Betel nut (BN), betel quid (BQ) and products derived from them are widely used as a socially endorsed masticatory product. The addictive nature of BN/BQ has resulted in its widespread usage making it the fourth most abused substance by humans. Progressively, several additives, including chewing tobacco, got added to simple BN preparations. This addictive practice has been shown to have strong etiological correlation with human susceptibility to cancer, particularly oral and oropharyngeal cancers.The PUBMED database was searched to retrieve all relevant published studies in English on BN and BQ, and its association with oral and oropharyngeal cancers. Only complete studies directly dealing with BN/BQ induced carcinogenesis using statistically valid and acceptable sample size were analyzed. Additional relevant information available from other sources was also considered.This systematic review attempts to put in perspective the consequences of this widespread habit of BN/BQ mastication, practiced by approximately 10% of the world population, on oral cancer with a clinical perspective. BN/BQ mastication seems to be significantly associated with susceptibility to oral and oropharyngeal cancers. Addition of tobacco to BN has been found to only marginally increase the cancer risk. Despite the widespread usage of BN/BQ and its strong association with human susceptibility to cancer, no serious strategy seems to exist to control this habit. The review, therefore, also looks at various preventive efforts being made by governments and highlights the multifaceted intervention strategies required to mitigate and/or control the habit of BN/BQ mastication. It makes the mouth red. They say they do it to comfort the heart, nor could live without it.”— Pigafetta, in Purchas,i. 38. [Circa 1521]Areca nut (AN), which is more commonly referred to as betel nut (BN). BN is masticated or chewed either alone or in combination with a wide variety of additives, which are often wrapped in the leaf of Piper betle , giving it the more common name, betel quid (BQ) The above quotation gives a vivid description of the Areca catechu. The Areca fruits may be sun dried for several weeks, fibrous shells removed and the hard, dry nuts, commonly called ‘supari’ in India, are ready for use. Such sun dried variety of BN is very hard, and is cut into small pieces to make it easier to masticate. Alternatively, the ripe BN are boiled for several hours in an aqueous solution containing the bark of the plant Eugenia jambolana, jaggery or brown sugar, and various edible oils, to ‘cure’ it. The cured fruits are sun dried for several weeks, fibrous shell removed and very hard, brown nuts, another variety of ‘supari’, are ready for use. In contrast, ripe, partly ripe or unripe Areca fruits are freshly picked, fibrous shells removed and the relatively soft nuts are ready for mastication  or ripe (orange/red) fruit from the tropical palm, tication . Occasiopaan’ in India. The components of BQ can vary widely between countries, regions, communities and individuals. However, the major constituents are BN, catechu (Acacia catechu), a resinous extract from the wood of the Acacia tree, slaked lime, various additives, such as grated coconut, aniseed, pepper mint, cardamom, cloves, perfumes and stimulants wrapped in betel leaf (kwai or tambul is primarily consumed only with betel leaf and slaked lime (gutkha or khaini) or without tobacco , have become increasingly popular gutkha often begins at a very young age. Gutkha contains large amounts of sweeteners to conceal the bitterness of tobacco, and children often consider it as a type of candy. Many people take gutkha to be harmless and mere ‘mouth freshener’ Gutkha and paan masala are consumed by very young and old alike, particularly in India, and also among migrant populations from these areas worldwide Areca fruit is not removed before consumption. Three main variants of BQ are prepared - husk without BN  being the most common, husk with other substances e.g. dried grapes, and husk with BN The BN is consumed either alone or as BQ in which case it is wrapped in a betel leaf along with slaked lime  and additives . The BQ tel leaf  [2]–[5].ked lime  [5]. BetSeveral studies have reported the effect of BN and its constituents on human health, especially as a possible cause of oral cancer (OC) The studies included in this review have been retrieved from the PUBMED database of the National Library of Medicine, National Institutes of Health, Bethesda, Maryland, by setting limits for papers published only from 1985 to 2012, using the keywords “Betel nut AND cancer”. The search was refined using the operator ‘AND’ in order to retrieve the desired results. This search yielded 675 records , it is believed that around 30,000 shops sold BN in the capital town during the reign of Khosrau II, the King of Persia during 590–628 AD. The use of BN by humans has been known since the 4th century AD in different parts of the world, including the South and South-East Asia, several Pacific islands, many regions of the former Soviet Union, parts of North America and Europe The earliest use of BN as a masticatory object by humans has been mentioned by Theophrastus in scripts dating around 430 BCE (Before Common Era), which described it as a component of the betel morsel. Chinese texts of 150 BCE, also mention BN as “gutkha have made it very attractive to youth, and an alarmingly high number of children and teenagers in India. According to one estimate, as many as one in three individuals regularly or occasionally chew gutkhaAreca fruit rather than the solid nut used by others. Although the raw Areca used in Hunan province is imported from Hainan in Mainland China as well as from Thailand, the vast majority of BQ products in China, including commercial forms, are manufactured locally. The Xiangtan city in Hunan province, where BQ chewing was reported to be very common , is also where most BQ production factories and workshops are located. The prevalence of BQ chewing in Hunan men was higher in the younger age groups, suggesting that Hunan province is an emerging region of BQ usage. The improving economy and easy access to BQ products there, supplemented by aggressive advertising campaigns, could be the factors responsible for the widespread use of this substance, particularly among young people. Men in the Eastern and South Asian study communities were deemed likely to combine chewing with smoking and drinking (5.6–13.6%). Low level of school education, alcohol drinking and tobacco smoking were identified as factors associated with BQ chewing It is estimated that currently 10% of the world population or nearly 700 million individuals might be consuming BN in different forms across the globe paan and gutkha, long after immigration gutkha outside of Asia, with imports having doubled in the last three decades. The sale and use of paan and gutkha are legal in the United States and they are readily available in ethnic enclaves, widely used by the large and rapidly growing South Asian communities South Asian immigrants in Australia, Europe, the United Kingdom, South and East Africa and the Malay Peninsula continue using BN products, including Areca palm tree and the methods of curing BN are main factors that contribute to the observed variation in the constituents The constituents of BN include crude fiber, carbohydrates, fats, polyphenols, alkaloids, tannins, proteins and water. Trace amounts of fluorine, sapogenins (glycosidic derivatives of steroids and triterpenoids) and free amino acids have also been reported in some forms. The relative amounts of these constituents are highly variable in produce of different regions as well as in the dry or raw/wet variety of BN. Geographical and climatic conditions of growth of the Alkaloids are reduced pyridines. BN contains several alkaloids, of which arecoline and arecaidine are biologically highly relevant. Arecoline  is the most abundant alkaloid of BN followed by arecaidine . Other alkaloids, such as guvacine (methyl ester of arecaidine), guvacoline (methyl ester of guvacine) and arecolinidine are also present in small to very small or trace amounts et al. have reported that betel nut extract (BNE) contains catechin based procyanidins which range from dimers to decamers and polymers Catechin, flavanoids, flavan-3:4-diols, leucocyanidins and hexahydroxyflavans are the prominent polyphenols found in BN Specific types of polyphenols that are capable of precipitating proteins are tannins. The predominant tannin of BN is gallotannic acid, which is present in the outer part of the nut. In addition, minor amounts of gallic acid, D-catechol and phiobatannin are also present in the inner part of the nut paan masala have been reported to contain sodium, magnesium, chlorine calcium, vanadium, manganese, copper and bromine. The copper content in samples of raw and processed BN was analysed and reported to be much higher than that found most frequently in other nuts consumed by humans. The mean concentration of copper in samples of processed, commercially available BN was 18±8.7 µg/g. In an Indian Food Report, the copper content of processed BN was found to be 2.5 times that of the raw BN BN and 2.−) and hydrogen peroxide (H2O2) at pH greater than 9.5 2.− and H2O2 formation from BQ ingredients, ROS are formed in the alkaline chewing mixture within the saliva of a chewer due to the addition of slaked lime Cellular metabolism of BN or BQ components may also generate reactive oxygen species (ROS), such as superoxide anion radicals (OVagbhata (4th century), and Bhavamista (13th century), BN has also been described as a ‘therapeutic agent’ th century In old Indian scripts such as et al. reported that 10 out of 11 current and former heavy BN users reported cessation withdrawal effects with the mean severity of Dependence Score of 7.3 consistent with the existence of a dependence syndrome among those who use BN products et al. reported that about two out of five persons using BN preparations without tobacco additives developed a recognizable pattern of dependent use, satisfying both Diagnostic and Statistical Manual of Mental Disorder, 4th edition  as well as International Classification of Diseases, 10th revision  criteria for current dependence BQ chewing has been claimed to produce a sense of well-being, euphoria, warm sensation of the body, sweating, salivation, palpitations, heightened alertness, improved concentration and relaxation, diminished hunger, improved digestion and an increased capacity to work paan masala chewers Prolonged as well as excessive usage of BN has been reported to exert significantly adverse effects on human health. There is enough evidence to suggest that BN products, even without tobacco, are associated with increased risk for the development of oral malignancy, such as oral squamous cell carcinoma (OSCC). A vast majority of BN users show precancerous clinical conditions, such as oral leukoplakia (OL)  as well In vitro studies have demonstrated that BN extracts containing arecoline inhibit growth and protein synthesis in cultured human periodontal fibroblasts. These findings suggest that BN may be cytotoxic to periodontal fibroblasts and may exacerbate preexisting periodontal disease as well as impair periodontal reattachment Another condition associated with prolonged BQ use, especially among elderly women, is betel chewer's mucosa (BCM), which is characterized by a brownish-red discoloration of the oral mucosa . BCM is in situ or invasive squamous cell carcinoma. The presence and degree of epithelial dysplasia is generally accepted as the best indicator of malignant potential of leukoplakia, although some clinicians believe that ploidy analysis may be more reliable. There also appears to be an increased risk of transformation associated with a non-homogeneous leukoplakia, especially one that is clinically erythroplakic, verrucous or nodular OL can be defined as a predominantly white patch or plaque on the oral mucosa. Based on clinical appearance, leukoplakia can be divided into several subtypes: homogeneous (white), speckled (red/white), nodular or verrucous leukoplakia It has been reported that the cessation of BN chewing resulted in resolution of 62% of leukoplakia, suggesting that BN on its own is a significant etiological factor in the development of leukoplakia. Further evidence of its relationship with BN chewing has come from the increased prevalence of this condition in subjects who suffer from OSF, which is associated strongly with the habit of BN chewing Sushrutha, a renowned Indian physician, who lived in 2500–3000 BC and described a condition resembling OSF which he referred to as ‘Vidari’ OSF is a chronic disorder characterized by fibrosis of the mucosa lining the upper digestive tract involving the oral cavity, oro- and hypopharynx and the upper third of the oesophagus. It was first described by Schwartz in 1952 as a fibrosing condition in five Indian women in Kenya and he called it as atrophica idiopathic atropica paan masala, gutkha, and mawa have higher concentrations of BN and appear to cause OSF more rapidly than self prepared conventional BQ, which contains smaller amounts of BN paan masala, gutkha and mawa has spawned an epidemic of OSF, particularly among young individuals in India It is now well accepted that all BN products, even those without tobacco, are associated with OSF, which has been established as a precancerous condition. When a paste made out of an instant BN preparation was painted into the oral cavity of albino rats, biopsies taken from the oral mucosa revealed mild to moderate loss of nuclear polarity and increase in keratoses, parakeratoses, inflammatory cell infiltration and vascularity et al. assessed the mRNA expression of histone methyltransferases, acetyltransferases, and demethylases in K-562 cells following exposure to arecoline. They observed that arecoline produced changes in the expression of several genes catalyzing histone methylation , acetylation (Atf2), and demethylation (JMJD6). Since H3K9 methylation is involved in maintaining the stability of heterochromatin structures and inactivating euchromatic gene expressions, this study indicates that arecoline-induced epigenetic changes play a role in the mechanisms underlying chemical-mediated cytotoxicity and genotoxicity A clear dose dependent relationship has been reported for both frequency and duration of chewing BN without tobacco in the development of OSF In vitro examination of effects of arecoline on both normal and OSF fibroblasts in culture revealed an augmented collagen synthesis by OSF fibroblasts as compared to normal fibroblasts. The reason for this elevation was thought to reflect the clonal selection of a particular cell population in the altered tissues or normal cells with somatic mutation that persists through several generations. This could be due to upregulation of pro-inflammatory and pro-fibrotic cytokines like interleukins , tumor necrosis factors , platelet-derived growth factors (PDGF), fibroblast growth factors (FGF) and keratinocyte growth factor-1 (KGF-1), among others, and downregulation of interferon gamma (IFN-γ) level, resulting in fibrosis. Additionally, activation of pro-collagen genes like COL1A1, COL3A1, COL6A1 and COL7A1 has also been reported in OSF in vitro suggesting that arecoline may have cytotoxic properties. The disparity of results from in vitro studies suggests that the BN may contain other agents in addition to arecoline, which are important in the pathogenesis of OSF through increased collagen synthesis In three separate but related studies, interleukin-6 [IL-6], keratinocyte growth factor-1 (KGF-1) and insulin-like growth factor-1 (IGF-1) expressions, which have all been implicated in tissue fibrogenesis, were significantly upregulated in persons with OSF due to BQ chewing and arecoline may be responsible for their enhanced expression Another mechanism envisions involvement of BN in the pathogenesis of OSF due to decreased collagen degradation through decreased obliteration, inhibition of phagocytosis or resistance to degradation 2 increased the collagen synthesis by the oral fibroblasts. However, the addition of CuCl2 neither increased the synthesis of non-collagenous proteins by the fibroblasts nor influenced their proliferation rate. These in vitro results support the hypothesis that Cu in BN acts as a mediator of OSF in vitro supposedly by upregulation of activity of a Cu-dependent enzyme, lysyl oxidase, which catalyses the cross linking of collagens and elastin Raised copper concentrations have been shown in products containing BN in comparison to other nut based snacks. It has also been observed that chewing BN for 5–30 min significantly raised the soluble Cu level in saliva. Study of buccal mucosal biopsies from patients with OSF indicated raised Cu level There exists an accumulated wealth of historical evidences suggesting that the BN may be involved in the development of OSCC  [24]. ReA causal association between tobacco and BQ chewing habits and oral mucosal diseases such as OL, OSF and OC has been established and heavy users have a significantly increased mortality rate gutkha and paan masalain vivo and in vitroA study of OSCC and concomitant oral habits undertaken in South African Indians revealed that 68% women with cheek cancer and 84% with tongue cancer only chewed BN without tobacco, snuff or smoking. The data show that the BN habit with or without tobacco is important in the development of OSCC and it has been suggested that elimination of this habit can reduce the risk in these women substantially (89–91%) if all other factors remained the same In vitro data suggest that arecoline is metabolized by carboxylesterase in mouse liver and kidney. Male Swiss albino mice fed BN powder or arecoline showed enhanced levels of the hepatic cytochrome P450 and b5 and decreased levels of hepatic GSH Salmonella typhimurium YG7108 expressing each form of human P450 together with NADPH-P450 reductase Several animal studies have confirmed that BN products and derivatives, such as arecoline and BN derived nitrosamines, also referred to as betel specific nitrosamines (BSNA), have the ability to induce neoplastic changes in experimental animals . Alkaloipaan masala without metabolic activation paan masala extract (EPME) were determined using the hairless skin of S/RVCri-ba or Bare mice and the forestomach and esophagus of ICRC mice as the target tissues. EPME promoted skin papilloma formation and enhanced the rate of conversion of papilloma to carcinoma. Induction of mild epidermal hyperplasia, dermal edema, increase in epidermal mitotic activity and the rate of epidermal and dermal DNA syntheses by EPME correlated well with its skin tumor promoting potential. In ICRC mice, EPME was inactive as a complete carcinogen, but effectively promoted the development of forestomach and esophageal papilloma and carcinoma in a concentration dependent manner indicating that habitual paan masala use may exert carcinogenic and co-carcinogenic influences paan masala revealed a significant dose dependent increase in lung adenocarcinoma but not in liver and stomach The frequency of SCE was found to be elevated in mouse BMC when mice were exposed to the aqueous extract of betel nut (AEBN) and its tannin in vitro. In manifestation of these effects arecoline, HEBN and EEBN were most potent jun independent of GSH depletion BNE was found to decrease cell survival, vital dye accumulation and membrane integrity of cultured human buccal epithelial cells in a dose dependent manner. BNE also caused formation of both DNA single strand breaks and DNA protein cross links Piper betle (IPB) also induced DNA strand breaks. In addition, BNE, IPB, the BN polyphenol, catechin as well as arecoline decreased cell survival and proliferation. In contrast, another component of BQ, the aqueous extract of lime, was found to increase cell proliferation n-butanol extracts of BN as well as betel leaf are reported to induce CA in human lymphocytes and Chinese hamster ovary (CHO) cells in vitro. AEBN also induced DNA strand breaks and enhanced cell proliferation in mouse kidney T1 cells in vitro2O2 level and actin filament disorganization The BNE and inflorescence of S-transferase (GST) activity have been demonstrated in cultured human oral keratinocytes and in fibroblasts treated with arecoline FANCG, also known as XRCC9 (tumor suppressor capable of correcting CA), CHAF1 and CHAF2 (encoding chromatin assembly factor I or CAF1) and BRCA1 (breast cancer susceptibility gene implicated in DNA damage response and DNA repair). Among them, at least the BRCA1 response was dose dependent. COX-2 and PTGS2, which are involved in cancer initiation and progression, were over expressed in HGF cells. HSP4A1 and DNAAJA1, which belong to the HSP70 family of stress-induced proteins and GDF15/MIC-1, were also upregulated by arecoline in dose dependent manner et al. established two oral cancer sublines chronically treated with BNE and used methods such as microarray and immunohistochemistry to screen and validate the genes exhibiting altered expressions in BNE sublines or in cancer tissues. They found that a total of 35 genes were differentially expressed in both sublines. Several functional pathways were significantly altered. Six genes were confirmed over 2-fold of changes, including Ches1. Functional analyses showed that overexpression of Ches1 suppressed cell growth and arrested cells in the G2/M phase. They thus concluded that loss of Ches1 may be attributed to BNE-induced oral carcinogenesis Arecoline alone has been reported to inhibit cell attachment, cell spreading and cell migration in a dose dependent manner in cultured human gingival fibroblasts (HGF) FANCG was confirmed to be a target of miR-23a by ectopic overexpression or knockdown of miR-23a. The correlation between miR-23a overexpression and BN-chewing habit was also reported in oral cancer patients. Thus, BNE-induced miR-23a was correlated with a reduced FANCG expression and DNA double strand break (DSB) repair, which might contribute to BNE-associated human malignancies via their secreted molecules Salmonella typhimurium tester strains TA 100 and TA 98. But this mutagenicity was potently inhibited by glutathione, N-acetylcysteine, and cysteine Salmonella typhimurium but not in Chinese hamster V79 cells. AEBN, on the other hand, induced mutations in Salmonella typhimurium and in Chinese hamster V79 cells besides inducing gene conversion in Saccharomyces cerevisiae as well as CA in CHO cells. BN tannin fraction induced gene conversion in Saccharomyces cerevisiaeSalmonella typhimurium strain TA 1535 revealed that arecoline, AEBN and HEBN were weak mutagens while AAEBN and EEBN were strong mutagens suggesting that the mutagenic potential of arecoline could be significantly enhanced by other constituents of BN hypoxanthine phosphoribosyltransferase (HPRT) locus in human keratinocytes, which also increased frequency of appearance of MN, intracellular levels of reactive oxygen species (ROS) and 8-hydroxyguanosine in the cells suggesting that stress caused by long term BNE exposure enhanced oxidative stress and genetic damage in human keratinocytes Treatment of normal human oral fibroblasts with BNE was also reported to alter miRNA expression profile. BNE-induced overexpression of miR-23a was found to be correlated with an increase of γ-H2AX, a DNA damage marker. Among BN chewers, the possible genomic damage caused by BN without tobacco was confirmed in cytogenetic studies. BNE has been shown to be cytotoxic and genotoxic to human buccal epithelial cells A study revealed that OSF was largely associated with BN and the exfoliated oral mucosal cells of such patients had significantly higher numbers of MNC. The patients also exhibited increased SCE in circulating blood lymphocytes indicating that the carcinogenic agents in BN produce damage not only in target tissue but also in other tissues et al. reported the upregulation of Asb6, a coupling protein to the APS adapter protein, which is involved in insulin signaling for glucose transportation, of normal keratinocytes and oral cancer cells under BNE treatment. They also demonstrated a positive correlation between Asb6 upregulation  and clinicopathological features such as poor survival status in OSCC patients Hung et al. revealed that the alkaline environment created in the oral cavity of BN chewers by lime may enhance nicotine-related oral carcinogenesis through a synergistic effect of nicotine and the alkalinity in inducing higher expression of phosphorylated AKT In a study pertaining to the contribution of combined BN chewing and cigarette smoking to the risk of OSCC in Taiwan, Wu A study involving patients of HNC suggested that BQ chewing may increase mitochondrial DNA (mtDNA) mutation in human oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation of these mutations during cell division could be important contributors to the early phase of OC et al. investigated the functional role of αvβ6 using oral keratinocyte-derived cells genetically modified to express high αvβ6 (VB6), and also NTERT-immortalized oral keratinocytes, which express low αvβ6 (OKF6/TERT-1). VB6 cells showed significant αvβ6-dependent activation of TGF-β1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up-regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, which revealed that the stroma of OSF contained myofibroblasts and that TGF-β1-dependent Smad signaling was detectable both in keratinocytes and in myofibroblasts. The investigators also found that arecoline, the major alkaloid of BN up-regulated keratinocyte αvβ6 expression. This was modulated through the M4 muscarinic acetylcholine receptor and was suppressed by the M4 antagonist, tropicamide. Arecoline-dependent αvβ6 up-regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. This study thus suggests that the pathogenesis of OSF may be epithelial-driven and involve arecoline-dependent up-regulation of αvβ6 integrin Alphavbeta6 (αvβ6) integrin is capable of promoting both tissue fibrosis and carcinoma invasion, and has been reported to be markedly up-regulated in OSF Heat shock protein 47 (HSP47) is a product of CBP2 gene located at chromosome 11q13.5, a region frequently amplified in human cancers. HSP47 expression was reported to be significantly higher in OSCC specimens than normal epithelium, while lower HSP47 expression was associated with lymph node metastasis. No significant difference in HSP47 expression was observed with respect to age, sex, tumor category, tumor stage and differentiation. Furthermore, arecoline was found to elevate HSP47 expression in a dose- and time-dependent manner in oral epithelial cell line OC2. This study therefore concluded that HSP47 could be used clinically as a marker for lymph node metastasis of oral carcinogenesis p53 gene is known to be mutated in a variety of human and experimental animal cancers. Similarly, change in cellular level of p53 protein is also known to occur. Accumulation of p53 protein or its stabilization is an important indicator of the presence of mutant p53 protein p53 mutation status of cancers associated with BN chewing have been widely contradictory. Exposure to BN and/or BQ with or without tobacco has been reported to result in high incidence of p53 mutations in Taiwanese, Thai, Sri-Lankan and North Indian Population; however, similar exposures resulted in low frequency of p53 mutations in the populations of India and Papua New Guinea, in other reports. Despite these conflicting observations, a common trend observed among the varying populations was p53 overexpression and nuclear accumulation of p53 protein  of exon 27 of the Brca2 gene in P, F1, F2 and F3 mice. In contrast, a mis-sense mutation (G→C) was observed in exon 11 of the Brca1 gene in F1, F2 and F3 mice, but not in P mice. Such a mutation would result in corresponding amino acid replacement Cys→Ser. In silico protein modeling revealed that the amino acid substitution was likely to cause structural alterations in the RAD50 binding region of the Brca1 protein, which is crucial for its function in error free repair of DNA single and double strand breaks. These observations clearly indicate that the p53, Brca1 and Brca2 tumor suppressor genes are intrinsically involved in the process of BN induced carcinogenesis in mice as well as in the transgenerational transmission of carcinogenic risk following AEBN exposure. The p53, Brca1 and Brca2 responses were abrogated in the mice exposed transgenerationally to AEBN resulting in significantly increased predisposition to cancer p53 gene, which apparently played a crucial role in BN associated cancer in mice, was not achieved through p53 mutation. The mechanism of p53 inactivation may also involve other routes, which requires to be investigated in the future. One possible alternative mechanism for p53 inactivation in BN induced carcinogenesis may be over expression of MDM2 protein, which has been shown in OSCC DNA sequence analyses revealed no mutation in exons 5 and 7 of the et al. have reported that the antioxidant capacity of the BNE procyanidins increased with the degree of polymerization. Further, they have also demonstrated that BNE which contains catechins based oligomeric and polymeric procyanidins, regulates COX-2 expression in vitro and possess anti-inflammatory potential in vivoToxicity studies relating to BN specific polyphenols and tannins are not conclusive with both carcinogenic and anti-carcinogenic effects being reported in literature. It is reported that ROS produced during autooxidation of BN polyphenols in the BQ chewer's saliva might be crucial in the initiation and promotion of OC XRCC4 in Taiwanese population and XRCC1 and XPD in Indian population, genes encoding detoxifying enzymes such as NAT2 encoding the most important phase II metabolic enzyme for BQ in Taiwanese population, GSTT1 and GSTM1 in Indian and Thai populations, and CYP2A6 in Sri-Lankan population and genes encoding matrix metalloproteinases, such as MMP9 in Taiwanese population and MMP3 in Asian population , determine the final risk of human cancers, especially OC, following the exposure to BN or BQ alone or in combination with additives, including tobacco. Mere exposure to BN or BQ may not commit the chewer to cancer. For any given level of exposure to BN carcinogens, only a proportion of exposed individuals will develop cancer, indicating the prevalence of inter-individual differences in susceptibility tion see  [139]–[1et al. reported that COX-2 protein as well as mRNA expression was significantly enhanced in OSCC as compared to non-cancerous matched tissue (NCMT). Hydroxychavicol, a unique ingredient in BQ, also induced COX-2 overexpression in NHOK, indicating the early involvement of COX-2 in BQ associated OC et al. also reported that in human BMF, COX2 mRNA increased in a manner dependent upon increase in the dose of arecoline Cyclooxygenase (COX), an inducible enzyme responsible for prostaglandin synthesis, plays an important role in certain inflammatory diseases and carcinogenesis. Tang et al. reported that BNE induced (a) rounding cell morphology and nuclear shrinkage in different types of carcinoma cells, (b) the cleavage of LC3-I, and (c) the emergence of AV and acidic vesicles et al. reported that BNE treatment induced autophagy among oral cancer cells characterized by LC3-II accumulation, genesis of autophagosomes and the appearance of EGFP-LC3 puncta Autophagy is a regulated self cannibalism, classified as type II programmed cell death, and is preceded by the inhibition of the mammalian target of rapamycin (mTOR). The hallmarks of autophagy are the cleavage of the precursor form of microtubule associated protein 1 light chain 3 (LC3-I) (molecular weight = 18 kDa) to the active form LC3-II (molecular weight = 16 kDa) and the emergence of autophagic vacuoles (AV) and acidic vesicles et al. conducted in Taiwan, 60 cases of oral and pharyngeal cancers, including lip, tongue, gum, mouth floor, buccal palate, oropharynx and hypopharynx cancers were evaluated by linking a retrospective leukoplakia cohort consisting of 435 patients recruited from hospitals between 1988 and 1998 to a population cancer registry Studies and follow-up programmes conducted over the past three decades indicate that the rate of malignant transformation of pre-cancer lesions shows a population dependent variability. A study conducted in Karachi, Pakistan (1996–98) on 79 cases of OSCC and 149 hospital controls showed that the risk for developing OC was 19 times higher  among cases of OSF than among subjects with no precancerous condition Thus, the presence of pre-cancer lesions appears to correlate well with malignant transformation and eventual development of OC . Besideset al. reported that an oral screening program conducted in a tertiary medical centre for detection of oral lesions and oral cancer was effective. The group suggests that individuals aged ≥40 years or who are habitual cigarette smokers, alcohol consumers, and BQ chewers should receive oral screening regularly so that potential oral cancer can be detected as early as possible It is a scientifically established fact that BN or BQ without or with various additives, including tobacco products, are clearly associated with increased risk of HNC, especially OC. In spite of this fact, it has remained a challenge for governments and policy makers to contain its usage and reduce the deleterious effects of BN or BQ on population due to its socio-cultural heritage and addictive nature. Global data on efforts to control usage of BN or BQ and its additives strongly suggest that no single strategy is likely to succeed in achieving the desired level of control on BN or BQ consumption. A concerted effort involving radical measures is required in this direction. It may involve health care professionals, media, policy makers, law enforcing agencies and the community at large. Even though no unified guideline exists due to variation in the socio-cultural groups, it appears practical to make sustained behavioral interventions, especially adolescents and young adults, for reducing the use of BN or BQ without or with additives. Lack of scientific information on its deleterious effects on health is conspicuous by its absence. In contrast, various advertisements try to glorify the opposite in order to allure most vulnerable section of the society, the adolescents and young adults. Once addicted, they become a long term consumers of BN or BQ. Media may play a very important role in this effort due to its widespread reach in all strata of the society, particularly the lower strata, which appears to be deeply affected by this practice. Governmental and non-governmental health care professionals may routinely assess and record the usage pattern in the society and rigorously inform the individuals, groups of people or patients about its potential hazard. Chang Unnecessary glorification of the BN or BQ products in media must be stopped by enacting appropriate laws. Visual representation of the diseases, particularly OC, caused by the use of these products and appropriate warning signs may be effective in this campaign. The industrial policies, including taxation, need revisit in order to limit such industrial activities to discourage marketing of such products. There is need to have strong political commitment towards this aim. Many countries have made some headway in this direction. A brief summary of such efforts is listed below:gutkha and paan masala or any similar product containing or not containing tobacco. In India, a warning label is now mandatory on packets of commercial BN and tobacco products, but there are no regulations about the size of the letters. Several states are at various stages of passing laws to ban gutkha or are in court after being challenged by the industry. A recommendation that gutkha should be banned nationwide has been made to the Government of India by the Central Committee on Food Safety http://www.tobaccojournal.com/Chewing_tobacco_plastic_pouches_banned.50432.0.html). On the occasion of World No-Tobacco day on the 31st May 2012, the state of Bihar joined two other states  in banning manufacture, storage, distribution and sale of any form of tobacco and nicotine containing paan masala and gutkha for a period of 1 year to begin with .On 1 August 2002, the Commissioner for Food and Drug Administration and Food  Authority, Maharashtra State, issued a gazette notification banning the manufacture, sale and storage of BN figures on the list of herbs that are unacceptable as a non-medicinal ingredient in oral use products. The sale of BN products has been banned in Canada as a result of the link between arecoline and mutagenic effects. The US FDA maintains an import alert within the USA, the main concerns being adulteration and addition of unsafe food additives. In 1976, the US Government announced a ban on interstate traffic of BN Within the European Union, excluding Sweden, there is legislation banning the sale of tobacco products for oral use. However, there are no specific laws regulating or banning the sale of BN products, even when mixed with smokeless tobacco, as chewing tobacco is excluded from the directive gutkha easily. Only a few BN products give specific health warnings on the dangers of chewing BN, although most carry the statutory health warning regarding added tobacco. Among 20 commercially processed and packaged BN products on sale in the United Kingdom, only three carried a health warning related to OC; none warned about OSF or potential addiction In the United Kingdom, there is no law to regulate the import or sale of products containing BN. Presently numerous BN preparations, with or without tobacco, are commercially available in shops. The Department of Trade and Industry classifies these products as sweets. Labelling and a list of ingredients on the packaging are sometimes non-existent. Several studies have shown that in most outlets the sales are unrestricted to minors and children under the age of 16 were able to purchase In the late 1970s, the Public Services of Papua New Guinea issued a ban on BQ chewing in government offices. Possession of BN in the California public school system is grounds for suspension. In Singapore, spitting in public places can lead to a fine, indirectly discouraging the practice of BQ and BN chewing paan masala, gutkha and the likes in several countries. This review reveals that BN has far reaching consequences on the general health of the masticator, especially in context with the oral health of the users gutkha and paan masala, is proving to be difficult because the habit is not associated with any social stigma and taboo. In fact, it is other way round in which this practice has largely been given social, religious or other sanctions in different regions of the world. Hence, strong multifaceted intervention is required to discourage or control the habit of BN/BQ mastication. Firstly, legislation against open sale and use of such products should be stricter and more states and countries should bring out such legislations sooner than later. Secondly, public awareness should be created regarding the harmful effects of these products among all sections of society, particularly among children, since the habit starts early in the majority of cases. Lastly, attempts need to be made for harm-reduction of BN/BQ/commercial substitutes. Multi-institutional transnational case controlled studies are also required in order to establish the exact etiopathogenesis and molecular changes of diseases caused by BN/BQ and/or its constituents.BN and products derived from it are widely used as a masticatory among various communities, and in several countries across the world, as a socially endorsed habit


Chlamydia trachomatis save on their lipid needs by incorporating sphingomyelins (SMs) made by their host ChlamydiaFifteen years ago, in a series of elegant studies, Hackstadt and colleagues showed that the obligate intracellular bacteria PLoS Pathogens show that non-vesicular traffic is also involved Throughout their developmental cycle, chlamydiae reside within a membrane-bounded compartment, the inclusion. How they acquire host lipids remains an open question. Possible mechanisms studied so far involve vesicular trafficking from host compartments, including vesicular traffic out of the Golgi apparatus, fusion with multivesicular body–derived vesicles, and engulfment of lipid droplets SMs are synthesized by the transfer of phosphorylcholine to a ceramide in a reaction catalyzed by SM synthases. When added to infected cells, the fluorescent probe C6-NBD-ceramide traffics through the Golgi apparatus and rapidly accumulates in the bacteria, in the form of SM and not ceramide trans-Golgi apparatus while SMS2 is predominantly associated with the plasma membrane. Elwell and colleagues show that both enzymes are in close proximity to the inclusion membrane, and propose that the recruitment of CERT, its ER binding partner VAPA, and SM synthases establish an “on-site SM factory” For what purpose does ceramide traffic to the inclusion? Bacteria accumulate an estimated 50% of SM synthesized from exogenously added ceramide Like CERT, other lipid transfer/binding proteins have been described as functional components of ER-Golgi membrane contact sites. Future studies need to address whether these non-vesicular lipid transfer systems are involved in the acquisition of phospholipids and sterols by the inclusion. Such a direct transfer could explain why transfer of host phospholipids to the bacteria was unaffected by brefeldin A, which inhibits Arf1-dependent vesicular transit through the Golgi apparatus. It is also consistent with the observation that traffic of glycoproteins out of the Golgi is not disrupted by infection PLoS Pathogens do not speak against this possibility, which can operate alongside non-vesicular traffic. In fact, Elwell et al. also provide data showing that depletion of the brefeldin A target GBF1 reproduces the effect of the drug on Chlamydia infection, implicating GBF1 in the vesicular route for SM acquisition In the presence of brefeldin A, SM acquisition by the bacteria is reduced and inclusions are smaller Chlamydia, CERT being important for bacterial replication and the vesicular pathway being essential for inclusion growth and stability Interestingly, while brefeldin A (or GBF1 depletion) only affect inclusion size, and not bacterial proliferation, CERT (or VAP) depletion have an impact on both Chlamydia trachomatis. Surprisingly, Derré and colleagues report that the guinea pig strain Chlamydia caviae does not recruit CERT to its inclusion, consistent with the absence of IncD in this strain Both studies were conducted on the human pathogen


To assess the role of the VDR in the aging mammary gland, we utilized 12, 14, and 16 month old VDR knockout (KO) and wild type (WT) mice for assessment of integrity of the epithelial and stromal compartments, steroid hormone levels and signaling pathways. Our data indicate that VDR ablation is associated with ductal ectasia of the primary mammary ducts, loss of secondary and tertiary ductal branches and atrophy of the mammary fat pad. In association with loss of the white adipose tissue compartment, smooth muscle actin staining is increased in glands from VDR KO mice, suggesting a change in the stromal microenviroment. Activation of caspase-3 and increased Bax expression in mammary tissue of VDR KO mice suggests that enhanced apoptosis may contribute to loss of ductal branching. These morphological changes in the glands of VDR KO mice are associated with ovarian failure and reduced serum 17β-estradiol. VDR KO mice also exhibit progressive loss of adipose tissue stores, hypoleptinemia and increased metabolic rate with age. These developmental studies indicate that, under normocalcemic conditions, loss of VDR signaling is associated with age-related estrogen deficiency, disruption of epithelial ductal branching, abnormal energy expenditure and atrophy of the mammary adipose compartment.The vitamin D The developmental changes associated with puberty, pregnancy, lactation and involution of the mammary gland have been extensively studied and signaling pathways initiated by both membrane and nuclear receptors are essential for coordination of these events Stromal-epithelial interactions are crucial for mammary gland development and maintenance, including the mammary fat pad, which provides signals that mediate ductal morphogenesis 3 receptor (VDR), whose ligand, 1,25-dihydroxyvitamin D3 , a derivative of vitamin D3, is expressed and dynamically regulated in mammary gland during the reproductive cycle We have previously shown that the nuclear vitamin D3 signaling, via VDR ablation, in the mammary gland might impact ductal epithelial cell turnover, leading to hyperplastic nodules that could lead to transformation. We demonstrate that VDR expression persists in the aging mammary gland of WT mice, but in contrast to expectations, abnormal energy metabolism in older VDR KO mice leads to atrophy of the mammary adipose compartment and apoptotic regression of the mammary epithelium. Thus, our studies suggest that VDR signaling is required for overall metabolic homeostasis and for maintenance of epithelial and stromal interactions in the mammary gland during the aging process.In the studies reported here, we used normocalcemic VDR KO mice to determine whether the changes we observed in the mammary gland during pubertal development and the reproductive cycle of VDR KO mice persist or are exacerbated with age. We hypothesized that chronic absence of vitamin D3 deficiency is prevalent in the elderly population and has been linked to an increased risk for breast cancer 3 signaling in aging mammary glands, we used real time PCR to assess VDR gene expression in mammary glands harvested from 12, 14 and 16 month old virgin mice. As shown in Vitamin DTo assess the long-term impact of VDR ablation on mammary gland ductal morphology, whole mounts of inguinal mammary glands were prepared from virgin WT and VDR KO mice sacrificed between 6 and 16 months of age. Although the gross morphology of mammary glands from WT and VDR KO mice at 6, 8 and 10 months were similar (data not shown), developmental differences were apparent in glands from VDR KO mice beginning at 12 months. Representative micrographs of inguinal mammary glands from 16 month old mice are shown in Counts of primary, secondary and tertiary branch points  indicate3 has been shown to regulate E-cadherin in mammary cells in vitro, we assessed the expression of several proteins associated with the epithelial tight junctions. No differences in E-cadherin, β-catenin or occludin abundance were detected by western blotting of tissue extracts from WT and VDR KO mice at any age  to determine whether VDR ablation altered the stromal compartment of the mammary gland. As shown in  any age .To further explore the effect of VDR signaling on the stromal compartment, the cellularity of the mammary fat pad was compared in WT and VDR KO mice. As shown in 2 level, indicating a hypermetabolic phenotype, beginning at 6 months of age, with significant (p<0.05) differences detected from 9 months of age on  was not affected by VDR ablation (data not shown). However, serum 17β-estradiol was significantly reduced in 12, 14, and 16 month old VDR KO mice compared to age-matched WT mice and compared to pubertal aged WT and VDR KO mice . Serum 1The severe adipose tissue atrophy, altered stromal architecture and reduced branching in glands from VDR KO mice suggested an imbalance in mammary cell turnover. Therefore, we measured the expression of Bax and caspase-3, genes associated with apoptosis, in lysates of mammary tissue from WT and VDR KO mice. As shown in Real time PCR was used to compare the expression of several genes linked to mammary gland differentiation and turnover, including beta casein, clusterin, fatty acid synthase, macrophage inhibitory factor, and TGFβRII. Expression of two genes, clusterin and TGFβRII, were found to be significantly up-regulated in VDR KO mice . InductiThese studies have revealed a novel, age-related contribution of the VDR in maintenance of mammary gland integrity and adiposity. Through comparative analysis of mammary gland development and gene expression in relation to age, we report that mice lacking the VDR exhibit ductal ectasia, reduced branching and progressive atrophy of the mammary fat pad with age. These changes do not become evident until the onset of the second year of life and are in sharp contrast to the effects of VDR ablation observed during puberty and pregnancy, when lack of the VDR is associated with accelerated branching and precocious alveolar development Due to the chronic and systemic nature of VDR ablation in these studies, the extent to which VDR signaling within the mammary gland is responsible for maintaining glandular homeostasis is unclear. In support of direct effects of VDR signaling, VDR expression remained high in the aging mammary gland, and positive VDR staining was observed in glandular epithelial and stromal cells as well as adipocytes. Although the effects of 1,25D and VDR on mammary epithelial cells have been well studied, little attention has been paid to the effects of VDR on fibroblasts or adipocytes. Thus, the dramatic changes in the stromal microenvironment of the mammary gland of VDR KO mice were unexpected. The absence of VDR signaling in the adipocytes and stromal fibroblasts may contribute to the regression of ductal branches, since stromal-derived growth factors and extracellular matrix proteins are crucial for normal mammary gland development and maintenance Regardless of mechanism, our data indicate an elevation in apoptotic signaling, including activation of the standard apoptotic markers Bax and Caspase-3 and up-regulation of clusterin and TGFβRII, in VDR KO mammary tissue. Since glandular homogenates were used for these assays, further studies will be needed to determine whether apoptosis is increased in the epithelium, the stroma or both compartments of aging VDRKO mice. We also observed an increase in AKT expression, which may be an adaptive response to the elevated apoptosis, since AKT activation of phosphodiesterase 3B limits cAMP production and reduces protein kinase A (PKA) activity. These changes inhibit lipolysis since PKA activity is required for phosphorylation and activation of hormone sensitive lipase Our work reports that the depletion of adipose stores observed in younger VDR KO mice Our studies also provide additional insight into the interactions between VDR, ovarian function and fertility. Neither fertility nor circulating estrogen is compromised in young VDRKO mice maintained on the high calcium rescue diet Our studies also identify a novel anti-inflammatory effect of VDR signaling in the mammary gland. Inspection of mammary gland whole mounts at high magnification revealed multiple dense lesions in VDR KO mice that were not present in WT mice. Although these lesions were initially thought to be hyperplastic epithelial nodules, Hematoxylin and Eosin Y staining indicated the presence of inflammatory cells, in support of chronic inflammation in VDR KO mammary tissue. Furthermore, the inguinal lymph nodes of VDR KO mice were significantly larger than those of WT mice. Our findings are consistent with reports that VDR KO mice exhibit altered cytokine profiles, T cell populations and antibody responses, and are highly susceptible to colonic inflammation with age 3 signaling regulates adipose tissue, ductal branching and ultimately breast maintenance. In the case of the mammary gland phenotype described here, both direct and indirect effects of VDR signaling in multiple cell types  likely contribute to these changes, and aging studies in conditional VDR and cyp27b1 knockout models will offer the best approach for clarification of the underlying mechanisms.In summary, chronic VDR ablation exerts global effects on energy metabolism and ovarian function that are associated with alterations in mammary gland, including atrophy of the fat pad, degeneration of epithelial ductal branching, enlarged lymph nodes and chronic inflammation. These progressive changes represent additional features of the accelerated aging phenotype of VDR KO mice, and are distinct from the effects of VDR ablation during puberty and pregnancy. Other phenotypic changes that become evident with age in VDRKO mice include alopecia 3/g . This diet prevents the mineral disturbances and impaired growth associated with VDR ablation Wild type and VDR KO mice For whole mount analysis, mammary glands were fixed in Carnoy's fixative and stained overnight in Carmine Alum. Samples were dehydrated, cleared in xylene, mounted, and examined on an Olympus SZX12 stereoscope. Whole mounts were used to calculate total area of the fat pad and the degree of branching morphogenesis . Primary branches were considered as ducts that arose in the nipple region and extended to the leading edge of the gland, secondary branch points were those that extended from primary ducts and tertiary branches were lateral branches that arose from secondary ducts. The fat pad and lymph node areas were determined by tracing around the outer edges of each tissue and calculating the area with AxioVision software .2) and carbon dioxide production (VCO2) for each mouse were measured once per hour for 3.5 minutes . The respiratory quotient (RQ) is the calculated ratio of CO2 produced to O2 consumed during a given time period. Oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure were recorded at 3, 6, 9, and 12 months of age in four separate recording periods. The four day measurements were averaged to produce a 24 hour measurement for each mouse, and the six individual mouse data sets were averaged to provide a representative data set for the 24 hour time period for each genotype.Indirect calorimetry was performed using an Oxymax system  in a room with controlled temperature and 12 hour lighting conditions. Mice were placed in individual chambers with free access to food and water. Six weight-normalized WT and VDR KO male mice were placed individually into metabolic isolator chambers, acclimated for three days, reweighed and recorded over a 96 hour time period. Airflow through the chambers was 0.6 L/min. Oxygen consumption  at a dilution of 1∶60, followed by incubation with anti-rat secondary antibody at a dilution of 1∶200. To detect estrogen receptor α (ER) and progesterone receptor (PgR), slides were pretreated using citrate buffer (pH-6.0) heated to boiling in a pressure cooker for 15-20 minutes and incubated for 1 hr with mouse ERα  or PgR  antibodies and detected with the M.O.M. kit (Vector Laboratories) according to manufacturer's directions. To detect smooth muscle actin, slides were digested using a 0.1% trypsin solution for 10 minutes and incubated with a mouse antibody to smooth muscle actin  for 1 hr at room temperature and detected with the M.O.M. kit as above. For all antibodies, sections were counterstained with Harris modified hematoxylin (Fisher Scientific).Blood was removed by cardiac puncture for analysis of serum calcium, leptin and estradiol. Calcium was determined with a colorimetric assay kit (Sigma) according to manufacturer's directions. Leptin was determined using an ELISA kit from Linco Research  and 17β-estradiol radioimmunoassays were conducted with a reagent kit from DiaSorin .Total RNA was isolated from 90–150 mg of frozen thoracic mammary gland with Trizol reagent (GibcoBRL). Independent mammary gland RNA preps from five mice of each genotype were made at the time points indicated in the figure legends. After concentration and purity of the RNA was determined by spectrophotometry, total RNA was reverse transcribed with Taqman Reverse Transcription Reagents . Three independent 1.5 ug cDNA stocks were generated from each RNA sample, and each was independently analyzed in duplicate (60 ng of cDNA/well) using the Taqman PCR Core Reagent Kit  and specific primer and probe sets. Gene expression levels were normalized against 18S RNA, and reported as relative gene expression. For data presentation, duplicate values from each run were averaged, and triplicate values were then averaged to generate one value for each animal. The final data is expressed as the mean ± standard error of five animals/time point.Thoracic mammary glands (100 mg) from three animals of each genotype per time point were homogenized in Laemlli buffer containing phosphatase and protease inhibitors www.graphpad.com).Data are presented as mean ± standard error, with the number of analyses for each mean indicated. Data were analyzed by Student's t test, and means were considered significantly different if a p value less than 0.05 was obtained. All statistical evaluations were performed with Instat software (GraphPad Software, Inc., San Diego California USA,


Arabidopsis SCW biosynthesis, and numerous studies have explored SCW-related transcription factors in other dicots and monocots. Whilst the general structure of the Arabidopsis network has been a topic of several reviews, they have not comprehensively represented the detailed protein–DNA and protein–protein interactions described in the literature, and an understanding of network dynamics and functionality has not yet been achieved for SCW formation. Furthermore the methodologies employed in studies of SCW transcriptional regulation have not received much attention, especially in the case of non-model organisms. In this review, we have reconstructed the most exhaustive literature-based network representations to date of SCW transcriptional regulation in Arabidopsis. We include a manipulable Cytoscape representation of the Arabidopsis SCW transcriptional network to aid in future studies, along with a list of supporting literature for each documented interaction. Amongst other topics, we discuss the various components of the network, its evolutionary conservation in plants, putative modules and dynamic mechanisms that may influence network function, and the approaches that have been employed in network inference. Future research should aim to better understand network function and its response to dynamic perturbations, whilst the development and application of genome-wide approaches such as ChIP-seq and systems genetics are in progress for the study of SCW transcriptional regulation in non-model organisms.The current status of lignocellulosic biomass as an invaluable resource in industry, agriculture, and health has spurred increased interest in understanding the transcriptional regulation of secondary cell wall (SCW) biosynthesis. The last decade of research has revealed an extensive network of NAC, MYB and other families of transcription factors regulating  Populus, Eucalyptus) , consisting of a cross-linked matrix of cellulose, hemicellulose and lignin biopolymers. The latter form the basic scaffold of fibers and vessels found in angiosperm xylem. In addition to providing mechanical support, SCWs facilitate critical biological processes, such as water and nutrient transport, anther dehiscence, silique shattering, plant organ movement and response to pathogens  differentiation systems  within the silique  and R2R3-type MYB (MYELOBLASTOSIS) family proteins, both characterized by conserved N-terminal DNA-binding domains and diverse C-termini that participate in transcriptional regulation  promotes xylem differentiation  differentiation in cultured Zinnia cells  , a C3H-type zinc finger gene C3H14 and homeobox TF KNAT7  genes in both primary and SCWs  and third tier  also directly activate structural genes. BP, ATHB18, a C2H2-type zinc finger protein At3g46080, MYB20, MYB69, MYB79, MYB85, and the functionally redundant pair MYB58/MYB63 are known only to directly or indirectly regulate lignin biosynthetic genes , are capable of ectopic SCW formation in Arabidopsis and can complement the snd1 nst1 double mutant  have evolved through changes in cis-element composition in their promoters, rather than the SHN DNA-binding domain, since AtSHN1 can switch from wax to SCW pathway regulation depending on the genetic background.The high degree of conservation in SCW-associated TF function between Carroll, , has occcis-elements in their promoters. General canonical binding sites for MYB and NAC domain TFs have been identified, and a number of cis-regulatory elements recognized by TFs involved in SCW regulation specifically have been described  was discovered in the promoters of SND1 direct targets, existing as several related variants in target gene promoters   is an SND1-activated NAC domain TF that may negatively regulate tracheary element growth  T-DNA mutant demonstrated a marked reduction in SCW formation in interfascicular and xylary fibers as well as a reduction in inflorescence stem growth, without otherwise affecting fiber cell specification  and metaxylem vessel formation at the stele centre  . Combined with the example above of the WRKY12 repressor that inhibits SCW initiation in some ground tissues in Arabidopsis, these data may point to an unexpected mechanism in which transcriptional activation of SCW deposition is a developmental program that is repressed in certain non-sclerified tissues, rather than simply induced in vascular tissues. Alternatively, co-factors required by these master regulators are not present in these non-vascular tissues, as evidenced by the failure of certain cells to ectopically deposit SCWs when the master regulators are overexpressed (see section Master Regulators).Some spatial specificity in SCW deposition can be explained by the presence of transcriptional repressors in non-sclerenchymatous cells. For example, The upstream regulators of SND1/NST1 and VND6/VND7 have not yet been reported, nor have the gene targets of xylem-regulating HD ZIPIII TFs , BEARSKIN1 (BRN1), and BRN2  deposition in the l et al. , where echimeric repressor silencing technology  has been widely used to identify TFs that interact directly with SCW-related promoters  offers many advantages that are particularly suited to non-model organisms where genomic information is available Table . It has Arabidopsis SCW biosynthesis, few attempts have yet been made to integrate such data with other -omics platforms. The integration of metabolomic data into transcriptional networks, for example, can link modifications of TFs and their interactions to phenotypic outputs. Two model Arabidopsis studies, one using multiple knockout mutants of lignin biosynthetic pathway enzymes  is severely underdeveloped compared to what is known about the network's connectivity. The immediate goal of future research is to comprehensively identify the physical interactions (protein–DNA and protein–protein) involved in SCW transcriptional regulation. This includes the identification of not only interacting partners of known TFs, but also their cell-type context that might influence the functions of TFs in different ways. This goal will allow us to identify TFs and transcriptional modules that regulate genes involved in the biosynthesis of specific SCW biopolymers. This, together with systems approaches, will also reveal to what degree regulation of different genes and metabolic pathways is independent. Currently, only the lignin pathway seems to be specifically targeted by TFs such as MYB58, MYB63, and MYB85, and it may not be possible to uncouple the transcriptional regulation of cellulose and hemicellulose biopolymers. However, two recent studies have used components of the SCW transcriptional network to engineer plants with favorable biofuel properties by restoring vessel wall integrity in xylan (Petersen et al., In this review we aimed to provide a comprehensive summary of what is currently known about Arabidopsis, for which knock-out lines are readily available to quantify network dynamics in response to genetic perturbations, will contribute extensively in this regard. For non-model organisms, Y1H and ChIP-seq are expected to be two key techniques used to identify protein–DNA interactions in the near future. However, systems genetics, which facilitates network reconstruction, modeling and quantification from perturbations caused by natural genetic variation, is gaining momentum in agronomically important species (Ingvarsson and Street, The ability to predict the regulatory outcome of perturbations in transcriptional networks through network modeling is invaluable to the field of biotechnology. A detailed knowledge of the strength of interaction for each edge connecting two nodes and a mathematical understanding of how the network responds to perturbations in expression, as well as genetic and environmental modulation, has not yet been attained. Systems biology experiments in Populus (Li et al., An understanding of the integration of intercellular signals, miRNAs, chromatin changes and temporal dynamics in transcription during xylem development remains a future challenge, marred by a limited understanding of regulatory mechanisms. For example, the occurrence of alternative splicing as a form of SND1 regulation in 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.


In response to extracellular stimuli, mitogen-activated protein kinases (MAPKs) modulate gene expression to maximize cell survival. Exposure of yeast to high osmolarity results in activation of the p38-related MAPK Hog1, which plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress. Hog1 not only regulates initiation but also modulates other steps of the transcription process. Recent work indicates that other yeast signalling MAPKs such as Mpk1 modulate transcriptional elongation in response to cell wall stress. Similarly, mammalian MAPKs have also been found associated to coding regions of stress-responsive genes. In this paper, significant progress in MAPK-regulated events that occur during the transcriptional elongation step is summarized, and future directions are discussed. We expect that the principles learned from these studies will provide a new understanding of the regulation of gene expression by signalling kinases. Saccharomyces cerevisiae, there are several MAPK cascades . The HOG (high osmolarity glycerol) pathway is activated in response to osmostress by two upstream independent branches that converge on the MAPKK Pbs2, which controls Hog1 MAPK activity see  2, 9–11–119–11. In yeast, these two MAPKs play an important role in controlling gene expression, and they both modulate transcription initiation and elongation steps of the transcription cycle. We will focus here on the role of Hog1 and Mpk1 in regulation of transcription elongation.Among other functions, Hog1 is a master regulator of reprogramming gene expression in response to osmostress. Upon stress, the yeast genome alters its expression pattern up to 20% depending on the strength and duration of the stress –24. A maIn response to cell wall stress, two known transcription factors, Rlm1 and SBF (Swi4 and Swi6), are controlled by Mpk1 by different mechanisms. Whereas Rlm1 is activated through direct phosphorylation –44, SBF The transcription cycle consists of several steps, and elongation is a critical phase of transcription susceptible of strong regulation –53. In aWhich are the tasks of Hog1 at coding regions is still an open question. Recruitment of the kinase to the open reading frames (ORFs) is essential for an increased association of RNA Pol II and proper mRNA production in response to osmostress . MoreoveBinding of Hog1 to the stress-responsive ORFs is restricted temporally. Although the initial recruitment of the MAPK and RNA Pol II is similar, Pol II association is observed for a longer period upon osmostress, whereas binding of Hog1 is restricted at the initial phase of elongation . This suNucleosome positioning of stress-responsive loci is altered dramatically in a Hog1 MAPK-dependent manner during osmostress . Hog1 phSingle-cell experiments have shown that Hog1 nuclear accumulation increases linearly with stimulus. However, at low stress levels, the transcriptional output shows two distinct subpopulations, one responding and the other one not. Of note, this bimodality is reflected in chromatin remodelling and depends on both the retention time and concentration of Hog1 in the nucleus . Thus, cFKS2 gene in response to cell wall stress although such binding does not require MAPK kinase activity [The role of MAPK in the modulation of transcriptional elongation is not restricted to Hog1. Other yeast signalling kinases, such as Fus3, PKA, or Mpk1, have been reported to associate to coding regions of activated genes . Mpk1 asactivity . This diFKS2 coding region during transcription? Mpk1 is tethered to the elongation complex through its interaction with RNA Pol II-associated complex Paf1C complex [paf1-4A mutant, which is unable to interact with Mpk1, is deficient for transcription elongation of the FKS2 gene and renders cells sensitive to cell wall stress. Actually, in this mutant, Mpk1 is recruited to the promoter but does not progress into the coding region of FKS2 in response to stress [FKS2 promoter-proximal region terminate under noninducing conditions [How does Mpk1 associate with the  complex , 63, 64.o stress . This iso stress  if such nditions , 66. Thenditions . TherefoBinding of signalling kinases to chromatin has been now shown in organisms other than yeast. Several reports support an essential role of p38 MAPK in the regulation of transcription upon inflammation and stress responses , 68 as wp38 controls skeletal muscle differentiation by regulating the sequential activation of myogenic regulatory factors and their transcriptional coactivators, including chromatin remodelling enzymes (reviewed in ). HoweveThe extracellular signal-regulated kinase (ERK) pathway also regulates gene expression. Erk1 is activated by progesterone and phosphorylates the progesterone receptor. Then, a complex of activated progesterone receptor, Erk1, and its target kinase Msk1 is recruited to the target promoters, where Msk1 phosphorylates histone H3 at serine 10 promoting chromatin remodelling and gene regulation . Onmpk1 mutant. Moreover, Erk5 is able to activate gene expression through Rlm1 as well as SBF transcription factors [When expressed in yeast, the human Erk5 MAPK is activated in response to cell wall stress and suppresses the phenotypic defects of a  factors , 37. The factors .Therefore, it is likely that MAPK regulation of transcription in higher eukaryotes is not restricted to initiation but also to elongation upon stress and that MAPK-driven mechanisms are conserved among eukaryotic cells.Proper adaptation to stress is critical for cell survival. There has been a number of signalling networks involved in stress signal transduction, and, amongst them, MAPK signalling networks stand out. One important piece of the different adaptive strategies consists of a massive reorganization of the gene expression capacity. Protein kinases not only phosphorylate target proteins, but also themselves become part of the transcriptional complex. This suggests a new scenario in which signalling kinases, rather than simply relaying the signal to the transcriptional effectors, may function as integral components of the transcriptional complexes that activate expression of distinct target genes see . ActuallWe are just beginning to understand the overlapping link between signal transduction pathways and modulation of transcriptional elongation. The molecular mechanisms of such relationship are far from fully being understood. For example, one unresolved question is how the MAPK Hog1 is recruited specifically to the osmo-responsive coding regions. Is there some special feature in the 3′UTR region of stress-dependent genes targeting the kinase? or is there some transcription elongation factor that specifically recruits the kinase? Another key question remaining in the field is which are the phosphorylation events mediated by MAPK on the transcriptional elongation machinery. Although it is clear that there are noncatalytic functions in transcription regulation by MAPK, activity of Hog1 is needed for the recruitment of the kinase onto the promoters and coding regions of osmostress-responsive genes. Recent findings shed light on the link between upstream signalling kinases and direct phosphorylation of histones and/or histone modifiers (reviewed in ). MoreovLast but not least, to what extent regulation of transcriptional elongation will apply to other yeast and mammalian signalling modules will be interesting avenues for future investigation. In any case, it is becoming clear that mechanisms, which regulate transcription, have been preserved from yeast to mammals.


Calonectris diomedea nesting in Berlengas and in the Selvagens, two colonies in contrasting oceanographic environments, separated by ca. 1200 km. Although no differences were found in winter distribution, there was a marked divergence in timing, route and the use of staging areas during the postbreeding (autumn) migration. Birds from Berlengas typically travelled to oceanic waters in the North Atlantic for an extended stopover, whereas those from Selvagens rarely did so. In the South Atlantic, birds from Selvagens spent more time in flight, perhaps because they had higher energy and nutrient requirements for feather replacement compared to birds from Berlengas, which moult more flight feathers during breeding. Stable isotope analyses of feathers suggested that this variation in activity patterns was unrelated to trophic ecology. Differences in migration routes and stopovers may expose populations to distinct threats, and should be taken into consideration when defining units for conservation purposes and developing appropriate management strategies.Although seabirds that are trans-equatorial migrants show apparently broad overlap among populations in the non-breeding season, such large-scale pattern may conceal subtle but nevertheless key differences in migratory behaviour. These specializations could reflect adaptation to different environments during the breeding season, carry-over effects from the breeding to the nonbreeding period, or asymmetries in competitive ability of birds of different origin. We compared the migratory and wintering behaviour of Cory's shearwaters  Trans-equatorial migrant seabirds travel huge distances across entire ocean basins, with apparent broad overlap between populations Birds from colonies close to each other (relative to distant wintering grounds) may display differences in migratory behaviour and wintering ecology due to (1) population specializations arising from local adaptation to breeding environments Recent developments in technology allow the year-round tracking of pelagic migrants, providing not only geographic position, but also information on activity patterns derived from immersion sensors Differences between populations improve our understanding not only of migration strategies, but also of demographic changes, as birds may be subjected to distinct threats in different areas, with implications for conservation Calonectris diomedea is a pelagic seabird that nests in the NE Atlantic and Mediterranean and winters in many areas of the Atlantic, mostly in the south C. (d.) diomedea are well differentiated morphologically and genetically from those in the Atlantic C.(d.) borealisC.(d.) borealis from the Azores and the Canaries, but sample sizes were too small to allow the detection of significant differences in the choice of migration routes or wintering areas.The Cory's shearwater C.(d.) borealis from two colonies – Berlengas, on the Portuguese continental shelf, and the Selvagens, a large offshore colony located north of the Canary islands, where a large-scale study on migration and at-sea behaviour is currently underway borealis off continental Europe, which, given major changes in climate and marine systems, may come to represent a significant conservation unit for the taxon. Unlike several others, this population has been increasing in recent decades, was estimated at 850 pairs in 2005 Here we present tracking data from migrant Cory's shearwater C.(d.) borealis from continental Europe; (2) compare the wintering areas of Berlengas and Selvagens birds; (3) assess if timing of migration and routes differ between colonies, (4) investigate if birds from the two colonies differ in their behaviour and trophic position in wintering grounds. Knowing that Cory's shearwaters from Berlenga are intermediate in body size and in geographic position of the colony between birds from the Mediterranean and from other Atlantic colonies Our main objectives were to: (1) provide the first tracking data on the migration of The deployment of MK7 loggers (see details below) did not take more than 10 minutes and on no occasion did it interfere with reproduction or have visible deleterious effects on study animals. All work was approved by the relevant authorities .In 2006/07, we tracked the migratory routes and wintering areas of 11 Cory's shearwaters from Berlengas  and 25 from Selvagens  using leg-mounted geolocators e.g. . LoggersTransEdit software  provide two positions per day based on light levels , with an accuracy of approximately 186±114 km 13C) and nitrogen (δ15N) isotope analyses. Sub-samples of 1.0±0.1 mg of finely cut pieces of feather were weighed into 8×5 mm tin capsules and combusted at 1000°C in a Euro EA Elemental Analyser. Resultant CO2 and N2 gases were analysed using a continuous-flow isotope ratio mass spectrometer IsoPrime (MicroMass). Replicate measurements of laboratory standards showed measurement errors of ±0.1‰ and ±0.3‰ for stable carbon and nitrogen isotope measurements, respectively. Laboratory standards were “Methionine OAS B2045” and “L-Cystine OAS B2035”, calibrated and verified every six months with the standards IAEA-CH6, IAEA-CH7, IAEA-600 e IAEA-N1 from the International Atomic Energy Agency, Vienna. Quality control samples (one of our randomly chosen samples) were run in triplicate before and after each sequence, and feather samples were run interspersed with laboratory standards (3 standards every 30 unknowns).Upon recapture of the study birds for logger recovery, we cut a small section of primary 8, which is known to be grown in the wintering grounds One of the loggers recovered from a bird at Berlengas ceased functioning before arrival at the wintering grounds, and hence only provided information on the postbreeding migration. Comparisons of timing and routes taken during the postbreeding migration only included individuals that wintered in the South Atlantic.Activity patterns during the winter period (namely the percentage of time spent on sea surface and the number of landings per hour) were derived from saltwater immersion data (wet/dry), registered by the geolocators with a 3 s precision. In addition, a night flight index was calculated as the percent of total time spent in flight that occurred in darkness, divided by the relative duration of the dark period in each day. This is a measure of the amount of flight undertaken during darkness rather than daylight, corrected for the overall duration darkness relative to daylight, i.e., a value of 1 corresponds to an allocation of the flight time during daylight and darkness proportional to the duration of each phase.th North parallel and the date of arrival at the colony were made using Mann-Whitney tests. Comparisons of the timings of other events, of mean activity indices per individual, and of isotope values were made using two-way ANOVAs, with wintering area  and nesting colony (Berlengas vs. Selvagens) as fixed factors.Comparisons of the date of departure, the date crossing the 10The non-breeding distributions of Cory's shearwaters from Berlengas and Selvagens are broadly similar between them . Sixty pIn 2006, departure dates for the postbreeding migration were earlier at Berlengas than at Selvagens . At leas13C values differed between wintering areas, but not between birds of different populations, whereas δ15N showed no significant variation according to origin or wintering area (Activity patterns in the wintering grounds in the South Atlantic differed between birds from different colonies (% time in the water), and between wintering areas  , Table 2borealis Cory's shearwaters nesting near continental Europe, and is the first published comparison of wintering site selection, timing of migration and choice of route, activity patterns and feeding ecology  of birds from different breeding populations in a long-distance migrant seabird.This study presents the first tracking data for borealis populations and the nominate birds in the western Mediterranean  and geographically, Cory's shearwaters from Berlengas are intermediate between other Our study is novel in comparing two populations, using adequate sample sizes, to reveal subtle, but nevertheless striking differences in migratory behaviour of a pelagic long-distance migrant. Berlengas and Selvagens are separated by <1200 km, which is a relatively short distance when considering that many Cory's shearwaters migrate >30,000 km between nesting seasons  targeting different types of prey. We note, however, that similar isotope values do not necessarily imply a similar diet, although that is the most likely scenario. An experimental approach would be recommended, to further assess this new hypothesis that the degree of moult progression in the breeding grounds affects bird behaviour in distant wintering areas.It is also interesting to note that, irrespective of the colony of origin, individuals adjusted their activity patterns to the areas in which they wintered, presumably as a response to local resource availability patterns, which confirms that Cory's shearwaters are flexible in several dimensions of their migratory behaviour, as reported here and elsewhere The difference in migration strategies between birds from different populations should also encourage their conservation as separate management units, as they may not be equally resilient to predicted changes in future climate and marine ecosystems.


Antibiotics have natural functions, mostly involving cell-to-cell signaling networks. The anthropogenic production of antibiotics, and its release in the microbiosphere results in a disturbance of these networks, antibiotic resistance tending to preserve its integrity. The cost of such adaptation is the emergence and dissemination of antibiotic resistance genes, and of all genetic and cellular vehicles in which these genes are located. Selection of the combinations of the different evolutionary units  is highly asymmetrical. Each unit of selection is a self-interested entity, exploiting the higher hierarchical unit for its own benefit, but in doing so the higher hierarchical unit might acquire critical traits for its spread because of the exploitation of the lower hierarchical unit. This interactive trade-off shapes the population biology of antibiotic resistance, a composed-complex array of the independent “population biologies.” Antibiotics modify the abundance and the interactive field of each of these units. Antibiotics increase the number and evolvability of “clinical” antibiotic resistance genes, but probably also many other genes with different primary functions but with a resistance phenotype present in the environmental resistome. Antibiotics influence the abundance, modularity, and spread of integrons, transposons, and plasmids, mostly acting on structures present before the antibiotic era. Antibiotics enrich particular bacterial lineages and clones and contribute to local clonalization processes. Antibiotics amplify particular genetic exchange communities sharing antibiotic resistance genes and platforms within microbiomes. In particular human or animal hosts, the microbiomic composition might facilitate the interactions between evolutionary units involved in antibiotic resistance. The understanding of antibiotic resistance implies expanding our knowledge on multi-level population biology of bacteria. Antibiotics produced by natural organisms play a role in their interactions shaping the lifestyle and homeostasis of bacterial populations and communities . Such inHowever, the production of natural antibiotics might only have functions unrelated with inter-bacterial antagonisms. Antagonism might arise in particular contexts as a side-effect of cell-to-cell signaling effects resulting in self-regulation of growth, virulence, sporulation, motility, mutagenesis, SOS stress response, phage induction, transformation, lateral gene transfer, intra-chromosomal recombination, or biofilm formation .Natural antibiotic resistance modulates the effect of the natural production of antibiotics, so antibiotic production and antibiotic resistance act as two complementary sides of the same process assuring the homeostasis of microbial populations and communities. In fact, communities with a cohesive habitat association might act as units in terms of antibiotic production and resistance. In these clusters, antibiotics are frequently produced by few bacterial organisms, whereas other members of the club are resistant .As in the case of natural antibiotic production, natural antibiotic resistance might not only focus on “defense against antibiotics” or “self-protection” in antibiotic producers. In fact, this “defense” is frequently a side-effect of other functions of the “natural resistance mechanisms,” including nutrition, metabolism, detoxification of noxious substances, and catabolic processes .The so-called “intrinsic resistome,” the ensemble of non-acquired genes and functions normally present in bacterial cells which influence the susceptibility to antibiotics, might account for 3% of the bacterial genome . ObviousThe main problem that we are examining in this manuscript derives from the huge escalation of the amount of antibiotics released into the microbial environments as an effect of anthropogenic action, greatly exceeding the amount of natural antibiotics signaling and controlling the homeostasis of the bacterial world. Moreover, the amount of antimicrobials of anthropogenic origin entering into the environment assures the presence of every possible antibiotic concentration in contact with bacteria. The consequences of such an extensive release of inhibitory and regulatory molecules have an important impact on the population biology and evolutionary biology of bacteria.The units of selection define the evolutionary individuals . But whaWhat we propose in this work is essentially a mental heuristic exercise. Let us imagine that we are aware of a kind of replicators called genes but we still ignore the existence of cells. We could observe changes in the frequency and variety of genes, and we might consider populations of genes, submitted to evolutionary dynamics and natural selection. If we were only conscious of the existence of transposons, we would establish population biology of transposons. If we considered plasmids, we would refer to the pan-plasmidome, the plasmid population harbored by a particular microbiome or a particular bacterial group . This won-) copy has the evolutionary freedom (liberation from purifying selection) to be modified, eventually leading to a variant or novel gene  occupying the same functional niches . AntibioblaTEM-1 in Escherichia coli, or blaZ in Staphylococcus aureus) might result only from a “founder effect”  appears to have been captured in separate events by different gene-capturing elements as ISEcp, ISBR1, or IS26? .How can we explain that the same resistance gene of plausible environmental origin , their functionality seems to be better preserved than could be expected. This might suggest that the current function of a number of classic “resistance genes” is something other than antibiotic resistance  target the terminal inverted repeats of Tn21 family transposons , favor the capture and mobilization of a full series of antibiotic resistance genes leading to complex multi-resistance class 1 integrons  of the ISnsposons . The outnsposons . Such monsposons . Also, I2 family , or IS4-s IS1999 . Note thntegrons .Have antibiotics increased the abundance of resistance transposable elements in the microbiosphere? Transposable elements encode an enzyme, transposase, which is required for excising and inserting the mobile element. Transposases . The wide spread and maintenance of different classes of transposable elements in bacterial populations has been obviously favored by antibiotic selection because of their association with antibiotic resistance genes. However, most of the contemporary antibiotic resistance transposable elements belong to a few families that have been detected in ancient isolates, often linked to alternative functional roles, thus suggesting antibiotic resistance might have overshadowed previous selection forces.5 or Tn10, confer growth rate advantages under different conditions of nutrient availability, enabling populations to rapidly adapt to different physiological conditions (7 able to selectively direct transposition into both mobile and stationary DNA pools (see later), avoids the occurrence of deleterious insertions and allows the host population or community to recruit genes through a variety of mobile DNAs, thus favoring the adaptation of diverse groups of bacteria to survive or adapt to different conditions . They are frequently located on plasmids associated with composite transposons containing antibiotic resistance genes  and one glutamate (E) residue), originally identified in the retrovirus integrase and having a role during the transfer of the DNA strand. Most IS families use this catalytic reaction for transposition with the exception of ISce genes .7 poses an unique fine-tuned regulated transposition array (TnsABCDE) involved in the regulation of transposition  and the mobilization of the element  that controls the activity of such transposase and often its target site selection. Members of the ATP-subunit superfamily comprise widespread AbR transposons that differ in the transposase  and the number of proteins in the transposition module  or Tn501  and all of them were already detectable in ancient bacteria from permafrost , and have converted this family in the “flagship” of floating resistance genes , and Tn3  can bind to “processivity factors” involved DNA replication; competition for this interaction could limit their proliferation. However, a benefit for the transposons appear to be derived from the fact that the interaction of transposases with processivity factors favors “target site selection,” so that activation of transposition with Tn7 (transposon excision and insertion) does not occur until an appropriate target has been identified, most frequently mobile plasmids, providing Tn7 with a means of spreading to a new host  poses an interesting question. Eventually, the biological cost of resistance plasmids for the host could be compensated by higher transmission . As Stoktransfer .Finally, we can consider bacteriophages as mobile mediators of inter-bacterial transfer of resistance genes. Also in this case antibiotics might modulate phage–bacteria population dynamics by processes as “phage-antibiotic synergy,” a non-SOS mechanism of virulent phage induction caused by exposure to sub-inhibitory concentrations of beta-lactams . AntibioE. coli ST131 (phylogroup B2), ST155 and ST393 (phylogroup B1), or ST69, ST405, and ST648 (phylogroup D); K. pneumoniae ST258, ST14 or ST37; Enterococcus faecium, ST18, ST17, ST78; Enterococcus faecalis ST6; S. aureus, ST45, ST5, ST8, ST30, or ST22. A number of these clones were ancient lineages, well-adapted to colonization and transmission between particular hosts, that acquired antibiotic resistance and consequently enhanced capabilities of dispersal  and Firmicutes  and Gammaproteobacteria has been recently shown under the same conditions  increases Proteobacteria even in a higher proportion , that makes the emergence and spread of resistance possible is also favored by poor sanitation, facilitating contact of human or animal sewage with the soil. Some of these effects might escalate with other anthropogenic effects as destruction of diversity in food animals  or even 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.