High blood pressure (BP) is a major public health problem in India and its prevalence is rapidly increasing among urban and rural populations. Reducing systolic and diastolic BP can decrease cardiovascular risk and this can be achieved by non-pharmacological (lifestyle measures) as well as pharmacological means. Lifestyle changes should be the initial approach to hypertension management and include dietary interventions , weight reduction, tobacco cessation, physical exercise, and stress management. A number of pharmaceutical agents, well evidenced by large randomized clinical trials, are available for initial treatment of high BP. These include older molecules such as thiazide diuretics and beta-blocking agents and newer molecules, dihydropyridine calcium channel blockers (CCB), angiotensin converting enzyme (ACE) inhibitors, and angiotensin receptor blockers (ARB). In view of the recent clinical trials data, some international guidelines suggest that CCB, ACE inhibitors or ARB and not beta-blockers or diuretics should be the initial therapy in hypertension management. Comprehensive hypertension management focuses on reducing overall cardiovascular risk by lifestyle measures, BP lowering and lipid management and should be the preferred initial treatment approach. High blood pressure (BP) is a major public health problem in India and elsewhere8There is, therefore, a need to lower BP in all groups of patients. This can be achieved by non-pharmacological (lifestyle measures) as well as pharmacological meansInitial management of hypertension uses a two-pronged approach, with emphasis on lifestyle measures and add-on drug managementTreatment is successful when multiple factors in the patient’s life are addressed, since essential hypertension is considered as a result of interactions between genes and environmentLifestyle measures, are a crucial step in hypertension management. Dietary Approaches to Stop Hypertension (DASH) studyNumerous short-term trials have documented that individuals can make these lifestyle changes which lower BP18Dietary modifications are mainstay for prevention and initial treatment of hypertensionEvidence from animal studies, epidemiological studies, clinical trials, and meta-analyses suggests that with increase in dietary salt (sodium chloride) intake, BP also rises. The most persuasive evidence about the effects of salt on BP comes from rigorously controlled, dose response trialsThe BP response to differences and changes in intake of dietary sodium intake is heterogeneousThe Indian Council of Medical Research (ICMR) recommends 1.5 g/d (65 mmol/d) sodium as an adequate intake level, primarily to ensure nutrient adequacyet alHigh potassium intake is associated with reduced BP. Although data from individual trials have been inconsistent, three meta-analyses of these trials have documented a significant inverse relationship between potassium intake and BP in non-hypertensive and hypertensive individualsBecause a high potassium intake can be achieved through diet rather than pills and because potassium derived from foods is also accompanied by a variety of other nutrients, the preferred strategy to increase potassium intake is to consume foods such as fruits and vegetables rich in potassium, rather than supplements. In the DASH trial, the two groups that increased fruit and vegetable consumption both lowered BP.Observational studies and clinical trials have documented a direct, dose-dependent relationship between alcohol intake and BP, particularly when the intake of alcohol increases above 2 drinks per dayDietary fibre consists of the indigestible components of food from plants. Evidence from observational studies and several clinical trials suggests that increased fibre intake may reduce BP16Many dietary factors have been evaluated for influence on hypertension control. These include dietary fish oil supplementation, fats other than omega-3 fatty acids, calcium, magnesium, carbohydrates and proteins. Although there are studies supporting their role in BP control, robust scientific evidence from randomized trials is lackingMultiple dietary factors are involved in hypertension and it is reasonable that a comprehensive dietary management would be the best approach to manage hypertension and to reduce overall cardiovascular risk.Dietary Approaches to Stop Hypertension (DASH) was a program by the National Institutes of Health, USA1622The OmniHeart triale.g., physical activity), established dietary risk factors , and other aspects of vegetarian diets . Limited trial evidence, indicates that non-dietary factors and established dietary risk factors are not fully responsible for the BP-lowering effects of vegetarian diets and that some other aspects of vegetarian diets lower BP.Vegetarian diets have been associated with low BP. In industrialized countries individuals who consume a vegetarian diet have markedly lower BP than do nonvegetarianset al2829In India, diets in many rural and urban populations are predominantly vegetarian38A substantial and consistent body of evidence from observational studies and clinical trials shows that weight is directly associated with BP2 or even <23 kg/m2 in Indians- as effective approach to prevent and treat hypertension. More importantly, in view of the well-recognized difficulties of sustaining weight loss, efforts to prevent weight gain among those who have normal body weight are critically important. Central or truncal obesity is also a major hypertension risk factor in Indians2 to 24.9 kg/m2, preferably < 23.0 kg/m2) and waist circumference (<90 cm in men and < 80 cm in women) is therefore recommended.With rare exception, clinical trials have documented that weight loss lowers BP. Importantly, reductions in BP occur before, and without, attainment of a desirable body weight. In one meta-analysis that aggregated results across 25 trials, mean systolic and diastolic BP reductions from an average weight loss of 5.1 kg were 4.4 and 3.6 mm Hg, respectivelyP<0.01)Smoking as a hypertension risk factor is not well defined. In western countries epidemiological studies have reported that smokers tend to have lower BP than non-smokersP<0.01)Several large epidemiological studies have reported an inverse relationship between BP and physical activityet al13The role of yogic practices in BP management is controversial. Yogic practices have been reported to reduce BP and multiple cardiovascular risk factors in many studies from India(i) when to treat and (ii) with which drug?Whilst non-pharmacological therapies show modest reductions in BP, all too often these treatments are not adopted and, even if they are, not sustained. Furthermore, in many people the BP remains high despite appropriate non-pharmacological measures especially if the initial BP at diagnosis was high. Under these circumstances pharmacological approaches must be considered. The questions are sign to monitor the patients’ clinical status; or a risk factor for atherosclerotic cardiovascular disease or as a disease and major contributor to death from cardiac, cerebrovascular, renal or peripheral vascular disease. Currently hypertension is defined as BP equal to or greater then 140/90 mm Hg based on the average of two or more correct BP measurement taken during two or more contacts with health care providerHigh BP can be viewed as a Another aspect of management is the time of the day to administer an antihypertensive medication. Attributes of cardiovascular system are characterized by predictable changes during 24 h cycle61The overall goal of treatment in hypertensive patients is to reduce the risk of cardiovascular morbidity and mortality by lowering BP and treating other modifiable risk factors. In general, the goal is to lower BP to below 140/90 mm Hg. In patients with heart failure, diabetes, or renal disease, the goal is to lower BP to below 130/85 mm Hg . In oldeThe initial drug management of hypertension is a contentious issue. The JNC-7 report emphasizes on beta-blockers and thiazide diuretics as suitable drugs for first line managementOne way to improve control could be to start early and utilize combination therapy. The JNC-7 recommends initiation of therapy with combination therapy rather than a single agent if BP is more than 20/10 mm Hg above the treatment goal as in stage II hypertensionPatients with diabetes mellitus and hypertension have twice the risk of cardiovascular disease as non-diabetic hypertensive patients. In addition, hypertension increases the risk of diabetic retinopathy and nephropathy136971Aggressive control of elevated BP can slow progression of renal damage and delay or prevent the development of end-stage diseaseThe debate continues as to whether the benefits of treating high BP are simply due to the quality of BP control or whether the choice of drug therapy adds to the benefit of BP lowering (beyond BP effect). The experimental basis of the benefits of renin-angiotensin system (RAS) blockade beyond BP lowering is compelling, since angiotensin II is implicated in many pathophysiological changes in the functional and structural disturbances in cardiovascular, cerebrovascular and renal systemsHypertension is a vascular disease and raised BP is associated with multiple risk factors. It has been shown that as the BP level increases the incidence of cardiometabolic abnormalities increase134In conclusion, hypertension is a major problem in India and the most prevalent chronic disease. Most of the subjects have mild to moderate hypertension and the initial strategies for management involve lifestyle changes focussing on reduction of dietary salt, fat and alcohol and increase in potassium and fruits and vegetables. Weight management and reduction in obesity and truncal obesity, regular physical exercise, tobacco cessation and stress management are important. Pharmacological treatment should be initiated after lifestyle interventions and choice of drug depends on age, the overall cardiovascular risk and co-morbidities. Management should focus on comprehensive risk reduction for better prognosis.


Telomerase is a highly specialized reverse transcriptase (RT) and the maintenance of telomeric length is determined by this specific enzyme. The human holoenzyme telomerase is a ribonucleoprotein composed by a catalytic subunit, hTERT, an RNA component, hTR, and a group of associated proteins. Telomerase is normally expressed in embryonic cells and is repressed during adulthood. The enzyme is reexpressed in around 85% of solid tumors. This observation makes it a potential target for developing drugs that could be developed for therapeutic purposes. The identification of the hTERT as a functional catalytic RT prompted studies of inhibiting telomerase with the HIV RT inhibitor azidothymidine (AZT). Previously, we have demonstrated that AZT binds preferentially to telomeres, inhibits telomerase and enhances tumor cell senescence, and apoptosis after AZT treatment in breast mammary adenocarcinoma cells. Since then, several studies have considered AZT for telomerase inhibition and have led to potential clinical strategies for anticancer therapy. This review covers present thinking of the inhibition of telomerase by AZT and future treatment protocols using the drug. Telomerase is a ribonucleoprotein that elongates the telomeres in eukaryotic cells. The enzyme is composed of a catalytic subunit, hTERT, a RNA component, hTR, and a group of associated proteins. Based on experiments in fibroblasts, in the absence of telomerase, cellular telomeres are seen to shorten with an increasing number of cell divisions. It was suggested that the lost of telomeres could account for cell senescence after a given number of duplications .Studies on fibroblasts cultures have indicated that telomeric length could be used to predict the replicative capacity of these cells . It was Telomerase is active in the germ line and stem cells but is inactive in most somatic cells. However, telomerase activity is found in most immortalized cell lines and in 85–90% of human tumors. It was also reported that telomerase activity was present in 90 of 101 biopsies that represented 12 human tumor types, but none in 50 normal somatic tissues .Figure 1) used in the treatment of AIDS, since it blocks the replication of human immunodeficiency virus-1 (HIV-1) by competitively inhibiting reverse transcriptase (RT). It is phosphorylated intracellularly to AZT-triphosphate (AZT-TP) by thymidine kinase, and is finally incorporated into viral DNA blocking chain elongation by RT  or zidovudine], is a thymidine analog . The digest was then loaded into Chroma Spin-1000 columns to isolate fragments of 1 kb and upward. The first fraction to be eluted was characterized as the telomeric fraction (TF) whereas the non-telomeric fraction (NTF) eluted later in second place. TF was comprised of longer sequences (>2 kb) compared to NTF. This was confirmed by a Southern blot using a human telomeric probe. Radioactivity associated with each fraction revealed a threefold increase in [3H]-AZT incorporated in the TF compared to NTF. No preferential telomeric binding was detected for [3H]-thymidine (Tdr) or [H-3]-5′-bromodeoxyuridine (BrdU) in similar experiments. The failure of thymidine or BrdU to preferentially incorporate into telomeric repeats supported the hypothesis that differential incorporation of AZT into TF was not a direct result of the telomeric sequence itself but most probably a phenomenon related to the specificity of a polymerase such as telomerase. Primary cultures of mouse dermal fibroblasts, cells with large telomeric repeats that lack telomerase, did not preferentially incorporate AZT into the TF. Bal 31 digested DNA consists of mononucleotides, including AZT, from both 3′ termini of the two strands of linear DNA. When the chromosomal ends of high molecular weight [3H]-AZT-DNA were digested with Bal 31, the radioactivity was double in the TF (short-term digest) compared with the NTF (longer digestions). Therefore incorporation of AZT in CHO immortalized cells but not in primary fibroblasts (that lack telomerase) indirectly showed that AZT incorporation could be telomerase-mediated.Briefly, telomeric DNA was separated from genomic DNA by digesting with restriction enzymes and size fractionation. We quantitatively compared the amount of [in vitro by AZT , small proline rich proteins (spr), and apoptosis, were analyzed after AZT treatment. The results were indistinguishable from the control untreated cells or the recovered cells with no morphological or biochemical changes attributable to senescence being observed. The fact that telomere length was decreased in HeLa cells after long-term exposure to AZT without any evidence of senescence could be the result of different mechanisms. Firstly, the number of AZT-treated passages could be insufficient for a senescence program to be triggered, and secondly telomeres shortening to a critical length could induce a compensatory mechanism of preservation so preventing further losses, known as alternative lengthening of telomeres or ALT  and human breast cancer cell line (MCF-7) treated with AZT. Similar results were observed in human endometrial carcinoma cells (HEC-1) whereas inhibition of telomerase activity and telomere length were seen to be dependent on the AZT concentration and duration of exposure. The same authors also found that in HEC-1 cells the effects of cisplatin were enhanced with a marked reduction in cell proliferation, appearance of morphological changes and senescent-like cells in the presence of AZT  is the clinical relevance of inhibiting telomerase in cancer patients. According to the paradigm currently proposed for telomeres and telomerases, it can be predicted that telomerase inhibition will not affect a tumor until its telomeres reach the critical size for entering senescence. This means that during anti-telomerase therapy, the tumor cells will continue to grow undergoing 20–30 divisions until the telomeres reach a critical size leading to tumor senescence. Therefore, concerns must be expressed when attempting to treat advanced tumors with AZT . HoweverTable 1 in non-viral tumors, AZT has been used in phase I and II clinical trials alone or in combination with other drugs for gastrointestinal cancers, pancreatic cancer, and other advanced malignancies with some tumor regression being reported (Azidothymidine is used to treat several virus-associated human cancers, including AIDS-related Kaposi sarcoma, Kaposi sarcoma-associated primary effusion lymphoma, Epstein–Barr-associated lymphoma, primary central nervous system lymphoma, and adult T cell leukemia . As showreported . AlthougThe 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


Human cytomegalovirus (HCMV) is a herpesvirus that is prevalent in the human population. HCMV has recently been implicated in different cancer forms where it may provide mechanisms for oncogenic transformation, oncomodulation and tumour cell immune evasion. Moreover, antiviral treatment against HCMV has been shown to inhibit tumour growth in preclinical models. Here we describe the possible involvement of HCMV in cancer and discuss the potential molecular impact expression of HCMV proteins have on tumour cells and the surrounding tumour microenvironment. The interplay between cancer cells and the surrounding microenvironment is essential for the growth and spread of a tumour. The development of malignant tumours requires a microenvironment that supports the uncontrolled proliferation and spread of cancer cells but also conditions that avoid destruction from the various arms of the immune system must be present. The immune system represents an important tool for the destruction of the majority of cancer cells and precancerous conditions in the human body. However, malignant growing tumours have in most, if not all, cases developed immune evasion strategies to avoid destruction by immune cells. One essential immune evasion strategy that can be induced or applied by tumour cells is the formation of an inflammatory microenvironment. Tumour cells can induce inflammation directly through oncogenes that induce transcriptional programs responsible for the production of pro-inflammatory eicosanoids, cytokines and chemokines that attract different cells of the immune system to the microenvironment. Also chronic inflammation caused by viral or microbial infections, autoimmune diseases, dietary products or inflammatory conditions caused by unknown reasons can create an inflammatory microenvironment that support tumour growth . Immune Although it has been both experimentally difficult and heavily debated, it is today well accepted that approximately 20% of the global cancer burden can be linked to infectious agents including viruses, bacteria and parasites . Recent Human cytomegalovirus (HCMV) is a beta-herpesvirus that is common in the human population. Although HCMV is not currently causally implicated in human cancer, a number of recent evidence suggests that HCMV may be specifically associated with some human malignancies. HCMV nucleic acids and proteins have been detected in 90-100% of glioblastomas and medulloblastomas, prostate, breast and colon cancers and in mucoepidermoid carcinomas of salivary glands -12. ConsWe have consistently observed that HCMV proteins are widespread and easily detected in a majority of tumour samples, whereas viral DNA is detected only in few cells within the tumour .The functional inhibition of the p53 and Rb families of tumour suppressor proteins by HCMV encoded proteins implicates that HCMV is able to promote cell cycle progression, increase DNA synthesis and block apoptosis resulting in increased chromosomal instability -43. In nHence, HCMV encodes for a number of different proteins that have profound effects on cellular processes leading to increased proliferation, inhibition of apoptosis, stimulation of cellular migration, the release stimulatory factors, induction chemotherapeutic resistance and increased telomerase activity.Symptoms of a primary HCMV infection are usually mild or asymptomatic in immunocompetent individuals but can cause severe disease in fetuses and immunocompromised patients such as transplant recipients and AIDS patients. The virus is spread through all bodily fluids and establishes a life-long latent/persistent infection. Reactivation from latency appears to be triggered by inflammation, which the virus can initiate by inducing cytokine and chemokine production and by enhancing the synthesis of pro-inflammatory eicosanoids. Indeed, the biological responses elicited by HCMV reactivation mimic those seen in leukocyte dysfunction, wound healing and chronic inflammation . HCMV reDuring evolution HCMV has coevolved with the human host and the virus has developed several immune evasion strategies to allow persistent infection and viral spread without harming its host. HCMV contains a 250 kb ds DNA genome that has 252 open reading frames and encodes approximately 200 proteins, of which only about 50 are essential for viral replication . Hence, We recently showed that HCMV nucleic acids and proteins are present in the majority of medulloblastoma primary tumours and cell lines. We also found that US28  was expressed in medulloblastoma and induced expression of COX-2 in these tumours . Microar2 (PGE2). Increased levels of prostaglandin E2 (PGE2) are perceived in malignancies of different origin, including brain tumors [2 exerts its physiological effects by interacting with a subfamily of four distinct G-protein–coupled receptors designated EP1, EP2, EP3, and EP4. PGE2 promotes tumour growth in an autocrine and/or paracrine manner by stimulating EP receptor signalling with subsequent enhancement of cellular proliferation, promotion of angiogenesis, inhibition of apoptosis and stimulation of invasion [2 is an important mediator for the interaction between tumour cells and cells in the tumor microenvironment where PGE2 contributes to the generation of a tumor promoting inflammatory microenvironment that suppress the activities from cells in the immune system [COX-2 is over- expressed in a number of different adult cancers of epithelial origin as well as in gliomas where high expression often is correlated with poor prognosis (reviewed in -60). In n tumors -68. PGE2invasion . In addie system .2 are required for efficient replication of HCMV by facilitating the production of the HCMV immediate-early 2 protein [2 and reduce the virus burden in HCMV infected cells [2 and the expression of HCMV proteins in medulloblastoma, as well as tumour growth in vitro and in vivo [Different nonsteroidal anti-inflammatory drugs (NSAIDs) which inhibit the enzymatic function of cyclooxygenases and the production of prostaglandins and other inflammatory mediators has been shown to be promising agents for the prevention and treatment of various cancers . Elevate protein . Daily a protein ; the ben protein . Interesed cells , 73; thu in vivo .in situ showed co-localization of US28 with phosphorylated STAT3, COX-2, VEGF and e-NOS, suggesting that US28 in addition to promoting an inflammatory microenvironment also contribute to tumour invasiveness and angiogenesis [US28 that induces the expression of COX-2 in HCMV infected cells can bind different chemokines, including CCL2, CCL5, and CX3CL1 , and supogenesis , 19. TakHCMV establishes latency in myeloid lineage cells, and reactivation is dependent on inflammation and differentiation of monocytes into macrophages of dendritic cells. HCMV can also persistently infect monocyte/macrophage lineage cells and induce a strong inflammatory response in these cells . In humaHCMV is a neurotropic virus that can persistently infect neural precursor cells. As a consequence HCMV is the major infectious cause of birth defects in infants, including sensori-neural hearing loss or neuronal migration disturbances during brain development, and in the most severe cases, microcephaly or anencephaly. We have demonstrated that HCMV can block the ability of neural progenitor cells to differentiate into neurons or astrocytes , 83. HCMMedulloblastoma and neuroblastoma are embryonal tumours of the central and peripheral nervous systems, respectively. Compared to adult tumours, paediatric tumours generally have a dramatically shortened latency period and harbour fewer genetic aberrations causing oncogene activation or loss of apoptotic regulators. The reason for these differences is that these malignancies probably arise from stem or progenitor cells which already possess proliferative capacity as a part of the normal developmental process . Medullo+ and/or CD15+ cells in both medulloblastomas and glioblastomas have been recognized as potential cancer stem cells [+ cell population expressed HCMV IE1 [Although the cellular origin of gliomas still is contended, recent evidence suggests that multipotent neural stem or progenitors of the subventricular zone (SVZ) are cells with the potential to form gliomas . Subpopuem cells , 93. In em cells . We haveem cells . We obseem cells , and unpHCMV IE1  and our The findings that several cancer forms are HCMV positive, including those with a neural origin that usually have a dismal prognosis, opens up the possibility to treat these cancers with anti-viral drugs against HCMV. In nude mice engrafted with human medulloblastoma cells, the antiviral drug valganciclovir, significantly inhibited tumour growth. Interestingly the treatment effect was extensively enhanced when valganciclovir was combined with the COX-2 specific inhibitor celecoxib , which iMedulloblastoma, neuroblastoma and glioblastoma tumors express high levels of COX-2 and NSAIDs, inhibitors of COX-2 and PGE2 production, have profound effects on the growth of these tumours , 94-96. In a randomized double-blinded phase II study we are currently evaluating antiviral drugs against HCMV as an adjuvant therapy for glioblastoma. Results from this study are expected to be ready soon. Also a phase I/II immunotherapy clinical trial of autologous HCMV pp65 RNA loaded dendritic cells has been initiated in which 13 patients with newly diagnosed glioblastoma multiforme were enrolled. Initial results from this study are promising. Patients exhibited a median progression-free survival of 15.4 months and overall survival of 20.6 months, numbers which are highly significant compared to historical controls .The promising preclinical and clinical results obtained using antiviral drugs against HCMV to treat tumours carrying HCMV should be extended to include larger controlled clinical trials. Also, developing drugs that specifically inhibit the functions of HCMV encoded US28 may be of future benefit in cancer treatment since the US28 protein may possess important functions in tumour initiation through the activation of intracellular signalling pathways, angiogenesis and effects on the tumour microenvironment.The presence and functions of HCMV in cancer is still debated and scepticism vestiges regarding the relationship between HCMV and cancer. This mainly originates from conflicting results regarding the detection of HCMV in tumour samples and since HCMV by itself not has been shown to transform normal cells into cancer cells . The lasAs of today HCMV has been detected in glioma, medulloblastoma, neuroblastoma, breast, prostate and colon cancer and mucoepidermoid tumors of the salivary gland. Although the exact molecular functions of HCMV in these tumours still need to be further investigated, the findings that antiviral HCMV treatment inhibit the growth of certain tumours (, Wolmer-


S. cerevisiae as a test bed in the search to predict and validate trait differences based on observations of regulatory variation. Malaysian yeast, when cultured in standard medium, activated regulatory programs that protect cells from the toxic effects of high iron. Malaysian yeast also showed a hyperactive regulatory response during culture in the presence of excess iron and had a unique growth defect in conditions of high iron. Molecular validation experiments pinpointed the iron metabolism factors AFT1, CCC1, and YAP5 as contributors to these molecular and cellular phenotypes; in genome-scale sequence analyses, a suite of iron toxicity response genes showed evidence for rapid protein evolution in Malaysian yeast. Our findings support a model in which iron metabolism has diverged in Malaysian yeast as a consequence of a change in selective pressure, with Malaysian alleles shifting the dynamic range of iron response to low-iron concentrations and weakening resistance to extreme iron toxicity. By dissecting the iron scarcity specialist behavior of Malaysian yeast, our work highlights the power of expression divergence as a signpost for biologically and evolutionarily relevant variation at the organismal level. Interpreting the phenotypic relevance of gene expression variation is one of the primary challenges of modern genomics.Comparative genomic studies have reported widespread variation in levels of gene expression within and between species. Using these data to infer organism-level trait divergence has proven to be a key challenge in the field. We have used a wild Malaysian population of  Heritable genetic differences at the molecular level are widespread between populations and species. Much of the divergence observed in DNA sequence, gene expression, and other molecular attributes may bear little relationship to macroscopic traits, and the outstanding challenge in the field is to detect signatures of biological relevance in catalogs of molecular data. This unbiased search for trait differences with an impact on fitness, motivated by screens of molecular observations alone, is often referred to as reverse ecology  and two wine/European  isolates used in this study were obtained from the National Collection of Yeast Cultures. For analysis of cis-regulatory variation in File S5, the following three Malaysian × wine/European hybrids were generated through single-cell mating between Malaysian and wine/European strains: UWOPS03.461.4 × RM11-1; UWOPS03.461.4 × BC187; and UWOPS05.217.3 × BC187.All strains used are listed in 600) in yeast peptone dextrose (YPD) medium  using Bowtie , in contrast to the 601 and 265 genes, respectively, reported at this significance level in File S2 and File S5.Read counts per gene were summed using custom Python scripts and normalized with the EDASeq package in R . For each regulon, we tabulated all genes whose differential expression between the populations was significant at adjusted P < 0.05, and taking the log2 fold change between the populations for each such gene as described, we summed these differential expression measures across the genes of the regulon. We then repeated this ratio sum procedure on each of 10,000 random gene groups of the same size as the regulon of interest, drawn from the total set of genes with significant differential expression. The proportion of such null gene groups with a sign sum as extreme as, or more extreme than, the ratio sum of the real regulon was taken to be the nominal empirical P for directional expression divergence. P values were corrected for multiple testing using the Benjamini-Hochberg method as implemented in the p.adjust function in R. For analysis of directional cis-regulatory variation in File S6, we repeated this group test procedure using as input, for a given gene, the log2 ratio of the expression of the Malaysian allele to that of the wine/European allele, measured in hybrids as described above.To screen regulons for directional expression divergence between Malaysian and wine/European yeast, coregulated gene clusters were obtained from ccc1 laboratory strain, compared to wild-type, in 3 mM FeSO4 from  were used to make reciprocal hemizygotes by deleting one allele of the gene of interest  with a URA3 cassette  . For each sample, cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies) and diluted to 1 ng/µL. Maxima SYBR green (Thermo Scientific) was used for quantitative PCR, with two to three technical replicates for each gene in each strain, on the Mx3000P system (Agilent). Cycle thresholds (Ct) for each replicate were obtained using the MxPro software (Agilent), and gene expression levels relative to ACT1 were calculated using the 2−ΔΔCt method .Two biological replicates of each parental strain, two biological replicates of each of two independent transformants of each t method . As a fua/Ks described by a/Ks values across all genes of the iron toxicity response regulon as defined. We then repeated this mean Ka/Ks calculation on each of 10,000 random gene groups of the same size as the iron toxicity response regulon, drawn from all genes for which SNP data were available. The proportion of such null gene groups with a mean as extreme as, or more extreme than, the mean of the real regulon was taken to be the empirical P value for protein evolutionary rate.Multiple sequence alignments were generated for each gene using MUSCLE  from genTable S1). We used the RNA-seq reads to verify and correct coding sequences from low-coverage genomes available for these strains (File S1). Mapping to the amended genomes and analysis of the complete set of profiles revealed 601 genes differentially expressed between the populations at a false discovery rate (FDR) of 5% (File S2).To survey gene expression programs in wild yeast, we took a comparative approach using three homozygous Malaysian isolates and two homozygotes from a distinct, well-defined wine/European yeast population (http://yeastgenome.org) and those defined on the basis of coregulation in classic analyses of laboratory yeast . This defect acted as a recessive trait, because hybrid strains showed resistance to high-iron treatment on par with that of their wine/European parents (File S3). We also assessed growth of other environmental yeast isolates and found all to be resistant to iron toxicity (File S4), with the exception of NCYC110, a member of the broadly stress-sensitive West African population . Likewise, sequence-based analyses revealed no striking promoter variation in iron metabolism genes (data not shown). To identify candidate trans-acting loci underlying growth and regulatory traits, we searched the genome for nonsynonymous coding polymorphisms unique to the Malaysian population in components of the iron metabolism gene network. Among the resulting gene hits, we focused on two known to be essential in laboratory yeast for growth in high-iron conditions .We next sought to dissect the genetic basis of divergence in iron metabolism regulation among Malaysian strains using a candidate gene framework. We expected that causative variants would likely act in  vacuole . We alsoanalysis . In all AFT1, YAP5, and CCC1 in high-iron conditions and in a complete medium control, along with the wild-type parents, and we measured growth rates of each strain in each case. The results revealed a 1.2-fold difference in iron resistance between strains bearing the Malaysian and wine/European alleles of CCC1 and a more modest (1.05-fold), although still significant, effect on iron resistance of variation in YAP5 (File S3). In each case, the Malaysian allele of the respective gene compromised growth in high-iron conditions, mirroring the known effects of engineered null mutations at these loci , consistent with the behavior of hypomorphs at these loci , reflecting haploinsufficiency of CCC1 with respect to iron resistance. As expected, variation in AFT1, which functions primarily in conditions of iron starvation (File S3). However, the Malaysian allele of AFT1 had a significant regulatory effect in reciprocal hemizygotes, driving upregulation of iron starvation genes during growth in complete medium  in a manner consistent with a gain-of-function mutation (YAP5 and CCC1. With respect to gene regulation, the Malaysian genome harbors alleles with both activating and repressing effects on iron metabolism, reflecting a complex genetic model that involves YAP5 and CCC1 as well as AFT1.Expression measurements also supported a model of losses of function at Malaysian orthologs of arvation , had litWe next aimed to investigate signatures of natural selection on iron metabolism in Malaysian yeast, complementing our expression-based and phenotype-based analyses with sequence-based tests. Using SNPs private to and conserved in each of five yeast populations characterized by AFT1, YAP5, and CCC1, in regulatory and growth phenotypes.Against a backdrop of hundreds of comparative transcriptomic studies in many species, the power of expression divergence as a predictor of trait variation has remained largely invalidated. To establish Malaysian yeast as a test bed for the reverse ecology approach, we generated expression profiles from cultures grown in standard conditions, and we used these data as a point of departure for a study of regulatory and growth phenotypes in the presence of excess iron. Although several elegant studies have characterized growth traits in yeast populations (YAP5 and CCC1 alleles as losses of function, and the sequence-based evidence for non-neutral evolution of the coding regions of iron resistance genes, support a model of specialization by Malaysian strains to a low-iron environment. In one evolutionary scenario, reduced exposure to high-iron conditions in the Malaysian niche would have relaxed the strength of purifying selection on the iron toxicity response, allowing Malaysian yeasts to accumulate mutations in this gene network. Additionally, given the gain-of-function behavior of the Malaysian allele of AFT1, it is tempting to speculate that negative regulatory control of this activator of the iron starvation response has been eliminated in Malaysian yeast to sidestep iron-sensing mechanisms and raise expression of iron transporters and scavengers as would be suitable for a constant, low-iron environment. By contrast, the avid iron uptake and functional vacuolar iron storage of wine/European strains would reflect a need for flexible iron homeostasis machinery that can respond more fully to environmental change. Taken together, our findings provide a case in which the rare and often deleterious mutations that litter wild yeast genomes (What are the evolutionary forces that have driven divergence of iron metabolism in Malaysian yeast? The behavior of the Malaysian YAP5 and AFT1 transcription factors drove downregulation of iron resistance genes and upregulation of iron starvation genes, respectively, contrasting with the high and low expressions of these gene sets, respectively, in Malaysian parental strains. Given the prevalence of transgressive segregation in the genetics of gene expression variation within species (YAP5 and AFT1 in the purebred context. However, our experiments do not rule out the possibility that alleles of YAP5 and AFT1 exert different effects in the parental background than they do in the hybrid. Future work will establish the potential for epistasis among our candidate loci and between each gene and the genetic background using the loci we have validated here as a springboard to deepen our mechanistic understanding of the regulatory and growth behaviors of Malaysian isolates.Although we successfully harnessed expression profiles to identify an evolutionarily relevant trait difference between yeast populations, our results leave open the question of the molecular basis of the expression patterns themselves. In reciprocal hemizygote experiments, the most striking regulatory effects of our candidate genes were in directions that opposed the patterns of divergence in parental homozygotes: Malaysian alleles at the  species , we favoOur work makes clear that expression profiles can be used as a powerful hypothesis generator for the study of organismal trait variation. This reverse ecology paradigm is likely to be most successful when coherent regulatory change in well-annotated pathways underlies, or responds to, change in a phenotype. With the increased availability of functional genomic resources in many taxa, the expression-based approach will likely prove to be broadly applicable in the genetic dissection of differences between populations and species.


RNASET2 gene to possess a remarkable in vivo tumor suppressor activity, although no in vitro features such as inhibition of cell proliferation, clonogenic potential, impaired growth in soft agar and increase in apoptotic rate could be detected. This is reminiscent of the behavior of genes belonging to the class of tumor antagonizing genes (TAG) which act mainly within the context of the microenvironment. Here we present transcriptional profiles analysis which indicates that investigations of the mechanisms of TAG biological functions require a comparison between the in vitro and in vivo expression patterns. Indeed several genes displaying a biological function potentially related to tumor suppression could not be validated by subsequent in vivo expression analysis. On the other hand the fact that we could find congruency for three genes both in vivo and in vitro adds a warning to a too much stringent categorization of this class of genes which relies on the sensitivity of the methodological approaches.Using the Hey3Met2 human ovarian cancer cell line, we previously found the  Among the gynaecological malignancies, ovarian cancer is considered to be the most lethal tumor, with an incidence of 42,000 new cases per year in Europe  and 22,0in vivo but not in vitro.We reckon that one of the main reasons for such regrettable scenario is a poor understanding of the biological bases of ovarian cancer pathogenesis. Indeed, one of the salient feature of these tumors is their high heterogeneity, both at the morphological and biological levels, which make the contribution of genetic lesions difficult to interpret in a causative way. Among human cancers located in the ovary, those derived from the ovarian surface epithelium are the most frequent  and all phila from viruses to humans, suggesting an evolutionary important function [We have recently reported a preliminary biological characterization of one of these genes, called RNASET2, which codes for an extracellular RNase highly conserved among the function .In a xenograph model for ovarian cancer, we found RNASET2 to carry out a strong oncosuppressive activity by recruiting cells of the monocyte/macrophage lineage into the tumour mass. This subset of cells represents the main component of the host immunological response . AlthougThis was basically the rationale that prompted us to further investigate the cell-autonomous expression profile induced by RNASET2 in ovarian cancer cells.RNASET2 gene to possess a remarkable in vivo tumor suppressor activity, irrespective of the protein’s catalytic activity [in vitro, the same cell clones did not show inhibition of cell proliferation, changes in the clonogenic potential, impaired growth in soft agar and increase in apoptotic rate.As previously reported, using the Hey3Met2 human ovarian cancer cell line, we found the activity . NoteworRNASET2 might represent a new member of the family of TAG/MSG (acting mostly in a non-cell autonomous fashion), we could not formally rule out a cell-autonomous effect elicited by this gene on the cancer cells, which might have escaped detection by our panel of in vitro standard assays. We therefore decided to carry out a more thorough analysis to shed light on this issue.As stated in the previous section, although these data strongly suggest that RNASET2-overexpressing Hey3Met2 cells and compared it with that of control cells. An Agilent Whole Human Genome Oligo Microarray was employed with total RNA extracted from Hey3Met2 clones transfected with wild-type or a catalytically mutant form of RNASET2 . Total RNA from Hey3Met2 cells, transfected with the empty vector, was used as a control.Accordingly, we defined the expression profile of the p-value smaller than 0.01. These genes were further organized by hierarchical clustering using squared Pearson correlation into 2 clusters, as shown in figure RNASET2 expression vectors. This might, hypothetically, suggest some influence of RNASET2 ribonucleolytic activity on the steady state level of these gene’s transcripts.Sixty-five genes were found to be modulated by at least one of the two RNASET2 protein forms tested , with a fold-induction or repression greater than 2 and a In order to define the cellular processes/pathways affected by RNASET2, all differentially expressed genes were cross-referenced to the Gene Ontology (GO) database to identify functional categories over-represented in the panel of genes associated with the two clusters.http://david.niaid.nih.gov) using DAVID [The GO analysis was done at GO FAT level of biological process and molecular function, whereas Expression Analysis Systematic Explorer (EASE) biological theme analysis was carried out online , a regulator of cancer cells invasiveness which was reported to be frequently overexpressed in colon, breast, prostate, pancreas, lung and ovarian cancer [MCAM, a well known marker of poor prognosis in epithelial ovarian cancer, whose gene product promotes the growth, invasion and metastasic potential of malignant cells [PLAU (PLasminogen Activator Urokinase-type), which promotes fibrinolysis and degradation of extracellular matrix [NNMT (nicotinamide N-methyltransferase), a serum tumor marker for colorectal cancer recently identified as novel regulator of cell migration [Since TAG/MSG genes are postulated to encode for products involved in the cross-talk between the cancer cell and the tumor microenvironment ,13, we sn cancer -16; AREGn cancer -19; MCAMnt cells -22; PLAUr matrix ; and NNMigration ,25.DMKN, a gene differentially regulated in inflammatory conditions, and whose role in activating Rab5 function was recently defined [Moreover, on the basis of their biological function we decided to validate a few genes that were also shown to be down-regulated after RNASET2 overexpression. The first is  defined . The gen defined -30.DDIT4 gene was chosen due to its role in RAS-mediated transformation of ovarian epithelial cells [DDIT4 expression vectors in immortalized human ovarian surface epithelial cells has been shown to render the cells tumorigenic in substitution for oncogenic Ras mutations.The al cells . SignifiCPA4 gene expression and prostate cancer aggressiveness, possibly due to its role in extracellular peptide processing [CPA4 in the panel of genes to be further investigated.Recent studies have suggested a relation between ocessing . We thusPTPRH gene, encoding a member of the protein tyrosine phosphatase (PTP) family [The ) family , was alsRNASET2 were also selected for validation: LMCD1, belonging to the category of the LIMD1 tumor suppressor gene, and DSE (dermatan sulfate epimerase), whose protein product elicits specific cytotoxic T lymphocytes responses in cancer patients [Finally, two genes that were up-regulated by patients ,35. TablDSE and PLAU genes, whose expression pattern in Hey3Met2 cells expressing the catalytically-mutant RNASET2 protein turned out to be down-regulated by microarray hybridization but upregulated by qPCR. However, for both genes the observed change in expression pattern with respect to control clones was below the chosen 2-fold threshold for significance . Moreover, for both genes the expression pattern observed by qPCR in Hey3Met2 cells expressing wild-type RNASET2 was in agreement with that observed by microarray hybridization.To validate these genes, realtime RT-PCR (qPCR) assays were performed on the same RNA samples used for microarray hybridization. In order to allow for cross-references of the expression data, the sequence of all primer pairs was designed to span the region of the cDNA corresponding to the hybridization probes placed on the microarray chip. As shown in figure CPA4, DMKN and MCAM genes that was observed by microarray hybridization turned out to be much more evident when assayed by qPCR. Of note, the fold-change expression for the RNASET2 gene turned out to be much higher by qPCR, in keeping with previous expression data obtained by western blot analysis carried out on the same clones (data not shown). We next asked whether some of these genes could have any relevance in RNASET2-mediated tumor suppression in vivo. To this end, total RNA extracted from xenograph tumors [in vitro setting, the in vivo tumor population is rather heterogeneous, comprising both human Hey3Met2 cancer cells and host-derived murine stromal cells. The results of this survey are shown in figure RNASET2-mediated changes in the expression levels that we previously observed in vitro were not confirmed for most tested genes in the in vivo setting. Indeed, significant changes in the expression pattern that were found to be in agreement with those previously observed in vitro could be reported for just three genes, namely LMCD1, DSE and RELB.As expected for using a more sensitive test, the observed fold-changes in the expression levels appeared to be slightly higher for several genes when tested by qPCR. For example, the RNASET2-mediated downregulation of the h tumors  was usedCPA4, MDK and AREG) are almost unresponsive to RNASET2 expression in vivo, others showed obvious changes in expression pattern mediated by RNASET2, but these changes were in the opposite direction with respect to those observed in vitro.As for the remaining genes, whereas some of them  polarization. In fact, tumor progression is thought to involve a gradual switch of macrophage polarization from M1 to M2 class, which is paralleled by a gradual inhibition of NK-κB activity [RNASET2 is apparently able to recruit specific subclass of stromal macrophages in the Hey3Met2 ovarian cancer model studied by our group [RELB expression levels in cells of the monocyte/macrophage lineage. Moreover, due to the observed downregulation of RELB in Hey3Mey2 cells expressing RNASET2, a cell-autonomous role for RNASET2 cannot be completely ruled out, since the RELB protein is known to promote cancer cell survival by inducing the expression of proteins with anti-apoptotic roles, such as Survivin and Bcl-2 [Accordingly, the ic cells ,29. Moreactivity . This rond Bcl-2 . A closeRNASET2-responsive gene that might be related to immune cell function is DSE. It encodes for dermatan sulfate epimerase, an enzyme involved in D-glucuronic acid to L-iduronic acid conversion in the dermatan sulphate biosynthetic pathway [DSE gene was originally identified on the basis of the ability of its gene product to be recognized with high efficiency by a subset of cytotoxic T lymphocytes in certain tumors [DSE was found to be altered in laryngeal cancer specimens when compared to normal tissue samples [DSE in our ovarian model.The second  pathway . The DSEn tumors . Moreove samples . Signifi samples , providiin vivo (LMCD1), its direct involvement in RNASET2-mediated tumor suppression is rather speculative. LMCD1 encodes for a poorly characterized cysteine-rich and LIM domain-containing protein, which has been so far implicated in cardiac hypertrophy [LIMD1 gene has been recently reported to display functional properties reminiscent of tumor antagonizing genes [As for the third gene whose change in expression level was confirmed ertrophy . Howeverertrophy  and, mosng genes .in vitro and in vivo expression patterns. Indeed, several genes displaying a biological function potentially related to tumor suppression, and thus defined as candidate based on their in vitro expression pattern, were not validated by subsequent in vivo expression profiling.Taken together, the results of our studies provides a clear indication that investigations of the molecular mechanisms through which TAG/MSG carry out their biological functions necessitate a thorough comparison between the RELB, LIMD1 and DSE) were observed both in vitro and in vivo deserves some comments. t We rekon that the results gathered in the present report highlight some of the limitations inherent in the conceptual process of categorizing relevant biological processes. In our case, the strict categorization of tumor antagonizing genes as genes endorsed with an asymmetric behavior (i.e. suppressing tumorigenicity without affecting in vitro growth) was clearly dependent on the sensitivity of the methodological approach employed. In fact, using the highly sensitive microarray analysis some genes’ features were detected in a congruent fashion in both the in vivo and in vitro.On the other hand, the fact that the same changes in the expression levels of a few genes  protecting against malignant growth yet undiscovered.


Recent discoveries indicate that many G-protein coupled receptors (GPCRs) and channels involved in pain modulation are able to form receptor heteromers. Receptor and channel heteromers often display distinct signaling characteristics, pharmacological properties and physiological function in comparison to monomer/homomer receptor or ion channel counterparts. It may be possible to capitalize on such unique properties to augment therapeutic efficacy while minimizing side effects. For example, drugs specifically targeting heteromers may have greater tissue specificity and analgesic efficacy. This review will focus on current progress in our understanding of roles of heteromeric GPCRs and channels in pain pathways as well as strategies for controlling pain pathways via targeting heteromeric receptors and channels. This approach may be instrumental in the discovery of novel classes of drugs and expand our repertoire of targets for pain pharmacotherapy. Receptors and channels are traditional targets for drug development in the battle against a variety of diseases. Current strategies for drug development assume that receptors and channels are monomers or homomers. Conventional models propose that monomeric G-protein coupled receptors (GPCRs) control the sequential activation of cellular signaling cascades and eventually channel-effectors. Similarly, channels acting as monomers (or homomers) possess defined pharmacological, biophysical and functional properties and are regulated in cell-specific ways. Evidence accumulated in recent years suggests that these models are too simplistic to explain the functional flexibility of these receptors and channels. The current view of receptor and channel organization suggests that a majority of receptors and especially channels are present as dimers or heteromers in cells ,3,4,5,6.versus heteromers in the plasma membrane of actual physiological cells is still unknown for the majority of receptors and channels. Furthermore, there are functional, regulatory and pharmacological outcomes of heteromerization that are beginning to be acknowledged for a set of receptors and channels. Nevertheless, this area of research is well worth the effort and investment, as targeting receptor/channel heteromers may provide a completely new therapeutic strategy for treatment of different pain conditions.The aim of this review is to present advantages as well as strategies for controlling pain pathways via targeting heteromeric receptors and channels. This approach may be instrumental in the discovery of novel drugs for pain therapy. Although the occurrence of receptor and channel heteromerization is now well recognized  may be restricted to fewer cell types than the expression of monomeric and homomeric subunits that comprise the heteromers. More specific expression patterns may provide for a reduction of side-effects in response to heteromeric selective drugs. For example, highly selective TRPV1 receptor antagonists were developed with the intent to block inflammation-induced thermal and mechanical hyperalgesia -DAMGO (selective MOR ligand) with little or no change in [3H]-DAMGO binding affinity [3H]-deltorphin II, was increased in the presence of the selective MOR antagonist, CTOP i. Thus, [Ca2+]i may influence ligand affinity and/or signaling or other properties of a TRPA1-TRPV1 complex. In summary, to understand the mechanisms underlying functional interaction between the TRPA1 and TRPV1 channels, TRPA1 and TRPV1 in vitro and in vivo characteristics need to be studied in the presence and absence of TRPV1 and TRPA1 channels, respectively.Functional interactions between TRPA1 and TRPV1 channels may take place within a heteromeric complex. There are three possible pathways that may underlie this interaction. (1) Bradykinin (BK) can indirectly activate TRPA1, possibly via generation of diacylglycerol (DAG) accumulation . TRPV1, Currently, there are multiple options for the screening of drugs which are dependent on the nature of the therapeutic target and on the strategy of the laboratory or company involved in drug discovery. The existence of receptor dimers, especially of receptor and channel heteromers, gives rise to concerns about the use of monomer-based strategies. At the same time, the existence of receptor heteromers opens exciting new possibilities for drug development. Traditionally, pharmaceutical companies have utilized single receptor screening methods to identify candidate drugs. Although this approach has been successful, many of the clinical effects imparted by the drugs discovered utilizing this method have not always been predicted by the results from screening against a single receptor subtype. Further, recent analysis of the mechanism of action of some clinically used drugs has demonstrated that these drugs may be more heteromer specific and thus offer unique advantages. The atypical antipsychotic, clozapine, is an example for how targeting a GPCR heteromer may produce therapeutically effective results. Originally identified as a mixed dopamine and serotonin receptor antagonist, recently clozapine has been shown to cause dissociation of dopamine D1-D2 receptor heteromers . This unEarly investigations in drug discovery should first identify if a drug target of interest consists of a receptor/channel monomer, homodimer or heterodimer. If receptor monomers or homodimers are the assumed target, and screening is based on second-messenger readouts, then no modifications in the current approach are required. Yet, if radioligand binding is used in the screening of drugs targeting receptor homodimers, then dimer-based binding data will differ from monomer-based models. Finally, drug discovery must consider that drug targets may in fact be receptor heteromers, with unique properties that differ from their constitutive homomers.2A receptor is one order of magnitude lower for the receptor monomer/homomer than for the A2A-A1 receptor heteromer [As mentioned above, receptor heteromers often display unique pharmacological properties in comparison with their individual receptors ,6,35,199eteromer . CompounReceptor heterodimers may be exploited to increase therapeutic efficacy in a variety ways. As described above, small molecules that specifically bind and activate or inhibit a heteromer could be developed. Secondly, monoclonal antibodies that inhibit heteromer formation could be designed. The success of Trastuzumab and Pertuzumab which demonstrate that monoclonal antibodies can inhibit dimerization of the HER2/neu receptor in breast cancer patients suggests that such an approach is viable . MoreoveFourth and finally, a heteromer specific blocker (or inhibitor) may be developed using a non-conventional strategy utilizing RNA inhibitors or aptamers from an RNA library ,203. ThiReceptors and channels can form heteromeric complexes on the plasma membrane. We are just now beginning to understand the characteristics of different receptor and channel heteromerization in cells involved in nociceptive processing. There are functional, regulatory and pharmacological outcomes of heteromerization that are beginning to be acknowledged for a set of receptors and channels engaged in pain. Thus, these heteromeric receptors and channels display unique and specific pharmacological, biophysical, regulatory and functional characteristics. Therefore, heteromer complexes may be considered to be novel and distinct entities with the potential for playing unique physiological roles during normal and pathological conditions. Heteromers provide valid and distinct targets for agonists/antagonists development. This concept is currently overlooked by pharmaceutical companies, which concentrate on a single or homomeric receptor/channel by using expression systems in which heteromers cannot occur. In summary this area of research is well worth the effort and investment, as targeting receptor/channel heteromers may provide a completely new therapeutic strategy for the treatment of different pain conditions.


Arabidopsis thaliana has a small Lon gene family of four members. Although these genes share common structural features, they have distinct properties in terms of gene expression profile, subcellular targeting and substrate recognition motifs. This supports the notion that their functions under different environmental conditions are not necessarily redundant. This article intends to unravel the biological role of Lon proteases in energy metabolism and plant growth through an evolutionary perspective. Given that plants are sessile organisms exposed to diverse environmental conditions and plant organelles are semi-autonomous, it is tempting to suggest that Lon genes in Arabidopsis are paralogs. Adaptive evolution through repetitive gene duplication events of a single archaic gene led to Lon genes with complementing sets of subfunctions providing to the organism rapid adaptability for canonical development under different environmental conditions. Lon1 function is adequately characterized being involved in mitochondrial biogenesis, modulating carbon metabolism, oxidative phosphorylation and energy supply, all prerequisites for seed germination and seedling establishment. Lon is not a stand-alone proteolytic machine in plant organelles. Lon in association with other nuclear-encoded ATP-dependent proteases builds up an elegant nevertheless, tight interconnected circuit. This circuitry channels properly and accurately, proteostasis and protein quality control among the distinct subcellular compartments namely mitochondria, chloroplasts, and peroxisomes.Lon is the first identified ATP-dependent protease highly conserved across all kingdoms. Model plant species  The paradox of aerobic life or the “Oxygen Paradox” argues that organisms do not survive in oxygen depleted environments, yet oxygen is inherently dangerous to their existence. This “dark side” of oxygen is attributed to the damage of biomolecules . Life in2 to O2 by the electron carriers, known as oxidative phosphorylation (OXPHOS), is leading to energy production in the form of ATP. However, through this process leakage of electron occurs, ultimately generating highly reactive species, causing severe cell damage. This side-effect in mitochondria led to the “free-radical theory,” conceived in 1956, speculating that endogenous oxygen radicals were generated within cells and resulted in a pattern of cumulative damage , consisting of a 20S protease core particle and two 19S regulatory caps modulating several aspects of plant development (+ protein degradation machineries categorized into the soluble Lon and Clp (caseinolytic protease) families and the membrane-integrated FtsH-class (filament-forming temperature-sensitive) proteases  with a typical Serine–Lysine catalytic dyad at the active center  the homologous Lon protease has a major role in mitochondrial proteostasis as this organism lacks Clp . The expression of Lon1 (At5g26860) is high in rapidly growing organs of embryonic origin and in high-energy dependent tissues, which have increased mitochondria population per cell to sustain increased energy requirements. Lon1 is predominantly expressed in germinating seeds, embryonic organs, including cotyledons and primary roots, and in organs with high growth rates such as developing inflorescences, while it was hardly detected in mature roots or stems of adult plants  shows the lowest level of expression, albeit Lon4 gene response is qualitatively similar to Lon1. Among the members of the Lon gene family, Lon2 (At5g47040) is highly expressed, while gene expression generally remains constant among the examined cell types and tissues without significant fluctuations. Due to the lack of experimental evidence to report the presence of gene transcripts, Lon3 (At3g05780) is presumed to be a pseudogene . Lon4 is dual-targeted to mitochondria and chloroplasts displaying an ambiguous presequence (The protein isoforms encoded by the nuclear sequence . The C-tsequence . Computasequence .+ Lon protease is a soluble single-ringed multimeric holoenzyme. Adjacent to the AAA+ module is the sensor- and substrate-discrimination (SSD) domain mainly involved in modulating selective substrate recognition by Lon so as the target protein to be degraded. In line with its highly selective mode of action, the SSD domain exhibits substantial interspecies and within the same species diversity  that is ubiquitously present in various deep-sea hydrothermal vent systems. TonLon is a 635-residue protein belonging to the B-Lon family having the protease domains arranged with a near perfect sixfold symmetry relative to the axial pore  and human (HsLon) Lon proteases and with the heptameric complex of yeast Pim1. Homology modeling confirmed that EcLon and HsLon share the same structural features but differ from the heptameric Pim1 complex . As reported by EcLon and HsLon, which are significantly shorter than Pim1 and Arabidopsis Lon sequences . The first group includes AtLon1 and AtLon3 that share similar structural properties with Pim1, all preserving a single pair of parallel β-sheets (depicted in red color) that is typical of EcLon and HsLon. AtLon2 and AtLon4 belong in the second group bearing between the α-helices (depicted in gray color) an internal loop polypeptide configuration (depicted in yellow color) with different secondary structure (depicted in purple color) compared to the members of the first group. Additionally, a surprising core structure is discovered in Pim1 and Arabidopsis Lon proteases likely originating from the hexameric EcLon and HsLon complexes . The alignment between the models of AtLon1 or AtLon4 with the core structure of HsLon show that AtLon1 internal geometry differs from AtLon4. The internal loop domain of AtLon4 shows a right-handed extension, whereas in AtLon1 is left-handed. This structural difference between the two major representative proteases of Arabidopsis suggests that Lon1 and Lon4 are gene paralogs performing specialized functions without being necessarily redundant. However, the possibility of recognizing similar protein targets under adverse environmental conditions that considerably modify the internal milieu of mitochondria and chloroplasts cannot be excluded. Future studies are required to assess the homology models of Arabidopsis Lon proteases and to solve the crystal structure of the holoenzyme, providing insights on Lon structural dynamics and functional versatility.As the SSD domain is the most variable domain among the equences . InteresAncient invasions by eubacteria gave rise through symbiosis to mitochondria and chloroplasts that have enormous impact on bioenergetic and metabolic homeostasis of plants . MitochoLon1 dual-targeting that is attributed to twin presequences, an ambiguous presequence confers Lon4 dual-targeting specificity . Upon removal of this base the reading frame becomes open encoding an N-terminal extension conferring Lon4 targeting specificity to chloroplasts similarly to the N-terminal extension of Lon1. Moreover, Lon1 and Lon4 are remarkably similar in terms of amino acid identity and similarity of the structural domains besides the SSD , Lon1 and Lon4 gene duplicates were at the molecular level preserved through adaptive evolution with complementing sets of subfunctions . This duplication event most likely occurred upon land plant evolution over 400 million years ago. Both ancestral paralogs evolved through adaptive evolution discrete SSD domains and thereby specialized roles in plant development and stress-response. Moreover, the ancestral Lon4 gene evolved the mitochondrial presequence into an ambiguous one. This evolutionary step was characterized by a single thymine insertion interrupting the reading frame of Lon4 to prevent the synthesis of the chloroplast transit peptide. This evolutionary process drifts protein dual-targeting from twin presequences to the ambiguous one. Considering that the cases of protein dual-targeting by ambiguous presequence prevail over twin presequences . The FFAs are imported into the peroxisome entering the reactions of β-oxidation and the glyoxylate cycle. Seedling establishment additionally depends on the mitochondrial tricarboxylic acid (TCA) cycle and on gluconeogenesis that operates in the cytosol.Seed germination and seedling establishment depend on the assembly or biogenesis of mitochondria and the mobilization of storage reserves. In oilseed species like Lon1 is involved in the biogenesis and maintenance of mitochondrial function to ensure the proper operation of such biochemical network. Transmission electron microscopy studies of lon1 mutants revealed the presence of mitochondria with abnormal morphology. The lon1 mitochondria are swollen, having a poorly developed internal membrane system composed of few discernible cristae  subunits COX4-1 and COX4-2  of Lon gene promoter leading to the induction of Lon expression for the degradation of COX4-1. At the same time, HIF-1α up-regulates an alternate isoform-COX4-2, which is assembled into the COX complex replacing COX4-1. In hypoxic cells, the COX4-2 containing complexes are better optimized for transporting electrons and increasing the efficiency of respiration. Additionally, Lon expression is enhanced in vitro by hypoxia or under endoplasmic reticulum (ER) stress and in vivo by brain ischemia  that is required for the mitochondrial unfolded protein response (UPRmt) cascade ]. In healthy cells, the UPRmt is not activated as ATFS-1 is compartmentalized away from the nucleus efficiently imported in mitochondria matrix, where is rapidly degraded by the Lon protease. Consequently, mitochondrial homeostasis is maintained by the conditional-dependent translocation of a transcription activator between the nucleus, where it activates the stress response cascade, and mitochondria where it is removed by Lon protease.Despite primary metabolism and energy supply through OXPHOS, mitochondria also play a crucial role in cell signaling and communication. In mammalian cells, Lon protease under hypoxic conditions optimizes the activity of the electron transport chain by modulating the equilibrium between cytochrome d COX4-2 . Under rischemia . Under h cascade . During + proteases, modulate protein quality control, constitutive metabolism and adaptive responses to cellular or environmental stress. Our understanding of the physiological role of Lon proteases in plants is still evolving, although great advancement is made in non-plant species. However, contrary to the bacterial, yeast and mammalian counterparts, Arabidopsis has a genetic pluralism in terms of Lon gene copies within the nuclear genome. This could be attributed to the presence of an additional organelle in plants, the chloroplast, and to the fact that plants are sessile organisms exposed to extreme environmental conditions. The Arabidopsis Lon genes could be considered paralogs that evolved distinct structural and functional features including gene regulation and expression, subcellular targeting localization and substrate recognition mechanisms. Moreover, Arabidopsis has the genetic and molecular tools to contribute toward better understanding of the functional role of Lon as key controller of proteostasis in organelles and in response to intrinsic or environmental cues. These Arabidopsis paralogs could be proven valuable assets to unravel the substrate recognition mechanisms and organelle-to-nucleus communication circuits. This knowledge might be of use to precisely comprehend the role of Lon in non-plant species including humans and thereby to improve life quality and expectancy.Protein misfolding and degradation, especially in mitochondria which are the main source for oxidants in the cell, are processes that determine protein fate causing mitochondrial dysfunction. Mitochondrial dysfunction has now been implicated in aging, cancer and in a variety of age-related degenerative diseases. Lon, in association with other AAAThe 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.


Understanding the factors that shape the distribution of tropical tree species at large scales is a central issue in ecology, conservation and forest management. The aims of this study were to (i) assess the importance of environmental factors relative to historical factors for tree species distributions in the semi-evergreen forests of the northern Congo basin; and to (ii) identify potential mechanisms explaining distribution patterns through a trait-based approach.2 spanning the borders of Cameroon, the Central African Republic, and the Republic of Congo using forest inventory data from 56,445 0.5-ha plots. Spatial variation of environmental  and historical factors (human disturbance) were quantified from maps and satellite records. Four key functional traits  were extracted from the literature. The geological substrate was of major importance for the distribution of the focal species, while climate and past human disturbances had a significant but lesser impact. Species distribution patterns were significantly related to functional traits. Species associated with sandy soils typical of sandstone and alluvium were characterized by slow growth rates, shade tolerance, evergreen leaves, and high wood density, traits allowing persistence on resource-poor soils. In contrast, fast-growing pioneer species rarely occurred on sandy soils, except for Lophira alata.We analyzed the distribution patterns of 31 common tree species in an area of more than 700,000 kmThe results indicate strong environmental filtering due to differential soil resource availability across geological substrates. Additionally, long-term human disturbances in resource-rich areas may have accentuated the observed patterns of species and trait distributions. Trait differences across geological substrates imply pronounced differences in population and ecosystem processes, and call for different conservation and management strategies. Identifying the factors that shape species distributions is a central issue in ecology. Since species distribution patterns underlie community composition, diversity and ecosystem function, an understanding of the factors forming these patterns is necessary for projecting consequences of climate and land use changes, and for designing effective conservation and forest management strategies.Many tropical tree species are differentially distributed with respect to environmental factors at continental, regional and local scales Few studies have yet related tropical tree species distribution patterns at large scales with processes that potentially determine them The aims of this study were (i) to assess the role of environmental and historical factors in shaping the distribution patterns of common tree species in the semi-evergreen forests of the northern Congo basin taking spatial auto-correlation into account; and (ii) to identify potential mechanisms  explaining distribution patterns through a trait-based approach. At regional scale, we expected tropical tree species with similar distribution patterns to converge in strategy because of establishment, survival and/or growth barriers imposed by environmental and/or historical factors.2 at the northern edge of the Congo basin and combined them with the spatial variation of factors potentially driving the tree distribution patterns as well as with values for functional traits indicative of resource use strategy. This is one of the first quantitative data sets on tropical forest composition assembled at this scale.In this study, we used abundance data for 31 common tree species in 56,445 0,5-ha plots spread over more than 700,000 kmThe study area was distributed over south-eastern Cameroon, southern Central African Republic and northern Republic of Congo . The extIn the study area, forest inventories were conducted by logging companies to quantify timber resources and their spatial variation. Data from forest inventories have so far been little exploited in the Congo basin despite the consistency of protocols and the vast spatial area covered but see We assigned the values of five environmental variables pertaining to climate , topography  and geology (8 distinct substrates) to each plot. Climatic data were obtained by spatial extrapolation of METEOSAT records . According to these data, mean annual rainfall varied between 1200 mm and 1700 mm over the study area. While the METEOSAT records tended to underestimate rainfall in comparison with available ground measurements, they were suitable for comparisons among sites. The climate in the study area is characterized by a pronounced dry season of varying duration. The length of the dry season (in months) with rainfall less than 50 mm was used to quantify the seasonality of rainfall, which ranged from 0 to 3 months. Topography was characterized by slope (0–112%) and altitude (270–1070 m), obtained from the Shuttle Radar Topography Mission (SRTM). A homogenized geological map of the study area was obtained through the comparative analysis of three national maps th century), we extracted information on land cover through digitalizing 15 topographical and land-use maps  by the French national mapping agency (IGN), published between 1959 and 1964 . Most of the plots experienced no or minor human disturbances, and were classified as dense forest (97.2%), seasonally flooded forest  or swamps . The remaining plots were classified on the maps as degraded forest  or savannas and plantations , and we considered them as disturbed for the purpose of this study. Additionally, we included buffer zones in which forest plots were considered to have been prone to recent human disturbance: 15, 10, 5 and 3 km around main towns, main villages, secondary villages and hamlets respectively, and of 1 km and 500 m around main roads and tracks, respectively . Combining information on land cover from old maps and on proximity to road and villages from recent maps (buffer zones), a total of 7,382 plots (13%) were classified as disturbed. For maps of environmental and historical factors see To assess recent human disturbance  of the plots ×species (n = 31) abundance matrix. CA, also known as reciprocal averaging, produces a simultaneous ordination of plots and species u is the spatially dependent error term and e represents the spatially independent error term. In this paper, we used the maximal distance of the k-nearest neighbours to define the neighbourhood of each plot. We selected k combining expert knowledge and the Bayesian Information Criterion (BIC). First, k should reflect the distance between two transects (2–3 km) so that two consecutive plots within a transect are strongly spatially correlated and that two close plots between transects should be more correlated than two distant plot within a transect. Second, we used the BIC to select among k = 1, k = 2, until k = 8. Best spatial models were found for k = 1 and a maximal distance of 4.5 km, that was in agreement with expert knowledge. The average number of neighbours was equal to 61.4, one plot had one neighbour and five plots had 129 neighbours.To identify the main determinants of species composition and patterns of species distribution, we first used linear models relating plot scores on the first three compositional axes to the set of explanatory variables pertaining to environment and history of human disturbances. As to be expected, the residuals of the linear models remained highly spatially autocorrelated for the three axes . We then used simultaneous autoregressive (SAR) models to deal with spatial autocorrelation. We specifically used spatial error models, which are recommended for spatially autocorrelated distribution data Finally, to quantify the relative impact of each environmental and historical variable on the three compositional axes, we used a simple forward approach based on the Likelihood Ratio test (LR test) and the BIC to select explanatory variables sequentially To assess differences in species composition among geological substrates, we compared plot scores on the first compositional axis for pairs of substrates using pairwise Wilcoxon tests at P<0.001. Additionally, we analyzed differences in stand structure  among substrates, to control for a possible confounding effect of stand age. We tested the relationship between species distribution patterns  and key functional traits. We used Kruskall-Wallis one-way analysis of variance for categorical traits (leaf phenology and shade tolerance) and Spearman correlation coefficients for quantitative traits (wood density and growth rate). We tested for significant differences at P<0.05 in species scores among shade tolerance categories using pairwise Wilcoxon tests. All analyses were performed within the R environment Staudtia kamerunensis (Myristicaceae) was the most abundant species with 46,424 trees inventoried (13.1% of the stems in the 31-species set) and was also the most frequent species , Musanga cecropioides (Urticaceae), Pycnanthus angolensis (Myristicaceae), and Entandrophragma cylindricum (Meliaceae), accounted for 10.3, 10.2, 8.3 and 8.0% of the trees in the 31-species set, respectively.The 31 focal species represented on average 17.5% of the stems and 25.3% of basal area in the plots. These common tree species occurred in immense areas of African rainforest, similar to the situation in upper Amazonian forests Lophira alata , and Terminalia, while the remaining 25 species encompassed a fourth group.In agreement with the patterns described above, a cluster analysis of the species scores identified four contrasting distribution patterns . LophiraGeological substrate, climate and the recent history of human disturbance were the main drivers of species distribution patterns in the study area, which were strongly spatially autocorrelated .Species distribution and community composition were strongly affected by the underlying geological substrate . Species2 in the centre of the study area played a dominant role in shaping distribution patterns  from the sandy soils typical of sandstone and alluvium. On the other hand, specific adaptations allow another set of species (the group composed of the 25 species and Lophira) to grow and reach high abundance on the resource-poor soils.The strong association between tree species distributions and geological substrates, on which soils develop that have distinct chemical and structural properties and consequently different nutrient and water availability, suggests that soil resource availability is important for these patterns. Dominant soils on both the sandstone and alluvium substrates were characterized as sandy loam Triplochiton, absent from sandstone, lowest score on the first compositional axis, and Lophira, abundant on sandstone, highest score), support their differential soil resource requirements, and the decisive role of nutrients in driving their distribution patterns. In experiments, high soil fertility strongly favoured the growth of Triplochiton, while the growth of Lophira was favoured by infertile soils, indicating an actual preference for low nutrient conditions in the latter species Lophira is likely to be considerably more drought sensitive than Triplochiton, as suggested by their large scale distribution patterns across a pronounced rainfall (1000–3400 mm) in Western Africa Lophira as an indicator of wet conditions in paleoecological studies Triplochiton and the high abundance of Lophira on sandy soils is consistent with nutrient requirements, but not with drought sensitivity, in shaping species distribution patterns in the northern Congo basin.Previous experimental and observational data on the two species with the most contrasting distribution patterns  in sites with a pronounced dry season  may have accentuated the observed patterns of species distributions across geological substrates. Specifically, forests growing on sandy soils may have experienced little human disturbances in the past because local people may have preferred fertile sites for slash and burn agriculture. Such differential land use would have favoured the abundance of fast growing pioneer species on fertile soils, and the persistence of slow growing species on less fertile sandy soils. Footprints of ancient human disturbances have indeed been demonstrated in tropical forests worldwide We examined whether species with contrasting distribution patterns exhibited characteristic suites of functional traits. We found that species scores on axis one, indicating species association with geological substrates, differed between species with different traits .Lophira, an evergreen pioneer with high wood density.The four traits examined were highly correlated with each other  and specEvergreen leaves tend to be favoured under low soil fertility and/or high soil moisture conditions, because they limit the loss of nutrients, but may increase water demand through year-round activity Wood density of tropical trees has been shown to decrease with both soil fertility and rainfall Lophira, were significantly less associated with sandy soils than shade-tolerant or non-pioneer light demanding species. Lower turnover rates characterizing weakly productive forests in resource poor environments may contribute to the low abundance and diversity of pioneer species on sandy soils Pioneer species, except Species associated with sandy soils exhibited low potential growth rates. Traits that enable plants to exploit low-resource environments, such as dense wood and evergreen leaves (see above) are physiologically linked to low growth rates vs. efficient conservation of resources The differential distribution of the four correlated traits, which are indicative of the trait syndromes of rapid acquisition The data are also consistent with a resource-related demographic trade-off, where low mortality rates (associated with low growth rates) allow persistence on low-resource soils, while high growth rates  give species an advantage on high-resource soils Overall, our results show that species with similar distribution patterns converge in strategy, suggesting that species that do not possess adequate functional traits are not able to survive, grow or reproduce in the community The observed trait distributions have consequences for ecosystem function, suggesting high biomass and carbon storage, but low productivity and turn-over rates of the forests on resource-poor sandy soils Figure S1Maps of environmental and historical factors. Spatial variation of five environmental factors: annual rainfall (A), dry season length (B), slope (C) and altitude (D) and geology (E), and one historical factor  were quantified from maps and satellite records. Climate and topography correspond to satellite records  while geology is a synthesis of three national maps. The recent history of disturbance combines information on forest cover from old maps with data on proximity to road and villages from recent maps (see (TIF)Click here for additional data file.Table S1S) for quantitative traits, Kruskal-Wallis chi-squared test (K-W χ2) for a mix of a quantitative and a categorical trait; and chi-squared (χ2) test for categorical traits.Results of the pairwise relationships between the four functional traits. To test the correlation between pairs of functional traits, we used Spearman correlation coefficient (r(DOCX)Click here for additional data file.