Cancer onset and progression have been linked to oxidative stress by increasing DNA mutations or inducing DNA damage, genome instability, and cell proliferation and therefore antioxidant agents could interfere with carcinogenesis. It is well known that conventional radio-/chemotherapies influence tumour outcome through ROS modulation. Since these antitumour treatments have important side effects, the challenge is to develop new anticancer therapeutic strategies more effective and less toxic for patients. To this purpose, many natural polyphenols have emerged as very promising anticancer bioactive compounds. Beside their well-known antioxidant activities, several polyphenols target epigenetic processes involved in cancer development through the modulation of oxidative stress. An alternative strategy to the cytotoxic treatment is an approach leading to cytostasis through the induction of therapy-induced senescence. Many anticancer polyphenols cause cellular growth arrest through the induction of a ROS-dependent premature senescence and are considered promising antitumour therapeutic tools. Furthermore, one of the most innovative and interesting topics is the evaluation of efficacy of prooxidant therapies on cancer stem cells (CSCs). Several ROS inducers-polyphenols can impact CSCs metabolisms and self-renewal related pathways. Natural polyphenol roles, mainly in chemoprevention and cancer therapies, are described and discussed in the light of the current literature data. Many epidemiological studies suggest that diet particularly rich in fruits and vegetables have cancer preventive properties –3. The bDuring the past few decades the growing interest in natural polyphenols has contributed to understanding these compounds in terms of their chemical and biological functions and beneficial effects on human health , 7. WithκB, JAK/STAT) which result in the inhibition of tumour progression [The protective mechanisms that block the initiation of carcinogenesis can be defined as chemoprevention, a concept that was originally introduced by Wattenberg . Interesgression , 16.Research on the anticancer activities of dietary polyphenols, identified new antitumour molecules that can be used in cancer prevention and treatment, both alone and in combination with current chemotherapy/radiotherapy –19.Cellular senescence is a physiological process of irreversible cell-cycle arrest that contributes to various physiological and pathological processes of aging . ReplicaInduction of cellular senescence can be considered a relevant mechanism of tumour suppression. The concept of prosenescence therapy has emerged over the past few years as a novel therapeutic approach to treat cancers. Emerging evidence has demonstrated that therapy-induced senescence (TIS) is a critical mechanism through which many anticancer drugs inhibit tumour progression . TIS mayEpigenetic alterations, such as DNA methylation, histone acetylation level, and gene expression miRNA-regulated cancer stem cells biology, and induction of premature senescence in tumour cells have been identified as relevant anticancer features of many dietary polyphenolic compounds –28. IncrIn this review, we will discuss the current progress in the study of polyphenols as very promising tools for the management of cancer prevention and treatment.http://who.int/cancer/en) 8.2 million people died of cancer in 2012; however 30% of cancer can be prevented and some of the most common cancers such as breast, colorectal, and cervical cancer are curable if treated promptly. Among many factors that cause cancer, oxidative stress is one of the most important and well-studied event that gives rise to the conditions leading to tumour onset and progression [Cancer is currently one of the most deadly diseases worldwide. According to a report by the World Health Organization (WHO)  expression and chromatin remodelling through either activation or inhibition of epigenetic-related enzymes such as HDACs, histone acetyltransferases (HATs), and DNMTs [Within the last few years most of the studies reporting on polyphenols have focused on their antioxidant properties . In addind DNMTs . In partnd DNMTs . FurtherCancer cells are distinguished by several distinct characteristics due to cumulative epigenetic changes of multiple genes and associated cell signalling pathways, some of which are linked to inflammation. Immune cells infiltrate tumours and are engaged in a cross talk with neoplastic cells; thus, inflammation might affect responses to cancer therapy . StudiesκB pathway through the activation of intrinsic HAT activity, resulting in the expression of proinflammatory mediators, but it can also inhibit HDAC function.Since several polyphenols can modulate both HDAC and HAT, there may be a common underlying mechanism. For instance, curcumin, a known antioxidant as well as a free radical, may regulate both acetylation and deacetylation through the modulation of oxidative stress. Rahman et al.  have shoκB, forkhead box class O (FOX), and histone proteins [The role of polyphenols in regulation of epigenetic pathways including sirtuin 1 (SIRT1) modulation has been investigated . Sirtuinproteins .Sulforaphane (SFN) is a bioactive polyphenol present in cruciferous vegetables such as broccoli, cabbage, and kale . It has Isothiocyanate, such as phenethyl isothiocyanate (PEITC), has been shown to inhibit carcinogenic process by growth arrest of many types of cancer cells through induction of apoptotic pathway . TreatmeCurcuma longa), has antioxidant and anti-inflammatory properties which have been associated with multiple health benefits including cancer prevention [β and COX2. These results suggest antioxidant and anti-inflammatory properties of curcumin [Curcumin, a polyphenol extracted from the most popular Indian turmeric spice  is a fundamental mechanism of epigenetic silencing in human cancers. EGCG has been reported to directly reactivate the WIF-1, through the promoter demethylation in lung cancer cell lines .EGCG modulated miRNAs in lung cancer and hepatocellular carcinoma where the expression of several miRNAs was changed , 99. OneCoffee and tea polyphenols are also demethylating agents in human breast cancer cell lines where caffeic acid or chlorogenic acid inhibited DNMT1 activity, in a concentration-dependent manner .Resveratrol (RV), a natural polyphenol found in blueberries, cranberries, nuts, red grapes, and wine, exerts anti-inflammatory and anticancer effects . It has Since anticancer biological activities are already demonstrated for RV and curcumin, to investigate the combined chemopreventive potential of these two polyphenols has been of great interest. It has been shown by Malhotra and colleagues  that curRV activates the protein deacetylase SIRT1 leading to the formation of inactive chromatin and changes in gene transcription . On the  BRCA1 is associated with lower levels of SIRTs expression. It has been reported that in in vitro and in vivo experimental models RV can increase the expression of BRCA1 by inhibiting Survivin expression and activating SIRT1. These findings suggest that resveratrol treatment serves as a potential strategy for targeted therapy for BRCA1-associated breast cancer [In breast cancer, the tumour suppressor genet cancer . Furthert cancer .α) and the estrogen receptor beta (ERβ). The ERα/ERβ ratio is a prognostic marker for breast tumours, and ERα expression could indicate the presence of malignant tumours. It has been reported that in human breast cancer cell lines genistein effects depend on ERα/ERβ ratio for oxidative stress regulation, mitochondrial functionality, and modulation of antioxidant enzymes, and sirtuins [ p16INK4a in human esophageal squamous carcinoma cell line [ BTG3 is transcriptionally downregulated. This inhibition is due to promoter CpG island methylation. The methylation-silenced BTG3 gene can be reactivated by genistein treatment that causes CpG demethylation, inhibition of DNMT activity, and induction of active histone modifications [Isoflavones are compounds found in soy beans and act like estrogens. Among them, genistein and daidzein have gained the most research attention. Many studies have reported that genistein can be used as a chemopreventive agent in several types of cancers, especially for hormone-dependent breast cancer . Genistesirtuins . Genisteell line . In renaications .MCM-2), target of miRNA-1296 [Moreover, genistein treatment has shown the ability to modulate miRNAs expression level. For instance, in prostate cancer cells, genistein caused an increase of miRNA-1296 and accumulation of cells in the S phase of the cell cycle along with a significant downregulation of minichromosome maintenance gene  p16INK4a, p21CIP/WAF1, and p27 [CIP/WAF1 can induce a senescence-like cell-cycle arrest, whereas depletion of p21CIP/WAF1 can delay senescence-associated arrest. Moreover, p16INK4a acts in Rb pathway by inhibiting the activation of CDK4 and CDK6 which is the initial step of Rb phosphorylation [INK4a is to keep Rb in its active, hypophosphorylated form, which blocks the expression of genes regulated by E2F transcription factors leading to a G1 cell-cycle arrest. Numerous studies have provided important insights into the p53/p21 and Rb/p16 pathways that promote cellular senescence: the first one is primarily responsible for senescence induced by telomere shortening or DNA damage; the second one is involved in mediated stress-induced premature senescence (SIPS).Importantly, the combined activity of p53 and pRb could determine whether cells enter senescence or cell death pathways , 127. Ho and p27  are actirylation . The funThe combination of prosenescence induction with already established treatment protocols in order to take into account the prosenescence approaches in the development of novel cancer therapies has been considered of interest. For instance, both neoadjuvant and adjuvant therapies have a more and more relevant role in the treatment of some neoplasias, including breast, prostate, and colon cancer, where such approaches significantly increase the disease-free survival and the overall survival of patients. In a neoadjuvant protocol, a prosenescence approach could be combined with traditional treatments in order to reduce tumour mass before surgery. Furthermore, senescence-inducing molecules such as natural compounds may be used in combination with radiotherapy in cancer patients who are not suitable for surgery because of their age or advanced stage of disease , 131. SuA number of promising prosenescence agents are currently under consideration for cancer clinical management . To thisSeveral anticancer polyphenolic compounds from fruit and vegetables induce cellular growth arrest largely through the induction of a ROS-dependent premature senescence. Among them, 20(S)-ginsenoside Rg3 , a compoPolyphenolic extracts from the edible part of artichoke (AEs) have been shown to be potential chemopreventive and anticancer dietary compounds. High doses of AEs induce apoptosis and decrease the invasive potential of the human breast cancer cell line, MDA-MB231 . ChronicLow doses treatment of RV exerts its anticancer and chemopreventive effects through the induction of premature senescence in lung cancer cells. This event correlates with increased DNA double strands breaks and ROS production through the upregulation of NAPDH oxidase-5 expression . in vivo using a nude mice xenograft model. RV exerted inhibitory activities on gastric development and significantly decreased the fraction of Ki67-positive cells in the nude mice tumour specimens. After the RV treatment, the induction of senescence and the changes in the expression of the regulators involved in the cell cycle and senescence pathways were similar to what was observed in vitro.Furthermore, low doses of RV treatment arrested gastric cancer cells in the G1 phase and led to senescence instead of apoptosis which is initiated by high doses treatment . The inhThe propensity of tumour cells to undergo senescence in response to low and chronic exposure of RV treatment and to apoptosis with high doses of RV was confirmed in C6 rat glioma cells and further investigated in cooperation with quercetin. Chronically administered, RV and quercetin in subapoptotic doses can induce senescence-like growth arrest. These results suggested that the combination of these agents could be a good candidate treatment for glioma tumours .In the few last years, many studies have highlighted the existence of CSCs in most solid and nonsolid tumours, including brain, head and neck, breast, colon, and leukaemia among others –143. Thea) inhibiting their self-renewal ability and chemoresistance related pathways, (b) inducing their differentiation [c) targeting some of their cell-surface molecular markers [d) impacting their energetic metabolism via inhibition of glycolysis [e) designing miRNA-based strategies to block cancer stemness [Recently, many strategies targeting cancer stem cells have been proposed, namely,  impycolysis  and/or bycolysis , and (e)stemness . Theoretstemness , oxidatistemness , chronicstemness , and a pstemness . Thus, mstemness .Novel anticancer strategies should be designed to selectively target cancer stem cells and to this purpose natural compounds might have a relevant role. We provide a revision of the most recent literature addressing the CSCs-regulation role of some of the most investigated polyphenols.(a) Role of Polyphenols in the Regulation/Inhibition of Cancer Stem Cells Self-Renewal. It has been shown that polyphenols can impact cancer stem cells self-renewal related pathways, such as Wnt/β-catenin, Hedgehog, and notch [κB and STAT3 [nd notch . In partnd notch . Beside nd STAT3 .κB, Hedgehog, and Wnt/β-catenin also contributing to the induction of epithelial-mesenchymal transition. For these properties, SFN has been proposed as an adjuvant of chemotherapy in several preclinical studies [ in vitro [ in vitro and in vivo models; such effect strongly depends on the activation of Hedgehog pathway for cancer stem cells self-renewal activity [Between cruciferous family natural compounds, SFN has been demonstrated to be capable of targeting cancer stem cells in different types of cancer, by regulating pathways such as NF- studies . Previouin vitro . Furtheractivity , 168. in vitro models, showed that quercetin, a polyphenol present in many fruit and vegetables [ in vivo experiments, quercetin inhibited cancer stem cells-derived xenografts, reducing the expression of proliferation, stemness, and angiogenesis related genes [Pancreatic CSCs studies carried outgetables , decreased genes .Remarkably, quercetin effects were amplified in the presence of SFN, suggesting the importance to combine different polyphenols for designing synergistic anticancer strategies , 170.The use of soy foods has been shown to be beneficial for the reduction of mammary tumour risk. The intake of these natural compounds was demonstrated to be beneficial for the modulation of body weight and adiposity associated with breast cancer both in humans and in animal models –174. Mor(b) Polyphenols Affecting Cancer Stem Cells Metabolism. Cancer stem cells, like physiological stem cells, are characterized by a hyperglycolitic metabolism [ p21CIP/WAF1, downregulation of the mammalian target of rapamycin pathway, and suppression of the cancer stem cells population [tabolism  and, in tabolism , 176. Thtabolism . To thistabolism . Moreovetabolism . Activatpulation . Moreovepulation .Hyperglycolytic cancer stem cells have an increased basal level of ROS, although they result as being more vulnerable than physiological cells to a further increase in oxidative damage elicited by prooxidant polyphenol action . One of Cancer stem cells are localized within specific niches, normally characterized by the presence of lower oxygen tension (hypoxia), inflammation, oxidative stress, and a lower pH. Polyphenols can be exploited to regulate the CSC niche, by targeting signalling pathways that are implicated in the maintenance of tumour microenvironmental features. Among the polyphenols targeting hypoxia, pterostilbene, a stilbene isolated from blueberries, has been recently reported to have anticancer properties.α, β-catenin, Twist1, and NF-κB and by a high ability to produce mammospheres, was present. By adding pterostilbene to the cell medium, the percentage of cancer stem cells was significantly reduced. The effects of such a polyphenol were confirmed in vivo experiments where tumorigenesis and metastasis were inhibited [Breast cancer cell lines such as MCF-7 and MDA-MB231 were cocultured with tumour-associated macrophages, known to enhance malignancy promoting metastasis. In these experimental conditions, a large subpopulation of cancer stem cells, characterized by an increased level of HIF1nhibited .(c) Polyphenols Targeting Proinflammation Signalling Pathways. The presence of chronic inflammation could be a characteristic of the neoplastic niche [κB pathway, which is involved in inflammation, cellular transformation, tumour cell proliferation, invasion/metastasis, and angiogenesis [κB signalling and thus have anti-inflammatory and anticancer properties [ic niche . Some poic niche . Flavonoogenesis . Moreoveoperties . Recentloperties .(d) Polyphenols Regulating the Peritumoural Acidic pH. Cancer stem cells extracellular microenvironment is often characterizated by an acidic status caused by CSCs metabolic dependence on aerobic glycolysis. Buffering the acidic cancer pH with the use of sodium bicarbonate inhibited tumour growth and cancer cell invasion in a preclinical animal model [al model , 190. ToFurthermore, several plant compounds have been shown to increase pH values by inhibiting proton pump activity and consequently elicited apoptosis in cancer . This miCompared to normal cells, cancer cells have an increased rate of ROS production and have aberrant regulation mechanisms to deal with their particular redox status. ROS have a well-defined role in promoting and maintaining tumorigenicity indicating that dietary antioxidants have an active role in preventing or reducing tumorigenesis. On the other hand, high levels of ROS can also be toxic to neoplastic cells and can potentially induce cell death. Accumulating evidence shows that ROS levels in tumour cells are crucial for designing advanced therapies and future challenge in anticancer treatments. To this purpose, increasing knowledge from epidemiological and experimental data supports the bright future of natural polyphenols as anticancer tools –194 Fig. The comMuch attention is currently focused on the role of natural polyphenols on modulating intracellular ROS levels leading to epigenetic modifications of pivotal genes in tumorigenesis. It is important to stress that DNA methylation and posttranslational histone modifications are crucial actors in epigenomic landscape playing a relevant role in the structure and function of chromatin. Several polyphenols were demonstrated to interfere with enzymes driving the epigenetic alterations which modulate inflammation process that might hesitate in cancer. As such, it will be a challenge for future anti-inflammatory therapies to deeply evaluate the anticancer role of polyphenols as epigenetic modulators. However, there are some concerns that anticancer therapies with polyphenol regulators of DNMT and HAT/HDAC may suffer from a lack of specificity. To overcome this limitation, an alternative strategy may be to synergistically combine nonselective epigenetic treatments with low doses of conventional targeted therapies which lead to less toxicity comparing to a high dose standard treatments. Furthermore, microRNAs molecules are promising actors in the epigenetic combination therapies, as their target specificity may bridge the gap between genetic and epigenetic changes. To this purpose, natural polyphenols may indirectly modulate the epigenome by affecting levels of microRNAs which target specific epigenetic modifier enzymes.The future of polyphenol-epigenomic therapy has several challenges ahead and it is a promising field for clinical cancer interventions.In developing novel anticancer strategies, prosenescence has a relevant role. The current knowledge of senescence, as a major mechanism of tumour suppression as well as a determinant of the outcome of cancer treatment, leads to the concept of prosenescence therapy, which could be an important alternative or addition to conventional chemo-/radiotherapy. To this aim, prosenescence-polyphenols treatment may minimize toxicity and side effects of conventional therapies in cancer patients. On the other hand, some investigators suggest caution in the clinical management of this therapy because the induction of senescence might give rise to quiescent tumour cells, mainly cancer stem cells, which represent a potential niche for cancer recurrence. Thus, deeper understanding of the biological mechanisms responsible for cellular senescence is required in order to better characterize the role of polyphenols in prosenescence therapy for more efficient management of cancer treatment in the future.The association of cancer stem cells and the resistance to chemo-/radiotherapy stimulate a critical consideration regarding the efficiency of prooxidant therapy on CSCs. Most conventional anticancer therapies are ineffective in killing this cell population. It is for this reason that there has been a growing interest to develop new strategies based on identifying agents able to directly target quiescent cancer stem cells. Since low ROS levels has been suggested to be critical for maintaining cellular stemness, an increase of these reactive species polyphenol-mediated might sensitize cancer stem cells to therapy. However, evaluating novel treatment approaches also require the development of assays or identification of biomarkers able to identify CSCs population in order to select and assess cancer patients.


To examine the extent to which state adoption of the Centers for Disease Control and Prevention (CDC) 2006 revisions to adult and adolescent HIV testing guidelines is associated with availability of other important prevention and medical services. We hypothesized that in states where the pretest counseling requirement for HIV testing was dropped from state legislation, substance use disorder treatment programs would have higher availability of HCV testing services than in states that had maintained this requirement.We analyzed a nationally representative sample of 383 opioid treatment programs from the 2005 and 2011 National Drug Abuse Treatment System Survey (NDATSS). Data were collected from program directors and clinical supervisors through telephone surveys. Multivariate logistic regression models were used to measure associations between state adoption of CDC recommended guidelines for HIV pretest counseling and availability of HCV testing services.The effects of HIV testing legislative changes on HCV testing practices varied by type of opioid treatment program. In states that had removed the requirement for HIV pretest counseling, buprenorphine-only programs were more likely to offer HCV testing to their patients. The positive spillover effect of HIV pretest counseling policies, however, did not extend to methadone programs and did not translate into increased availability of on-site HCV testing in either program type.Our findings highlight potential positive spillover effects of HIV testing policies on HCV testing practices. They also suggest that maximizing the benefits of HIV policies may require other initiatives, including resources and programmatic efforts that support systematic integration with other services and effective implementation. To increase HIV testing, promote earlier detection of HIV infection, counsel and link infected persons to HIV clinical and prevention services, the Centers for Disease Control and Prevention (CDC) published revised recommendations for HIV testing in health care settings for adults and adolescents in 2006 . These rWhile CDC recommendations may influence state laws, the authority to set or modify HIV testing policies is under the jurisdiction of states. States must decide to amend laws to conform or at a minimum to not conflict with CDC recommendations . By 2011The effects of HIV-related legislative changes on the availability of other medical services, on the other hand, have seldom been investigated, even though such effects may be present. The elimination of HIV pretest counseling, for example, may considerably reduce the need for human and financial resources dedicated to HIV testing. These resources could then be redirected towards offering new preventive services or to improving the quality of existing services , 14. SimServices addressing infection with the Hepatitis-C Virus (HCV) may be particularly affected by changes in HIV-related legislation. This is so because both viruses partially share routes of transmission and co-infection is common , 16. HIVSuch “spillover effects” are important because they may optimize the efficiency and effectiveness of service delivery, through integration of care, and the availability of comprehensive services. These models of care have the potential to increase access, quality, and health outcomes , 20. SpiIn this paper, we examined whether the adoption of state legislation compatible with the 2006 CDC guidelines on HIV testing was associated with subsequent improvements in the availability of HCV testing services. We focus on the elimination of the requirement for HIV pretest counseling in opioid treatment programs (OTPs). OTPs are physical facilities with resources dedicated specifically to treating opiate dependence through methadone or buprenorphine (excluding primary care or physician offices). OTPs treat approximately 26% of all individuals enrolled in substance use disorder (SUD) treatment programs the Unites States . The preWe hypothesized that in states where the pretest counseling requirement for HIV testing was dropped from state legislation, OTPs would have higher availability of HCV testing services than in states that had maintained this requirement. Indeed, simplifying HIV testing protocols may help OTPs invest time and resources in the provision of HCV testing services to their clients. An increasing number of OTPs are buprenorphine-only programs: they represented 14 % of all OTPs in 2005 vs. 27 % in 2011 . Buprenon = 187) and 2011 (n = 196) NDATSS.We analyzed data from the National Drug Abuse Treatment System Survey (NDATSS). The NDATSS is a nationally-representative survey, which examines the organizational structures and operating characteristics of the nation’s outpatient SUD treatment programs . BecauseHCV testing practices in OTPs were ascertained from NDATSS questions that asked whether OTPs offered any option for HCV testing to their patients, and if so, whether such services were provided on-site or off-site. We constructed two dependent variables from answers to these questions. First, we created a binary variable (0–1) set to 1 if the program offered any HCV testing option to their patients (either on-site or off-site) and 0 otherwise. Given the greater coordination and investment that may be required to offer on-site HCV testing compared to off-site testing, a second binary variable examined the availability of on-site HCV testing services, among OTPs that offered any HCV testing option to their patients. OTPs providing on-site HCV testing were coded 1, and those providing only off-site services were coded 0.A variable describing state-level HIV-related legislation was constructed based on three previous studies of states’ HIV testing laws. Wolf et al.  and MahaOTPs that treat a large proportion of patients with characteristics indicating high-risk for HIV or HCV may have greater likelihood of offering preventive services. Patient factors accounted for in the models therefore included prevalence of injection drug users (IDU) among patients in each program and total number of OTP patients in the past year and race/ethnicity . Organiz2 test for categorical variables and the t test for continuous variables, and we computed unadjusted odds ratios. Second, we estimated multivariate logistic regression models to examine associations between state-level HIV-related legislation and availability of HCV testing services in OTPs, controlling for program and patient characteristics [We first reviewed state policies about HIV pretest counseling in 2005 and 2011 and assessed their compatibility with the 2006 CDC guidelines. We then described differences in OTP characteristics between programs offering any HCV testing services (either on-site or off-site) to their patients vs. OTPs in which no HCV testing services were offered. We used the Pearson χeristics . Third, In all models, we addressed issues of unobserved heterogeneity  by including state fixed-effects . This stOnly 4 states included in NDATSS had already adopted HIV-related legislation that eliminated the requirement for pretest counseling at the time of the 2005 NDATSS wave. These included Georgia, Illinois, Maryland, and New Mexico. On the other hand, most states had adopted such legislation by 2011: only six states or jurisdictions  in the 2011 NDATSS had retained legislation that did not permit dropping the pretest counseling risk (Table p < 0.01). In multivariate analyses with controls for organizational characteristics and state-level fixed effects, however, there were no significant differences in HCV testing availability between states whose legislation permitted dropping the HIV pretest counseling requirement and other states .Table p-value <0.05). In programs offering methadone-only or methadone and buprenorphine to their patients, 80 % of OTPs located in states that had dropped the requirement for HIV pretest counseling offered HCV testing services vs. 87 % in states that still required pretest counseling. In OTPs that offered only buprenorphine, on the other hand, 88 % of OTPs in states that had dropped the pretest counseling requirement offered HCV testing services vs. only 68 % in states that had maintained this requirement (p < 0.05).The association between state-level HIV-related legislation and the availability of HCV testing services differed significantly by type of treatment program . However, in multivariate analyses, there were no significant differences in the likelihood of offering on-site testing between these two types of states .In bivariate analyses, among OTPs that offered HCV testing to their patients, the type of HCV testing services offered  was associated with the prevalence of IDUs among patients, funding from the federal government, more favorable staff-to-patient ratios, OTP ownership, hospital affiliation and method of treatment Table . OTPs loAmong OTPs that offered HCV testing services to their patients, there was no interaction between HIV-related legislation and the type of treatment program Fig.  in deterIn this paper, we examined the potential spillover effects of state-level HIV testing policies on the availability of HCV testing in opioid treatment programs. We hypothesized that in states where the pretest counseling requirement for HIV testing was dropped from state legislation, OTPs would have higher availability of HCV testing services than in states that had maintained this requirement. Indeed, simplifying the procedures for HIV testing may help OTPs mobilize financial and/or human resources for the provision of other medical services, such as HCV testing.Using data from two recent rounds of the NDTASS, we found that this was not uniformly the case. In OTPs that offered only buprenorphine treatment to their patients for substance abuse treatment, legislative changes that permitted dropping the pretest counseling requirement for HIV testing were associated with significantly higher availability of HCV testing. On the other hand, in OTPs that offered methadone to their patients for substance abuse treatment, there were no differences in HCV testing availability between states with and without a legal requirement for HIV-related pretest counseling.There are several reasons why the elimination of pretest counseling for HIV testing may affect HCV testing in OTPs that offer only buprenorphine. First, this may be related to the type of patient population served by buprenorphine-only programs , 38, 39.Second, we suggest that the removal of the requirement to provide pretest counseling for HIV may have reduced concerns of stigma among the patient population and providers of buprenorphine-only programs. Patients of these programs may indeed be more likely to associate pretest counseling messages with stigmatized behaviors such as injecting drug use, thus reducing their demand for testing services , 43. StrFinally, there may be aspects of the operating environment of buprenorphine-only programs that make them more responsive to legislative changes than methadone programs. Whereas methadone maintenance programs must be certified by SAMHSA, licensed at the state level and provide directly observed therapy (in some case patients may be eligible for take-home methadone), buprenorphine-only programs solely need to have on staff a health care provider who has obtained DEA approval to prescribe controlled substances. The less structured operational requirements of buprenorphine-only programs may thus make it easier for these programs to invest resources in HCV testing following the adoption of legislative changes. This may account for the observed differences in the association between the removal of the HIV pre-test counseling requirement and HCV testing practices across different types of OTPs.Our results also indicated that HIV-related legislation did not translate into an increase in the offer of on-site testing among OTPs that offered HCV testing. This is likely sub-optimal since on-site services are significantly more effective at improving uptake of testing among patients of drug treatment programs than off-site services , 45. ChaSeveral other factors may further improve the adoption of HCV testing in OTPs. This includes the 2012 CDC recommendation to expand HCV testing to include persons born between 1945 and 1965 , or the Our results improve on previous evaluations of the effects of HIV-related legislation , 58, 59 While HIV testing policies may have a spillover effect on HCV testing practices, increasing the availability of HCV testing will require a multi-pronged approach. Consent and counseling guidelines focused on HCV may inform state policies on the issue, but state adoption of existing guidelines and their direct effects on HCV testing practices has not been examined , 55. FutThis study has several limitations. First, the NDATSS had limited questions on patients of OTPs. We therefore could not measure the relationship between HIV pretest counseling policies and uptake of HCV testing services among patients. Second, we also could not determine the influence of length of time since elimination of pretest counseling on HCV testing practices. Specifically, we do not know how long after a State changed its legislation, the OTPs in that State changed their HIV testing practices, including removal of the pre-test counseling requirement. This is important because an OTP with more experience implementing the guidelines, e.g., a program operating under elimination of pretest counseling for a year, may have better knowledge about the influence of the policy on operating procedures, and may therefore be more likely to offer HCV testing services. On the other hand, a program that has newly eliminated pretest counseling may be less likely to offer HCV testing services. Third, we performed separate analyses to assess the associations between State legislation on HIV testing and a) HCV testing (either on-site or off-site), as well as b) on-site HCV testing in OTPs. This was necessary because of limited sample size. Future studies should rely on larger samples, and should employ other methodological approaches  to further extend our findings. Lastly, the NDATSS data do not permit assessing whether the relation between HIV testing policies and HCV testing practices is causal. Even though we included state-level fixed-effects in our models, these only control for time-invariant characteristics of states that may confound the relationship between legislative changes and HCV testing. They do not account for a) characteristics of states that changed between 2005 and 2011, and b) unobserved characteristics of OTPs. Addressing this issue requires panel data on OTPs and their practices, i.e., a sample of OTPs that are repeatedly surveyed over time. Unfortunately, only a small number of OTPs in the NDATSS were interviewed both in 2005 and 2011, thus precluding such robust causal analyses. In addition, our data does not permit determining whether OTPs adopted HCV testing before or after the State in which they are located changed its HIV-related legislation. This is so because we only know whether surveyed OTPs offered HCV testing in 2005 or 2011. We do not know when exactly they first adopted HCV testing. As a result, we can only identify associations between HIV-related state laws and HCV testing practices in OTPs.Our results highlight some of the implications of HIV policies for the availability of non-HIV services. Buprenorphine-only programs were more likely to offer HCV testing services in states that mandated the elimination of HIV pretest counseling, but this was not the case for methadone programs. Changes in legislation related to HIV pretest counseling also were not associated with the availability of on-site HCV testing. Our findings underscore several areas for future research. There is a need for a better understanding of the mechanisms  or pathways  through which HIV pretest counseling policies influence non-HIV services. Additionally, studies should investigate the impact of HIV testing policies on HCV testing services, considering state guidelines for HCV testing. Lastly, the longer-term effect of eliminating HIV pretest counseling on clients and their use of HIV-related services, and whether programs introducing HCV testing after the elimination of HIV pretest counseling provide pre-test counseling for HCV testing should be examined in future studies. These analyses will provide a more in-depth understanding of the role of policy interventions in shaping HCV testing practices, promote the effective integration of HIV and HCV services, and increase the availability of HCV testing in substance abuse treatment programs and other health care settings.ACA, affordable care act; CARF, commission on accreditation of rehabilitation facilities; CDC, centers for disease control and prevention; DEA, drug enforcement administration; EIA, enzyme immunoassays; FDA, food and drug administration; HCV, hepatitis c virus; HIV, human immunodeficiency virus (HIV); IDU, injection drug users; NDATSS, national drug abuse treatment system survey; OTPs, opioid treatment programs; RNA, ribonucleic acid; SAMHSA, substance abuse and mental health services administration; STI, sexually transmitted infections; SUD, substance use disorder


A fundamental understanding of behavior requires predicting when and what an individual will choose. However, the actual temporal and sequential dynamics of successive choices made among multiple alternatives remain unclear.In the current study, we tested the hypothesis that there is a general bursting property in both the timing and sequential patterns of foraging decisions. We conducted a foraging experiment in which rats chose among four different foods over a continuous two-week time period. Regarding when choices were made, we found bursts of rapidly occurring actions separated by time-varying inactive periods, partially based on a circadian rhythm.Regarding what was chosen, we found sequential dynamics in affective choices characterized by two key features: (a) a highly biased choice distribution; and (b) preferential attachment, in which the animals were more likely to choose what they had previously chosen. To capture the temporal dynamics, we propose a dual-state model consisting of active and inactive states. We also introduce a satiation-attainment process for bursty activity, and a non-homogeneous Poisson process for longer inactivity between bursts. For the sequential dynamics, we propose a dual-control model consisting of goal-directed and habit systems, based on outcome valuation and choice history, respectively.This study provides insights into how the bursty nature of behavior emerges from the interaction of different underlying systems, leading to heavy tails in the distribution of behavior over time and choices.


Understanding the metabolism of persister cells poses challenges but also paves the way for the development of anti-persister compounds.Persister cells form a multi-drug tolerant subpopulation within an isogenic culture of bacteria that are genetically susceptible to antibiotics. Studies with different Gram negative and Gram positive bacteria have identified a large number of genes associated with the persister state. In contrast, the revelation of persister metabolism has only been addressed recently. We here summarize metabolic aspects of persisters, which includes an overview about the bifunctional role of selected carbohydrates as both triggers for the exit from the drug tolerant state and metabolites which persisters feed on. Also alarmones as indicators for starvation have been shown to influence persister levels via different signaling cascades involving the activation of toxin-antitoxin systems and other regulatory factors. Finally, recent data obtained by  S. aureus with penicillin was found to leave a few survivor cells behind  drug-tolerant Lewis, . Persist Bigger, . Notably Bigger, . Consist Bigger,  and thei Bigger, . This co Bigger, . Retarde Bigger, , as does Bigger, . Molecul Bigger, . Akin toE. coli  systems act as key regulators in this regard Lewis, . These sE. coli mutants lacking ubiF or sucB, encoding for enzymes involved in ubiquinone biosynthesis, or the TCA cycle, respectively, showed decreased persister levels compared to the wild-type strain  and impedes energy production causing an elevated persister level , which attack essential cellular functions  or transketolase A (tktA) lead to accumulation of dihydroxyacetone phosphate (DHAP) which is finally converted to methylglyoxal. Interestingly, the glyoxylate shunt is upregulated in B. cenocepacia persister cells, bypassing NADH production and possible ROS formation via the TCA cycle, illustrating an additional protective mechanism. Another link between persister level and ROS formation in biofilm was established in P. aeruginosa, where mutants defective in the stringent response were more susceptible toward antibiotic treatment (Nguyen et al., P. aeruginosa in biofilms conferring increased tolerance toward ciprofloxacin (Zhang et al., S. aureus cells, in which mutants lacking the TCA cycle enzymes aconitase or succinate dehydrogenase showed an enhanced stationary-phase survival level (Somerville et al., S. epidermidis isolates with enhanced survival during β-lactam treatment (Thomas et al., S. aureus (Thomas et al., alsSD operon encoding for an α-acetolactate synthase/decarboxylase leading to acetoin synthesis from pyruvate, thereby reducing the amount of acetate by CidC. These data illustrate the strong connection of TCA cycle dependent ROS formation and its negative influence on the long-term survival of cells thereby requiring alternative metabolic pathways to avoid their production.Recent studies provided first insights into the metabolic state of persisters associated with biofilms that provide a protective niche for bacteria against antibiotics and other harmful conditions (Mah and O'Toole, E. coli as well as S. aureus persisters. Subsequent to this finding on metabolite enabled killing, the non-susceptibility of persisters toward aminoglycosides treatment aided in the identification of the most utilizable substrates for such cells (Orman and Brynildsen, P. aeruginosa biofilms with a combination of mannitol and tobramycin (Barraud et al., S. epidermidis led to a higher level of dormant cells in a biofilm, presumably due to the accumulation of acidic degradation products resulting from glucose metabolism (Cerca et al., The importance of persisters in bacterial infections is more and more corroborated (Fauvart et al., Recent studies highlight the importance of investigating the two interconnected fields of persister state and metabolic activity in bacteria in more detail. Adaptation of the metabolism is a key prerequisite for persisters to cope with hostile conditions. In particular, the modulation of TCA cycle activity appears as a hallmark in persister metabolism. This regulation must be precisely controlled to avoid ROS formation with potentially destructive implications for persister cells. Multiple lines of evidence suggest that the metabolism of persisters can be tuned to alter their susceptibility toward antibiotics or to trigger programmed cell-death-like processes (Rice and Bayles, 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.


Cancer is a traitorous archenemy that threatens our survival. Its ability to evade detection and adapt to various cancer therapies means that it is a moving target that becomes increasingly difficult to attack. Through technological advancements, we have developed sophisticated weapons to fight off tumor growth and invasion. However, if we are to stand a chance in this war against cancer, advanced tactics will be required to maximize the use of our available resources. Oncolytic viruses (OVs) are multi-functional cancer-fighters that can be engineered to suit many different strategies; in particular, their retooling can facilitate increased capacity for direct tumor killing (oncolytic virotherapy) and elicit adaptive antitumor immune responses (oncolytic immunotherapy). However, administration of these modified OVs alone, rarely induces successful regression of established tumors. This may be attributed to host antiviral immunity that acts to eliminate viral particles, as well as the capacity for tumors to adapt to therapeutic selective pressure. It has been shown that various chemotherapeutic drugs with distinct functional properties can potentiate the antitumor efficacy of OVs. In this review, we summarize the chemotherapeutic combinatorial strategies used to optimize virally induced destruction of tumors. With a particular focus on pharmaceutical immunomodulators, we discuss how specific therapeutic contexts may alter the effects of these synergistic combinations and their implications for future clinical use. Do not repeat the tactics, which have gained you one victory, but let your methods be regulated by the infinite variety of circumstances.–Sun Tzu, The Art of WarOncolytic viruses (OVs) can selectively infect, replicate in, and kill tumor cells with minimal impact on normal tissue. These tumor-specific properties, called oncotropism, is dependent on the expression of surface receptors that allow viral binding and entry, as well as, the permissiveness of the tumor cell toward viral replication. Genetic manipulation of the viral genome aims to improve the inherent therapeutic value of OVs by enhancing their capacity for targeted tumor killing , 2. ThroThe oncotropism of viruses is guided by cell surface receptors that enable viral binding and entry, and the permissiveness of the infected cell to viral replication. Surface receptors that are recognized by different types of viruses can be specific to neoplastic cells. These viruses target receptors characteristic of malignant phenotypes, such as Poliovirus that binds CD-155 that is almost exclusively present in high grade glioma cells , 11, andSelective retargeting of viruses to tumor cells can also be generated in viruses without innate oncolytic abilities. Adenovirus (Ad)-based vectors are a good demonstration of this approach, since they possess a wide tropism, but a lytic life cycle that can be exploited for oncolytic virotherapy . One metViral oncolysis directly destroys tumor cells through either their lytic replication cycle or the expression of endogenous cytotoxic gene products . To furtThe various mechanisms through which OVs are capable of lysing cancer cells result in the release of tumor associated antigens (TAAs), proinflammatory cytokines, chemokines, and other danger signals, which facilitates immune cell recruitment and activation within tumors. In particular, activation and maturation of dendritic cells (DCs) and other antigen presenting cells (APCs) allow for efficient cross-presentation to T cells, and subsequent initiation of antitumor and antiviral immune responses , 43. Howin vivo  cell activity and potent cytotoxic T lymphocyte (CTL) responses that are necessary for clearing virus-infected cells . Additiofunction .Altogether, these immunological barriers pose a particular problem for repeat administration of OVs, by further promoting the development of adaptive antiviral immunity and reducing of its oncolytic effects. Moreover, a large fraction of the population has previously been exposed to the naturally occurring viruses that are commonly employed for generating therapeutic strains. Therefore, the infectious potential of recognized OVs  becomes limited by high levels of neutralizing antibodies , 82. The+ T cells  are anti-inflammatory agents that can modulate immune responses to viral infection. By impeding the type I IFN response, a major component of the cellular innate antiviral response, HDACi’s can enhance the spread and antitumor effects of OVs . In addiing rats . Global ing rats . Host lying rats .+ T cells in murine tumor models , overexpressing anti-inflammatory cytokines , suppressing proliferation and cytokine production by T cells and NK cells, and inducing apoptosis of CD8 T cells . Further T cells –129. Num T cells –134. Com T cells , 136. No T cells . Moreove T cells . In cont T cells . Therefo′-aza-2′-deoxycytidine and 5-Fu have been shown to enhance tumor antigen expression . In fact, targeting certain pathways with chemotherapy will also, by association, compromise the replicative capacity of OVs. As a result, discernable conflicts between virus-enabled therapeutic strategies and drug-enabled therapeutic strategies may limit the extent to which the two can be combined. For example, viruses require actively dividing cells to maximize their replicative efficiency, while many anticancer agents are either cytotoxic or cytostatic with death-inducing or anti-proliferative effects, respectively . Further134.5 deleted) in pancreatic cancer cell lines , was unable to induce robust tumor-specific immunity because the host immune response was predominantly redirected toward viral antigens expressed on the vector. Therefore, by adopting a heterologous prime-boost system whereby mice were initially primed with recombinant Ad-hDCT and boosted with VSV-hDCT, substantive immunity was generated against the tumor, while the antiviral response to VSV was dampened . The HDAWar strategy dictates methods in which to arrange and maneuver military forces during armed conflicts. Using the available resources and landscape to your advantage is a key aspect to defeating the enemy. The analogy of OVs as fighters, “targeting” cancer cells and being “armed” with various genes, is commonplace in the literature. Its ability to induce antitumor immune responses is akin to the call for air support, bringing in additional fighters that can help to identify and target enemy forces. The introduction of chemotherapeutic drugs to the battlefield is then, chemical warfare; a wide-spread, indiscriminate weapon. With our various forces at hand, how do we determine the best strategy to defeat our opponents? As with any war strategy game, finding the best approach begins with knowing the enemy (type of cancer), knowing our forces , their strengths and weaknesses (function), and finally how they interact with each other on the battlefield (combination therapy). Before you make a move, you postulate various scenarios in which your opponent may attack, but also how you can take the advantage. In a similar fashion, to identify the most suitable approach to OV-drug combination therapies, we should adopt a broader perspective to the treatment of cancer. Then and only then, will we not only win some battles, but we may also win the war.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.


Researchers at Okayama University, Japan showed lactate dehydrogenase (LDH) inhibition suppresses neuronal excitation in vitro, reduces EEG discharges and seizures in rodent models, and may provide a novel mechanism for anticonvulsant medications in human patients. Researchers at Okayama University, Japan showed lactate dehydrogenase (LDH) inhibition suppresses neuronal excitation in vitro, reduces EEG discharges and seizures in rodent models, and may provide a novel mechanism for anticonvulsant medications in human patients. Using basal ganglia brain slices to investigate the mechanism of the ketogenic diet (KD), they found neurons became hyperpolarized not from the ketone bodies per se, but from the reduced glucose concentration that accompany the switch to ketone bodies-based cerebral metabolism. Then through a series of experiments, they pinpointed pyruvate and oxaloacetate as the specific molecules that affect the K-ATP channel and membrane potential: less glucose leads to less pyruvate and oxaloacetate, which opens the K-ATP channels and hyperpolarizes the neurons. In vitro they reproduced this ketone bodies-induced neuronal suppression by inhibiting LDH, the enzyme that reversibly converts lactate to pyruvate. The researchers also inhibited LDH in vivo to reduce seizures and EEG paroxysmal discharges in pilocarpine and kainate rodent models. They then screened 20 current anticonvulsants and found that stiripentol alone had significant LDH inhibitor action. Lastly, they showed that isosafrole, a stiripentol analog and a more potent LDH inhibitor, had anticonvulsant activity and was well tolerated in rodent models. COMMENTARY. The authors report two important new findings: (1) the neuronal inhibition associated with the KD correlate with reduced pyruvate and oxaloacetate levels, and 2) LDH inhibitors that lower pyruvate and oxaloacetate levels work as anticonvulsants in vitro and in vivo models. Despite this shared mechanism, KD and LDH inhibitors have an important difference: LDH inhibitors do not provide an alternative fuel to make up for the lost pyruvate and oxaloacetate. LDH inhibition, therefore, may be more akin to selective fasting than the ketogenic diet. It's easier to imagine using LDH inhibitors to enhance the action of the ketogenic diet than to replace it. Either way, LDH inhibitors now join 2-deoxy-D-glucose [ LDH inhiMany hypotheses have been proposed for the anticonvulsant mechanism of the ketogenic diet. They include changes in the water and electrolyte balance, changes in cerebral energy reserve, direct anticonvulsant effects of ketone bodies and fatty acids, and indirect action by increasing GABA/glutamate ratio –5. The n


Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder with defective structure and/or function of motile cilia/flagella, causing chronic upper and lower respiratory tract infections, fertility problems, and disorders of organ laterality. Diagnosing PCD requires a combined approach utilizing characteristic phenotypes and complementary methods for detection of defects of ciliary function and ultrastructure, measurement of nasal nitric oxide and genetic testing. Currently, biallelic mutations in 31 different genes have been linked to PCD allowing a genetic diagnosis in approximately ~ 60% of cases. Management includes surveillance of pulmonary function, imaging, and microbiology of upper and lower airways in addition to daily airway clearance and prompt antibiotic treatment of infections. Early referral to specialized centers that use a multidisciplinary approach is likely to improve outcomes. Currently, evidence-based knowledge on PCD care is missing let alone management guidelines. Research and clinical investigators, supported by European and North American patient support groups, have joined forces under the name of BESTCILIA, a European Commission funded consortium dedicated to improve PCD care and knowledge. Core programs of this network include the establishment of an international PCD registry, the generation of disease specific PCD quality of life questionnaires, and the first randomized controlled trial in PCD. The term primary ciliary dyskinesia PCD MIM 244400)) has been used for a clinically and genetically heterogeneous group of recessive disorders with defective ciliary motility resulting in chronic upper and lower respiratory tract disease. Organ laterality defects occur in approximately ~50% of cases, usually situs inversus totalis (Kartagener’s syndrome). In 1976, Afzelius reported that PCD is characterized by ultrastructural defects of cilia leading to ‘immotile’ cilia [44400) ha4400) hasThe PCD prevalence is difficult to determine  and is iPCD is both under-diagnosed and diagnosed too late . TherefoUp to 85% of individuals with PCD have a history of unexplained neonatal respiratory distress . SymptomIn many PCD variants, ciliary dysfunction also involves cilia of the embryonic node which are essential for establishing the left-right asymmetry of visceral organs. Dysfunction of these nodal cilia results in a randomization of left-right body asymmetry. Hence, 40% to 50% of PCD individuals present with situs inversus totalis, a condition known as Kartagener’s syndrome, and a smaller subset of individuals (at least 6.3%) display complex situs anomalies associated with congenital heart disease . Of noteAs the structure of sperm flagella is similar to the ciliary axoneme, many PCD variants are associated with male infertility. By contrast, it is yet unclear to what extent women with PCD are affected by subfertility due to dysfunction of cilia in the fallopian tubes.Measuring nasal nitric oxide (nNO) is a very robust screening test for PCD as values are very low in most PCD patients -21. nNO in vitro ciliogenesis in respiratory epithelial cell cultures. Although the ciliary beating phenotype may be altered after cell culture, primary abnormalities are still present [High-speed video microscopy (HVMA) of ciliary beat pattern and frequency of respiratory epithelial cells obtained by trans-nasal brushing currently is the first-line diagnostic test in many centers. Cilia can be observed at high-resolution in real time and with slow-motion replay . HVMA is present ,30.DNAH11 mutations [Analysis of ciliary cross-sections by TEM has been used traditionally to confirm a PCD diagnosis. However, as this approach cannot be used to identify an increasing number of PCD variants at least 30%; ,32) with0%; ,32 wutations ,36 usualDuring the last decade, high resolution IF has been introduced as an additional tool to investigate the subcellular localization of the ciliary proteins in human respiratory epithelia . MeanwhiPCD is an autosomal recessive disorder. Mutations in a rapidly expanding number (currently 31) of genes are disease-causing  or radiN [RSPH1 ,40, RSPHA, RSPH9 ) as well [MCIDAS , CCNO [4l course . Males wmotility . Subjectmotility ,4.Modern high-throughput genetic technologies allow identification of disease-causing biallelic mutations in approximately ~60% of patients. Although not yet implemented for routine diagnostics, next generation sequencing already is cost-efficient and effective in diagnosing PCD compared to traditional sequential Sanger sequencing of single genes. However, it has to be kept in mind that every year novel genetic defects are discovered and therefore genetic testing cannot be used to rule out a PCD diagnosis. In addition, expertise is necessary to distinguish mutations from rare polymorphisms.Given the heterogeneity of possible findings associated with PCD, there is no uniform approach in diagnosing PCD. Currently, we consider a PCD diagnosis confirmed if the following diagnostic criteria are fulfilled: (1) clinical presentation consistent with PCD; and (2) confirmation of the diagnosis by at least two of the following methods: unequivocally abnormal HVMA finding, unequivocally abnormal TEM finding, unequivocally abnormal IF finding, abnormally low nNO concentration/production and demonstration of unequivocal biallelic disease-causing mutations by genotyping. In cases where only HVMA and nNO concentration/production are abnormal, HVMA should be repeated at least three times and show the same abnormal results each time. Individuals with typical clinical symptoms and only one abnormal diagnostic test are usually considered to have a possible PCD diagnosis with exceptions made on an individual basis .Of note, this approach is provisional, as it is very likely that further research will continue to modify our understanding of different PCD phenotypes.There is a lack of evidence-based management guidelines for PCD. Randomized controlled trials have not yet been performed in this condition. Thus, therapies are deduced from other diseases with defective mucociliary clearance, notably CF and non-CF bronchiectasis. The PCD task force of the European Respiratory Society has issued recommendations for the management of PCD highlighting the importance of routine airway clearance techniques, the use of antibiotics to control infection, and the avoidance of harmful agents such as active and passive smoke .http://www.bestcilia.eu/). Core programs of this network include: (1) A cross-sectional observational trial for answering pertinent questions on clinical phenotype, severity, prognosis, and effect of treatments on outcomes; (2) An international prospective PCD registry for systematic data collection on incidence, clinical presentation, treatments, and course of the disease. This will allow to monitor trends in management and outcomes and to recruit patients for trials. (3) The introduction of standardized diagnostic testing for PCD in three European countries , where this is currently not available. This approach will be paradigmatic for other countries how to implement sophisticated diagnostic facilities; (4) The development of PCD-specific health-related quality of life questionnaires (QoL-PCD) as an outcome measure in clinical trials. QoL-PCD is furthermore particularly useful to track changes over time and therefore to assess changes in disease impact on daily life. Therefore, QoL-PCD will be integrated into the longitudinal international PCD registry; (5) The first randomized controlled clinical trial ever performed in PCD to analyze the efficacy and safety of long-term use of azithromycin.To overcome limitations in PCD care, European and North American investigators and clinicians, as well as patient support groups, have joined forces in BESTCILIA, a European Commission funded consortium dedicated to improve PCD care and knowledge  should be considered at larger intervals . MagnetiPossibly, determination of lung clearance index (LCI) using multiple breath washout may evolve as a tool to monitor PCD lung disease accurately and non-invasively. It correlates well with HRCT findings in CF and is more sensitive to early changes in lung physiology. However, a recent study has failed to demonstrate a correlation between LCI and HRCT scores in PCD .Monitoring of upper airways disease includes regular hearing tests that should be performed at least every 6 months in young children and every year in adults. PCD individuals are at an increased risk to develop sleep disordered breathing, particularly due to obstructive sleep apnea syndrome ,67. EvenDaily airway clearance and aggressive antibiotic treatment of respiratory tract infections are treatment cornerstones of PCD lung disease. Review of individualized, age-appropriate airway clearance therapies should be part of the regular follow-up visits. No individual technique has proven superior. Although of unproven benefit, inhalation of hypertonic saline to improve cough clearance is widely used as is use of bronchodilators. Physical exercise effectuates general health improvements and therefore is encouraged. The role of inhaled rhDNase, a medication commonly used in CF, is unclear. In non CF-bronchiectasis, rhDNase has been shown ineffective and therefore it is not regularly used in PCD . ConsistHaemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, and Streptococcus pneumoniae [Pseudomonas aeruginosa is found particularly in adults with advanced disease. It is unclear, if chronic Pseudomonas aeruginosa infection has similar detrimental effects on lung function in PCD as in CF. However, as prompt eradication of potentially harmful bacteria remains a core goal in PCD management, protocols based on those used in CF are recommended. Chronic Pseudomonas aeruginosa infection is often treated with nebulized antibiotics. Some centers advocate the use of long-term prophylactic antibiotics. Currently, there is no evidence for this approach. Empirical knowledge, however, supports long-term antibiotic use in children requiring frequent courses of antibiotics. Currently, a BESTCILIA trial is recruiting patients to test the effects of long-term Azithromycin use.Antibiotic treatment of respiratory tract infections should be instituted promptly and adjusted to microbiological findings. Fever is not a reliable symptom in many PCD individuals. We recommend antibiotics if patients note an increase in sputum quantity or a change in sputum viscosity or color. The most commonly cultured pathogens are eumoniae . ChronicLung surgery in PCD is usually not recommended; in selected cases of localized disease refractory to conservative management, lobectomy can be considered. Bilateral lung transplantation is a treatment option for end-stage lung failure.Impaired mucociliary clearance in the upper airways affects the nasal cavity, the paranasal sinuses, and the middle ear. Persistent nasal discharge and blockage is treated by nasal irrigation. Chronic rhinosinusitis may be treated additionally by sinonasal inhalation of hypertonic saline solution and, when infective exacerbations occur, with antibiotics. Sinus surgery for nasal polyposis bears a high risk of early recurrence and is therefore performed only in cases of severely blocked passages. Similar to the treatment of lower airways disease, topical corticosteroids are useful only in individuals with additional allergic rhinosinusitis. The role of ventilation tubes for treatment of conductive hearing loss due to chronic otitis media with effusion is controversial. Due to the impaired mucociliary clearance, PCD patients have a higher risk of developing chronic mucopurulent discharge after ventilation tube insertion ,70. TherCongenital heart disease, when present, usually requires corrective or palliative surgery. Male or female infertility is managed with adequate reproductive techniques. However, as men with PCD are not always infertile and women only have a slightly decreased fertility , birth control measures are necessary if there is no wish to have children.Many PCD individuals receive suboptimal management because there are no evidence-based treatment guidelines. Establishing the diagnosis requires several complementary tests. A multidisciplinary management approach is well recognized to benefit long-term outcomes. Regular surveillance should include lung function testing, microbiological studies, and review of airway clearing techniques. Currently, management is mainly deduced from evidence from other suppurative lung diseases.


Both acute and chronic stress profoundly affect hippocampally-dependent learning and memory: moderate stress generally enhances, while chronic or extreme stress can impair, neural and cognitive processes. Within the brain, stress elevates both norepinephrine and glucocorticoids, and both affect several genomic and signaling cascades responsible for modulating memory strength. Memories formed at times of stress can be extremely strong, yet stress can also impair memory to the point of amnesia. Often overlooked in consideration of the impact of stress on cognitive processes, and specifically memory, is the important contribution of glia as a target for stress-induced changes. Astrocytes, microglia, and oligodendrocytes all have unique contributions to learning and memory. Furthermore, these three types of glia express receptors for both norepinephrine and glucocorticoids and are hence immediate targets of stress hormone actions. It is becoming increasingly clear that inflammatory cytokines and immunomodulatory molecules released by glia during stress may promote many of the behavioral effects of acute and chronic stress. In this review, the role of traditional genomic and rapid hormonal mechanisms working in concert with glia to affect stress-induced learning and memory will be emphasized. Astrocytes primarily express β1, α1, and α2  is perhaps the best-characterized cytokine released in response to stress. Following chronic mild stress, mice have impaired memory on both object location and object recognition memory. Cognitive impairments are accompanied by increased plasma IL-1β, plasma tumor necrosis factor-alpha, and IL-6 . Following chronic stress, tryptophan can be directed toward the KP (Miura et al., GCs have well-established anti-inflammatory effects that decrease microglial activation. GCs provide master control over several inflammatory and anti-inflammatory factors Figure . MicroglChronic stressors can attenuate or prevent the anti-inflammatory effects of GR activation on microglia. Microglia can readily become sensitized to GC over-secretion to the point where GCs no longer prevent pro-inflammatory microglial activity, but rather promote it, as is the case in neurodegenerative disease and obesity (Munhoz et al., NE has several effects on microglia that may contribute to cognitive deficits following stress Figure . GeneralWhile most attention in this field has been given to the impact of astrocytes and microglia on cognitive function, there is evidence that oligodendrocytes are also affected by stress and could modulate learning and memory. Oligodendrocytes' list of known functions extends beyond axon myelination, and now includes direct modulation of neuronal function (de Hoz and Simons, Although stress and/or GC administration almost ubiquitously decreases neurogenesis (Anacker et al., Glia are increasingly recognized as critical regulators of cognitive processes; understanding of the multiple cell types involved in behavioral regulation and the molecular processes involved continues to expand. Glia are quickly becoming pharmacologically-relevant cellular targets for treatments of a variety of psychiatric disorders Koyama,  and offeJP-L, DO, and EM all planned, drafted, and edited the manuscript.Merlin Trust 2015 Excellence award to ECM; American Diabetes Association 7-12-BS-126 to ECM.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. The reviewer Professor Leif Hertz and handling Editor Professor Ye Chen declared past collaboration and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


In Austria occurrence of 40 different species of mosquitoes belonging to 6 genera has been described. AGES in 2011 initiated a nation-wide mosquitoe-surveillance program to identify invading species. As part of this project, pools of Culex, Aedes, Anopheles and Culisetta species collected in different parts of the country were analyzed by PCR analysis; in addition seroepidemiological testing for bunyaviruses was performed in selected regions. In order to be able to detect also new strains, a broad spectrum CODEHOP (Consensus-Degenerate Hybrid Oligonucleotide Primer) approach was used initially; presumably due to the low sensitivity of degenerated primer designs and due to dilution in insect pools, we did not pick up any new isolates so far using Orthobunyavirus, Phlebovirus or Nairovirus group-specific primers. Two bunyaviruses were found in Culex pipiens from the southern province Carynthia in 2012, detected with non-degenerate multiplex PCR, the sequences highly homologous to Italian Tahynavirus (TAHV) isolates. 245 sera collected for a study of zoonotic infections in hunters, veterinarians, farmers, and abattoir workers , were tested by ELISA tests, using inactivated Crimean Congo virus (CCHF) from Bulgaria as well as recombinant CCHF nucleoprotein produced in baculovirus. In addition, cell culture derived TAHV lysates as well as TAHV infected cells were used as antigens in ELISA and immunofluorescence (IF) under an experimental setup. No seropositivity for CCHF or related nairoviruses was found in these Austrian human sera originating from risk groups for zoonotic infection.


Although hippocampal theta, a 4-12 Hz rhythm associated with episodic memory, has been studied extensively, the cellular mechanisms underlying its generation are unclear. OLM cells have been considered pacemakers of local CA1 theta , make thin vitro, and used these properties to constrain Izhikevich-type models of BCs/AACs, BSCs, and OLM interneurons. We constructed our network model with these individual cell models, and constrained network size, connectivity, and synaptic properties with experimental data. Experimental excitatory postsynaptic currents (EPSCs) recorded during endogenous CA1 theta oscillations were used to drive the various cell model populations, and we used a simple local field potential (LFP) model to integrate the effects of cell firing. To determine how the interactions between OLM cells and BSCs affect local theta rhythms, we explored how specific features of the network affected model LFP power. In addition, we simulated optogenetic experiments by silencing the OLM cell model population during the network rhythm.We derived cellular properties from patch clamp recordings of fast-spiking parvalbumin-positive (PV+) interneurons – likely comprising basket cells (BCs), axo-axonic cells (AACs), and BSCs – and of somatostatin-positive putative OLM cells in the CA1 region of an intact hippocampus Spike characteristics and firing behaviors in our network models approximated those determined experimentally. Our models distinguish between regimes in which OLM cells minimally or strongly affect the power of network oscillations, and predict that the dis-inhibitory effect of OLM cells on BSC to pyramidal cell interactions plays a critical role in the power of network theta oscillations. When OLM to BSC model connections are not too strong, the OLM cells’ direct influence on pyramidal cells balances with its indirect dis-inhibitory effect (through the BSCs). In this case, when the OLM cell population is silenced, there is a compensatory effect on network power, and thus minimal change in power. However, when these OLM to BSC connections are stronger, the dis-inhibition of pyramidal cells does not balance with their direct influence, and thus silencing OLM cells has a stronger effect. This does not change when we consider various distributions of strengths in which the cell populations affect the LFP. Thus, our network models are able to make particular predictions that can be tested with optogenetics.


This review aims at presenting a current view on the physiopathologic mechanismsassociated with temporomandibular disorders (TMDs). While joint pain is characterizedby a well-defined inflammatory process mediated by tumor necrosis factor-α andinterleukin, chronic muscle pain presents with enigmatic physiopathologic mechanisms,being considered a functional pain syndrome similar to fibromyalgia, irritable bowelsyndrome, interstitial cystitis and chronic fatigue syndrome. Central sensitizationis the common factor unifying these conditions, and may be influenced by theautonomic nervous system and genetic polymorphisms. Thus, TMDs symptoms should beunderstood as a complex response which might get worse or improve depending on anindividual's adaptation. Pain is the most commonsymptom usually concentrated in masticatory muscles and/or temporomandibular joints(TMJs), but exacerbated by mandibular movement and stomatognathic functions.Despite extensive literature on the topic, there is a need for further prospective,controlled, randomized, long-term clinical trials in order to establish a concrete causeand effect relationship. On the other hand, significant advance has been made withregard to the physiopathologic mechanisms associated with this condition. -The pathophysiology of TMJ pain is better understood than masticatory muscle pain; thus,the former is a convenient starting point to discuss TMDs pain mechanisms.,,,,TMJ inflammation results in the release of various proinflammatory cytokines,particularly tumor necrosis factor-α (TNF-α) and interleukins,Cytokines are mainly produced by macrophages penetrating into the synovium. Synoviuminflammation affects the viscosity of synovial fluid and leads to insufficientlubrication and nourishment of cartilage and disc.,Inflammatory mediators stimulate the nociceptors of TMJ and increase the release of CGRP and substance P, which result in swelling, redness anda rise in temperature. This process is known as neurogenic inflammation.,,Chronic TMD, especially myofascial TMD, is considered a functional pain syndrome similarto fibromyalgia, irritable bowel syndrome, interstitial cystitis and chronic fatiguesyndrome. These conditions appear to have common etiological factors which explain thegreat comorbidity of symptoms. It is interesting to notice that functional disorderstend not only to cumulatively affect an individual, but also to present centralsensitization and amplified pain perception. Such disorders have similar treatmentresponse, and may be treated with antidepressant drugs and cognitive behavioral therapy.The pathophysiologic mechanisms of such pain conditions remain unknown. However, it isbelieved that amplified pain perception, alterations in brain activity as well as inimmune and neuroendocrine activities, and genetic predisposition may be involved.Further studies should be capable of revealing a predominant or unifying mechanism thatexplains such functional alterations.In cases of functional pain syndrome, pain is no longer a protection factor. Pain isspontaneously felt and might be triggered by innocuous stimuli ; it might beexcessive and prolonged and occurring in response to nociceptive stimuli ;and it might spread beyond the injured site .The phenomenon of peripheral sensitization occurs as a result of inflammatory responseprovoked by a tissue injury. Should that be the case, allodynia and hyperalgesia occurdue to inflammatory mediators released at the site of lesion. For instance, whenever atooth is extracted, the site of inflammation presents with increased sensitivity topressure  mediated by sensitized nociceptors. Nevertheless, such reactionis expected to restore within a reasonable period of time due to decreased nociceptorsactivity and consequent decreased afferent activity of the dorsal horn. However, theinflammatory process and its consequent afferent activity might be intense enough so asto establish a central process.,,C-fibers are the first nociceptors involved in central sensitization onset, as theyproduce slow synaptic currents and repetitive stimuli, thereby increasing depolarizationin the spinal cord dorsal horn as a result of activating the calcium channels thatdepend on binders. Initially, calcium channels are opened, quickly and for a shortperiod of time, by AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)receptors. This process allows calcium ions to penetrate the cell and favorsdepolarization of wide dynamic range neurons which can respond to a large variety ofstimuli. In addition to activating ionotropic receptors, glutamate and substance P alsoactivate metabotropic receptors, thereby releasing more calcium to intracell vesicles,increasing the concentration of calcium ion and, as a result, activating protein-kinaseenzymes that phosphorylate the N-methyl-D-aspartate (NMDA) receptor. In normalconditions, the channel bound to the NMDA receptor is blocked by magnesium ions. Oncethis receptor is activated, it is phosphorylated and magnesium ions are released,thereby opening the channel and allowing calcium ions to enter the cell. Unlike whathappens with the AMPA receptor, activation of the NMDA receptor is enduring and hardlyever reverted.,Although activation of NMDA receptors seem to play a major role in centralsensitization, a single molecular mechanism responsible for the process has not yet beenidentified, as it might be mediated by different processes capable of producing avariety of alterations in the somatosensory system. Of the many alterations, thefollowing apply: increased excitability of neuronal membrane, facilitation of synapsesand decreased inhibitory influence of dorsal horn neurons. Thus, central sensitizationmight lead to pain despite absence of pathologies or peripheral pain stimuli and,therefore, it should target the central nervous system, not the peripheral one.,,Lorduy et al,,The relationship established between anxiety or stress and TMD is simply explained bythe greater contraction of masticatory muscles happening as a result of TMD, sincemuscle hyperactivity is one of the most frequent mechanisms influencing myofascialpain.,,Mild negative emotions might favor the occurrence of pain. In this context, wheneverdamage is unpredictable, pain plays an important role in detecting risky situations soas to preserve tissue integrity. It is an adaptive means of promoting environmentalmonitoring, a sensory monitoring mechanism to increase threat detection.In situations involving intense negative emotions, whenever thread is imminent andpredictable, fighting and escapement reactions establish hypoalgesia as a defensemechanism. An interesting experiment revealed that Vietnam veterans with post-traumaticstress disorder (PTSD) reported 30% less pain when stimulated by heat after beingexposed to battle videos. There was no reduction in pain intensity when veterans weresubject to naloxone, an opioid receptor antagonist. Results clearly revealed analgesiainduction mediated by opioid and induced by stress in PTSD patients.,The amygdala detects danger by causing fear and anxiety and, as a result, putting us instate of alert. The connections between amygdala and periaqueductal gray are involved inthe modulation of emotion-mediated nociception. An unregulated hazard detection circuitin functional pain syndrome patients might decrease the threshold of negative, intense,long-term emotional experiences. Thus, unlike healthy patients, intense negativeemotions might lead to hyperalgesia, not hypoalgesia.,Diffuse noxious inhibitory controls (DNIC) are the main endogenous pain inhibitorysystems. The literature asserts that a nociceptive stimulus suppresses anothernociceptive stimulus ("pain inhibits pain" mechanism) provided that body surfaces understimulation are at a certain distance. Patients with fibromyalgia subjected to coldpressor tests are less likely to respond to DNIC, thereby making endogenous paininhibitory system deficiency explicit.Robinson et al-Psychological stress is known for inducing adaptive responses of physiological systems,including increased hypothalamo-hypophyseal adrenal system activity. These responsesinduce cortisol secretion by the adrenal cortex and increase sympathetic adrenalmedullary system (SAM) activity which, in turn, isolates adrenalin and noradrenalinethrough peripheral sympathetic nerve endings and adrenal medulla.-,Emerging evidence suggests that unregulated autonomic nervous system contributes to TMDdevelopment and chronicity.,,,,,Chalaye et al,,Unregulated central in TMD patients causes the autonomic nervous system to react less tophysical or psychological stress, since the sympathetic tone is high even at rest. Thischaracteristic has been associated with COMT gene variables (SNPs or single-nucleotidepolymorphisms),The different physiopathologic mechanisms involved in the multifactorial nature of TMDssuggest that distinct genetic loci are connected in such a way that each locus producesminor effects and interacts with environmental exposure.TMD symptoms should be understood as a complex individual response with uniquecomplaints and which might get worse or improve depending on an individual's geneticcomposition.Due to multiple etiological factors and different individual adaptation,multidisciplinary therapy should be encouraged. Likewise, future research elucidatingneurobehavioral processes underlining chronic pain should also be encouraged.


Complex microbial communities are an integral part of the Earth's ecosystem and of our bodies in health and disease. In the last two decades, culture-independent approaches have provided new insights into their structure and function, with the exponentially decreasing cost of high-throughput sequencing resulting in broadly available tools for microbial surveys. However, the field remains far from reaching a technological plateau, as both computational techniques and nucleotide sequencing platforms for microbial genomic and transcriptional content continue to improve. Current microbiome analyses are thus starting to adopt multiple and complementary meta'omic approaches, leading to unprecedented opportunities to comprehensively and accurately characterize microbial communities and their interactions with their environments and hosts. This diversity of available assays, analysis methods, and public data is in turn beginning to enable microbiome-based predictive and modeling tools. We thus review here the technological and computational meta'omics approaches that are already available, those that are under active development, their success in biological discovery, and several outstanding challenges. Microbes and their biochemical activities are an essential component of virtually all ecosystems on earth, shaping environments ranging from deep marine sites to our own body. For example, marine microbial communities are responsible for half of the oxygen produced on our planet , and theAlthough the ubiquity and complexity of microbial communities have been well studied for decades, advances in high-throughput sequencing have provided new tools that supplement culture-based approaches both in their molecular detail and in their accessibility to a broad scientific community. The first culture-independent approaches were based on low-throughput sequencing of the bacterial 16S ribosomal rRNA gene , and theCitrobacter rodentium infections  and theceptible . Pathogeceptible . Findingceptible  and by wceptible . Conversceptible  or metabceptible , are sticeptible  and the ceptible . In partA meta'omic study typically aims to identify a panel of microbial organisms, genes, variants, pathways, or metabolic functions characterizing the microbial community populating an uncultured sample. Metagenomics as a term can refer loosely to the field as a whole and to the specific sequencing of whole-community DNA, and it is naturally complemented by metatranscriptomics (cDNA sequencing) and functional technologies, such as metaproteomics and community metabolomics . MetagenWMS sequencing is based on extracting DNA or RNA from the community in its entirety, followed by library construction and short-read sequencing of the entire mixture of genomes or transcripts. The resulting millions of short random DNA/cDNA fragments can then be assembled  or used individually as markers for specific organisms and metabolic functions. Compared with rRNA amplicon sequencing, shotgun meta'omics typically provides insight into functionality of microbes and their biological processes, including horizontal gene transfer, sequence variants and evolutionary variability, and genome plasticity. It allows organisms to be identified with increased taxonomic resolution , as the The Illumina platform is currently preferred for meta'omic sequencing, and is also supplanting the Roche 454 platform widely used in microbial community analysis for rRNA gene surveys . EmerginMetagenomic sequencing, if performed at a sufficiently high coverage, can in some cases allow reconstruction of complete genomes of organisms in a community . In pracHowever, recent years have seen an explosion of metagenome-specific assemblers, which use strategies to tease apart sequencing artifacts from true biological ambiguity within communities. Only a subset of these includes MetaVelvet , khmer , metAMOSWhole-genome assembly from metagenomes is impossible in most cases, and such assemblers instead aim to provide the largest reliable and useful contigs achievable from their input sequence reads . Althougde novo binning of microbial sequences (using intrinsic sequence properties) or by identifying them using information from sequenced microbial genome databases  is the typical primary objective of amplicon sequencing investigations . Metagenomic shotgun sequencing can provide comparable information, often at better resolution, either by All of these approaches rely in some way on reference genome catalogs. Although those sequenced for microbial organisms are biased towards model organisms and pathogens, large-scale efforts like the Human Microbiome Project ) and theIntrinsic binning approaches for community profiling ), interpExtrinsic or homology-based classification  adopts 3The most extrinsic methods for binning and community profiling instead use whole-genome searches of metagenomic sequences against the entire catalog of reference genomes. This can be performed with varying levels of sensitivity by using homology search  or mappMicrobial communities can be seen not only as groups of individual microbes, but also as collections of biochemical functions affecting and responding to an environment or host organism. Metagenomics can thus also identify the genes and pathways carried by a microbial community, and metatranscriptomics can profile their expressed function. Just as several alternatives for microbial profiling are described above, two broad classes of functional community profiling depend either on genes identified within longer assembled contigs or on assembly-free read-based approaches. Assembly-based methods are sensitive to the challenges outlined above, such as a bias towards higher-abundance community members or sequences that are easy to assemble. Assembly-free functional inference, by mapping sequences to annotated reference genomes or functional databases, can be more sensitive (including a greater proportion of reads or microbes) but less specific in its functional identifications.Assembly-based metagenome annotation can be performed by adapting pipelines for annotation of single microbial genomes. Metagenomic contigs are thus scanned for identifying protein-coding genes (CDSs), as well as CRISPR repeats, noncoding RNAs, and tRNA. Functional characterization can then be performed assigning full CDSs (rather than single-sequencing reads) to functional categories by means of orthology relations with sequences in well-characterized functional databases, such as NCBI nr , the KEGFunctional profiling using reference information can be based either on reference genome read mapping (at the nucleotide level) or on translated protein database searches. For the former, reads mapped to reference genomes as discussed above can then be sent through an additional second mapping from loci to annotated functions. For the latter, functional databases of diverse protein families as listed above can be leveraged to identify function by translated homology search. Like reference genomes, these databases are generally also enriched for functional information from model organisms and pathogens. Thus, the greater the enrichment of such organisms in a community, the more characterized functional annotations are likely to be retrieved. Examples of pipelines, including functional annotation by translated mapping, include MG-RAST , MEGAN , and HUMIt is important to note that whole-community functional profiling is not yet a mature area, and neither gene annotations within reference genomes nor those in protein databases are well tuned to whole-community metabolism. For example, MetaCyc  and SEEDKnowledge of the microbes and gene products within communities is an important step toward understanding their ecology, environmental responses, and interorganismal interactions . MicrobiTo assess meta'omic profiles more accurately, recent association approaches have been developed specifically for intermicrobe co-occurrence and co-exclusion detection in microbial communities. For example, in vitro culture and in vivo dynamics, will be needed to ensure the correctness of these challenging models; some studies of the latter with respect to natural long-term dynamics  remains the most practical current approach in whole-community analysis.The major challenge faced in metatranscriptomics is the isolation of microbial mRNA, which usually makes up only a small percentage of total microbial RNA and an even smaller proportion of total RNA if host nucleotides are present. Eukaryotic genes and genomes are sufficiently large as to rapidly swamp smaller microbial transcripts, and even in nonhost-associated communities over 90% of microbial transcripts are typically ribosomal rRNA. The difficulty of isolating prokaryotic mRNA is further compounded by its lack of the 3′-end poly (A) tail that marks eukaryotic mRNA . High-quSingle-cell sequencing provides an alternative approach to accessing novel information about uncultured microbes . AlthougCurrent single-cell approaches first isolate single microbial cells by sorting them, lyse them separately, amplify and label them separately, and sequence the resulting pool. The subsequent analysis of single-cell sequence data thus relies much more heavily than do meta'omics on assembly, but fortunately in a less-challenging setting. IDBA-UD  and SmasMeta'omics provides an important tool for studying evolution within microbial communities, which can occur on two very different time scales. Over the course of days, weeks, or the years of a host's lifetime, microbial genome plasticity allows remarkably rapid acquisitions of novel mutations and laterally transferred genes. Over the course of millennia, however, the overall structure of host-associated communities, their phylogenetic composition, and their microbial pan-genomes can evolve more slowly in tandem with their hosts' physiology and immune systems .in vivo  is a synthetic community transferrable to gnotobiotic mice that has been in use as an experimental system for years (Given the complexity of most ‘wild' microbial communities, one of the most promising approaches for such validation has been in the construction of model microbial communities. These have been successful both entirely emostats , with onemostats . Recent ifficile . In vivoor years . The eigor years .Although technologies and analyses are constantly improving, WMS sequencing is currently reaching maturity in the sense that validated, standardized experimental and bioinformatic procedures are available to answer typical biological questions of interest . These iAn exciting next step in microbial community systems biology will be the opportunity to integrate and meta-analyze multiple data sets. This is already starting to be the case with large 16S and, gradually, metagenomic data sets defining healthy human microbial baselines . Just asin vivo will continue to improve our understanding of microbial community systems biology.The degree to which microbial community activity and structure is dynamic over time has perhaps been underappreciated, and an additional component necessary for whole-community modeling will be the combination of longitudinal surveys  with sysStep-by-step guide into the computational pipeline of Figure 4