Child stunting and anemia are intractable public health problems in developing countries and have profound short- and long-term consequences. The Sanitation Hygiene Infant Nutrition Efficacy (SHINE) trial is motivated by the premise that environmental enteric dysfunction (EED) is a major underlying cause of both stunting and anemia, that chronic inflammation is the central characteristic of EED mediating these adverse effects, and that EED is primarily caused by high fecal ingestion due to living in conditions of poor water, sanitation, and hygiene (WASH). SHINE is a proof-of-concept, 2 × 2 factorial, cluster-randomized, community-based trial in 2 rural districts of Zimbabwe that will test the independent and combined effects of protecting babies from fecal ingestion  and optimizing nutritional adequacy of infant diet  on length and hemoglobin at 18 months of age. Within SHINE we will measure 2 causal pathways. The program impact pathway comprises the series of processes and behaviors linking implementation of the interventions with the 2 child health primary outcomes; it will be modeled using measures of fidelity of intervention delivery and household uptake of promoted behaviors and practices. We will also measure a range of household and individual characteristics, social interactions, and maternal capabilities for childcare, which we hypothesize will explain heterogeneity along these pathways. The biomedical pathway comprises the infant biologic responses to the WASH and IYCF interventions that ultimately result in attained stature and hemoglobin concentration at 18 months of age; it will be elucidated by measuring biomarkers of intestinal structure and function ; microbial translocation; systemic inflammation; and hormonal determinants of growth and anemia among a subgroup of infants enrolled in an EED substudy. This article describes the rationale, design, and methods underlying the SHINE trial.Clinical Trials Registration. NCT01824940. In a pooled analysis of 9 studies that together collected diarrhea and growth data on 1393 children, the odds of stunting at 24 months increased multiplicatively by 2.5% per episode of diarrhea, and 25% of all stunting among 24-month-old children was attributable to having 5 or more episodes of diarrhea in the first 2 years of life . In otheLike stunting, childhood anemia is a major public health problem in Africa and Asia  and a prA major cause of child stunting and anemia is environmental enteric dysfunction (EED). EED is a subclinical disorder of the small intestine, which is virtually ubiquitous among asymptomatic people living in low-income settings throughout the world –38. EED The primary cause of EED is infant ingestion of fecal microbes due to living in conditions of poor quality and quantity of water, sanitation, and hygiene (WASH).The Sanitation Hygiene Infant Nutrition Efficacy (SHINE) trial is motivated by a 2-part premise :A majorStudies of stillborn fetuses have established that EED is not present at birth . SeminalThe chronic immune activation that is characteristic of EED also may cause anemia through 2 possible pathways. First, elevated proinflammatory cytokines trigger increased hepatic synthesis of hepcidin, which suppresses iron absorption and utilization , 57, leaEvidence linking poor WASH with EED in humans is limited to observational studies. Among 3- to 5-year-old children in Malawi, those in households with latrines and a greater quantity of water had lower  LM ratios, and children who washed their hands more often or washed their hands with soap had a more normal sucrose-lactulose ratio (an indicator of gastric permeability) . SimilarNumerous observational studies have reported a strong association between the level of environmental WASH and child linear growth , 64–70; z score (HAZ) of 0.26 and that this effect was stronger among households with sanitation  vs without sanitation  [Adequate sanitation may be a particularly important requirement for normal growth. In the Esrey analysis, the association with growth was stronger for sanitation than water services . In the , +0.21) .Controlled trials in animal husbandry provide strong evidence that poor WASH restricts animal growth and that this effect is mediated by inflammation. In a factorial trial in which chicks were randomized to live either in cages with accumulated feces, dust, and dander (“dirty chicks”) or steam-cleaned cages (“clean chicks”), and to receive either antibiotics or no antibiotics, the clean chicks grew better than dirty chicks. However, the dirty chicks receiving antibiotics grew as well as the clean chicks not receiving antibiotics; overall, antibiotics had no growth effect in clean chicks . The pooabnormal T cells are hyperreactive to a normal exposure , EED mabacteria . Bacteribacteria , germ-frChildhood stunting and anemia are 2 highly prevalent, frequently overlapping problems among children aged <2 years in Africa and Asia, which result in immediate and long-term morbidity and cognitive deficits, lost human capacity, and reduced adult economic productivity. Moreover, both problems are only partially responsive to current public health interventions. SHINE is motivated by the premise that EED is a major underlying cause of stunting and anemia, that chronic inflammation is the central characteristic of EED mediating these adverse effects, and that EED is primarily caused by high fecal ingestion due to living conditions of poor WASH. SHINE will also investigate whether these hypothesized effects of improved WASH will be additive to those of concurrently improved infant feeding in reducing child stunting and anemia. Accordingly, SHINE is designed as a proof-of-concept, 2 × 2 factorial trial to assess the independent and combined effects of protecting babies from fecal ingestion  and optimizing nutritional adequacy of infant diet  on length and hemoglobin at 18 months of age. Within SHINE we are measuring 2 causal pathways. The program impact pathway comprises the series of processes and behaviors linking implementation of the interventions with the 2 child health primary outcomes; it will be modeled using measures of fidelity of intervention delivery and household uptake of promoted behaviors and practices . We are The WASH Benefits Trials in Bangladesh and Kenya  are ongoThe SHINE trial addresses one primary objective and multiple secondary objectives Table . The 4 tTable 1 of Supplementary Appendix) so that women in active arms receive more information than women in the SOC arm. Each randomized intervention comprises 5 core modules, focused on a key message and delivered to the mother at a specified relevant fetal or infant age. A separate team of research staff employed by the trial make data collection home visits at baseline (approximately 14 weeks' gestation), at 32 weeks’ gestation, and at 1, 3, 6, 12, and 18 months postpartum to measure fidelity of intervention delivery, uptake of the interventions, and trial outcomes  of the Zimbabwe Ministry of Health and Child Care (MoHCC). More than 5000 pregnant women were identified in a surveillance system of early pregnancy detection using urine pregnancy tests and enrolled into the trial between November 2012 and March 2015. All enrolled women receive the same number (15) of visits from their VHW between enrollment and 18 months postpartum, during which standard-of-care (SOC) primary healthcare messages are delivered. During some of these 15 visits, VHWs in clusters randomized to WASH, IYCF, and WASH + IYCF also deliver lessons specific to their arm . As much as possible, we chose messages, commodities, and delivery systems that could be economically sustainable at scale, but we prioritized potential biological efficacy over cost.SHINE interventions were based on formative research  andWe chose to implement interventions through the existing MoHCC VHWs rather than creating a separate intervention delivery system. This decision was motivated partly by Zimbabwe's historically strong community health system, but primarily taken because VHWs (who are nominated by and live in the communities they serve) could achieve the greatest coverage and acceptability within these rural communities. Beginning in 2010, vacant VHW posts were filled  and new VHWs completed the standard MoHCC 5-month training program  . VHWs weAll interventions (including latrine construction) were pilot-tested in a district adjacent to the study area among 40 households with children <18 months. VHWs in the pilot district were trained to implement the study to assess the training requirements and support needs of this cadre before training the 360 VHWs in the SHINE districts.VHWs are trained through the MoHCC curriculum to respond to a broad spectrum of health problems. The curriculum instructs VHWs to visit pregnant women and infants frequently, but the precise content or purpose of each visit is not specified. Consequently, the SHINE SOC intervention was designed to standardize the number of visits  and the content of primary healthcare messages across treatment arms. Four of these visits promote exclusive breastfeeding (EBF) from birth to 6 months using modules designed to overcome contextual barriers to EBF , 92. OthWithin 6 weeks of enrollment (approximately 20 weeks’ gestation) into the WASH and WASH + IYCF arms of the trial (see companion article ), a BlaiFor the IYCF intervention (see companion paper ), IYCF mSchistosoma hematobium infection , but very low prevalence of soil-transmitted helminth infections  [The study site comprises the contiguous districts of Chirumanzu and Shurugwi (combined population of 158 821 in 2010) , 300 km hurugwi) .The study area was divided into 212 clusters, defined as the catchment area of 1–4 VHWs. Digital maps of administrative boundaries of the study area were obtained from the Central Statistics Office and, using Google Earth, all homesteads and key landmarks  were plotted. Using large-scale maps, each of the 360 VHWs circled the houses they serve. Cluster-specific numbers of households, reproductive-age women, and <2-year-old children were gathered from VHW registers. Boundaries were finalized by grouping up to 4 VHWs into the same cluster if (1) they were working habitually as a team; (2) their catchment areas included homesteads within close proximity of each other; or (3) to achieve approximately the same number of households, pregnant women, and young children per cluster. About half of the clusters comprise the catchment area of a single VHW; on average, there are 1.6 VHWs per cluster.In 2011–2012, we assessed cluster-specific water access and sanitation coverage so that these factors could be balanced across treatment arms during randomization (see companion article ). A georCommunity engagement began 5 years prior to trial enrollment through discussion with leaders, strengthening of the PMTCT of HIV program, and VHW network strengthening. During a 3-month period before launching SHINE, we held 45 meetings throughout the study area, each attended by 50–75 influential community members and led by local government and district-level Zimbabwe MoHCC authorities. Following these consultations, Memoranda of Understanding were signed between each district, the MoHCC and Zvitambo, detailing SHINE activities and defining the tripartite roles.We established 4 study hubs in the study area, strategically sited such that all households in the study area could be reached by motorbike in <2 hours from the nearest hub; all were established within MoHCC facilities. Each hub is staffed with approximately 25 people and includes specimen processing laboratories with −80°C archiving freezers, internet connection, offices, and warehousing.The enrollment goal was a total sample size of 4800 pregnant women, 1200 in each of the 4 trial arms, each of which is comprised of 53 clusters. The primary outcomes are LAZ and hemoglobin level at 18 months of age, stratified by maternal HIV status. The main trial inference will be based on outcomes of infants of HIV-negative women, of whom we expect at least 4080 based on an estimated 15% HIV prevalence among pregnant women at the time of designing the trial. Although the primary outcomes are continuous, the sample size is based on dichotomous versions of these, as we wanted to provide answers regarding stunting and anemia per se. Allowing for 20% loss of evaluable infants at 18 months , we expect about 816 measurements in each of the 4 study arms. With type I error of 5% (2-sided test), and power of 90%, and control group stunting (HAZ < −2) of 30%, we will be able to detect a reduction of about 8 percentage points  for the marginal effect of either of the interventions, even in the absence of an effect of the other intervention .z scores, assuming a within-randomized cluster variance of 1.25. For hemoglobin, assuming a standard deviation at 18 months of 128 g/L [Calculations were based on an assumed coefficient of variation of the true proportion stunted at 18 months of 0.43 , and an effective loss of 33% of sample size due to cluster size variability, resulting in a design effect of 2.5. To be conservative, we used the same coefficient of variation for anemia. The largest design effect reported by Katz for stunting in several countries was 2.6 . Using t 128 g/L , we willA highly constrained randomization technique was used to allocate clusters (stratified by district) to treatments . A GAUSSTable 2 in the Supplementary Appendix.The randomization was stratified by district to achieve the same approximate number of clusters within each of the 4 study arms within each of the 2 districts. Although overall there were 53 clusters in each study arm, exact balance within each district was not possible, because 111 clusters were formed in one district and 101 in the other, neither of which was a multiple of 4. Forming appropriate clusters was deemed more important than achieving exact balance. The constraint criteria and the achieved values of the constraining variables by study arm are shown in VHWs were trained to visit all 15- to 49-year-old women in their catchment area once every 5 weeks, to offer a urine pregnancy test to those who had missed a menses, and to refer pregnancies to antenatal clinic and (with the woman's assent) to SHINE. We enrolled all consenting pregnant women permanently residing in the rural parts of the study districts during the period of trial recruitment; women living in employer-provided housing or renting accommodation were excluded because their residence was unlikely to be permanent and they could not authorize construction of a latrine at the household. We strove to enroll women after gestational week 8 (to minimize miscarriage risk after enrollment) but before week 14 .All research visits are conducted in study households by hub-based data collectors (DCs) who travel by motorcycle. Forty-three primary care nurses were recruited as DCs and given a 14-week training course in research ethics and Good Clinical Practice (GCP) (1 week); motorcycle riding (3 weeks); computing and data entry (2 weeks); HIV testing and counseling (3 weeks); SHINE questionnaire content and interviewing techniques (2 weeks); maternal and child anthropometry (1 week); clinical assessment and referral of infants according to Integrated Management of Childhood Illness  guidelinFollowing referral of a pregnant woman to SHINE, a DC conducts a visit to confirm the woman's pregnancy (by repeat urine test) and eligibility for the trial, and to obtain written informed consent. Illiterate women provide a thumbprint in the presence of a witness. Subsequently, DCs make 2 antenatal visits, at approximately 10–20 weeks (baseline) and 32 weeks, and 5 postnatal visits when the infant is 1, 3, 6, 12, and 18 months of age (Table Table 3 in the Supplementary Appendix). Maternal and household factors are reassessed at subsequent research visits as detailed in Table Maternal and household data collected at baseline include household composition, socioeconomic status, current WASH facilities and practices, and current IYCF practices if the household has a child <2 years old. The woman is interviewed regarding her antenatal care for the current pregnancy and recent morbidity; maternal capacities and depression are assessed (see companion article ). InformTable 3 of Supplementary Appendix 1) of infant scales and stadiometers used in trial research visits were installed by the trial in all 43 health institutions offering delivery services to women in the study area; anthropometry training is repeated every 6 months for health institution staff. For home deliveries, a visit is conducted to collect infant anthropometric measurements if SHINE learns of the delivery within 7 days. Birth dates are combined with last menstrual period date to calculate gestational age at birth and to categorize preterm birth.Birth information including date, mode, complications, weight, length, and head circumference are transcribed from the institutional delivery register  and also from the mother's handheld antenatal care card. The same models . HIV-infected women are advised to take their infants to the local clinic to undertake early infant diagnosis and commence cotrimoxazole prophylaxis at 6 weeks of age; blood samples from HIV-exposed infants collected from 3 months of age onward are additionally tested for HIV by DNA polymerase chain reaction in the Zvitambo laboratory and results returned to women with appropriate counseling, unless the mother does not wish to learn her infant's HIV status. In the case of maternal or infant deaths, a verbal autopsy is conducted with a family member and cause of death adjudicated by 2 study clinicians according to World Health Organization criteria [Samples are transported in cooler boxes by motorcycle to 1 of the 4 hub laboratories where they are processed and archived in −80°C freezers; every 2 weeks, samples are transferred from hub laboratories for long-term archiving in the Zvitambo Institute for Maternal and Child Health Research (Zvitambo) laboratory in Harare. Because both districts are known to have a high prevalence of schistosomiasis among school-aged children, women are tested for criteria .A subsample of at least 1000 HIV-unexposed infants  and all consenting HIV-exposed infants are being recruited into the EED substudy (see companion article ). The suSeveral observational studies are being pursued within the SHINE study population, drawing upon the infrastructure, dataset, and sample archive. Maternal schistosomiasis (assessed by urine microscopy) and maternal and infant biomarkers of mycotoxin exposure (see companion article ) are beiWe also will investigate the possibility that maternal EED and/or mycotoxin exposure during pregnancy contributes to adverse birth outcomes, including miscarriage, stillbirth, preterm delivery, fetal stunting, low birth weight, and neonatal death. Pregnancy losses are ascertained prospectively and data collected on risk factors and outcomes in a specific questionnaire. These studies are planned as case-control designs; the randomized interventions will be treated as covariates in analysis.DCs electronically collect questionnaire and observation data at all research visits using netbooks; questionnaires were programmed in Microsoft Access and SQL database and include quality control functions . Paper forms are available if technology fails in the field. At the close of each working day, all new data in the netbooks are copied onto 2 computers in the hub office: one accumulates all research data and the other is linked to the internet and automatically transfers data overnight to the main server in the Zvitambo office in Harare.Blood, fecal, saliva, and urine samples are labeled with preprinted bar-code labels at the time of collection and transported to hub laboratories. After transfer to the Zvitambo laboratory in Harare, specimens are logged into laboratory information system prior to further processing, analysis, and archival.P < .05 from a Wald test of the product term, we will also present results using a regression model with 3 terms to represent the 4 study arms.As a cluster-randomized trial, SHINE outcomes, in general, will be analyzed using statistical methods that account for within-cluster correlation. With a relatively large number of clusters (212) and for some analyses only a few events or prevalent cases per cluster, generalized estimating equations (GEEs) will be used, specifically the Stata GEE regression procedure, with exchangeable correlation structure within each cluster, for both continuous and discrete response variables. Primary analyses will contain 2 dummy variables representing each intervention; they will be unadjusted for other covariates. A log link function with Bernoulli response will be used for prevalence outcomes, to aid in interpretability by directly producing risk ratio estimates. Although the study is not powered to detect a statistical interaction effect between the 2 interventions, it will be estimated. If there is a statistically significant interaction, defined as In addition to these (unadjusted) primary analyses, we will report a set of adjusted estimates, conditional on baseline covariates described elsewhere . The goaReporting of results will follow the guidelines established in the extended CONSORT guidance for cluster-randomized trials .Most analyses, including the primary ones, are modified intent-to-treat at the child level. “Modified” refers to the requirement of an 18-month follow-up visit to determine the child's anthropometric status. Loss to follow-up is anything that prevents a child's 18-month visit from being completed, including pregnancy loss, infant death, refusal for further participation, and a permanent move outside of Zimbabwe. The analyses will be conducted according to the mother's assigned study arm based on her residence at the time of her enrollment, regardless of subsequent migration.Secondary analyses may require varying levels of contact and/or adherence with a focus on the following modified per-protocol population : 18-montAll research procedures are conducted in accordance with the Declaration of Helsinki and according to International Conference on Harmonisation GCP guidelines. The Medical Research Council of Zimbabwe and the Institutional Review Board of the Johns Hopkins Bloomberg School of Public Health provided initial and ongoing review and approval of the study protocol. Adverse events and serious adverse events are reported to both boards, according to their respective reporting requirements. An independent data safety and monitoring board (DSMB) was formed, comprising 2 physicians from the University of Zimbabwe and a statistician from the United Kingdom. The DSMB oversees implementation of the trial and monitors the safety and efficacy of interventions during quarterly reviews of interim data. A monitoring plan outlines the scope of internal and external monitoring for all trial activities. Internal monitoring is conducted quarterly at each field hub, and findings requiring corrective action are categorized as critical, major, or minor, with appropriate timelines for resolution. An external monitor appointed by the trial visits each hub every 6 months to conduct an inspection using his or her own monitoring and reporting tools.Supplementary materials are available at Clinical Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.


By tracking the incorporation of the 13C from lactate tracer we found that gluconeogenesis (GNG) from lactate accounted for 67.1 ± 6.9%, of whole-body glucose appearance rate (Ra) in TBI, which was compared to 15.2 ± 2.8%  in healthy, well-nourished controls. Standard of care treatment of TBI patients in state-of-the-art facilities by talented and dedicated heath care professionals reveals presence of a catabolic Body Energy State (BES). Results are interpreted to mean that additional nutritive support is required to fuel the body and brain following TBI. Use of a diagnostic to monitor BES to provide health care professionals with actionable data in providing nutritive formulations to fuel the body and brain and achieve exquisite glycemic control are discussed. In particular, the advantages of using inorganic and organic lactate salts, esters and other compounds are examined. To date, several investigations on brain-injured patients with intact hepatic and renal functions show that compared to dextrose + insulin treatment, exogenous lactate infusion results in normal glycemia.Because it is the product of glycolysis and main substrate for mitochondrial respiration, lactate is the central metabolic intermediate in cerebral energy substrate delivery. Our recent studies on healthy controls and patients following traumatic brain injury (TBI) using [6,6- As described by editor Schurr in the introductory chapter, the conclusion that glycolysis makes lactate is a central issue; by definition, glycolysis makes lactate Schurr, . WhetherImportance of the fact that lactate can be produced in particular cellular compartments and from those sites can enter the interstitium and vasculature from where it can reach adjacent or anatomically distributed cells, organs, and tissues to affect important functions was recognized in 1984 Brooks, . The excIn humans and other mammals the extent of lactate production is typically overlooked because removal  rate balances production (appearance) rate. In other words, with typical concentration measurement technology low and stable blood lactate levels belie high turnover  rates. And, even when arterial-venous [a-v] concentration differences and tissue blood flow measurements are available, in the absence of isotope tracer technology turnover rate is unknown because of rapid turnover within the tissue bed of interest. Restated, basing conclusions about the dynamics of lactate or any other metabolite solely on concentration data is intellectually equivalent to taking the US census in the morning and evening of the same day, and then upon seeing no significant change in population numbers concluding that nothing happened in the population during that day. Happily, many entered the population, and regrettably also, some exited. Hence, concentration data provide an incomplete view of lactate turnover (flux) except in conditions of sudden and large changes when homeostatic imbalances in the relationship between production and removal are indicated.In resting or working muscle  equals the arterial-venous difference for glucose  times CBF: CMRgluc = (AVDgluc) (CBF). Similarly, for lactate (CMRlac) equals the arterial-venous difference ([a-v]lac) times CBF: CMRlac = (AVDlac) (CBF). As already noted, in skeletal .Glucose uptake and glycolysis are features of normal cerebral metabolism, but trauma affects CMRgluc and glycolysis in specific ways. In particular, there is a relative increase in glycolysis, a “hyper-glycolysis” that is a recognized feature of cerebral metabolism immediately after injury  as well as glucose uptake and use (CMRgluc). Hence, the presence of hyperglycolysis is indicated by a fall in the normal molar ratio (MR), or “Metabolic Ratio” of 6/1 for CMRO2/CMRgluc, the stoichiometry for complete glucose oxidation being given as: 6 O2 + C6H12O2 → 6 CO2 + 6 H2O. The fall in cerebral MR following injury is indicative of a relative increase in the role of glycolysis leading to cerebral lactate production , whereas typical circulating blood [lactate] is <1 mM (<9 mg%), and [pyruvate] and [β-OH butyrate] are in the μM range (<1 mg%). Restated, vascular glucose mass is 10-fold or more greater than the circulating alternative CHO energy sources combined. Hence, it is to be expected that maintaining euglycemia from exogenous nutritional support is of major importance in the neural ICU. This can perhaps be illustrated best by the better outcomes of TBI patients as a result of careful assessment and early enteral and parenteral nutritional support  necessary for cellular uptake  and [3-13C]lactate to track whole-body and cerebral glucose-lactate interactions in TBI. Patients were studied as soon as permission of legal representatives could be obtained, usually 96–140 h after injury at which time numerous parameters were approaching control levels. The time lag had to do with mechanics of securing permission of patients' legal representatives to conduct studies on patients receiving standard of care treatment. Consequently, at the time of study arterial glucose , whereas consistent with Figure Along the way to studying cerebral glucose-lactate interactions in patients following TBI an unexpected result emerged on our TBI patients given standard of care treatment in state of the art facilities by the dedicated and highly trained health care professionals. This was the herculean effort of the body to mobilize energy resources for the injured brain , but trends toward indicating a hypermetabolic state, or, in comparison to exercising controls, a moderate intensity of exercise similar to a brisk walk. Again the intra-patient variability demonstrated that some patients were more hypermetabolic than others, and therefore would require increased nutritional support as their body energy stores are depleted more rapidly. However, even though arterial [lactate] was similar in control subjects and patients, blood lactate turnover (Ra shown) was elevated by ≈90% in TBI patients compared to controls  in treated TBI patients was similar to values in controls Figure . Due to s Figure . Becauses Figure  and the s Figure  as is fo13C into blood glucose from infused [3-13C]lactate, the rate of gluconeogenesis (GNG) from lactate and the percentage of circulating glucose formed from lactate could be measured. Compared to values of 15.2% of glucose Ra from lactate in control subjects, most (67.1%) circulating glucose in TBI patients came from lactate . Like decreased CMRO2, decreased CMRgluc following TBI may reflect the presence of intrinsic cerebral mechanisms to allow the injured brain to “rest” in recovery. Alternatively, decreased cerebral metabolism following injury may be representative of an injury-induced metabolic crisis, the latter being associated with poor patient outcomes  Figure  and simu% Figure  show tha% Figure .With the benefit of simultaneous CBF as well as arterial and jugular bulb concentration and isotope enrichment measurements for glucose and lactate it has been possible to partition total cerebral carbohydrate uptake in healthy controls and TBI patients Figure . These rIn healthy controls cerebral glucose uptake accounts for 15–25% of blood glucose disposal  will participate NIH,  in efforThe 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.


This enzymatic complex creates a transmembrane gradient of sodium that is used by the cell to sustain ionic homeostasis, nutrient transport, ATP synthesis, flagellum rotation and other essential processes. Comparative genomics data demonstrate that the nqr operon, which encodes all Na+-NQR subunits, is found in a large variety of bacterial lineages with different habitats and metabolic strategies. Here we studied the distribution, origin and evolution of this enzymatic complex. The molecular phylogenetic analyses and the organizations of the nqr operon indicate that Na+-NQR evolved within the Chlorobi/Bacteroidetes group, after the duplication and subsequent neofunctionalization of the operon that encodes the homolog RNF complex. Subsequently, the nqr operon dispersed through multiple horizontal transfer events to other bacterial lineages such as Chlamydiae, Planctomyces and α, β, γ and δ -proteobacteria. Considering the biochemical properties of the Na+-NQR complex and its physiological role in different bacteria, we propose a detailed scenario to explain the molecular mechanisms that gave rise to its novel redox- dependent sodium -pumping activity. Our model postulates that the evolution of the Na+-NQR complex involved a functional divergence from its RNF homolog, following the duplication of the rnf operon, the loss of the rnfB gene and the recruitment of the reductase subunit of an aromatic monooxygenase.The sodium -pumping NADH: ubiquinone oxidoreductase (Na Thus, Na+-NQR coordinates the operation of the entire metabolism with the mechanisms used for osmotic regulation. Na+-NQR is commonly associated with the aerobic respiratory metabolism of pathogenic bacteria, such as Vibrio choleraeVibrio harveyiKlebsiella pneumoniaeHaemophilus influenzae+-NQR has an important role in infection, regulating the production of virulence factors The sodium -dependent NADH: ubiquinone oxidoreductase  rnf operon, most of which are homologous to the subunits of Na+-NQR  directly as a redox cofactor +-NQR is completely novel. Moreover, Na+-NQR produces several flavin radicals during the catalytic cycle, and surprisingly also contains stable flavin semiquinone radical species in different inactive forms of the enzyme +-NQR presents a mechanism, like no other reported so far, to stabilize the unpaired electron of the flavin. Furthermore, the binding of the two FMN molecules to NqrB and NqrC subunits involves a phosphoester bond 2O reductase expression, NOsR and NirI bc1 complex and photosystem II) c oxidase and complex I) +-NQR does not follow these ion -pumping strategies. Instead, ion translocation by this complex involves a series of conformational states that allow the capture of sodium in the internal side of the membrane and its release on the opposite side +-NQR has evolved separately from other redox enzymes and ion transporters, which has led to the arising of novel enzymatic strategies. In order to gain insights into the evolution and phylogenetic history of Na+-NQR, we analyzed the distribution and organization of the nqr operon in diverse pathogenic and non -pathogenic bacteria. Our results allow us to hypothesize and discuss a series of molecular events that may have been important in the origin, evolution and spreading of Na+-NQR, as well as to postulate some ecological factors that could have been important in its spreading and evolution. The data indicate that Na+-NQR appeared originally in the Chlorobi/Bacteroidetes (C/B) phyla, after a duplication of the rnf operon. The subsequent loss on the rnfB gene prompted significant changes in the mechanism of the enzyme, which together with the recruitment of the reductase subunit of the aromatic  monooxygenases (AMOr protein) underlie the origin of Na+-NQR. The current distribution of this complex in diverse bacterial lineages can be explained by at least two ancestral independent horizontal gene transfer (HGT) events, to proteobacteria, and Chlamydiae and Planctomyces, and at least two posterior HGT events, from Chlorobi to δ-proteobacteria and from Pasteurellales (γ-proteobacteria) to Neisseriales (β-proteobacteria).Na+-NQR subunits of Vibrio cholerae (O1 El Tor strain N16961) orthologous sequences were identified in the RefSeq GenBank database. After an exhaustive similarity search , protein sequences were retrieved, covering all major bacterial groups where Na+-NQR subunits were identified  to estimate the pairwise percentage of sequence identity (PID) between Na+-NQR and RNF putative homolog pairs, and NqrF with the AMOr protein. We carried out two independent intraspecific sequence comparisons using the Na+-NQR and RNF proteins from both Chlorobium phaeobacteroides (Chlorobi) and Anaerophaga thermohalophila (Bacteroidetes), respectively. A single randomly selected AMOr sequence  was aligned independently against the Chlorobium and Anaerophaga nqrF subunit.Using the amino acid sequences of the Naentified . The nqrkaryotes . Our finial taxa . The six+-NQR/RNF pairs are in the “twilight zone” A. thermohalophila  +-NQR subunit with no homologous counterpart in the RNF complex. Our PSI blast similarity analysis showed that the AMOr subunit is the closest homolog to NqrF, sharing the FAD, NADH and 2Fe-2center binding sites To gain additional insights into the evolution of the Na+-NQR subunit alignment was selected according to ProtTest. The Le and Gascuel (LG) substitution model The best amino acid substitution model for each Na+-NQR complexes are among the few respiratory enzymes that couple their redox activities with the ability to create an electrochemical gradient of sodium , Bacteroidetes and Chlamydiae, the latter are obligate intracellular parasites +-NQR plays an essential role in the physiology of many pathogens. In fact, the expression of Na+-NQR increases greatly during the infection of the small intestine by V. choleraenqr operon reduces drastically the infectivity of these cells +-NQR activity regulates the expression of the V. cholerae virulence factors, such as the cholera toxin (CTX) and the major subunit of the toxin co-regulated pillus (TcpA), through the modulation of the ToxR, TcpP, and ToxT regulatory cascade Na+-NQR include Plactomycetes, Chlorobi, as well as α, δ, free- living β and “early diverging” γ-proteobacteria. All these types of bacteria have different metabolic strategies, including photoautotrophic or chemolitotrophic anaerobes, chemorganothrophs, as well as strict aerobic bacteria. In these organisms, Na+-NQR pumping activity could be used both to generate a gradient of sodium, coupled to the activity of the oxidative phosphorylation, and to maintain the intracellular ionic composition. Indeed, in the extreme haloalkalophile Desulfurivibrio alkaliphilus  bacteria, such as δ -proteobacteria, especially in marine species. In these bacteria Na+-NQR might participate in osmotic protection, but may have other roles in the metabolism, especially in menaquinone reduction. Menaquinol is the main electron donor in the first stage of sulfate -based respiration. The Qmo complex catalyzes the menaquinol -mediated reduction of the activated sulfate (APS), which is converted to sulfite. Further reduction of sulfite to sulfuric acid requires electrons provided by periplasmic hydrogenases +-NQR could have an important role in this process.The Acetobacterium woodii (anaerobic acetogenic), RNF catalyzes the reduction of NAD (−320 mV), using the redox equivalents of Ferredoxin (−500 mV) Rhodobacter capsulatus (purple non -sulfur photosynthetic), and probably in other nitrogen fixing bacteria, RNF uses the electrochemical gradient to drive the backwards reaction, reducing ferredoxin, using NADH as electron donor E. coli RNF transfers the electrons from NADH, to the low molecular weight SoxR protein, which is a transcription factor that controls the expression of proteins involved in oxidative stress protection +-NQR.One of the distinguishing aspects of RNF is that its function can vary enormously, depending on the metabolic adaptations of the bacteria in which is found. For instance, in nqr operon is found only in certain bacterial lineages, while the rnf operon is widely distributed , defining the RNF clades as outgroups (see +-NQR/RNF homolog-pair details). The NqrF and RnfB subunits are complex-specific and they have no homologous counterpart in RNF or Na+-NQR, respectively. Thereafter, a multiple alignment NqrF/AMOr was generated as well. Each of the four rooted ML trees . The phylogenetic analysis suggests that other bacterial lineages acquired the Na+-NQR complex via multiple HGT events (see below).The fact that the tributed , suggestML trees –S5 consinqr operon is split in three fragments Vibrio cholerae) and NqrF (404 residues) ML trees show considerable resolution in different nodes and it is likely that the catalytic roles of NqrB and NqrF have restrained the substitution rate in the catalytic regions, but allow higher variability in non-essential regions, producing a relatively consistent phylogenetic signal. The topologies of the ML trees of the short subunits NqrD  and other species, including Candidatus Accumulibacter phosphatis (not shown), as well as Nitrosomonadales  and Methylophilales  contain Na+-NQR, which is closely related to α -proteobacteria and “basal” γ -proteobacteria, matching relatively well their phylogenetic history nqr operon, indicating that the common ancestor of these groups lost the genes coding for Na+-NQR. Thus, it is possible that Neisseriales, which are pathogenic, reacquired the nqr operon from pathogenic γ-proteobacteria  branch consistently with Chlorobi/Bacteroidetes . This to+-NQR was likely spread to Chlamydiae and Planctomyces, through one or two lateral ancient HGT events. Phylogenetic trees obtained with 16S rRNA have located Chlamydia and Planctomyces as distantly related groups +-NQR may have been transferred from C/B to the common ancestor of Chlamydiae and Planctomyces, and that Chlamydiae Na+-NQR would have evolved faster, including a fragmentation of the nqr operon, than the Planctomyces homologs, explaining the Chlamydiae long branch and the separation of both lineages in the trees.The ML trees of NqrB and NqrF also show that Na+-NQR to different bacterial lineages could have been a limited iron availability, which is now reflected on the particular cofactor composition of the “modern” Na+-NQR complex. One of the most intriguing aspects of this enzyme is the preferential use of flavin molecules compared to iron containing cofactors +-NQR contains only one 2Fe-2S center and four flavin molecules. Iron has been considered one of the key elements involved in the origin of life, probably forming part of the first cofactors in enzymatic reactions +-NQR is well represented An important ecological aspect that may have contributed to the evolution and spread of Narnf and nqr operons in the major bacterial lineages suggest that Na+-NQR appeared from the duplication of the rnf operon in the C/B common ancestor. After its putative duplication, the original rnf operon kept its function, as sodium -pumping ferredoxin: NADH oxidoreductase, while the paralogue copy lost a key gene  remained essentially identical. Examination of the rnf operon organization in different bacteria offers further evidence to support this hypothesis -X-L-X3-(L/I)-X3-(A/S), implying that it is not only an membrane anchor site, but also should be able to interact with the transmembrane segments of other subunits. Remarkably, helix I of the RnfB subunit resembles the transmembrane helix of NqrF, sharing a very similar structure A-(V/I)-X2-L-X2-(L/I)-X6-(V/I/L)-(I/L)-X2-(A). The wheel projections of both helices show that positions 5, 8, 15, 16 and 19 have similar residues and are conserved in both transmembrane segments and are concentrated in a relatively small face of the helical cylinder , compared to the homologous region of RnfD (transmembrane helix II) from different bacteria. Helix III in NqrB subunit contains two conserved glycine residues that are involved in the formation of the ubiquinone binding site and whose mutation, even to other small residues, such as alanine or valine, disrupts completely the interaction with ubiquinone +-NQR originally evolved as a NADH: menaquinone reductase, since ubiquinone (Em = +90 mV) is absent in both Chlorobi Em = −80 mV). The ability to use menaquinone might be one of the key elements that determined the spread to other types of bacteria, for instance to δ-proteobacteria and planctomyces, which also contain menaquinone +-NQR to use ubiquinone appeared later on evolution, probably first in aerobic α-proteobacteria and was inherited to β and γ-proteobacteria, all of which contain exclusively ubiquinone +-NQR should be essentially the same with the two redox electron acceptors, since the reduction of the quinone molecule is not involved directly in sodium translocation +-NQR. Our group described that riboflavin is a functional cofactor in Na+-NQR, delivering the electrons to the final substrate +-NQR did not require the acquisition of riboflavin, but merely an adaptation in the function of RNF.Moreover, another important part of Na+-NQR. The forward reaction catalyzed by RNF involves the electron transfer from Ferredoxin (Em = −500 mV) to NADH (Em = −320 mM) +-NQR, the electrons move downhill from NADH to ubiquinone (Em = +90mV), following a pathway in which most redox centers have midpoint potentials higher than NADH +-NQR, a significant change in the redox potential should have occurred, increasing the potentials probably by 50–490 mV, depending on the specific cofactor. In most proteins, the control of the redox potential of the centers is achieved by modifying the environment surrounding the cofactors. For instance, classic studies on Myoglobin have shown that the mutation of valine 68, which is close to the heme group, to aspartate or asparagine decreases the midpoint potential by 200 or 80 mV, respectively +-NQR the environment of the flavins was modified, replacing negatively charged residues and decreasing the polarity of the flavin environment, which will increase the midpoint potential of the three flavin cofactors.In addition to the subunit acquisition and motif evolution, the adjustment in the midpoint potentials of the redox centers should have been one of the most important aspects necessary to complete the transition from RNF into Na+-NQR subunits in different bacterial lineages suggest that the nqr operon, and subsequently the Na+-NQR complex, is the outcome of an ancestral duplication of the homologous rnf operon. Here, we present evidence that suggest a series of molecular events, comprising a specific partial gene loss, a gene fusion and enzymatic recruitment, that occurred during the divergence and neo-fuctionalization of the ancestral versatile RNF complex into the specialized sodium pump that became the modern Na+-NQR assemblage. Moreover, our phylogenic survey indicates that the putative duplication of the rnf operon occurred in the C/B group, and later the ancestral nqr operon dispersed via independent HGT events into different proteobacterial groups, and Chlamydiae and Planctomyces. This model is consistent with the idea that genes with related functions and organized in clusters  are prone material for successful HGT, given that the encoded products, as an effective functional unit, are potentially able to confer novel metabolic traits, such as sodium pumping coupled to electron transport. Our analyses highlight the major relevance that duplication of genes, or clusters of genes, has in the emergence and consolidation of evolutionary innovations. In this case underlying the diversification and expansion of the bacterial metabolic capabilities under particular ecologic scenarios, such as the use of sodium gradients across membranes to generate electromotive forces when proton “inverted”  gradients prevail in the surroundings. Our model posits that the emergence, and later independent recruitments, of the Na+-NQR allowed different types of bacteria with diverse metabolic adaptations to take advantage of the abundance of Na+ ions in particular habitats, such as marine, alkaline and intracellular environments. Finally, our work invites to thoroughly investigate the presence of paralogous operons in prokaryotic genomes to study the magnitude that duplication of gene clusters have had in the diversification and expansion of the microbial primary metabolism.The distribution and the phylogenetic history of the NaFigure S1Anaerophaga thermohalophila (Bacteroidetes) RnfC and NqrA. Figure shows the secondary structure prediction consensus of eight algorithms, using the Network Protein Sequence Analysis software. The conserved residues of RnfC involved in NADH and FAD binding sites, as well as the cysteine residues involved in the 4Fe-4S centers attachement are highlighted in black.Alignment of NqrA and RnfC subunits. Secondary structure prediction of (PDF)Click here for additional data file.Figure S2+-NQR complex and the homolog RnfD subunit from the RNF complex. The tree was rooted using the RnfD clade as outgroup. Numbers near nodes indicate RaxML bootstrap branch support values (when ≥50%). Branch lengths are proportional to the number of substitutions per site. NCBI GI numbers precede the taxa identification.Rooted phylogenetic tree of NqrB subunit. Maximum Likelihood phylogenetic analysis of the NqrB subunit of the Na(PDF)Click here for additional data file.Figure S3+-NQR complex and sequences of the reductase subunit of aromatic monooxigenases (AMOr), which are the closes know homologues of NqrF. The tree was rooted using the AMOr (benC/ToL) proteins as outgroup. Numbers near nodes indicate RaxML bootstrap branch support values (when ≥50%). Branch lengths are proportional to the number of substitutions per site. NCBI GI numbers precede the taxa identification.Rooted phylogenetic tree of NqrF subunit. Maximum Likelihood phylogenetic analysis of the NqrF subunit of the Na(PDF)Click here for additional data file.Figure S4+-NQR complex and the homolog RnfE subunit from the RNF complex. The RnfE branch was defined as outgroup. Numbers near nodes indicate RaxML bootstrap branch support values (when ≥50%). Branch lengths are proportional to the number of substitutions per site. NCBI GI numbers precede the taxa identification.Rooted phylogenetic tree of NqrD subunit. Maximum Likelihood phylogenetic analysis of the NqrD subunit of the Na(PDF)Click here for additional data file.Figure S5+-NQR complex and the homolog RnfA subunit from the RNF complex. The tree was rooted using the RnfA branch (100% BS) as outgroup. Numbers near nodes indicate RaxML bootstrap branch support values (when ≥50%). Branch lengths are proportional to the number of substitutions per site. NCBI GI numbers precede the taxa identification.Rooted phylogenetic tree of NqrE subunit. Maximum Likelihood phylogenetic analysis of the NqrE subunit of the Na(PDF)Click here for additional data file.Figure S6+-NQR complex. Numbers near nodes indicate RaxML bootstrap branch support values (when ≥50%). Branch lengths are proportional to the number of substitutions per site.Unrooted phylogenetic tree of NqrD subunit. Maximum Likelihood tree of the subunit D (NqrD) of the Na(PDF)Click here for additional data file.Figure S7+-NQR complex. Numbers near nodes indicate RaxML bootstrap branch support values (when ≥50%). Branch lengths are proportional to the number of substitutions per site.Unrooted phylogenetic tree of NqrE subunit. Maximum Likelihood tree of the subunit NqrE of the Na(PDF)Click here for additional data file.Figure S8+-NQR complex. Numbers near nodes indicate RaxML bootstrap branch support values (when ≥50%). Branch lengths are proportional to the number of substitutions per site.Unrooted phylogenetic tree of NqrA subunit. Maximum Likelihood tree of the subunit A (NqrA) of the Na(PDF)Click here for additional data file.Figure S9+-NQR complex. Numbers near nodes indicate RaxML bootstrap branch support values (when ≥50%). Branch lengths are proportional to the number of substitutions per site.Unrooted phylogenetic tree of NqrC subunit. Maximum Likelihood tree of the subunit NqrC of the Na(PDF)Click here for additional data file.Table S1+-NQR complex.List of the analyzed bacterial taxa harboring Na(PDF)Click here for additional data file.Table S2+-NQR subunits with the closest homologues in RNF and AMOr.Similarity profiles of the Na(PDF)Click here for additional data file.


Histone deacetylases (HDACs) induce deacetylation of both histone and non-histone proteins and play a critical role in the modulation of physiological and pathological gene expression. Pharmacological inhibition of HDAC has been reported to attenuate progression of renal fibrogenesis in obstructed kidney and reduce cyst formation in polycystic kidney disease. HDAC inhibitors (HDACis) are also able to ameliorate renal lesions in diabetes nephropathy, lupus nephritis, aristolochic acid nephropathy, and transplant nephropathy. The beneficial effects of HDACis are associated with their anti-fibrosis, anti-inflammation, and immunosuppressant effects. In this review, we summarize recent advances on the treatment of various chronic kidney diseases with HDACis in pre-clinical models. Histone and non-histone protein acetylation has been widely studied in the field of cancer research  are critically connected with yeast RPD3 gene. Class II HDACs  are related to yeast Hda1 gene. Class III HDACs (SIRT1-7) are homologous to silent information regulator 2 (Sir2) and have no sequence similarity to class I and II HDACs; these Sir2 proteins, also called sirtuins are unaffected by known class I/II HDACis. Class IV (HDAC11) has conserved residues in its catalytic regions that are shared by both class I and II HDACs. Class I and II HDACs need ZnCurrently, the expression profiles and distribution of HDACs in the kidney have not been completely clarified. It has been documented that class I HDAC isoforms are expressed in the cortex of developmental kidney, renal fibroblasts, and renal tubular cells , cyclic peptides , aliphatic acids , and benzamides (entinostat) , either alone or in complex with the gene product of PKD1, polycystin-1 (PC1), functions as a calcium-permeable cation channel and regulates intracellular Ca2+ levels, alteration of signaling pathways regulated by calcium such as cAMP-dependent B-Raf and ERK  activation resulted in abnormal proliferation of tubule epithelial cells  is very common hereditary kidney diseases in humans, affecting 1/500 in the United States Gabow, . In ADPKPkd2−/− mouse embryos. Cao et al.   is characterized by ECM protein accumulation in glomerular mesangium and tubulointerstitium with thickening of glomerular and tubular basement membranes, ultimately progressing to glomerulosclerosis and tubulo-interstitial fibrosis  is a very common autoimmune disease  decrease the rate of acute rejection in renal transplantation patients, but side effects such as nephrotoxicity, neurotoxicity, and diabetogenicity (Shapiro et al., Numerous studies have shown that treatment with HDACis is able to inhibit activation and proliferation of cultured renal interstitial fibroblasts and attenuate renal fibrosis in animal models. HDAC inhibition is also beneficial for other chronic kidney diseases caused by the diverse etiologies, as listed in Table The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TERT), the most common noncoding mutations in cancer, give rise to monoallelic expression of TERT. Through deep RNA sequencing, we find that TERT activation in human cancer cell lines can occur in either mono- or biallelic manner. Without exception, hotspot TERT promoter mutations lead to the re-expression of only one allele, accounting for approximately half of the observed cases of monoallelic TERT expression. Furthermore, we show that monoallelic TERT expression is highly prevalent in certain tumor types and widespread across a broad spectrum of cancers. Taken together, these observations provide insights into the mechanisms of TERT activation and the ramifications of noncoding mutations in cancer.Here we report that promoter mutations in telomerase ( TERT) is generally restricted to the germline and stem cells. However, TERT activation, necessary for the maintenance of unlimited replicative potential, occurs commonly in many cancers. Recurrent mutations in the TERT promoter region were first discovered in melanoma and have since been found to be the most common somatic mutations in many cancers.4 Two mutually exclusive cytidine-to-thymidine mutations, C228T and C250T , positioned at 124 and 146 base pairs upstream of the ATG translational start site of TERT, respectively, are found in ~70% of melanomas, 80–90% of glioblastoma multiforme, 60% of hepatocellular carcinoma, 60% of bladder cancer, 70% of basal cell carcinoma, 50% of cutaneous squamous cell carcinoma and up to 30% of thyroid cancers. Other recurrent but much less frequent mutations in the TERT promoter  have also been observed.5 All of these mutations create putative consensus ETS transcription factor binding sites (GGAA/T) and are hypothesized as a mechanism of TERT activation.2 Subsequent studies in glioblastoma and bladder cancer demonstrated that TERT promoter mutations correlate with higher TERT mRNA and protein expression and elevated telomerase activity.6 Furthermore, recent data demonstrate that the mutant TERT promoter can be bound and activated by GABP, an ETS transcription factor.7 The recurrent TERT promoter mutations also prevent silencing of TERT upon differentiation of stem cells.8 For this study, we used whole-genome DNA and deep RNA sequencing data from 329 cancer cell lines representing a wide range of tumor types from the Cancer Cell Line Encyclopedia (CCLE)10 to interrogate the TERT locus and determine the modes of TERT activation.In healthy tissues, expression of the telomerase gene   in the DNA sequences of expressed exonic and untranslated regions of the TERT gene. Cell lines with insufficient RNA sequencing coverage of anchor SNPs  and cell lines with high-level copy-number amplification or deletion of the TERT locus were excluded from downstream analyses. In total, 88 CCLE lines met these criteria and had at least one heterozygous SNP that would allow evaluation of an allelic bias in RNA expression  and overall, nearly half (48.7% 19/39) of the cell lines that showed monoallelic expression could be accounted for by the presence of the recurrent TERT promoter mutations  at position chr5:1 294 086 and the promoter mutation C228T (chr5:1 295 228 C>T) in four unrelated cell lines. Using Sanger sequencing, we found that the allele that showed monoallelic expression at this position was in cis with the mutant promoter allele and not the wild-type promoter allele in all lines tested  in lines with an unexplained monoallelic pattern of TERT activation as compared with lines with biallelic expression of TERT (cis-acting genetic alterations do not appear to explain monoallelic expression of TERT in the cancer cell lines we analyzed that do not harbor a hotspot mutation in the TERT promoter. However, we cannot rule out the possibility that some of the substitutions found within the promoter or that genomic rearrangements beyond the upstream 10 kb could potentially contribute to monoallelic expression.Although all cell lines with  of TERT . These dTERT expression is a crucial step in the progression of many cancers and understanding the molecular mechanisms of such activation is important for the understanding of tumorigenesis. Recent genome-wide analyses have highlighted that noncoding mutations in the TERT promoter are among the most common mutations in human cancer.14 We observed that the monoallelic mode of TERT activation is widespread, with half of cases explained by the known promoter mutations and certain lineages showing almost exclusive monoallelic expression of TERT. These observations highlight the heterogeneity of molecular mechanisms to activate TERT, some of which are still to be discovered. Here we show that the integration of DNA and RNA sequencing to determine monoallelic expression can be used to identify and characterize cis-acting noncoding mutations in cancer. While our results establish TERT monoallelic expression in cell lines across many tumor types, confirmation of these results in primary tumors will be needed. Whether other mechanisms such as random monoallelic expression or epigenetic mechanisms account for the non-promoter-mutant-associated TERT monoallelic expression remains an active area of investigation.17 Aside from TERT, recent studies demonstrate that other promoter regions may be recurrently mutated in cancer.20 Our findings suggest that somatic monoallelic activation of TERT is a common mechanism of TERT expression and that TERT promoter mutations drive monoallelic expression of TERT.Activation of


Previous randomized controlled trials (RCTs) have reported conflicting results for the efficacy of sitagliptin and sulfonylurea therapy in patients with type 2 diabetes mellitus showing inadequate glycemic control with metformin monotherapy. To clarify these findings, a meta-analysis was conducted of the outcomes of all published RCTs comparing sitagliptin with sulfonylureas in the treatment of type 2 diabetes mellitus. Standard medical databases were searched to identify relevant English- and Chinese-language RCTs. RCT results were compared regarding the mean change in glycated hemoglobin (HbA1c) level; the proportion achieving <7% HbAlc; and a change in body weight. No significant differences were found between the metformin plus sitagliptin and metformin plus sulfonylurea groups regarding HbAlc or the proportion achieving <7% HbAlc, while the metformin plus sitagliptin group experienced fewer hypoglycemic events (P<0.00001) and a greater reduction in body weight (P<0.00001). Metformin plus sitagliptin therapy may decrease HbAlc values in patients with type 2 diabetes mellitus who are not achieving their glycemic targets with metformin monotherapy in a manner similar to metformin plus sulfonylurea therapy, whilst posing a lower risk of hypoglycemia, and yielding a more beneficial effect on body weight. Type 2 diabetes mellitus has become a worldwide epidemic with a prevalence that has tripled in the last 30 years, and is predicted to affect >350 million individuals by 2025 . DespiteMetformin, a commonly used oral antihyperglycemic agent used as a monotherapy and in combination with other antihyperglycemic agents, was introduced in the 1950s for the treatment of type 2 diabetes mellitus. Metformin has many advantages, including that it neither promotes weight gain nor causes hypoglycemia, it exerts beneficial effects on cardiovascular risk  and is wThe Medline, Embase, Cochrane Library, Chinese National Knowledge Infrastructure and Chinese Biomedical Literature databases were systematically searched to identify studies published in English between January 2000 and December 2012 or published in Chinese between January 1996 and December 2012 using the following search terms: Type 2 diabetes mellitus, type II diabetes mellitus, diabetes mellitus type 2, metformin, sitagliptin, sulfonylurea, glibenclamide, gliclazide, glipizide controlled-release tablets, gliquidone, glimepiride, dipeptidyl peptidase-4 and clinical trial. Following retrieval of the relevant articles, a manual search of the references was performed to identify the relevant trials. Attempts were also made to contact investigators for unpublished data and the full text of articles when deemed necessary for clarification or for more information. Two investigators reviewed all potentially relevant articles independently to determine whether they met all the inclusion criteria and none of the exclusion criteria.Studies were included in the analysis if they met all the following inclusion criteria: i) Use of a prospective RCT; ii) comparison of combined metformin and sitagliptin therapy with combined metformin and sulfonylurea therapy in the treatment groups; iii) treatment of patients for ≥12 weeks; iv) inclusion of patients who had not been achieving their glycemic targets with metformin monotherapy; and iv) reporting of outcomes in terms of HbA1c values. Trials were excluded if they met one or both of the following exclusion criteria: i) Evaluation of the addition of more than one drug to metformin monotherapy and/or ii) inclusion of participants using background therapies other than metformin monotherapy. Methodological quality assessment was conducted using the Cochrane Collaboration’s tool for assessing the risk of bias in randomized trials. The RCTs were assessed for quality according to the criteria of i) method of randomization; ii) allocation concealment; iii) blinding of participants; iv) addressing of incomplete data; v) freedom of selective reporting; vi) comparability of groups at baseline; and vii) sample size calculation. The trials were independently reviewed and graded by two investigators who resolved any disagreements through discussion.The primary outcome measure was the mean change in HbA1c values from baseline to study endpoint. Secondary outcomes included the proportion of participants achieving <7% HbA1c body weight, and the occurrence of hypoglycemia.Two investigators independently reviewed the titles, abstracts and full texts of articles for inclusion using standardized data extraction forms. Validity assessment was performed using the Jadad scale . Disagre2 statistic, with I2>50% considered an indication of significant heterogeneity between two trials. A random-effects model was used in the presence of heterogeneity and a fixed-effects model in the absence of heterogeneity. The weighted mean difference (WMD) or odds ratio and its 95% confidence interval (CI) for each outcome relative to the control were calculated for continuous and dichotomous variables, respectively. Studies were excluded from the meta-analysis if insufficient information was provided to enable standard error calculation.The analyses were performed using Review Manager  and Stata  software. The heterogeneity of treatment effects among the studies was formally tested with Cochrane’s test at a significance level of P<0.1 and determination of the IThe database search results are summarized in The results of the meta-analyses conducted in this study strongly indicate that the addition of sitagliptin therapy to the regimen of patients with type 2 diabetes mellitus who are currently undergoing metformin monotherapy but failing to achieve their glycemic targets can result in a reduction in HbA1c values similar to that resulting from the addition of sulfonylurea therapy to metformin monotherapy. Metformin plus sulfonylurea therapy, however, was not found to lower the risk of hypoglycemia to the same extent as metformin plus sitagliptin therapy. Since sulfonylurea stimulation of insulin secretion is not strictly glucose dependent , sulfonyThe results also suggest that metformin plus sitagliptin therapy results in greater weight loss compared with metformin plus sulfonylurea therapy. Regarding the underlying mechanism, sitagliptin can increase endogenous GLP-1, which, by delaying gastric-emptying, increases satiety, resulting in significant weight loss . This beThe present study had two primary strengths and three major limitations that should be considered when reviewing the results. Regarding its strengths, the study authors ensured that only high-quality evidence was examined by limiting the inclusion of studies to only double-blind RCTs. Furthermore, as the first systematic review, to the best of our knowledge, to compare the efficacy and safety of sitagliptin and sulfonylurea therapies in the treatment of patients with type 2 diabetes mellitus experiencing inadequate glycemic control with metformin monotherapy, this study filled an important research gap. Regarding its limitations, the study examined RCTs that were conducted for varying lengths of time , which may affect the extent to which the results can be compared. In addition, as the RCTs did not report economic indicators, the present study was not able conduct the relevant economic analysis. Such analysis is important, as cost is a significant consideration in therapeutic decision making in order to support the allocation of sufficient healthcare resources for the treatment of type 2 diabetes mellitus and its complications, particularly in light of its increasing global prevalence. Finally, as none of the RCTs were designed to compare the cardiovascular endpoints of the two study groups, any conclusions regarding outcomes, such as cardiovascular morbidity or mortality, should be considered with caution and only be accepted following confirmation by additional investigation by more RCTs.In conclusion, the addition of sitagliptin therapy to the regimen of patients with type 2 diabetes mellitus who are not achieving their glycemic targets with metformin monotherapy may result in a reduction in HbA1c values in a manner similar to the addition of sulfonylurea therapy to metformin monotherapy while posing a lower risk of hypoglycemia and resulting in a greater loss of weight.


Rhodobacter, Pseudoxanthobacter, Escherichia, Alcaligenes, Roseobacter, Ochrobactrum, Agromyce, Sphingomonas and Paracoccus. The phylogenetic tree shows that most of the identified populations were evolutionarily related to the Alphaproteobacteria (91.66% of sequences). The minerals formed were studied by X-ray diffraction, scanning electron microscopy (SEM), and energy dispersive X-ray microanalysis (EDX). All of these strains formed phosphate crystals and precipitated struvite (MgNH4PO4·6H2O), bobierrite [Mg3(PO4)2·8H2O] and baricite [(MgFe)3(PO4)2·8H2O]. The results obtained in this study show that struvite and spherulite crystals did not show any cell marks. Moreover, phosphate precipitation was observed in the bacterial mass but also near the colonies. Our results suggest that the microbial population contributed to phosphate precipitation by changing the media as a consequence of their metabolic activity. Moreover, the results of this research suggest that bacteria play an active role in the mineral precipitation of soluble phosphate from urban wastewater in submerged fixed-film bioreactors.The ability of bacteria isolated from a fixed-film bioreactor to precipitate phosphate crystals for the treatment of domestic wastewater in both artificial and natural media was studied. When this was demonstrated in artificial solid media for crystal formation, precipitation took place rapidly, and crystal formation began 3 days after inoculation. The percentage of phosphate-forming bacteria was slightly higher than 75%. Twelve major colonies with phosphate precipitation capacity were the dominant heterotrophic platable bacteria growing aerobically in artificial media. According to their taxonomic affiliations , the 12 strains belonged to the following genera of Gram-negative bacteria:  It is widely accepted that microorganisms contribute to the bioprecipitation of a wide variety of minerals, including carbonates, phosphates, sulphides, oxides, and silicates . Differe4PO4·6H2O) is not a very abundant mineral in Nature; it has only been found in association with organic matter decomposition in locations such as barns, sheds, cemetery soils, guano deposits, manures, sediments rich in organic remains, and in kidney stones  and baricite [(MgFe)3(PO4)2·8H2O] . Moreover, the formation of both phosphates (bobierrite and baricite) took place at the same time as that of struvite crystals. These data suggest that the precipitation of bobierrite by microorganisms may be more common than expected. This is the first report on bacterial precipitation of baricite by bacteria in laboratory cultures.Our study also showed a significant number of strains (50%) with the ability to precipitate spherulites of the phosphate compounds bobierrite [Mg)2·8H2O] . These m  for the formation of struvite crystals.Rivadeneyra et al.  proposedAlphagammabacteria are ubiquitous microorganisms in natural habitats and our results suggest that these microorganisms can also be identified as a component of the microbial population in the biofilms formed in submerged fixed-film bioreactors used for the treatment of urban wastewater. These bacteria lack special growth requirements. Furthermore, they can easily grow in low or rich-nutrient environments and produce exopolysaccharides (EPS) characteristics which are advantageous to the growth and formation of stable biofilms in submerged filters. Moreover, the results of this work have demonstrated the potential capacity of these bacterial populations in the mineral precipitation of soluble phosphate from urban wastewater. Future research will focus on the specific in situ quantification of these organisms in biofilters by fluorescent in situ hibridization (FISH).The influence of microbial composition in the biofilm is a significant factor that affects the application of submerged fixed-film bioreactors for water treatment . In thisRhodobacter sp., Agrobacterium sp., Paracoccus sp., Roseobacter sp., and Sphingomonas sp., have an important function in the formation of struvite studied in vitro and that consequently, this is a biomineralisation process. However, the significance of this biological process for nutrient removal in wastewater treatment technology must be studied in greater depth in future research.Our study showed that struvite and spherulite crystals were free of mineralised cells. Optical microscopic observation of the plates containing SS1 and SS2 media showed that the majority of the struvite crystals were not only in the microbial masses but also near the colonies. These results suggest that the crystals were not formed by the aggregation of mineralised cells, and in those cases, bacterial cells contributed to this formation only by changing the media as a consequence of their metabolic activity. Thus, all the results of this study, which are obviously limited to cultivable bacteria isolated in laboratory media, verify that heterotrophic bacteria such as The results of this study, which are limited to platable bacteria, suggest that bacterial activity has an importance role in the formation of phosphate crystals such as struvite and spherulites in submerged fixed-film bioreactors, and that consequently, this is a biomineralisation process. The precipitation of phosphate crystals by bacteria isolated from a submerged fixed-film bioreactor used for the treatment of urban wastewater, which contains a high concentration of soluble minerals and metabolisable organic matter, is mainly as struvite. However, for the study of biomineralisation by heterotrophic bacteria from submerged biofilter systems used for urban wastewater treatment, it is advisable to use media derived from urban wastewater supplemented with a high concentration of magnesium, since environmental conditions present in the bioreactor are not optimal for the precipitation of minerals. Moreover, the results of this research suggest that heterotrophic bacteria populations play an active role in mineral precipitation of soluble phosphate from urban wastewater.


CIT2 gene, encoding the peroxisomal isoform of citrate synthase, as a typical target of the RTG pathway.Mitochondria, key organelles of eukaryotic cells that are responsible for essential metabolic processes, generation of energy, cellular redox state and many other processes, are deeply involved in cellular homeostasis and influence overall cell/organism physiology. Defects in mitochondria can lead to a number of different disorders in mammals . ChangesATO1 and ATO2 genes, involved in ammonia production and metabolic reprogramming of these cells [CIT2 expression and related metabolic reprogramming that may lead to production of glutamate/glutamine potentially released from these L cells and consumed by neighboring U cells. Increased intracellular glutamine concentration and/or glutaminolysis could then be involved in the activation of TORC1 signaling in U cells. In lower L cells the functionality of RTG signaling is essential for a viability of these cells, therefore we call this branch the “viability branch”. Neither ATO nor CIT2 genes are expressed in lower L cells. Importantly, all three of the Rtg activators, as well as the Mks1p repressor, are essential for each of the three branches of RTG signaling, despite the fact that these branches lead to expression of different gene targets and affect different cellular processes (Figure Recently, we have provided evidence that the RTG response in yeast is more complex than previously assumed, involves a number of yet to be identified regulatory elements and affects different cellular processes and the subsequent fate of differentiated yeast cells . In diffse cells . Contrarse cells , 7. Two The observed heterogeneity of RTG signaling within yeast colonies, that contributes to diversification of specifically localized cell subpopulations, resembles the diversity of mitochondrial retrograde signaling in mammals, which includes a number of regulatory events under different conditions and in different cells. This signaling is regulated through a variety of largely unknown factors. Future identification of new upstream and downstream elements involved in the regulation of RTG signaling in specialized cell types, developing within relatively simple yeast colonies that metabolically resemble tumor-affected organisms, may thus facilitate the identification of similar elements of retrograde signaling involved in cellular differentiation in mammals.


Both COPD and lung cancer are major worldwide health concerns owing to cigarette smoking, and represent a huge, worldwide, preventable disease burden. Whilst the majority of smokers will not develop either COPD or lung cancer, they are closely related diseases, occurring as co-morbidities at a higher rate than if they were independently triggered by smoking.Lung cancer and COPD may be different aspects of the same disease, with the same underlying predispositions, whether this is an underlying genetic predisposition, telomere shortening, mitochondrial dysfunction or premature aging. In the majority of smokers, the burden of smoking may be dealt with by the body’s defense mechanisms: anti-oxidants such as superoxide dismutases, anti-proteases and DNA repair mechanisms. However, in the case of both diseases these fail, leading to cancer if mutations occur or COPD if damage to the cell and proteins becomes too great.Alternatively COPD could be a driving factor in lung cancer, by increasing oxidative stress and the resulting DNA damage, chronic exposure to pro-inflammatory cytokines, repression of the DNA repair mechanisms and increased cellular proliferation. Understanding the mechanisms that drive these processes in primary cells from patients with these diseases along with better disease models is essential for the development of new treatments. Additional mutations can further transform the benign tumor to an invasive cancer, a process marked by metastatis (spread), invasiveness and anaplasia (loss of cell type specific features) 3COPD is a progressive and ultimately fatal deterioration of lung function over time Damage to the lungs in COPD is caused by oxidative stress (both exogenous from smoking and endogenous), inflammatory cytokine release, protease activity  and autoantibody expression 4COPD and lung cancer are caused by cigarette smoking and there is increasing evidence linking the two diseases beyond a common etiology. COPD is an independent risk factor for lung carcinoma, particularly for squamous cell carcinoma 5The probability of developing cancer increases with age 615 free radicals per puff Cigarettes contain approximately 106.1The free radical hypothesis of aging proposes that RNOS drives the accumulation of cell and DNA damage RNOS also plays a role in cancer promotion and progression 6.2RNOS also stimulates the production of inflammatory mediators either directly RNOS can alter protein structure and function by modifying amino acid residues, inducing protein dimerization, and interacting with Fe-S moieties or other metal complexes. In COPD these post-translational mechanisms include the nitration of histone deacetylase (HDAC) 2, leading to its inactivation and degradation resulting in the prolonged inflammatory phenotype seen in patients 7Telomeres are repetitive nucleotide sequences located at the ends of the chromosomes which protect against the progressive shortening that occurs with DNA replication. Telomere shortening is associated with cell aging and senescence; with cells unable to divide once their telomere shorten beyond a crucial point—termed the Hayflick limit 7 cells) can emerge, which maintains telomere length through cell divisions. Both COPD and lung cancer are associated with shortened telomere length, and importantly telomere shortening can be driven by cigarette smoking Some cells, however, are able to bypass senescence through inactivation of the Rb and p53 signaling pathways, enter ‘crisis’ and continue to replicate Several studies have shown a link between short telomere length and the development of lung cancer 8CHRNA3 and CHRNA5 SNPs (15q) and regions at 4q31 (HHIP), 4q24 (FAM13A) and 5q (HTR4). Nicotine addiction may explain the overlap in risk loci between lung cancer, smoking behavior and COPD VEGFR1, which promotes inflammation, EMT and tumor growth, is a susceptible locus for both COPD and lung cancer IL10 are associated with increased rates of lung and other cancer There is evidence for familial susceptibility in both COPD and lung cancer. This familial susceptibility appears to be linked and not just associated with the common consumption of cigarettes; implying that the underlying genetic predisposition to both diseases may be the same or reflect the link between the immune system, inflammation and cancer 9In addition to genetic changes, epigenetic changes including DNA methylation, covalent histone modifications, microRNAs (miRNAs) expression and nucleosome remodeling have also been reported to play an important role in the development of cancer Unsupervised hierarchical clustering of DNA methylation patterns results in 3 lung cancer clusters CCDC37 and MAP1B, was significantly associated with both COPD and lung cancer Recently an epigenome wide association study (EWAS) has been carried out to examine links between gene methylation in COPD and lung cancer Although alterations to DNA methylation have been examined in COPD these are mostly linked to hypomethylation of immune-modulatory genes DNA coiling around histones is a dynamic process controlled by histone acetylation and methylation and alterations to the activity to the enzymes which control this process can have a large impact on gene expression. Histones are acetylated by histone acetyl transferase proteins (HAT), which unwinds the DNA allowing transcription: acetyl groups are removed by HDACs resulting in gene silencing. Cigarette smoke reduces the expression and activity of HDAC2 both at the protein and mRNA level Non-coding RNAs include microRNAs (miRNA), which are small non-coding, single stranded RNA molecules, of 19–25 nucleotides in length miR-1 has been linked to cigarette smoking-related conditions such as heart disease and cancer miR-21 has been shown to play a role in both inflammation and cancer miR-146a has been shown to downregulate both inflammation and cancer cell proliferation The interactions of miRNAs and mRNAs and their role in disease is not yet fully understood, but may be potential drivers and biomarkers of disease 10COPD and lung cancer share many common pathways for activation, and inflammation and cancer are closely linked. As almost all cancerous tissues show inflammation and a number of inflammatory diseases can predispose to cancer Mitochondria are the major cellular source of RNOS Inflammatory mediators can influence the cancer micro-environment, and the expression of cytokines is vital to drive the immune response to prevent cancer formation. For example IFNγ knockout mice are more susceptible to carcinogens than wild-type mice Increased IL-17 expression is associated with the severity of COPD IL1, IL6, IL8 and TNFα as well that of key components of the cell cycle including the cyclins D1, D2, D3, E1 and various cyclin dependent kinases (CDKs) Chronic inflammation is associated with the overexpression of the transcription factor NF-κB, which is a key mediator of inflammation-induced carcinogenesis Another common pathway to inflammation and proliferation is the Wnt pathway. Canonically the Wnt proteins are extracellular messenger proteins that bind to the disheveled (Dsh) receptor on the cells; this in turn leads to a signaling cascade that ultimately leads to the buildup of the transcription factor β-catenin. Activation of Wnt proteins and β-catenin is increased in COPD patients The chronic inflammation in COPD also causes lung damage which results in cell division in an effort to restore homeostasis. The increased rate of cell division, especially if paired with increased DNA damage due to smoking per se, may also contribute to the development of lung cancer. The lungs of COPD patients are hypoxic due to air trapping and reduced air flow. Hypoxic conditions stimulate the activation of the transcription factor hypoxia inducible factor (HIF) 1-alpha. HIF-1α induction is also seen in cancer cells due to the local hypoxic environment. HIF-1α regulates over 200 genes and activates glycolysis, immortalization through telomerase activation, stoppage of differentiation and can inhibit apoptosis The physiological conditions within the lung caused by COPD, rather than the chronic inflammation underlying the disease 11Lung cancer remains one of the most fatal forms of cancer, both due to lack of diagnosis and lack of effective treatments. Screening of COPD patients for the development of cancer, for example through CT scans, has been suggested as a potential method enable early detection and thereby to improve outcomes. However, this approach of increased surveillance is hampered by the lack of sensitivity of treatment and the large numbers of false positive diagnoses that result It is increasingly recognized that, due to the range of drivers of cancer, personalized medicine approaches based on the individual’s cancer are important. For example patients can now be tested for alterations in Epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF) and, when appropriate treated with specifically targeted drugs for example EGFR inhibitor Erlotinib It may therefore be possible to, in future, treat COPD patients with specifically targeted therapies to reduce the risk of the patients developing lung cancer. For example, owing to the important role of oxidative stress in both diseases it has been suggested that anti-oxidant therapy, for example vitamin C, vitamin E or N-acetyl cysteine (NAC) The potential problems with the use of antioxidants to prevent the development of lung cancer in all at risk patients highlights the heterogeneity of both diseases and therefore the need to develop specific, targeted treatments for the most at risk sub-populations of COPD patients to prevent them from developing lung cancer. In order for such an approach to be feasible however we will need to increase our knowledge of both diseases and their links and identify biomarkers to both improve screening for the most at risk patients and develop effective treatments.12Whilst the exact mechanism(s) underlying the increased incidence of lung cancer in patients with COPD is currently unknown, the two diseases are closely linked at a molecular level. Further research to elucidate the relationship between these two diseases may provide not only insights into the their development but may also create the possibility of cross-over treatments being developed, whereby anti-inflammatories developed for COPD may be beneficial in lung cancer and anti-cancer drugs may play a role in the future treatment of COPD.ALD has no conflicts of interest. IMA has received consultancies, honoraria, and travel and research grants from GSK, AstraZeneca, Johnson & Johnson, Chiesi, Pfizer, Boehringer Ingelheim, Novartis and Vectura.


Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research. Notably, if excision repair were to happen when the impediment for the replicative polymerase is encountered, while being present on ssDNA , a double strand break (DSB) would be formed proximal to the fork. DSBs are extremely dangerous as their inappropriate repair is a leading cause for chromosomal rearrangements Here we will summarize basic concepts of DDT, with a focus on recently emerged principles that govern the deployment, location and timing of DDT pathways, and discuss recent findings related to factors and structures that mediate damage bypass, which inform about the underlying mechanisms. We will highlight areas of future research and topics of debate, which will also bring into focus that DDT is at the nexus of various DNA metabolism pathways.2Two modes or strategies of damage bypass or DNA damage tolerance (DDT) are highly conserved throughout the eukaryotic kingdom RAD6 and RAD18, which encode for ubiquitin conjugating and ubiquitin ligase activities, respectively. TLS polymerases also belong to the RAD6 pathway, but soon it became evident that the RAD18 pathway contained activities different from TLS polymerases that were required to mediate damage bypass and gap filling via a recombination-like mechanism to the sister chromatid RAD18-RAD6 genes, but genetically different from HR, was called template switching, although the mechanism and activities involved were puzzling. The Rad5 ubiquitin ligase and Mms2-Ubc13 ubiquitin-conjugating complex were later identified to belong to the template switch recombination branch of the RAD18 pathway. Thus, three main genetic ways mediating DDT emerged: TLS, template switch, and HR, the latter also called the “salvage pathway”  pathway, in addition to the Rad51- and Rad52-mediated homologous recombination (HR) pathway , is mono- and polyubiquitylated by factors belonging to the RAD6 pathway RAD6-RAD18 pathway. Specifically, replication stress leads to exposure of ssDNA and recruitment of the ssDNA-binding protein, Rad18 Following this genetic categorization of DDT, efforts were made to understand how the choice between these pathways took place and on solving the puzzle as to how protein ubiquitylation mediated by the in vivo, how dynamic these modifications are, if preference for an initial type of PCNA modification is induced by distinct replication stress cues, and whether the initial modification counteracts or rather supports subsequent modifications of the clamp with other types of ubiquitin or SUMO moieties. In other words, does the first modification on PCNA function to “lock” an individual clamp to serve in a specific DDT module , or do these modifications often cooperate or act sequentially to each other to allow fluidity and cooperation between different DDT pathways, as in the case of complex lesions?As PCNA modifications can both mediate and block specific DDT pathways  and temporally (in the context of cell cycle and replication timing) 3in vivo and in vitrosgs1 are also epistatic with the ones caused by mutations in factors belonging to the error-free branch of the Rad18 pathway Although template switch is a recombination process, as revealed from examining the final outcome of the bypass reaction However, in backgrounds defective in PCNA SUMOylation, recombination structures involving SCJs that are independent of Rad18 and PCNA polyubiquitylation activities, but have identical migration properties in 2D gel electrophoresis, mediate DDT via template switch versus the salvage pathway proximal to early origins of replication, our knowledge on DDT should improve in the next years to reveal factors that contribute to one or both pathways of recombination-mediated DDT, unveil their interplay with PCNA modifications, and allow deductions on their temporal preference of action. Already in regard to template switching, the repertoire of factors contributing to the formation or safeguarding the stability of the intermediates extended significantly, revealing for instance that template switching is prevalently initiated at DNA gaps enabled by replicative helicase-coupled repriming, that the strand invasion process is facilitated by gap extension, and that DNA polymerase delta plays a major role in the DNA synthesis step of this reaction Because current 2D gel combined with genetic approaches allow the distinction of the recombination structures arising sgs1 revealed the same types of intermediates in both backgrounds, but with a relative enrichment in structural intermediates requiring Sgs1 action for resolution sgs1 background is suitable to uncover activities that physiologically contribute to this process. Notably, during this recombination process of template switching, various structures may become targeted by nucleases, in which case deleterious outcomes in terms of crossovers are expected  expected . In thisTo what extent the salvage pathway is different from template switching in terms of contributing factors, the architecture of the DNA intermediates involved, and accuracy of the outcome remains largely unknown. If the salvage pathway represents the minimal set of a HR rescue team operating from S to G2/M to which additional factors are added earlier in S phase by means of enzymatic activities that induce or are coincident with PCNA polyubiquitylation is currently elusive. Is PCNA polyubiquitylation making the recombination reaction of gap filling more accurate? The reason of why PCNA poyubiquitylation itself is required for template switching is rather unclear. Are the polyubiquitin chains on PCNA functioning as a sort of glue that may locally concentrate factors required to mediate or facilitate this recombination reaction or the proximity to the sister chromatid? What would be those additional factors required for template switch-mediated DDT, but not for the salvage pathway? Which are the factors and intermediates common to template switch and the salvage pathway? And finally, is there a rationale in terms of genome integrity in preferring template switching in S phase or in postponing the salvage pathway to later times in the cell cycle? Does PCNA polyubiquitylation ensure that this recombination process takes place preferentially behind replication forks rather than at forks where HR-mediated rescue is generally error-prone 4When the replicative polymerase encounters a blocking lesion or DNA structure, a transient stalling will occur. If the damage is placed on the lagging strand, synthesis of a new Okazaki fragment will soon cause the obstructing damage to be contained in a small DNA gap behind the replication fork. In such circumstances, the lesion bypass will take place in the context of post-replicative gap filling using either TLS or recombination modes . Stallin4.1Yeast and mammalian cells replicating in the presence of DNA damage accumulate gaps behind replication forks, and these gaps are subsequently filled in a manner that depends on DDT activities  4.2Certain replication stress contexts that perturb replication are not amenable to repriming. These are for instance lesions, such as interstrand crosslinks, that block the progression of the replicative helicase, or regions containing multiple intrinsic replication obstacles, such as DNA-bound proteins, highly transcribed genes or heterochromatin with repressed genes. Such DNA lesions and chromatin organization patterns block fork progression and greatly increase the chances of fork fragility. Rescue by HR in those contexts has often proved to be error-prone and instigate homology-driven genome instability One well-studied mechanism of replication fork stabilization is the one provided by the replication checkpoint . The chesmc5/6 mutants are capable to boost checkpoint activation, and likely relates to local regulation of HR activities. In smc5/6 mutants, recombination structures that are instigated by prolonged replication fork pausing and genetically depend on the conserved Tof1/Csm3 (Tim-Tipin) replication fork-pausing complex accumulate at NPSs in vitro-activities, is to prevent Mph1-mediated fork regression A paradigm for chromosomal loci predisposed to fragility are common fragile sites (CFSs) and regions containing natural pausing site (NPS) elements. The replication checkpoint is crucial for preventing CFS breakage via mechanisms that still require elucidation.However, besides the potential roles of reversed forks to mediate (toxic) DDT , recent 5DDT mechanisms are crucial to prevent fork breakage and to promote replication completion, by promoting damage bypass DNA synthesis either at the fork or postreplicatively. The temporal coordination of DDT pathways that ensure preferential usage early during replication to error-free postreplicative template switching is still not well understood, but likely involves regulation of replication factors by posttranslational modifications and fork stabilization mechanisms triggered by local chromatin and genomic features that block replication fork progression. Understanding how these DDT pathways are deployed and interrelate to each other is crucial for our knowledge of genome integrity mechanisms and replication stress responses. Moreover, from a practical point of view, as cells treated with chemotherapeutic agents crucially depend on DDT for their proliferation, understanding DDT mechanisms, their preferential deployment in different replication stress contexts and their impact on chromosome structure may inform therapeutic approaches by providing targeted inhibition. While many challenging questions remain, improved tools, approaches and working models hold promise to bring insight into the mechanism and intermediate products through which recombination-mediated DDT affects genome integrity.The authors declare no conflict of interest.