Published reports that high calcium intakes in free-living adults have relatively little or no beneficial impact on bone mineral density (BMD) and fracture rates suggest that current recommendations of calcium for adults may be set too high. Because even healthy kidneys have limited capability of eliminating excessive calcium in the diet, the likelihood of soft-tissue calcification may increase in older adults who take calcium supplements, particularly in those with age or disease-related reduction in renal function. The maintenance of BMD and bone health continues to be an important goal of adequate dietary calcium consumption, but eliminating potential risks of CVDs from excessive calcium intakes needs to be factored into policy recommendations for calcium by adults.Concern has recently arisen about the potential adverse effects of excessive calcium intakes,  Twenty per cent or more of adult US citizens have calcium intakes that exceed the Recommended Dietary Allowances (RDAs) . Calciumi.e., >2500 mg of calcium per day, may result in hypercalcemia and the calcium-alkali syndrome [i.e., bolus consumption of large amounts in one dose, may be more likely to contribute to arterial calcification than smaller doses from foods over a day, especially in older adults [Extremely high intakes of calcium, syndrome . Whetherr adults . The eviThis review focuses on the confluence of recent research from a number of disciplines that point to potential pathophysiological events within the arterial wall that may result from excessive calcium intakes, especially from calcium supplements. The multi-organ complexity of arterial calcification requires knowledge on many fronts to gain better understandings of the possible linkages among high calcium intake and arterial calcification and potentially adverse effects on cardiovascular health.i.e., smoking, diabetes, hypertension and dyslipidemia. A growing atheroma encroaches on the arterial lumen and may lead to ischemic events including myocardial infarction and stroke if the artery becomes occluded or the plaque ruptures. The calcium component of mineralizing atherosclerotic plaques is deposited by an actively regulated process that resembles normal osteogenesis and constitutes a form of ectopic calcification. Along with the lipid component, plaque calcium contributes to luminal narrowing. Taking advantage of the fact that most atherosclerotic plaques contain calcium, rapid sequence computed tomography (CT) can be utilized to measure total atherosclerotic calcium burden by a calcium scoring technique [Atherosclerosis is driven by the deposition of soft sub-intimal lipid-laden plaques, which tend to calcify. Beyond the normal aging process, atherosclerosis is influenced by traditional Framingham risk factors, echnique . This meechnique ,16. Arteechnique . Demer [echnique  has suggWhether high calcium intakes, with or without calcium supplements, contribute to the calcification process in atheroma-laden arteries remains unknown. Recent cross-sectional data from a multiethnic population of older Americans ingesting a broad daily range of calcium failed to show a relationship between high calcium intakes (~1400 mg/day) and coronary artery calcification . ProspecYet, retrospective studies ,10,11,12Clearly, resolution of these two divergent views is needed.i.e., the RDA, has little or no additional benefit of calcium on BMD. i.e., approximating 400 to 500 mg per day may be sufficient to maintain BMD in individuals adapted to such a low intake [Calcium is a threshold nutrient, which means that an adult intake greater than an estimated arbitrary upper limit, w intake . This adw intake , and basw intake  the EAR w intake , and supw intake ,25,26,27A wide range of calcium intakes exists among adult males and females in the USA. A recent survey of NHANES (2004–05) intakes revealed that a majority of North Americans is meeting RDAs from a combination of food and supplemental calcium . RDAs ofDietary supplements of calcium contribute to high calcium intakes in substantial numbers of older Americans ,3. In twi.e., >1500 mg/day, are at risk of developing net calcium retention [i.e., >age-specific RDAs, the question arises: Where does the extra calcium go in the bodies of these adult individuals? Certainly, modest amounts of calcium are buffered temporarily in the bone fluid compartment or bone envelope (see below under Parathyroid Hormone Regulation) and some is lost in urine. Over prolonged periods of high calcium intake, however, the retained additional calcium is likely to be deposited in soft tissues and pre-existing atherosclerotic plaques  rather than in structural bone as calcium apatite. Concern has arisen that a positive calcium balance in these individuals may be expressed as soft tissue and vascular pathologic calcifications. If this hypothesis is confirmed, calcium loading may be considered as a potentially modifiable accelerant of atherosclerosis as well as the stiffening of arteries, i.e., arteriosclerosis.Zero calcium balance is appropriate for the adult skeleton and is achieved across a wide range of calcium intakes, even during low intakes because hormonal 1,25-dihydroxyvitamin D regulation of calcium absorption in the small intestine is enhanced. Very high calcium intakes may escape this regulatory system and, thus, contribute to an inappropriate positive calcium balance. A primary role of the kidney is to promote calcium reabsorption and retention . Therefore, calcium balance cannot always be achieved by means of an increase of urinary excretion in those with high calcium intakes. The small intestine, however, is better adapted than the kidneys to maintain calcium balance over the entire range of customary calcium intakes . Becauseetention . TolerabThe ionic concentration of calcium in the blood and other extracellular fluids (ECF) is tightly regulated on a minute-to-minute basis by parathyroid hormone (PTH) and it is maintained at 1.25 mM across a very wide range of dietary calcium intakes, except following supplement consumption, over a 3 to 4 h period . In conti.e., supplements, to be temporarily stored in the bone envelope, a readily available “bank” of calcium and phosphate ions [Dietary calcium is too variable and intestinal absorption regulation too slow to defend this critical physiologic calcium ion set point at 1.25 mM concentration. Although the skeleton contains >99 percent of body calcium stores, only surface bone consisting of the ionizable mineral phase  is a readily available source of calcium for these minute-to-minute regulatory requirements . Insteadate ions . Since uate ions , the kidate ions .The regulation of calcium balance of the body needs to be distinguished from the physiological regulators of the serum calcium set-point in ECF. Conceptually, calcium balance is more difficult to understand, primarily because quantitative data is derived from balance studies which are time-consuming, labor-intensive, and not readily available in the clinic. Nevertheless, numerous studies in normal subjects as well as in patients with CKD have provided useful information on this important, albeit frequently under-recognized, physiologic concept ,35.An external calcium balance study takes 7–10 days of inpatient study during which time the calcium content of all ingested food and supplements is measured on a daily basis. Calcium excretion is measured in feces and urine on a daily basis. An appropriate calcium balance in an adult is zero, that is, no net calcium retention occurs . During years of linear skeletal growth, calcium is retained at sites of bone matrix and forms new calcium apatite. After epiphyseal closure, a typical adult ingesting 1000 mg/day of calcium will excrete approximately 800 mg/day in stool and 200 mg/day in urine, thus achieving appropriate zero calcium balance. During periods of very low dietary calcium intake, zero balance is maintained by the action of 1,25-dihydroxyvitamin D which increases intestinal calcium absorption. In addition, an increased secretion of PTH helps defend calcium balance by reducing urinary calcium excretion.A recent balance study in normal subjects ingesting high amounts of calcium  demonstrated a positive calcium balance of 450 mg/day . In thisIn contrast, studies in patients with CKD have generated data supporting an association between high calcium intake and cardiovascular calcification . ApproxiBone growth early in life requires protein, energy, and the micronutrients, including calcium and phosphorus, to permit successive phases of bone formation followed by bone resorption until the final bone “model” is achieved. Physical activity is well known to enhance the growth process and contribute to improved bone micro-architecture. Model formation ends when the growth plates of the long bones close. Remodeling begins typically following increases in ovarian estrogen synthesis in females and androgen production in males and this process continues to the end of life.per se.Bone tissue remains dynamic at all stages of the life cycle, though in late life the rate of bone turnover declines; bone mass also declines because the rate of new bone formation is less than the rate of bone resorption, a situation opposite that of the early growth years of life. Once the model is complete, normal adult bone turnover shifts first to bone resorption and then to bone formation second, processes governed by various factors made by cells in bone marrow or elsewhere, but not in bone Too much calcium in the diet may suppress PTH secretion and thereby reduce the pulsatile anabolic action of PTH on bone  and, henCalcium supplements showed a slight improvement in BMD of adults in a meta-analysis , but no Growth hormones that orchestrate the growth of the skeleton, muscle, and other organ systems require a supply of moderately high quality protein and sufficient energy to foster primarily the appropriate increments in height. Less than optimal intakes of either result in shorter height and often disproportionate relative growth in the torso compared to the legs, largely independent of calcium intake. If energy and protein are subsequently provided in optimal amounts during childhood, height acceleration may contribute to reasonably normal or even great height, as currently witnessed among many children in diverse nations of the world. Micronutrients, including calcium, are also required in sufficient amounts for growth, but far too often inadequate amounts are consumed.The co-existence of osteoporosis and variable rates of loss of BMD with increasing rates of arterial calcification occurs in older adults ,16. ThisCalcium intakes that consistently exceed the RDA of older adults, especially when a substantial amount of the calcium is derived from bolus intakes of calcium supplements, may accelerate arterial calcification and raise the risk of cardiovascular events. This risk is much greater in patients with CKD because of their likelihood of calcifying arteries even in the absence of calcium loading. Whether arterial calcification occurs in older adults with truly normal renal function who consume high amounts of calcium in their diets, including supplements, has not yet been determined. Further prospective investigations, especially randomized control trials, on the calcium-CVD relationship, especially in those with high calcium intakes that approach or exceed the ULs, are clearly needed. High calcium intakes from a supplement bolus have now been generally established to have little or no clinical benefit on BMD or bone-fracture rate of non-institutionalized and non-homebound older adults. Health professionals and policy makers need to re-consider their advice regarding healthy patterns of daily calcium consumption in order to maintain skeletal benefits while reducing or even eliminating potential exposure to adverse cardiovascular effects from excessive calcium intakes, and they need to continue monitoring calcium recommendations, especially when calcium is consumed from non-food sources.


Boiga irregularis), whereas the nearby island of Saipan has a relatively intact vertebrate frugivore community. We captured seed rain along transects extending from intact into degraded forest and compared the species richness, density and condition of the seed rain from native bird-dispersed tree species between the two islands. Considering seeds from native bird-dispersed species, approximately 1.66 seeds landed per 26 days in each square meter of degraded forest on Saipan, whereas zero seeds landed per 26 days per square meter in degraded forest on Guam. Additionally, on Saipan, 69% of native bird-dispersed seeds in intact forest and 77% of seeds in degraded forest lacked fleshy fruit pulp, suggesting ingestion by birds, compared to 0% of all seeds on Guam. Our results show an absence of seed rain in degraded forests on Guam, correlated with the absence of birds, whereas on Saipan, frugivorous birds regularly disperse seeds into degraded forests, providing a mechanism for re-colonization by native plants. These results suggest that loss of frugivores will slow regeneration of degraded forests on Guam.In healthy forests, vertebrate frugivores move seeds from intact to degraded forests, aiding in the passive regeneration of degraded forests. Yet vertebrate frugivores are declining around the world, and little is known about the impact of this loss on regeneration of degraded areas. Here, we use a unique natural experiment to assess how complete vertebrate frugivore loss affects native seed rain in degraded forest. All native vertebrate frugivores (which were primarily avian frugivores) have been functionally extirpated from the island of Guam by the invasive brown tree snake ( Between one-third and one-half of Earth's land surface has been heavily influenced by humans Vertebrate frugivores assist in passive regeneration by transporting seeds to degraded areas from nearby intact forest Frugivores play an important role in the regeneration of degraded forests. Primates Despite the evidence that vertebrates play a role in regeneration of secondary forests, conservation or restoration of vertebrate frugivores is frequently ignored as a strategy for restoration of degraded lands e.g. . The worBoiga irregularis) was accidentally introduced to the island of Guam in the late 1940s, causing a widespread loss of Guam's native forest bird species by the mid to late 1980s The brown tree snake (We investigated whether the species richness and density of bird-dispersed seeds varies between degraded forests on Saipan (with birds) and Guam (no birds). We also asked whether the proportion of seeds lacking fleshy fruit pulp (primarily due to handling by birds) differs between forests with birds (Saipan) and those without (Guam). Ours is the first study of which we are aware that utilizes a comparison between similar forests with and without avian frugivores to investigate the regeneration potential of degraded forests.All field studies described here were conducted with the use of the necessary field permits. We obtained permission for the use of our study sites from the Government of Guam Forestry Division (for study sites on Guam), and from the Commonwealth of the Northern Mariana Islands Division of Fish and Wildlife (for study sites on Saipan). The field studies did not involve any protected or endangered species.This study was conducted on the Micronesian Islands of Guam  and Saipan . Both islands are at the southern end of the Mariana Island chain , and havLeucaena leucocephala (Family: Fabaceae). Today, both islands have large areas of degraded forest composed of nearly monotypic stands of L. leucocephala with a sparse understory The primary forest type on Guam and Saipan is karst forest, which grows on a rugged karst limestone substrate; there are approximately 40 tree and shrub species in this forest type, but 8–12 species dominate. Large swaths of karst forest were destroyed on both Guam and Saipan in the 1930s and 40s as a result of World War II, and clearing has continued for development since then. When land is cleared, the rugged karst substrate is lost, and replaced by a topographically homogenous, red dirt substrate, which is typically colonized by the non-native leguminous tree, L. leucocephala. Degraded forest has a low (4–8 m in height) canopy that allows significant light to penetrate to the forest floor, whereas the intact karst forest is taller (7–13 m in height), creating a multi-storied, low-light environment. Narrow bands of transitional forest, characterized by a mixture of L. leucocephala and native forest trees, form the ecotone between degraded and intact forest.In this study, we define degraded forests as forests dominated by Gallicolumba xanthonura), Mariana Fruit-dove (Ptilinopus roseicapilla), Mariana Crow (Corvus kubaryi), Guam Rail , Bridled White-eye (Zosterops conspicillatus) and Micronesian Starling (Aplonis opaca) Cleptornis marchei) is present on Saipan. Historically, the native Marianas Fruit Bat (Pteropus mariannus) was an important frugivore in forests on Guam and Saipan; however, populations of bats on both islands are functionally absent due to hunting and, on Guam, predation by the brown tree snake Birds and bats are the primary seed dispersers in the Mariana Islands. Prior to the introduction of the brown tree snake, six frugivorous birds dispersed seeds on Guam: the White-throated Ground-dove (Streptopelia bitorquata), is found on both Guam and Saipan, typically along roadsides; it is rarely seen in intact karst forest on Guam, and thus is unlikely to be playing a significant role in seed dispersal. Introduced rats, primarily Rattus diardii (sensu Sus scrofa) and Philippine deer (Rusa marianna) are present on Guam. While all three species consume fruit and seeds, their role in seed dispersal is unclear. In other locations, invasive rat species such as Rattus diardii and Rattus rattus are primarily considered seed predators Morinda citrifolia (native), Ficus prolixa (native), and Carica papaya , as well as several non-native herbaceous weed species . In this study, we capture seed rain exclusively from flying frugivores in order to quantify the effect of native frugivore loss on seed rain into degraded forests, and thus, we do not measure dispersal by feral pigs or rats.No non-native species have taken over the entire functional role of the native seed dispersers on Guam, although a small number of tree species may be dispersed by invasive mammals. One non-native avian frugivore, the Philippine Turtle-dove  were constructed using polyvinyl hoops with screen door netting added to make a basket, and suspended from trees at a height of 1.3 m. By hanging the traps, we measured only dispersal by volant frugivores, and not by terrestrial frugivores. Beginning with a trap in the transitional forest, three parallel transects were established a minimum of 10 m apart from one another, and a trap was placed every 10 m along each transect into degraded forest out to a distance of 100 m. To sample the intact forest seed rain, four additional seed traps were set up in the adjacent intact karst forest 10 m from the start of each transect. These four traps were arranged in a 5-m by 5-m square, with a trap at each corner; we placed traps in this arrangement because a small number of transects contained a small cliff or other boundary within the native forest which prevented us from creating 40-meter straight line seed trap arrays. The square array of seed traps at the base of each transect ensured all native forest areas were sampled using the same method. Since the forest canopy is low, seed traps spaced 5 meters apart are unlikely to be underneath the same tree canopy and thus can be considered independent samples. We considered seed traps to be ‘intact forest traps’ if they were within intact forest or in the transitional zone because native seeds could fall into any of these traps without the help of birds. Traps were designated as ‘degraded forest traps’ if they were located in degraded forest (i.e. traps at 10–100 m from the intact/degraded forest boundary).At each site, we set up seed traps in intact karst forest and along three parallel transects from the intact forest/degraded forest boundary into degraded forest. Circular seed traps  function of distance (We investigated whether forest type (intact vs. degraded) and island (Guam vs. Saipan) affected the species richness and density of native bird-dispersed seeds, and the proportion of native de-pulped seeds. We only used data on native bird-dispersed tree species in our analyses, to evaluate the role of birds in the regeneration of native tree species in degraded forest. Thus, we excluded data from two non-native, bird-dispersed naturalized species, distance . The samPremna obtusifolia, and only eight of the 180 degraded forest traps (4%) were located within 10 m of these fruiting adults. Five of these eight traps were located on Guam, where their contribution would have increased the density and richness of native bird-dispersed seeds in degraded forest, making their omission a conservative approach. Results were qualitatively similar even when including these traps.To account for the few remnant native trees in degraded forest, we excluded seeds from traps in degraded forest within 10 m of a fruiting remnant native tree. The only native species we found fruiting in the degraded forest was To determine whether the species richness and density of native bird-dispersed seeds found in degraded forest seed traps was greater on Saipan than Guam, we used generalized linear mixed effects models with a Poisson error distribution i.e. fewest explanatory variables) within two AIC units of the best-fitting model For each analysis, we fit five models; (1) null (intercept only); (2) island effects only; (3) forest type effects only; (4) island and forest type effects; and (5) island, forest type, and their interaction. We identified the best fitting model with Akaike's Information Criterion (AIC) values. We chose the simplest model , and a higher proportion of seeds with pulp removed in degraded than in intact karst forest on Saipan.We used two separate analyses to determine whether the proportion of bird-dispersed seeds with the pulp removed (likely by birds) differed between the two islands, and between intact and degraded forest on Saipan. We used generalized mixed effects models for both analyses, specifying a binomial error distribution and site as a random effect. We used AIC values to identify best fitting models. We expected to find a higher proportion of seeds with pulp removed on Saipan than on Guam . The proportion of bird-dispersed seeds with pulp removed was slightly higher in degraded forest than intact karst forest on Saipan (0.77 vs. 0.69), but the AIC value for the model including forest type as an explanatory variable was within 1.5 AIC units of the null model (including only an intercept), indicating that forest type does not significantly improve model fit.2 from bird-dispersed tree species were dispersed into degraded forests on Guam, indicating a loss of seed dispersal services on this island. In contrast, approximately 1.66 native bird-dispersed seeds/m2 are dispersed into degraded forest on Saipan per 26 days. While this is a small number of seeds, these seeds provide the potential for regeneration of native trees within the degraded forest. There are few wind-dispersed tree species in the Marianas, and Mariana Fruit Bats are functionally extinct on Guam and Saipan, leaving birds and gravity as the primary modes of dispersal. This is the second study to document the loss of an ecosystem function once provided by birds on Guam; the previous demonstration focused on birds as pollinators Avian frugivores act as critical links between intact karst forest and degraded forest in the Mariana Islands. We found increased species richness and density in the seed rain landing in degraded forest on Saipan compared with Guam, likely due to the absence of frugivorous birds in the forests on Guam. Zero seeds/mPremna obtusifolia, Psychotria mariana, and Melanolepis multiglandulosa. These species produce large fruit crops, and birds have been observed eating all three species. M. multiglandulosa is a pioneer species in this system, as it is only found in gaps and edges of forests.Vertebrate dispersers play a key role in catalyzing regeneration of native forest. In systems with intact frugivore communities, vertebrate dispersal from native to degraded forest is evident from the abundance of seeds and seedlings from fleshy-fruited species found in degraded forest Initial dispersal events from intact to degraded forests are important because they can initiate the regeneration of degraded areas. Once a native fleshy-fruited tree becomes established in degraded forest, it provides habitat, food, and perching and roosting sites for frugivores, which in turn will bring more seeds from the nearby intact forest While the focus of this study is on seed dispersal into degraded forests, we also found significant differences in seed rain in intact karst forests . Seed trPremna obtusifolia and Psychotria mariana showed increased germination of handled seeds over whole fruit . Thus, the loss of birds on Guam may affect germination due to the loss of seed handling for at least these two species, regardless of whether the seeds land in degraded or intact karst forest. However, this positive effect of bird handling may not be a general phenomenon in Mariana plants, and thus requires additional study. Un-dispersed seeds may also experience lower levels of germination due to increased natural enemy attack near the parent trees (i.e. Janzen-Connell effects) In addition to moving seeds into degraded forests, birds may influence the likelihood that seeds germinate. When birds ingest fruit, they remove the fleshy fruit pulp and scarify the seed, processes that enhance germination in some species, but do not affect or even reduce seed germination in others L. leucocephala-dominated forests L. leucocephala trees. These areas were likely bulldozed many years ago changing the substrate from karst to soil, then were initially colonized by L. leucocephala, which has since been gradually replaced by native forest species, resulting in a forest with a similar composition and diversity to intact karst forest. Finally, L. leucocephala does not appear to invade intact karst forest successfully, perhaps because it does not compete well with native trees in undisturbed forest L. leucocephala is indeed a poor competitor with native trees, then karst forest regeneration in areas currently dominated by L. leucocephala may be successful without labor-intensive management of L. leucocephala once native seedlings get established.There are at least two aspects of the present study that limit our capacity to predict the full impact of birds on degraded forest regeneration. First, we did not assess rates of germination from the seed bank in degraded forest. It is possible that native species were at one point deposited in the seed bank by birds, but are waiting for the right conditions to germinate e.g. Intsia bijuga, Premna obtusifolia, Eugenia spp.), and provides habitat for wildlife . In addition, intact forests are highly productive, and often store more carbon than degraded forests due to the abundance of large, old-growth trees as opposed to small, successional trees found in degraded forests, as well as a higher canopy with understory, mid-story and canopy tree levels Bird populations worldwide are in decline due to habitat loss, climate change, introduced disease, and invasive species Land managers should explicitly consider the role of seed dispersers in degraded forest regeneration. In locations where frugivore populations are healthy, conservation of intact forest and frugivore communities will ensure that passive regeneration of degraded forest is not limited by dispersal. In locations where frugivore communities are reduced or absent, conservation of intact forest, restoration of frugivore communities, and intensive management may all be needed to restore or replace the ecosystem services provided by frugivores. Conservation of remnant intact forest patches should be prioritized because these remnants serve as a seed source for degraded forests; forest regeneration is most likely to occur in areas where both older remnant forest stands and seed dispersers—such as avian frugivores—persist nearby The island of Guam provides the worst-case scenario for the impact of frugivore loss on seed rain in degraded forest, and shows a stark view of the disruption of ecosystem services provided by frugivores, evident here as the complete cessation of seed rain into degraded forests. The unequivocal absence of adequate frugivore populations on Guam both demands immediate action, and provides an opportunity for managers to test and advance new methods for addressing seed dispersal disruptions.Figure S1Seed rain in degraded forest with respect to distance from intact karst forest boundary.(TIFF)Click here for additional data file.Table S1Summary of tree species found in forest surveys or seed traps, with biogeographic status.(DOC)Click here for additional data file.


In 1898, the Golgi apparatus was discovered by light microscopy, and since the 1950s, the ultrastructure composition is known by electron microscopic investigation. The complex three-dimensional morphology fascinated researchers and was sometimes even the driving force to develop novel visualization techniques. However, the highly dynamic membrane systems of Golgi apparatus are delicate and prone to fixation artifacts. Therefore, the understanding of Golgi morphology and its function has been improved significantly with the development of better preparation methods. Nowadays, cryo-fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in amorphous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy and tomography. Even though the overall morphology of vitrified Golgi stacks is comparable to well-prepared and resin-embedded samples, previously unknown structural details can be observed solely based on their native density. At this point, any further improvement of sample preparation would gain novel insights, perhaps not in terms of general morphology, but on fine structural details of this dynamic organelle. Interestingly, our actual concept of Golgi architecture is also based on metallic impregnation methods, but at the electron microscopic level, as reviewed recently by Klumperman  sections, as introduced in the early 1950s (Sjöstrand 4), whereas the permanganate fixation showed slight advantages on Golgi morphology , the ‘glassy’ sample will not flow in the timescale of a realistic experiment, even viewed at electron microscopic magnifications —as introduced by Riehle and Moor , well-contrasted plastic sections give even substantial advantages compared to vitreous sections. At higher magnification, known structures like vitrified clathrin coats give gentle contrast in their cellular environment, compared to purified samples in cryo-electron microscopy  milling for serial block face imaging in the scanning electron microscope (SEM) (Heymann et al. Single-particle cryo-electron microscopy is a technique in structural biology that is widely used to solve the three-dimensional structures of isolated macromolecular assemblies close to their biological conditions. The technique started from relatively simple ‘negatively stained’ material deposited on electron microscope grids, as described in a more than 1,300 times cited article (Brenner and Horne trans-Golgi network to endosomes and from plasma membrane to endosomes during endocytosis, as well as in numerous specialized pathways with physiological relevance. Clathrin was the first membrane coat described and its characterization defined the prototype for membrane coat function that applied to other intracellular and Golgi-derived coats (Brodsky Clathrin-coated vesicles are important structures of membrane trafficking in cells, in particular of cargo transport from  Brodsky . Our cur Brodsky , 2006. F Brodsky .The intracellular transport of cargo and lipids from endoplasmic reticulum to the Golgi apparatus is mediated via vesicles generated by a set of cytoplasmic coat proteins known as the COPII coat. Building on a catalog of yeast mutants and in vitro reconstitution of ER–Golgi transport events, the COPII coat was initially defined more than 20 years ago (Fromme and Schekman An impressive example of studying Golgi-related mechanisms by in vitro approaches is the assembly of the SNARE complex on membrane fusion. Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. Recently, the SNARE fusion machinery was arrested in a cell-free reaction, and the fusion intermediates were identified by cryo-electron microscopy (Hernandez et al. Dictyostelium discoideum (Beck et al. In sub-tomogram averaging, features of cryo-electron tomography are combined with single-particle reconstruction procedures to provide 3D information and structural information of macromolecular complexes in situ. Multiple copies of certain macromolecular complexes are identified in cryo-electron tomograms. Then, these sub-tomograms containing the complex of interest are extracted from the larger original data set, aligned and averaged to obtain an isotropic 3D structure of the complex. One of the first examples was the reconstruction of nuclear pores in All the high-resolution studies mentioned above using single-particle reconstruction, in vitro reconstitution, and/or sub-tomogram averaging clearly gave great insights into protein functions, arrangements of protein complexes, and protein–membrane interactions. Hence, the determined 3D shapes could be used as patterns for finding similar protein complex shapes in reconstructed 3D volumes of vitrified Golgi stacks in their cellular context. The basic idea of ‘visual proteomics’ to map molecular landscapes inside unperturbed cellular environments into a quantitative description of macromolecular interactions that underlie cellular functions (Nickell et al. This review shows that describing the secretory pathway in its native state by cryo-electron microscopy has already started. Regarding that functional elements like macromolecular complexes interact at Golgi apparatus in distances far below light microscopic resolution, and that vesicles or cisternae of different compositions are homogenized during biochemical isolation or fractionation, it is obvious that high-resolution imaging technologies like cryo-electron microscopy are necessary to decipher Golgi functionality. Most likely, this will not be solved using cellular cryo-electron microscopy as stand-alone technique, but rather in conjunction with various other technologies such as structural biology, biochemistry, light microscopy, and mass spectrometry-based proteomics.In quantitative proteomic analyses, more than 1,400 different proteins have been reported to be involved in the early secretory pathway (Gilchrist et al. Current and new labeling techniques are on their way to find applications in vitreous samples (Bouchet-Marquis et al. Even since electron microscopy gained substantial understanding on the complex Golgi morphology in the last six decades, we still cannot delineate a completely unperturbed ultrastructure of a functioning Golgi apparatus inside a living cell. The combination of electron and cryo-electron microscopy with novel super-resolution light microscopic approaches of live cells might help to answer such fundamental questions, which seemed so straight forward from the beginning.


The evolution of the plant immune response has resulted in a highly effective defense system that is able to resist potential attack by microbial pathogens. The primary immune response is referred to as pathogen associated molecular pattern (PAMP) triggered immunity and has evolved to recognize common features of microbial pathogens. In response to the delivery of pathogen effector proteins, plants acquired R proteins to fight against pathogen attack. R-dependent defense response is important in understanding the biochemical and cellular mechanisms and underlying these interactions will enable molecular and transgenic approaches for crops with increased biotic resistance. Proteomic analyses are particularly useful for understanding the mechanisms of host plant against the pathogen attack. Recent advances in the field of proteome analyses have initiated a new research area, i.e., the analysis of more complex microbial communities and their interaction with plant. Such areas hold great potential to elucidate, not only the interactions between bacteria and their host plants, but also of bacteria-bacteria interactions between different bacterial taxa, symbiotic, pathogenic bacteria, and commensal bacteria. During biotic stress, plant hormonal signaling pathways prioritizes defense over other cellular functions. Some plant pathogens take advantage of hormone dependent regulatory system by mimicking hormones that interfere with host immune responses to promote virulence (vir). In this review, it is discussed the cross talk that plays important role in response to pathogens attack with different infection strategies using proteomic approaches. Unlike plant and animal cells, most bacteria are exposed to a constantly changing physical and chemical environment. Phylogenetic diversity of plant-associated bacteria (PAB) can categorize them in to commensals (acquire nutrients from the plant without damaging), mutualists , and pathogens (damage plant) , intracellular proteins containing a region of a LRRs and a nucleotide binding site , intracellular proteins containing a region of homology to the Toll/IL-1R proteins in addition to LRRs and a nucleotide binding site , and proteins with LRRs that appear to encode membrane-bound extracellular proteins  , which recognize pathogen associated molecular pattern (PAMP) and is basis of plant innate immunity  gels, resulting in a few hundred identified proteins  Tu, peptidoglycan, lipopolysaccharide, and bacterial cold shock proteins are important PAMPS and plant responses induced by them are referred to as “basal” defenses , jasmonic acid (JA) and ethylene, have emerged as key players in the signaling networks involved in plant immunity. Rhamnolipids are glycolipids produced by bacteria and are involved in surface motility and biofilm development and are considered as PAMPS. Ethylene is found to be involved in rhamnolipid-induced resistance to P. syringae use T3SS to deliver up to 40 effector proteins into host cells, inhibiting basal host defense responses, such as HR  were essential components of two complex bacterial machineries: the flagellum, which drives cell motility and the non-flagellar T3SS (NF-T3SS), which delivers effectors into eukaryotic cells Mudgett, . P. syriP. syringae via jasmonate signaling pathway , suggesting that AvrXa21/Xa21 constitutes a PAMP/PRR perception system , differential expression of protein is not restricted to the envelope, but affects general metabolism such as membrane lipid composition, protein folding, carbohydrate catabolism, and protein degradation  MS resulted in the identification of 87 proteins known to be involved in degradative activities and important for the infection of susceptible plant hosts . Proteome analysis of pathogenic bacteria X. campestris pv. campestris in association with B. oleracea and Pseudomonas savastanoi pv. savastanoi resulted in comprehensive expression analysis including stress and metabolic proteins , organic acids (threonic acid), sugars , and polyols (mannitol) were reported to be more abundant in symbiotic roots  and a pathogen (P. syringae DC3000) in the presence of root exudates from A. thaliana and Medicago sativa was studied. SOD is known to be necessary for symbiotic properties in S. meliloti and had higher expression in interaction with M. sativa  are involved in pathogenesis process , in a secrotomic study of P. atrosepticum, was analyzed  in some bacterial strains. OMVs are an ideal structure to transport hydrophobic compounds like membrane proteins into host cells or quorum signals to neighboring cells  pathway was analyzed. In Gram-negative bacteria the Tat system is part of the T2SS  vir factor is structural component of a type T6SSs, components of the T4 bacteriophage tail tube and is required to puncture the host membrane in the context of phage infection  via T2SS in A. thaliana in response to P. syringae  is known to be involved in tagging toxic endogenous substrates with glutathione conjugation to transport toxic substrates into the vacuole through a glutathione pump  had been reported to promote systemic resistance in different host plants as well. Stimulation of antifungal material  and proteins in lipoxygenase pathway in tomato were found after infection with P. putida (Akram et al., Chloroplasts may be key players in plant defense, as the loss of integrity of PSII may lead to the HR-associated oxidative burst, thereby restricting pathogen growth. Proteins related to photosynthesis, defense, protein destination and storage had been down regulated in response to Arabidopsis mutant strain, it was found that in the infection by avirulent P. syringae there is accumulation of defense, redox as well as stress proteins, while in virulent infection only defense proteins were expressed. This imparts importance of stress proteins in plant defense signaling. Induction of defense enzymes involved in phenyl propanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restriction of invasion of F. oxysporum f. sp. lycopersici in tomato roots. Induction of defense proteins and chemicals in tomato by P. fluorescens isolate against F. oxysporum was studied. Defense-related proteins such as PR-9, PR-2, PR-5, and 46 kDa chitinase increased in bacterized tomato root tissues (Ramamoorthy et al., Holzmeister et al.  used 2-DThis review summarizes the use of proteomic approaches to understand the molecular and cellular processes that govern host responses such as PTI, PRR, and ETI. Detailed global comparison of response pathways using proteomics has allowed the identification of novel proteins whose biological role warrants in-depth biochemical and cellular elucidation. Finally understanding of biotic stress responses may identify promising novel targets for the development of cultivars with improved disease resistance. Various types of bacteria can trigger rapid responses in plant cell cultures and defense responses in intact plant tissues (Jones and Dangl, To fully elucidate microbial metabolism and its responses to environmental factors, it will be necessary to go beyond the information obtained from proteomic studies alone. There is need of integration of data resulting from the functional characterization and quantification of molecules representing from genes, transcripts and proteins to metabolites (Delmotte et al., 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.


Recent clinical trials with selective inhibitors of the BRAF and MEK kinases have shown promising results in patients with tumors harboring BRAF V600 mutations. However, as has been observed previously with similarly successful targeted therapies, acquired resistance to these agents is an emerging problem that limits their clinical benefit. Several recent studies from our laboratory and others have investigated the causes of acquired resistance to BRAF and MEK inhibitors, and multiple resistance mechanisms have been identified. Here, we review these mechanisms and suggest that they can be broadly grouped into two main classes: ERK-dependent and ERK-independent. We also propose distinct therapeutic strategies that might be employed to overcome each class of acquired resistance. The RAF-MEK-ERK pathway regulates many important cellular processes (reviewed in -3). ClasConsistent with its role as a key regulatory pathway for cell survival and proliferation, RAF-MEK-ERK signaling is frequently dysregulated in cancer. RAF-MEK-ERK signaling can be driven by aberrant activation of growth factor receptor tyrosine kinases (RTKs) or by oncogenic mutations of intracellular components of this pathway. Indeed, activating RAS mutations  are the most common oncogenic mutations observed thus far in human cancer . SimilarPreclinical data has demonstrated that most BRAF mutant human tumor-derived cell lines are exquisitely sensitive to pharmacologic inhibition of RAF-MEK-ERK signaling. Thus, selective BRAF and MEK kinase inhibitors potently block cell proliferation and induce apoptosis in BRAF mutant cancer models and show high selectivity for cancers with BRAF mutations -12. As aOne potential reason that BRAF inhibitors have shown higher response rates than MEK inhibitors in BRAF V600 mutant melanomas relates to a unique characteristic of RAF signaling that was elucidated during the past year by several elegant studies -22. ThesWhile the initial response rates seen in BRAF mutant melanomas with BRAF and MEK inhibitors are encouraging, previous experience with similarly effective targeted therapies predicts that acquired drug resistance will be a major factor limiting the clinical benefit of these agents. Indeed, despite dramatic initial responses, the median time to progression of patients treated with PLX4032 was 7 months . UnderstPreclinical modeling of acquired drug resistance has been a useful tool for predicting the resistance mechanisms that emerge in patients receiving targeted cancer therapies. Previously, this approach has predicted the resistance mechanisms that occur clinically in many instances, including erlotinib-resistance in EGFR mutant lung cancer, imatinib-resistance in BCR-ABL translocated leukemia, resistance to Smoothened inhibitors in Patched1-deficient medulloblastoma, and resistance to ALK inhibitors in ALK-translocated lung cancers -28. In sTo date, preclinical modeling has identified multiple potential mechanisms of acquired resistance to BRAF or MEK inhibitors Fig. , and somThe vast majority of acquired resistance mechanisms to BRAF or MEK inhibitors that have been identified to date lead to reactivation of ERK signaling despite the presence of inhibitor. This finding underscores the importance of ERK signaling for the continued proliferation and survival of BRAF mutant cancer cells. In general, mechanisms of acquired resistance to targeted therapies commonly employ either mutation or amplification of the drug target itself or alterations which do not involve the drug target but that activate parallel or downstream signaling pathways to circumvent the activity of the drug -34. In rEmery et al identified a MEK1 point mutation in a resistant focus of disease in a patient with V600E mutant melanoma who had originally responded to the MEK inhibitor AZD6244 . ThroughTo date, no secondary BRAF mutations have been identified in BRAF inhibitor-resistant pre-clinical models or in biopsies from patients with clinically acquired BRAF inhibitor resistance. Nazarian et al screened twelve tumor biopsies from patients with clinically acquired resistance to PLX4032 and did not observe any secondary BRAF mutations . “Ultra-de novo resistance to BRAF and MEK inhibitors in the clinic.While secondary mutations in BRAF have not been identified as a cause of BRAF or MEK inhibitor resistance, our laboratory recently identified selective amplification of the mutant BRAF allele as the mechanism underlying acquired resistance in two independent BRAF mutant colorectal cancer cell lines selected for resistance to the MEK inhibitor AZD6244 . BRAF amWe found that the mechanism by which BRAF amplification led to BRAF and MEK inhibitor resistance hinged upon hyperactivation of MEK. We observed that the levels of phosphorylated MEK (P-MEK) in resistant cells were 5 to 6 times higher than the basal levels seen in parental cells. Careful evaluation of the dose-response relationship between BRAF inhibitor treatment and phosophorylation of MEK and ERK revealed that, in resistant cells, levels of P-MEK could be reduced by ~50% before any noticeable decrease in P-ERK levels was observed. This was in stark contrast to parental cells, in which a ~50% decrease in P-MEK levels led to a ~50% decrease in P-ERK levels. These findings suggested that the high levels of P-MEK in resistant cells (driven by BRAF amplification) were in excess of levels required for near-maximal ERK phosphorylation. As a result, a much higher concentration of BRAF or MEK inhibitor was required to fully suppress ERK phosphorylation in resistant cells, either by reducing excess P-MEK levels (as in the case of the BRAF inhibitor) or by inhibiting excess MEK activity (as in the case of the MEK inhibitor). However, if resistant cells were treated with a low dose of BRAF inhibitor sufficient to reduce levels of P-MEK to amounts observed under basal conditions in parental cells, the ability of MEK inhibitors to suppress P-ERK was completely restored. Accordingly, while resistant cells were insensitive to BRAF or MEK inhibitors individually, combined BRAF and MEK inhibition fully overcame resistance and induced dramatic apoptosis and growth inhibition in these cells. Furthermore, combined BRAF and MEK inhibition was also more effective in parental cells, suggesting a possible broader utility for combinatorial targeting of the RAF-MEK pathway in BRAF mutant cancers.This mechanism underlying the resistance to BRAF and MEK inhibitors caused by BRAF amplification has potential implications for other models of resistance in BRAF mutant tumors. Since excess levels of activated and phosphoryated MEK underlie the mechanism of resistance to BRAF and MEK inhibitors, it is possible that other changes that lead to similar degrees of MEK hyperactivation could cause a similar mode of resistance. For example, excessive upstream input from receptor tyrosine kinases (RTKs), RAS or RAF proteins, or other activators of MEK, could also potentially lead to MEK hyperactivation and result in similar resistance to BRAF or MEK inhibitors.Montagut et al identified elevated CRAF activity as a mechanism of resistance to the BRAF inhibitor AZ628 in pre-clinical studies . In AZ62Nazarian et al recently identified NRAS mutations as a mechanism of acquired resistance to the BRAF inhibitor PLX4032 . NRAS muMAP3K8. These data suggest that activation of MEK-ERK signaling by COT represents a novel RAF-independent mechanism of MEK activation. Interestingly, COT levels were observed to increase in cell lines treated with BRAF inhibitors, suggesting that COT may be involved with feedback regulation of MEK activity. Consistent with these findings, in the biopsies of two of three patients taken during treatment with PLX4032, COT transcript levels were elevated relative to pre-treatment biopsies. In one patient who was biopsied post-relapse, levels of COT transcript were elevated relative to pre-treatment and on-treatment biopsies, suggesting that COT may contribute to acquired resistance to BRAF inhibitors in the clinic. The authors also identified two cell lines with copy number gains at the MAP3K8 locus that expressed high levels of COT. These cell lines were resistant to PLX4720. Interestingly, even though COT appears to activate ERK through MEK, these cell lines and cell lines overxpressing exogenous COT were also resistant to MEK inhibitors. One possible explanation for this observation is that high COT levels might contribute to MEK inhibitor resistance by causing MEK hyperactivation, similar to the mechanism observed for BRAF amplification [Johannessen et al used an ORF expression library encoding approximately three-quarters of the human “kinome” to identify kinases that confer resistance to the BRAF inhibitor PLX4720 when they are overexpressed in sensitive BRAF V600E melanoma cell lines . Nine kification . AccordiAs discussed above, the majority of the resistance mechanisms to BRAF and MEK inhibition that have been identified lead to ERK reactivation and retain dependence on ERK signaling. While this underscores the importance of the RAF-MEK-ERK pathway in melanoma proliferation and survival, in the last few months several examples of resistance mechanisms have been reported that do not rely on sustained ERK signaling, indicating that other “ERK-independent” pathways can compensate for loss of ERK activity and can maintain the tumorigenicity of BRAF V600E melanoma in the absence of ERK activation.In the recent study by Nazarian et al, discussed above, of the three BRAF mutant melanoma cell lines made resistant to PLX4032, one resistant cell line was found to have acquired an NRAS mutation and maintained P-ERK levels despite the presence of BRAF inhibitor . In the Villanueva et al recently identified another RTK-driven resistance mechanism through in vitro modeling of BRAF inhibitor resistance in BRAF V600E melanoma cell lines . In cellBecause RTKs can signal through multiple RAF isoforms by activation of RAS proteins, and since RTKs activate multiple signaling pathways in addition to RAF-MEK-ERK, the authors investigated whether resistant cells showed differences in RTK phosphorylation relative to parental cells. More than one RTK exhibiting differential phosphorylation was identified by phospho-RTK array analysis, including IGF1R. However, pharmacologic inhibition of IGF1R decreased proliferation of resistant cells, and combined inhibition of IGF1R and MEK induced dramatic apoptosis, suggesting that ERK-independent survival signaling was mediated by IGF1R in resistant cells. While surface expression of IGF1R was found to be increased in resistant cells, the exact mechanism leading to increased IGF1R activation was not identified. In addition to activating the RAF-MEK-ERK pathway, IGF1R and other RTKs are known to activate PI3K-AKT signaling, which is known to be an important regulator of cell survival and proliferation , 47. ResTaken together, these two models of resistance driven by RTKs indicate that RTK-mediated resistance to BRAF or MEK inhibition highlights the complexity of signaling in resistant cells. Although in each resistance model, a dominant RTK was identified (e.g. PDGFRβ or IGF1R) that contributed to the majority of the signaling changes and to the decrease in drug sensitivity observed in resistant cells, there was evidence in each model that additional signaling pathways, perhaps involving other RTKs, were contributing to resistance. First, in each model, multiple RTKs were found to be elevated or to display increased phosphorylation, suggesting that other RTKs could contribute in some way to resistance. Second, in each model, inhibition of the dominant RTK, either using pharmacologic inhibitors or RNAi-mediated knockdown, was not sufficient to completely reverse all of the changes in signaling and sensitivity seen in the resistant cells.In the study by Nazarian et al, PDGFRβ knockdown blocked proliferation, but failed to induce apoptosis, even in the presence of BRAF inhibitor . FurtherOne potential strategy to overcome this problem would be to target common signaling nodes activated by RTKs in resistant cells, rather than attempting to target specific RTKs. In addition to activating RAF-MEK signaling, the PI3K-AKT pathway is a major signaling output of RTKs, and several studies have shown that inhibiting PI3K signaling in combination with RAF-MEK signaling is sufficient to induce apoptosis and suppress proliferation in RTK-driven cancer models -50. AccoWhile the recent clinical successes of BRAF and MEK inhibitors in BRAF mutant cancers are encouraging, many of the responses to therapy have been short-lived due to rapid development of acquired resistance . As a reHowever, in order to apply this basic therapeutic strategy most effectively, it would first be necessary to identify whether the mechanism driving a given patient's resistant tumor is ERK-dependent or ERK-independent. For this reason, routinely obtaining biopsies from recurrent tumors in patients treated with BRAF or MEK inhibitors will likely be important for the selection of the most appropriate therapy post-relapse, as has been done for acquired resistance to other targeted therapies . ParticuStill, while it might be feasible to screen tissue from resistant tumors for a small set of the most common individual alterations leading to resistance, the above studies demonstrate that resistance can arise through numerous specific molecular events. Therefore, especially as more specific mechanisms of resistance are defined, it may become unreasonable or even impossible to identify the specific change driving tumor resistance in every patient. As a result, an alternative approach might be to assess resistant tumor specimens for common indicators of ERK-dependent or ERK-independent resistance, perhaps by assessing biomarkers of RAF-MEK-ERK or PI3K-AKT signaling in a biopsy taken while the patient remains on treatment. For example, if P-ERK levels remain suppressed in a resistant tumor biopsy taken in a patient who remains on therapy , this fFinally, it is intriguing to speculate that anticipation of acquired resistance to BRAF or MEK inhibitors could lead to strategies to prevent resistance from emerging. Moreover, since it is possible for multiple distinct resistance mechanisms to arise in the same patient, employing combination strategies aimed at preventing resistance as part of a patient's initial therapy could have advantages. Recent studies have suggested that combined BRAF and MEK inhibition is more effective in treatment-naïve BRAF mutant cancers than treatment with either inhibitor alone , 54. Fur


Patients with cirrhosis of liver are at risk of developing serious bacterial infections due to altered immune defenses. Despite the widespread use of broad spectrum antibiotics, bacterial infection is responsible for up to a quarter of the deaths of patients with liver disease. Cirrhotic patients with gastrointestinal bleed have a considerably higher incidence of bacterial infections particularly spontaneous bacterial peritonitis. High index of suspicion is required to identify infections at an early stage in the absence of classical signs and symptoms. Energetic use of antibacterial treatment and supportive care has decreased the morbidity and mortality over the years; however, use of antibiotics has to be judicious, as their indiscriminate use can lead to antibiotic resistance with potentially disastrous consequences. Preventive strategies are still in evolution and involve use of antibiotic prophylaxis in patients with gastrointestinal bleeding and spontaneous bacterial infections and selective decontamination of the gut and oropharynx. Bacterial infections are a common, recurrent complication of cirrhosis associated with poor outcome . Decompeγ-receptor-mediated clearance of antibody-coated bacteria. Patients with alcoholic cirrhosis have depressed neutrophil phagocytic and intracellular killing of microorganisms [The mechanisms of increased susceptibility to infections in cirrhosis are unclear. Numerous mechanisms implicated in altered and diminished immunity include increased shunting of blood away from the liver, qualitative dysfunction of the reticuloendothelial system, decreased opsonisation capacity of the ascitic fluid, and increased intestinal permeability of bacteria and associated endotoxins . It has rganisms , 14.Escherichia coli, in about 60%, Gram-positive cocci (GPC) in about 30%–35%, and mixed in the last 5%–10%. Nosocomial infections behave differently with 60% GPC and 30%–35% positive for GNB, as a result of the use of therapeutic procedures and previous antibiotic therapies [Escherichia coli, the most frequently isolated bacteria are Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae. In cirrhotics less virulent organisms cause infections suggesting that, in advanced cirrhosis, bacteria do not need to develop strategies to circumvent host defenses and invade the host [Escherichia coli are the causative agents in spontaneous bacterial peritonitis (SBP) and urinary tract infections, Gram-positive bacteria (GPB) predominate in pneumonia (Streptococcus pneumoniae) and procedure-associated bacteremia (Staphylococcus aureus). Fungal infections especially Candida species are involved in up to 15% of severe sepsis in cirrhosis [The most common infections in cirrhotics are spontaneous bacterial peritonitis (SBP) (25%), followed by urinary tract infection (20%), pneumonia 15%), bacteremia following a therapeutic procedure, cellulitis, and spontaneous bacteremia [5%, bactethe host . While Girrhosis .Most of the available information on bacterial infections in cirrhosis refers to SBP, an entity that is essentially unique to the cirrhotic patient and shares its pathogenesis and management with spontaneous bacteremia and spontaneous bacterial empyema. Gram-positive infections in cirrhosis such as pneumonia or secondary bacteremia are managed according to conventional criteria.Spontaneous bacterial peritonitis (SBP), that is, spontaneous infection of ascitic fluid without any apparent intra-abdominal source of infection, is the most characteristic infective complication in cirrhosis . The one3, independent of ascites bacteriological culture results [3, and, therefore, although median sensitivity results are ~85%, there is lack of sufficient data for its use in clinical practice unless larger trials validate these observations.As SBP may pass unrecognized, diagnostic paracentesis should be done in all cirrhotics with ascites on admission to hospital, in-patients with ascites who develop signs of sepsis, hepatic encephalopathy, renal impairment, or altered gastrointestinal motility, and all ascitic patients with a gastrointestinal bleed . SBP is  results . The use results –32. MostCefotaxime is the most widely studied cephalosporin in patients with SBP and is suitable for empirical therapy for this condition. Prior to 1985, treatment of the condition was suboptimal. A landmark study comparing the combination ampicillin/tobramycin with cefotaxime showed that cefotaxime significantly increased the resolution of bacterial infections, including SBP in cirrhotic patients . FollowiThe gut appears to be the main source of bacteria that cause SBP and other Gram-negative infections in cirrhosis. Given that SBP is thought to result from the translocation of enteric GNB, the ideal agent should be safe, affordable, and effective at eliminating GNB from the gut while preserving the protective anaerobic flora. Bacterial translocation, the phenomenon by which viable microorganisms from the intestinal lumen migrate to mesenteric lymph nodes and other extraintestinal sites, has been postulated as one of the main mechanisms in the pathogenesis of these infections. Therefore, prophylaxis has been based on the oral administration of nonabsorbable or poorly absorbed antibiotics that will eliminate or reduce the concentration of Gram-negative gut bacteria without affecting Gram-positive organisms or anaerobes, the so-called selective intestinal decontamination. Given the high cost and inevitable risk of developing resistant organisms, the use of prophylactic antibiotics must be strictly restricted to those at highest risk of SBP.Staphylococcal infections due to methicillin-resistant strains [Long-term administration of orally administered norfloxacin, a poorly absorbed quinolone, has been shown to produce a marked reduction of GNB from the fecal flora of cirrhotic patients with no significant effects on GPC or anaerobic bacteria . The dev strains . TherefoThree patient populations considered at high risk and in whom prophylactic antibiotic therapy has been recommended are patients with prior history of SBP, patients admitted with gastrointestinal bleed, and patients with low total protein content in ascitic fluid. The 1-year and 2-year probabilities of survival after an episode of SBP are of 30–50% and 25–30%, respectively, . TherefoAll cirrhotic patients who develop an upper gastrointestinal bleed are at risk of a variety of bacterial infections, including SBP, within the first few days following the bleed. Bacteria of enteric origin are most commonly implicated, and the development of infection is associated with a poor prognosis –47. AmonAscitic fluid total protein has been shown to be an independent predictor of SBP. The risk of developing SBP in these patients depends largely on ascites protein content. Patients with an ascites protein >1.0 g/dL will not develop SBP in a follow-up period of 2 years, while patients with a low (<1.0 g/dL) ascites protein have a 1-year probability of developing SBP of around 20%. A prospective study in cirrhotic patients during hospitalization found that 15% of patients with ascitic protein <1.0 g/dL developed SBP compared to 2% of those with ascitic protein >1.0 g/dL. The incidence was greatest in those with Child C liver disease and in those who did not receive short-term prophylaxis if admitted with a gastrointestinal bleed. Two non-placebo-controlled studies, which showed a benefit of antibiotic prophylaxis in patients with low ascites protein, included patients with and without prior episodes of SBP and cannot be considered as reliable determinants of primary prophylaxis , 53. OraIn patients with SBP, there is a risk that their systemic hemodynamic parameters can deteriorate, with further arterial and splanchnic vasodilatation. These patients are, therefore, at high risk of developing renal insufficiency . The devLactobacillus has been reported to correct bacterial overgrowth, stabilize mucosal barrier function, and decrease bacterial translocation in rat models of acute liver injury and failure. However, the administration of Lactobacillus acidophilus- and Lactobacillus GG-fermented diets to animals with portal hypertension and cirrhosis failed to show any reduction in bacterial translocation or in ascites infection rates [Recent research in the use of certain probiotic agents has shown promise in decreasing cytokine release and improving neutrophil function in cirrhotic patients , 62. Theon rates , 64.Lactobacillus plantarum 299 and fiber had less posttransplant infections than groups receiving selective bowel decontamination. The second study used Synbiotic 2000 in post-OLT patients for 14 days and also found a lower 30-day infection rate. Importantly, no serious adverse effects were noted in either study. As it is a cheap and feasible alternative to selective intestinal decontamination, further studies are needed to evaluate the effect of this combination in other cirrhotic populations.Two prospective randomized studies demonstrated the efficacy of probiotics in reducing postorthotopic liver transplantation (OLT) infections , 66. In Urinary tract infections are the most frequent infective complications in cirrhosis. As in the noncirrhotic population, cirrhotics with indwelling catheters are highly predisposed to develop urinary tract infections. The incidence is markedly higher in female than in male cirrhotics . Urinary Streptococcus pneumonia being the causative organism in most lower respiratory tract infections [Haemophilus influenzae, or by Gram-negative bacilli, particularly Klebsiella pneumoniae, mycoplasma and legionella species [Pneumonias are the third most common infections in patients of cirrhosis after SBP and urinary tract infections. Community-acquired infections are the most frequent, although hospitalized patients admitted to intensive care units have high incidence of nosocomial pneumonias due to predisposing factors such as tracheal intubation, esophageal tamponade, or hepatic encephalopathy. Alcoholics are predisposed to chest infections,fections . A signi species –72. AntiHospital-acquired pneumonia is predominantly caused by Gram-negative bacilli and staphylococci , 72. AltStaphylococcus aureus and Streptococcus pyogenes are the most frequent causative organisms [Soft tissue infections, particularly lymphangitis of the lower extremities and abdominal wall, are relatively frequent in cirrhotic patients with ankle edema or ascites. rganisms . EmpiricStreptococcus pneumoniae, Escherichia coli, and Listeria are the commonest pathogens implicated. Signs of meningeal irritation including nuchal rigidity may be a delayed or even absent clinical sign. Mortality is significantly high and may reach up to 80% in Child-Pugh stage C [More commonly reported in alcoholic cirrhosis with high overall is one month case fatality rate exceeding 50%.  stage C , 76. Patients with hepatic dysfunction have an increased risk for bacteremia and sepsis . Bacteri Given the degree of immune dysfunction and the morbidity of infections, patients with significant cirrhosis who present with, or with probable, bacteremia or sepsis should undergo rapid diagnostic testing and should receive intravenous antibiotics that treat the likely organisms as soon as possible. In septic patients, early antibiotic initiation with the appropriate agents significantly improves outcomes, and this effect is especially important in immune-compromised patients , 81. These infections are common in critically ill patients with cirrhosis. These patients may benefit from appropriate hand hygiene, use of chlorhexidine for skin preparation, use of full-barrier precautions during the insertion of central venous catheters, use of the subclavian vein as the preferred site for insertion of the catheter, and the removal of unnecessary central venous catheters .Patients with chronic liver diseases sustain impairment to their immune systems, which worsens over time and with disease progression. These defects in their host defense lead to augmented risks of bacterial infections and increased morbidity when they are incurred. Providers caring for patients with hepatic dysfunction should have a heightened surveillance for infectious diseases and suspect that one is present with any acute change in a patient's status. With early diagnosis and proper antibiotic treatment, the mortality of bacterial infections has decreased significantly over the years.


The criminal justice system bears a disproportionate burden of the HIV epidemic. Continuity of care is critical for HAART-based prevention of HIV-related morbidity and mortality. This paper describes four major challenges to successful management of HIV in the criminal justice system: relapse to substance use, homelessness, mental illness, and loss of medical and social benefits. Each of these areas constitutes a competing priority upon release that demands immediate attention and diverts time, energy, and valuable resources away from engagement in care and adherence to HAART. Numerous gaps exist in scientific knowledge about these issues and potential solutions. In illuminating these knowledge deficits, we present a contemporary research agenda for the management of HIV in correctional systems. Future empirical research should focus on these critical issues in HIV-infected prisoners and releasees while interventional research should incorporate evidence-based solutions into the criminal justice setting. The criminal justice setting provides vast opportunities for early diagnosis, prevention, and treatment of HIV , 2. One Correctional facilities thus bear a disproportionate burden of the HIV epidemic in the USA with a prevalence rate of HIV 3–5 times higher than surrounding communities . Though Despite these obstacles, management of HIV in correctional settings has been shown to be feasible, acceptable,  and highUnfortunately, benefits of prison-based HAART are rarely sustained upon return to communities , 20, 21.There is a paucity of data evaluating barriers to sustained antiretroviral therapy for HIV-infected persons transitioning from the CJS to the community. In this review, we outline major obstacles to continuous HIV care among populations who interface with the CJS, both during incarceration and after release. Major challenges include relapse to substance use, housing instability, comorbid mental illness, and coverage gaps in medical and social benefits. We briefly describe the significance of each of these issues in general CJS or in HIV-infected nonincarcerated populations and then explore potential areas for future empirical and interventional research. Rigorous scientific evaluation of these “knowledge gaps” is critical for progress in the field. Furthermore, success of programs designed to facilitate transition between prisons and communities hinges on bringing evidence-based solutions into the criminal justice setting.HIV, HAART, adherence, outcomes, prisoners, prison/jail, incarcerated, inmates, criminal justice, corrections, transition, linkage, and released. These were combined with terms relevant to each topic of interest: substance, addiction, drugs, alcohol, dependence/use/abuse, homelessness, housing, mental illness, depression, insurance, Medicaid, and benefits. Continuity of care was defined in terms of HAART adherence and enrollment in HIV care upon release. Additional references from seminal papers were reviewed to broaden the search and assure that important contributions were not overlooked. Articles were included if they were written in English and were published in peer-reviewed journals between 1990–2011.A search strategy was undertaken using PubMed, OvidSP, and MEDLINE. The following key terms were used: Drug use and crime are inextricably linked. In the United States, where the “war on drugs” has devolved into a “war on drug users,”  drug-relSUDs have a seemingly profound impact on the health outcomes of HIV-infected prisoners, for whom addiction-related lapses in HAART continuity threaten virologic suppression after release. With some notable exceptions described here, there have been relatively few published studies regarding the effect of SUDs on HIV outcomes in released prisoners. A 2008 study of HIV-infected reincarcerated jail detainees examined HAART adherence between the time of prison release and reincarceration. Findings revealed that those subjects who discontinued HAART were three times more likely to have also relapsed to marijuana or injection drug use between incarcerations . Among HExtrapolating from studies of community-based subjects with HIV outside of the CJS, alcohol use is associated with decreased initiation of and adherence to HAART as well as increased rates of virologic failure . SimilarDespite overwhelming demonstrated need, substance abuse treatment programs during incarceration are relatively nonexistent . As few  Treatment of SUDs is clearly beneficial for relapse prevention but may also improve engagement in HIV care. Indeed, two recent interventions , 47 haveSubstance use disorders are often concurrent and synergistic with mental health disorders and it may be difficult to disentangle the two issues. Over three-quarters of jail inmates with mental health disorders meet criteria for substance dependence or abuse . Mental Little is known about the effects of mental health disorders on HIV outcomes in dually diagnosed persons in the CJS. In non-CJS settings, however, treatment of psychiatric comorbidities has a profound impact on adherence to antiretroviral therapy. Several studies have examined the association of depression with HAART nonadherence in community-based cohorts. Among HIV-infected women, depression has been associated with decreased virologic response to HAART, higher likelihood of immunologic failure, and higher risk for all-cause mortality . The lonFurthermore, relatively few studies have examined associations between serious mental illness (SMI) and HAART adherence despite significant epidemiologic overlap between HIV and SMI. SMI includes the diagnoses of schizophrenia, schizoaffective disorder, bipolar depression, and major depression with psychotic features. Adherence to psychiatric medications among SMI patients ranges between 40–60%, suggesting that adherence to HAART in these populations might also be problematic . This haManagement of mental illness in correctional settings is an understudied but critical issue for people living with HIV. Among depressed HIV-infected patients in a non-correctional managed care setting, those who were treated with selective serotonin reuptake inhibitors for depression were more likely to be >95% adherent to HAART medications than those with untreated depression . In addiUnfortunately, even if inmates are engaged in psychiatric care during incarceration, treatment of mental illness in the CJS is often disrupted after release to communities. Upon initial release, suicide is common among former inmates, ranking among the top 5 causes of death in one study of released prisoners in Washington state and indicating inadequate treatment of mental health disorders during this critical transition period . System-Methods to coordinate psychiatric services upon release from correctional settings are necessary. Formal case management services that enhance linkages to psychiatric care may secondarily improve adherence to HAART, and research is currently ongoing in this field . Other sA relative lack of mental health services in the community combined with the “War on Drugs” has resulted in the shuttling of mentally ill persons between homelessness and incarceration. Incarceration increases the risk of homelessness through loss of employment, loss of housing, and disruption of social support or community resources , 62. ConAmong PLWHA, homelessness and unstable housing have been associated with poor HIV outcomes including worse adherence to HAART, fewer ambulatory care visits, and increased risk of death –70. A caThe effects of homelessness on HAART adherence are often confounded by the presence of comorbid mental illness or SUDs. Homeless or unstably housed individuals are less likely to be adherent to HAART if they have recent substance use or higher depression scores . A randoAlthough these studies have demonstrated the negative impact of homelessness on HAART adherence, there is no scientific literature concerning the impact of homelessness on recently incarcerated, HIV-infected populations. Given the high prevalence of homelessness after release from prison and the demonstrated beneficial effects of housing on social, psychiatric, addiction and HIV-related outcomes, future research should focus on correlates of risk of homelessness among recently-released prisoners and jail detainees.Obtaining housing constitutes a top priority often expressed by HIV-infected released prisoners. Many prisoners return to communities plagued by poverty, unemployment, and violence—the same communities from which they were incarcerated. They must attempt to navigate this environment while simultaneously managing the more personal stresses of reuniting with parents, partners, and children after incarceration. Ideally, these relationships would support positive health promotion. All too often, however, these relationships are afflicted with addiction and interpersonal violence that only exacerbate stress and interfere with health-seeking behaviors. Returning prisoners must also attempt to manage comorbid medical illnesses, like diabetes and hypertension, which can become immediately life-threatening when undertreated, especially in the context of homelessness. These overlapping stresses  may be important to individuals' health after prison release and likely contribute to prison recidivism. Unfortunately, there have been few published studies to date around homelessness and HIV-related continuity of care after release, thus limiting our discussion on these topics. Future studies should address the impact of homelessness and social instability, as more broadly defined, on healthcare utilization patterns of PLWHA after release from prison.Despite the strong association between homelessness and incarceration, strategies addressing the housing needs of HIV-infected inmates upon release have not been well studied. A possible strategy that may improve comorbid substance use or psychiatric disorders as well as recidivism is a Housing First approach. Contrary to traditional substance abuse or mental health treatment programs which require strict adherence to treatment in order to maintain housing, Housing First programs provide immediate access to permanent housing without any prerequisites for psychiatric treatment or sobriety. Among homeless or marginally housed persons with serious mental illness, Housing First programs have been successful in improving residential status and decreasing use of inpatient, emergency, and criminal justice system services , 74. AltOther possible interventions to improve HIV outcomes among homeless or marginally housed releasees involve improving engagement to care through increased supportive services. In San Francisco, a clinic specializing in supporting recently released inmates in the transition back to the community was noted to successfully engage a population in which 38% were homeless . The cli Fortunately, homelessness is a modifiable risk factor that could be prevented with effective discharge planning services. Transitional case management programs should proactively address housing instability as part of a comprehensive package of social services, recognizing the extreme vulnerability during this period of movement between prisons and communities. Provision of housing is, like many of the other issues addressed here, not a stand-alone problem.In the current USA healthcare system, where medical care is predominantly funded by third-party payors, gaps in insurance coverage during incarceration contribute to fragmentation of care upon release. By virtue of their socioeconomic or medical disability status, most inmates with health insurance have Medicaid prior to incarceration. Medicaid prohibits any use of federal funds for medical or psychiatric care of inmates in jails and prisons. Most states (>90%) terminate Medicaid coverage during incarceration . For thoAs a result, many inmates are left with gaps in medical and social benefit coverage upon release from prison. Reapplication procedures for Medicaid can be initiated as early as 45–90 days prior to expected release  but requFor HIV-infected released prisoners, continuity of medical coverage is especially crucial for continued provision of HAART. In the past, coverage for antiretrovirals was dismal: in a 1994 study, 19% of HIV-infected prisoners reported trying to get rearrested just to obtain prison-based HIV care that was seemingly not available to them in the community after release . FortunaBeyond the availability of medication assistance programs, there remains a significant knowledge gap about why and how released prisoners experience decreased persistence in HIV care. Certainly the problem is multifactorial and complex: this population is often marginalized from the mainstream medical community because of active drug use, mental illness, or HIV-related stigma prior to and following incarceration. Even in Canada, in which a nationalized healthcare system removes significant insurance-related barriers to care, released prisoners report problems obtaining appointments with an HIV clinician after return to the community . These aIn spite of significant safety nets for people living with HIV, there remain gaps in medical coverage after release that present barriers to continuous HIV care. These should be addressed by future interventions. Delays in completing and processing ADAP applications are a limiting factor in HIV-infected recently released prisoners benefiting from the program. In a cross-sectional mixed methods study of 105 HIV-infected recently released prisoners in Florida, 19% reported that the paperwork required to obtain medical care, insurance, and HAART presented a major obstacle to care . Of 177 Each year, almost 10 million people are released from USA jails and prisons to the community , 44. A sThe numerous outstanding knowledge gaps and solution gaps concerning HIV outcomes in criminal justice populations also present exciting academic opportunities for future research. We have attempted to shed light on these gaps in support of a contemporary research agenda. Evidence-based examinations of the problems and solutions facing criminal justice populations are critical to progress in the management of HIV in correctional systems.Our review of the literature suggests that linkages from prison-based to community HIV care are critical for maintaining the benefits of HAART achieved during incarceration. There is ongoing debate in the criminal justice field about how best to design transitional interventions to address these complex issues that otherwise disrupt care for returning prisoners. Options proposed thus far include intensive case management, supportive housing, opioid substitution therapy, directly administered antiretroviral therapy, medication adherence support, or combinations therein. We advocate that, regardless of methodology, the most effective interventions require significant breadth to address all of the barriers that manifest during transfer from prison to the community. Unfortunately, there are significant logistical constraints to introducing evidence-based interventions into correctional systems either during incarceration or prior to release. Inmates are often transitory, cycling frequently and rapidly between jails and communities or among various correctional facilities. This migratory pattern provides a narrow window in which to introduce and assess any given intervention. Constraints may also be imposed by a correctional system itself that enforces strict privacy protection measures  and is subject to state, or federally based budgetary limitations. Even outside prison walls, and thus, technically outside of correctional systems, released prisoners may be difficult subjects in longitudinal studies because they often experience high rates of attrition. Strategies for collaboration between academic centers of research and correctional systems have been recently proposed elsewhere .Despite these challenges, empirical and interventional research is strongly needed to improve the management of HIV both during and following incarceration. Future research must work to develop geographically and culturally appropriate interventions that are highly tailored to individual needs. Perhaps most difficult, future research should attempt to deconstruct structural barriers to persistence in HIV care that include termination of medical/social benefits during incarceration, minimal linkages to community-based psychiatric and HIV primary care, and often-absent treatment for substance use disorders. As providers and clinical researchers, we must act as advocates for this often silenced and marginalized population living with HIV.


Pluripotent stem cells, i.e., embryonic stem (ES) and induced pluripotent stem (iPS) cells, can indefinitely proliferate without commitment and differentiate into all cell lineages. ES cells are derived from the inner cell mass of the preimplantation blastocyst, whereas iPS cells are generated from somatic cells by overexpression of a few transcription factors. Many studies have demonstrated that mouse and human iPS cells are highly similar but not identical to their respective ES cell counterparts. The potential to generate basically any differentiated cell types from these cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. ES cells and iPS cells also provide useful models to study connexin expression and gap-junctional intercellular communication (GJIC) during cell differentiation and reprogramming. In 1996, we reported connexin expression and GJIC in mouse ES cells. Because a substantial number of papers on these subjects have been published since our report, this Mini Review summarizes currently available data on connexin expression and GJIC in ES cells and iPS cells during undifferentiated state, differentiation, and reprogramming. Gap junctions are cell–cell communicating junctions that consist of multimeric proteins called connexins and mediate the exchange of low-molecular-weight metabolites and ions between contacting cells  and by microinjection of small-molecular-weight fluorescent dyes such as Lucifer yellow into a single cell and observation of the subsequent dye spread into the surrounding cells (dye coupling)  cells and induced pluripotent stem (iPS) cells, possess the ability to proliferate indefinitely without commitment in vitro differentiation system using ES cells and iPS cells also provides a useful model to study connexin expression and GJIC during the early stage of cell differentiation ?What roles do connexin expression and/or GJIC play in reprogramming?Main questions about connexin expression and GJIC in ES/iPS cells that have been addressed thus far can be summarized as below:Table Table de novo within each differentiated cell type in vitro, forming communication compartments within a histotype. Human iPS cells they utilized were derived from human umbilical vein endothelial cells (HUVECs) by lentiviral transfection with four transcription factors: KLF4, OCT4, SOX2, and C-MYC. To evaluate changes in GJIC during late stages of reprogramming, incompletely reprogrammed endo-iPSC10 cells at passage 6 and completely reprogrammed cells of the same line at passage 26 were studied. Incompletely reprogrammed iPS cells were characterized by residual expression of endothelial-specific genes including Cx37 and reduced expression of pluripotency-related genes. In addition, they compared expression of connexins in HUVEC, endo-iPS-10, 12, and human ES cells and found that only Cx37 and Cx43 expression varied significantly in the examined cell types. In incompletely reprogrammed iPS cells, Cx37 and Cx43 were expressed at the level similar to HUVEC. In faithfully reprogrammed iPS cells, cells lacked characteristics of parental HUVEC Cx37 expression, whereas Cx43 expression increased three- to five-fold.Using human iPS cells, Sharovskaya et al.  reportedKe et al.  demonstrTaken together, these results suggest that Cx43 may represent a pluripotency marker of iPS cells and may play an important role in the reprogramming process.in vitro. They have shown that these human ES cell- and iPS cell-derived cardiomyocytes are capable of maturing to a phenotype that more closely resembles adult cardiomyocytes in both structure and function. A robust induction of key cardiac structural markers including Cx43 has been demonstrated in late-stage ES cell- and iPS cell-derived cardiomyocytes. These findings suggest that ES cell- and iPS cell-derived cardiomyocytes are capable of slowly maturing to more closely resemble the phenotype of adult cardiomyocytes and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.Lundy et al.  recentlyIt seems reasonable to conclude that mRNAs encoding almost all of the connexins are expressed in ES/iPS cells. At protein level, however, expression of only a few connexins, such as Cx43, Cx45, Cx31, and Cx40, has been confirmed. Many studies have shown that undifferentiated ES/iPS cells communicate with each other via gap junctions at a high level. Several studies using Cx43 RNAi demonstrated that Cx43 contributes substantially to a high level of GJIC in undifferentiated ES/iPS cells.Concerning changes in connexin expression and GJIC during differentiation of ES/iPS cells, it has been shown that expression of tissue-related connexins, such as Cx40, Cx43, Cx45, and Cx37 in the cardiomyocyte, is upregulated and that GJIC between pluripotent and differentiated cells is disrupted, resulting in formation of “communication compartments.” Regarding changes in connexin expression and GJIC during induction of pluripotency in somatic cells, the studies mentioned here have demonstrated that GJIC is re-established and Cx43 expression is upregulated during reprogramming to pluripotency.Cx43-mediated GJIC has been shown to play an important role in maintenance of pluripotency. In fact, pharmacological blockers of GJIC and Cx43 downregulation by siRNA have been shown to induce a loss of their pluripotent state in mouse ES cells (Todorova et al., Because the literature on connexin expression and GJIC in iPS cells is limited, it is difficult to conclude whether there are differences between ES and iPS cells at present. However, available data suggest that human ES and iPS cells share a similar feature concerning expression and function of connexins.Although important roles of connexin expression and/or GJIC in ES/iPS cells can be currently perceived, many critical questions including precise mechanisms by which connexin expression influences pluripotency and reprogramming remain to be clarified.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 vitro and animal studies have shown that malignant cell lines and tumors induced by exposure to inorganic arsenic include more cancer stem cells than other tumor types. Arsenic is known to transform normal stem cells into cancer stem cells, but a new study shows that arsenic-induced malignant cells may do so even without direct arsenic exposure . This may explain why arsenic-induced tumors contain so many cancer stem cells.Millions of people are exposed to inorganic arsenic, a human carcinogen, in drinking water. In normal tissues, stem cells are the source of replacements for damaged or dead cells. Similarly, cancer stem cells are a source of new malignant cells that allow tumors to grow and spread, and they are integral to tumor initiation, progression, and metastasis. The cells’ origins and formation are not entirely clear, but evidence suggests they may transform from normal stem cells under the influence of inflammatory cytokines and other factors.With the prostate identified as a possible target tissue of arsenic, the authors of the current study created malignant epithelial cells (MECs) by exposing a normal human prostatic epithelial cell line, RWPE-1, to sodium arsenite. The RWPE-1 line was also used as the source for normal stem cells, which were co-cultured with either control RWPE-1 cells or MECs. A semipermeable membrane between the cell types in the co-culture system prevented direct cell-to-cell contact but allowed secreted factors to pass freely between cells. The system contained no detectable arsenic.During the weeks of co-culture and afterward, the investigators assessed the development of a cancer stem cell phenotype, which was defined by the secretion of matrix metalloproteinases (MMPs). Cellular gene expression was assessed at both the transcription and translation levels to determine which factors were involved in the changed phenotype. Finally, normal stem cells cultured alone were treated with interleukin-6 (an inflammatory cytokine secreted by MECs) and then analyzed for MMP activity and gene expression.PTEN tumor suppressor gene expression decreased, similar to previous observations in prostatic cancer stem cells. Changing patterns of gene expression over time, including early loss and later reactivation of several genes, were consistent with changes observed in cancer stem cells during oncogenesis. Similar MMP and gene-expression changes were induced in normal stem cells cultured alone and treated with interleukin-6, suggesting that inflammatory cytokines produced by malignant cells might cause the transformation to cancer stem cells.Test results indicated that normal stem cells in noncontact co-culture with MECs developed a cancer stem cell phenotype. MMP activity and invasive capacity increased, while The results suggest that MECs may recruit normal stem cells to become cancer stem cells, potentially affecting the growth and spread of cancer, and that interleukin-6 may be one of the recruitment signals. It is unknown whether MECs transformed by carcinogens other than arsenic have a similar ability to alter normal stem cells; more investigation of this mechanism is needed.


Although the role of thyroid hormone during embryonic development has long been recognized, its role later in adult life remains largely unknown. However, several lines of evidence show that thyroid hormone is crucial to the response to stress and to poststress recovery and repair. Along this line, TH administration in almost every tissue resulted in tissue repair after various injuries including ischemia, chemical insults, induction of inflammation, or exposure to radiation. This novel action may be of therapeutic relevance, and thyroid hormone may constitute a paradigm for pharmacologic-induced tissue repair/regeneration. Although the role of thyroid hormone (TH) during development has long been recognized, its role later in adult life remains largely unknown . A growiThe most important challenge that living organisms faced during species evolution was the ability to adapt to the transition from the aquatic environment, a condition of low oxygen, to the ground, an oxygen-rich state. This required a gene programming that would enable organ protection and remodeling during this transition. Interestingly, studies on amphibians revealed that thyroid-hormone-regulated gene programming is critical for the metamorphosis of tadpoles into juvenile frogs . SeveralThe potential role of thyroid hormone in stress response has been, until now, underestimated. However, thyroid hormone signaling is altered during various stressful stimuli and thyroid hormone is crucial to poststress recovery and injury repair –9. InterAccumulating experimental evidence shows that thyroid hormone may play a critical role in the repair after injury in almost every tissue and organ as shown in Thyroid hormone appears to be a common player for the organ development and response to stress. Thyroid hormone was crucial for species evolution, and thyroid-hormone-regulated mechanisms have been evolutionary conserved and play an important role early during development. However, recent research has revealed that thyroid hormone has a reparative role later in adult life. This novel action may be of therapeutic relevance, and thyroid hormone may constitute a paradigm for pharmacologic induced tissue repair/regeneration.