for clinical management of LASV is general supportive care with management of symptoms. There is no approved antiviral treatment; however, evidence indicates that Lassa responds to ribavirin if administered at early onset of symptoms. Oral ribavirin has been used for postexposure prophylaxis for persons at high risk of secondary infection.Favipiravir is another broad-spectrum RNA inhibitor (licensed for influenza) that has broad-spectrum activity against RNA viruses and has been shown to decrease Lassa viremia in animal models.in vitro studies.Small interfering RNA particles (siRNA) can been shown to inhibit LASV replication system for Monoclonal antibodies specific for LASV neutralisation cloned from West African Lassa fever survivorsCurrently, there is no vaccine that protects against LASV, and there are no vaccines available for use in animals to reduce zoonotic transmission.A number of different platform technologies have been evaluated as potential Lassa fever vaccines, including virus-like particles,Laboratory diagnosis of LASV infection is made by detection of the virus (culture), LASV RNA, LASV-specific IgG or IgM antibody response or LASV antigens shed during replication. LASV RNA is detected using a nucleic acid amplification test, which can include techniques such as PCR, loop-mediated isothermal amplification (LAMP) and strand displacement assays. Antibodies and antigens can be detected by indirect immunofluorescence assay test (IFA or IIFT), western blot (WB), ELISA or rapid diagnostic test (RDT) formats. Active infections can be diagnosed by virus isolation, PCR, LASV antigen positivity or IgM, along with clinical symptoms consistent with Lassa fever.Diagnostic test types differ in complexity, infrastructure requirements and appropriateness for a rapid response. An overview of the implementation requirements for the range of diagnostics available for LASV is shown in The majority of international laboratories use in-house LASV assays see , with reLassa lineage diversity must be considered in the choice of PCR primers and serology antigens, as test sensitivity relies on homology between the unknown specimen and the molecular probes or proteins used for detection. As more lineages and clades are revealed, and as mutations occur, tests will need to be revalidated and enriched to encompass variations observed at the nucleotide (20%–25%) and amino acid (8%–12%) levels.LASV can be found in many body fluid compartments during infection besides blood, including urine,3–104 copies/mL, which clears within 21 days for survivors.8 copies/mL typically progress to multiorgan failure and shock, with viremia increasing to 108–109 copies/mL days before death;3 TCID50/mL.Viral titre is highly predictive of disease outcome.Fatality has also been linked to suppressed or diminished IgM response throughout infection, supporting the hypothesis that an early and vigorous humoral response is critical to surviving infection.Molecular diagnostic tests  are designed to detect highly conserved regions of the pathogen genome and are commonly used as the most sensitive method to detect active infection. Molecular diagnostic tests for LASV generally target the S genome segment encoding the GPC or the NP regions . Tests t10.1136/bmjgh-2018-001116.supp2Supplementary dataSerological tests can be used to detect IgM and IgG antibodies raised against LASV antigens as well as direct capture and detection of LASV antigens. LASV NP, GP and Z proteins have been shown to be immunogenic.LASV antigen detection has been demonstrated to be a robust method for detection of active infection. While genetic sequence diversity and minor genetic mutations typically have less of an impact on protein sequence, early antigen tests have shown some variation in LASV lineage sensitivity. In tandem, detection of LASV NP antigen and anti-LASV IgM demonstrated 88% sensitivity and 90% specificity for early stage infection, sufficient for diagnosis in ~90% of PCR-positive cases.Serology test platforms for LASV include IFA/IIFT, ELISA, WB, multiplex bead assays and one RDT . IFA tes10.1136/bmjgh-2018-001116.supp3Supplementary dataLASV RDTs, which leverage the same antibody/antigen capture agents as an ELISA but packaged in a stripped-down lateral flow format, can play an important role for patient care and outbreak response in outlying laboratories and clinics. A dipstick-based RDT for LASV has been developed that detects NP from fingerstick whole blood specimens.At the early stages, the symptoms of Lassa fever can mimic other endemic diseases such as malaria, typhoid fever and other VHFs. A syndromic approach that tests for pathogens based on a syndrome such as VHF, using multiplex panels to quickly identify or eliminate likely pathogens from a single specimen, could be more effective in expediting LASV outbreak detection.Several groups have demonstrated multiplex PCR assays for differential detection of VHF including Lassa. Real-time RT-PCR assays were developed for 28 VHFs to be processed as 4-plex reactionsA multiplex bead-based immunoassay platform for differential diagnosis using antigens and IgM for Lassa and Ebola demonstrated greater detection sensitivity for LASV GP antigen and IgM  than conventional ELISA.As laboratory-developed protocols are generally not manufactured for distribution or regulated by an international agency, there remains a need for commercial pan-LASV tests with assured and reproducible quality that can be procured by any international laboratory or agency. The ideal LASV diagnostic test would detect all known LASV lineages with high sensitivity. While PCR enables the most sensitive platform for early detection, the high genetic diversity of LASV may result in false negatives for older tests, and to date, only a few attempts at pan-LASV PCR probe sets have been demonstrated. And while antigen tests are typically less dependent on minor genetic mutations, early results have shown some variation in lineage sensitivity, with further work underway to demonstrate pan-Lassa validation.Validation and quality assessment can be valuable in evaluating the entire testing process, including sample preparation, method of amplification or capture, detection methods and lineage sensitivity. Even for identical protocols, laboratory proficiency can significantly impact the quality of results. In 2004 and 2015, external quality assessments (EQAs) were conducted by the European Network for Diagnostics of Imported Viral Diseases using well-characterised Lassa panels for 24 international laboratories.Sourcing specimens for clinical validation can be a major roadblock for both diagnostic development and quality control. International reference standards and proficiency panels could assist development and validation of diagnostic tests and also help improve laboratory proficiency and EQA efforts towards quality. Several international reference institutes, including the WHO International Biological Reference custodian laboratories, could be sources for specimens for development and validation see . As new 10.1136/bmjgh-2018-001116.supp1Supplementary dataDiagnostics are essential for the recognition and control of outbreaks of LASV, one of the most widespread and genetically diverse agents of VHF. Improved LASV tests are needed for endemic clinical management, outbreak response and vaccine and therapeutic clinical trials. Surveillance across all LASV lineages and regions will continue to be important to rapidly identify ‘hot spots’ for intervention and containment, and to monitor genetic and geographical shifts in both human and animal reservoir populations. For vaccine development, diagnostics are fundamental to detecting and differentiating an infection challenge from vaccine immunity. Finally, there is strong consensus that early diagnosis and treatment increases the survival rate from LASV infection.This review has identified test resources for Lassa molecular diagnostics and serology and further described a large number of in-house LASV tests used across the international community. However, several gaps identified in the 2016 WHO R&D Blueprint remain.Given the challenges identified for LASV detection in endemic and often low-resourced settings, rapid on-site diagnosis of suspect cases may bridge the gap. A range of assays have been already developed for commercial point-of-care platforms, thus it is possible that current LASV assays could be adapted into a cartridge-based format. The combination of rapid, point-of-care antigen detection with a point-of-care confirmatory test has been suggested as an ideal implementation for early case detection and outbreak response.Each of the aforementioned diagnostic use scenarios have different performance requirements for text complexity, sensitivity, specificity and turnaround time. Development of new and improved LASV diagnostics could be facilitated by a clearly defined set of use cases to describe where and how diagnostic tests are most needed, coupled with TPPs with detailed performance characteristics for the highest priority molecular and immunodiagnostics.


Metabolic syndrome (MetS) is a mounting epidemic worldwide. MetS can start in early life, in a microenvironment that is now known as the developmental origins of health and disease (DOHaD). The concept of DOHaD also offers opportunities for reprogramming strategies that aim to reverse programming processes in early life. Resveratrol, a polyphenolic compound has a wide spectrum of beneficial effects on human health. In this review, we first summarize the epidemiological and experimental evidence supporting the developmental programming of MetS. This review also presents an overview of the evidence linking different molecular targets of resveratrol to developmental programming of MetS-related disorders. This will be followed by studies documenting resveratrol as a reprogramming agent to protect against MetS-related disorders. Further clinical studies are required in order to bridge the gap between animal models and clinical trials in order to establish the effective dose and therapeutic duration for resveratrol as a reprogramming therapy on MetS disorders from developmental origins. Metabolic syndrome (MetS) is a critical challenge for public health. Worldwide, two thirds of the deaths resulting from non-communicable diseases are mainly caused by MetS and related disorders . MetS isSeveral epidemiological studies indicate that fetuses exposed to suboptimal conditions increase the risk of MetS and related disorders. The first key evidence for developmental origins of MetS comes from the data gathered after severe famines, such as the Dutch Hunger Winter. Offspring born following famine exposure are prone to develop different phenotypes of MetS, such as obesity, diabetes and hypertension ,14,15. TSo far, a number of nutritional, pharmacological and genetic models have been used to examine the mechanisms underlying MetS of developmental origins ,4,5,19. Resveratrol, a polyphenolic compound  produced by several plants, is a naturally occurring phytoalexin ,24. A grThe works published in recent years confirm the link between molecular targets and signaling pathways of resveratrol and pathophysiology of MetS-related disorders. The decrease in endothelial nitric oxide synthase (eNOS) activity and NO bioavailability are crucial mechanisms underlying the endothelial dysfunction that occurs in MetS. Resveratrol has been reported to increase expression and activity of eNOS via activation of SIRT1 . AsymmetResveratrol inhibited the NF-κB inflammation pathway through activation of SIRT1 . Indepenn = 763 subjects) [n = 158 subjects) [Currently, a number of clinical trials show that resveratrol was well tolerated and had a beneficial influence in the disease biomarkers of MetS and related disorders ,36, amonubjects)  and NAFLubjects) .In humans, resveratrol seems to have no obvious toxicity . HoweverIn the current review, limited information is available about the use of large animals to study the role of resveratrol on developmental programming of MetS-related disorders. Two reports in nonhuman primates shown that resveratrol supplementation during pregnancy improved maternal and placental phenotype with beneficial effects in the fetal liver of mothers exposed to Western diets ,45. ConvAs shown in Oxidative stress, an imbalance between pro-oxidant and antioxidant factors, is considered a key mechanism that drives the developmental programming of MetS ,4,5. TheNutrient-sensing signals regulate cell metabolism and growth in response to variable nutrient levels. Therefore, nutrient-sensing signals have a unique role in the regulation of fetal growth and development. Several nutrient-sensing signals are also molecular targets of resveratrol, such as AMPK and SIRT1 . Activatl-arginine availability, decreased NOS expression/activity, inactivation due to oxidative stress and inhibition by ADMA (an endogenous NOS inhibitor). Among the reasons for NO-ROS imbalance, increasing attention has been given to ADMA [NO deficiency is a well-known pathogenetic mechanism of endothelial dysfunction that results from diverse cardiovascular risk factors such as hyperlipidemia, diabetes mellitus, obesity and hypertension . NO defi to ADMA . Indeed, to ADMA . As revi to ADMA . Convers to ADMA ,63. Resv to ADMA . Our pre to ADMA . It woulThe RAS is both a target of and contributor to MetS . The RASThere are other potential mechanisms related to MetS programming by which resveratrol might act: (1) by activating Nrf2, (2) by inhibiting NF-κB inflammation pathway and (3) by epigenetic regulation. Although Nrf2 activation and NF-κB inhibition have been reported to be protective in other developmental programming models ,72,73, tResveratrol supplementation as a reprogramming strategy against the development of MetS is a critical challenge and will become even more urgent in the near future as the growing epidemic of MetS and related disorders threaten humanity. The primordial studies in short-lived animal models, with controlled interventions across their life span provided key results revealing potential common mechanisms linking molecular targets of resveratrol to programming processes underlying MetS of developmental origins. Regardless of numerous beneficial effects of resveratrol, one major concern is its poor solubility and low intestinal absorption rate . Thus, tIn the current review, the beneficial effects of resveratrol are attributed to several mechanisms that are known to interrelate with each other. Since resveratrol has a wide range of activities, it is critical to determine if maternal resveratrol therapy may generate off-target effects in adult offspring. During the preparation of the current review, we observed that multiple MetS-related phenotypes and target organs are not examined simultaneously in the same model. Additionally, the follow-up periods after the cessation of resveratrol supplementation in most cited reprogramming studies were relatively short. We, therefore, must determine the long-term programming effects of resveratrol in different programming models and clarify whether there is an organ-specific response. Current evidence of the reprogramming effects of resveratrol is still in its infancy. Further research and more detailed clinical studies are required in order to ensure the beneficial effects of resveratrol on MetS-related disorders and for ascertaining the critical window, drug dose and therapeutic duration for reprogramming therapy.