This process culminates in a heterogeneous tumor, consisting of multiple subpopulations of cancer cells that often do not contain the same underlying mutations. Continuous selective pressure permits outgrowth of clones that harbor lesions that are capable of enhancing disease progression, including those that contribute to therapy resistance, metastasis and relapse. Clonal evolution and the resulting intratumoral heterogeneity pose a substantial challenge to biomarker identification, personalized cancer therapies and the discovery of underlying driver mutations in cancer. The purpose of this Review is to highlight the unique strengths of zebrafish cancer models in assessing the roles that intratumoral heterogeneity and clonal evolution play in cancer, including transgenesis, imaging technologies, high-throughput cell transplantation approaches and  These data sets have provided a rich resource for identifying putative drivers of tumor progression. However, these experiments often identify hundreds of candidate oncogenes and tumor suppressors, and cannot differentiate between driver lesions that are required for tumor initiation, progression or continued growth and passenger mutations that impart no selective advantage to the cancer cell. Cross-species comparisons between tumor subtypes can narrow the list of potential drivers because genetic mutations that are common to cancers arising in diverse species are more likely to contribute to tumor progression. Over 70% of human protein-coding genes are related to zebrafish genes , yet, be disease . aCGH an disease . In thisand phf6 . Cross-sand phf6 . In parttp53-deficient melanoma , TALENs (transcription activator-like effector nucleases) and CRISPR-Cas  induce targeted mutations in genes through the generation of double-strand breaks and error-prone repair by non-homologous end joining . It was xP sites . Unlike xP sites , which wxP sites , which a architecture of the tumor, pattern of heterogeneity and tumor microenvironment are maintained. In this regard, heterogeneous zebrafish tumors can be transplanted into multiple recipients, which allows for large-scale in vivo multi-drug screens against genotypes of interest  and the appropriate oncogene into a Zebrabow zebrafish strain can therefore be used to generate a tumor in which different clones are individually fluorescently labeled. Using fluorescent imaging techniques, tumors can then be followed in vivo to assess clonal expansion and competition. Subsequent drug treatment of these animals, as well as careful genetic analysis of clones before and after drug treatment, will provide insight into how clones within a heterogeneous tumor respond to and become resistant to therapy over time.Although recapitulating heterogeneity through cell transplantation has its merits, an ideal system would allow for direct visualization of intratumoral heterogeneity, longitudinal analysis of clonal dominance over time, and real-time analysis of the effects of drug treatment on clone number and type. Zebrabow technology, developed from the Brainbow system used to label mouse neurons , has theproteins . These cproteins . Co-injein vivo screening tool that can both complement and focus studies in mouse models and human cells. Understanding intratumoral heterogeneity will provide crucial biomarkers to predict cancer progression, therapy response and relapse. The ultimate hope is that identifying mutations associated with clonal heterogeneity and disease progression will lead to new personalized medicine approaches in which treatments will be tailored to the unique spectrum of mutations that define the individual clones within a patient’s tumor.Clonal evolution and intratumoral heterogeneity have far-reaching clinical consequences, yet research examining this important issue is still in the early stages. Next-generation sequencing data from longitudinal patient samples will continue to be crucial, but research must also be extended into more extensive characterization of the mechanisms of clonal evolution, the identification of dominant drivers and the acquisition of drug resistance in the context of intratumoral heterogeneity. Transgenesis, transplantation assays, single-cell functional assays and direct imaging studies using zebrafish cancer models can serve as an economical, large-scale


In this review article we discuss current knowledge about iron in the skin and the cutaneous wound healing process. Iron plays a key role in both oxidative stress and photo-induced skin damage. The main causes of oxidative stress in the skin include reactive oxygen species (ROS) generated in the skin by ultraviolet (UVA) 320–400 nm portion of the UVA spectrum and biologically available iron. We also discuss the relationships between iron deficiency, anemia and cutaneous wound healing. Studies looking at this fall into two distinct groups. Early studies investigated the effect of anemia on wound healing using a variety of experimental methodology to establish anemia or iron deficiency and focused on wound-strength rather than effect on macroscopic healing or re-epithelialization. More recent animal studies have investigated novel treatments aimed at correcting the effects of systemic iron deficiency and localized iron overload. Iron overload is associated with local cutaneous iron deposition, which has numerous deleterious effects in chronic venous disease and hereditary hemochromatosis. Iron plays a key role in chronic ulceration and conditions such as rheumatoid arthritis (RA) and Lupus Erythematosus are associated with both anemia of chronic disease and dysregulation of local cutaneous iron hemostasis. Iron is a potential therapeutic target in the skin by application of topical iron chelators and novel pharmacological agents, and in delayed cutaneous wound healing by treatment of iron deficiency or underlying systemic inflammation. Iron is a vital co-factor for proteins and enzymes involved in energy metabolism, respiration, DNA synthesis, cell cycle arrest and apoptosis. Over the past 10 years, major advances have been made in understanding the genetics of iron metabolism and this has led to identification of a number of new proteins, including hepcidin, an acute phase protein that is the master regulator of iron absorption and utilization, often activated in chronic diseases .Historically, it has long been known that iron is essential for healthy skin, mucous membranes, hair and nails. Clinical features of iron deficiency include skin pallor, pruritus, and predisposition to skin infection , angular chelitis, swollen tongue, fragile nails, kolionychia, and dry brittle hair.Figure 1). Iron is not actively excreted from the body, however the skin is a key organ in iron hemostasis as iron is lost through the skin by desquamation . Current theories regarding the underlying mechanisms of desquamation include active dissolution of desmosomes involved in keratinocyte cell–cell adhesion, by hydrolytic protease digestion  and Fe3+ (electron acceptor). Intracellular labile iron can undergo redox cycling between its most stable oxidation states (Fe2+/Fe3+) and react with ROS such as superoxide anion, hydrogen peroxide, giving rise to hydroxyl radicals via the Fenton reaction or superoxide-driven Fenton chemistry (The main causes of oxidative stress in the skin are reactive oxygen species (ROS) generated in the skin by ultraviolet (UVA) 320–400 nm portion of the UVA spectrum. Iron plays a key role in oxidative stress processes, as it is a transition metal, which exists in two stable states, Fehemistry .Exposure of skin fibroblasts to UVA can generate ROS that promote oxidative damage in lysosomal, mitrochondrial, nuclear, and plasma membranes. Ultimately loss of plasma membrane integrity together with mitrochondial ATP depletion results in necrotic cell death . It is tFigure 3.Wound healing is a dynamic and highly regulated process consisting of cellular, humoral and molecular mechanisms . The norIn current literature, animal studies fall into two distinct groups. Early studies investigating the effect of anemia on wound healing used a variety of experimental methodology to establish anemia or iron deficiency. They focused on wound strength studies rather than initial macroscopic healing or histological studies of re-epithelialization. More recent studies have investigated novel treatments aimed at correcting the effect of systemic iron deficiency and topical application of iron-chelators to reduce iron at the specific site of inflammation and in particular their effect on pro inflammatory macrophages.Early initial experimental rodent studies used powdered milk diet to establish iron deficiency. 2) in rabbits made anemic by bleeding and re-transfusing plasma. Additionally, connective tissue weight was greater at lower packed cell volumes, an initial finding that suggested a deleterious effect of hypoxia on collagen synthesis.Investigation into the impact of anemia and blood volume on wound healing strength by There are various mechanisms by which iron deficiency may impair wound healing. Current evidence favors a key role played by hypoxia. Hypoxia-inducible factor-1 (HIF-1) contributes to all stages of wound healing  and positive regulators of HIF-1, such as prolyl-4-hydroxylase inhibitors, have been shown to be beneficial in enhancing diabetic healing . Furtherin vitro study. Recent interest in lactoferrin, an iron-binding glycoprotein secreted from glandular epithelial cells, has focused on its role in promoting cutaneous wound healing by enhancing the initial inflammatory phase, and cell proliferation and migration. Of note, the functional role of iron in the wound healing process has not undergone detailed Table 1. Different iron chelators have been applied in studies using a variety of wound healing models.There is considerable variability in iron chelator structure, mechanism of action and their consequent applications. The most widely used iron chelator used to treat iron over-load is deferoxamine, see Early studies of porcine flap necrosis found that intramuscular injection of deferoxamine decreased the percentage of flap necrosis . This stIron chelation results in increased VEGF and HIF 1-α and positive effect on angiogenesis The effect of iron chelation on granulation tissue formation and angiogenesis has not been demonstrated in cutaneous wound healing studies, although there have been some studies of bone tissue in the context of fracture healing . LocalizThe incorporation of iron chelators in novel wound dressing for human chronic wound treatment has also been described. Human studies in patients with anemia have focused on wound strength. These studies have involved small case-series of patients with a variety of acute surgical conditions. To date, human studies have not demonstrated the specific effects of iron deficiency and anemia on the histological stages of chronic wound healing. Clinical studies by our group have found an association between diabetic foot ulceration (DFU) severity and hemoglobin (Hb) decline. DFU is a complex condition, characterized by poor wound healing. Over half all severe DFU patients have IDA . ClinicaChronic inflammatory conditions such as rheumatoid arthritis (RA) and Lupus Erythematosus are associated with dysregulation of local cutaneous iron hemostasis.RA is a progressive inflammatory autoimmune disease, with joint articular and systemic effects including development of ulceration and poor wound healing. The release of cytokines, especially TNF-α, IL-6, and IL-1, causes synovial inflammation. Pro-inflammatory cytokines also promote the development of systemic effects, including production of acute-phase proteins (such as CRP) which in turn may contribute to development dysregulation of iron homeostasis and anemia . Indeed,Clinically, RA leg ulcers are typically associated with venous insufficiency, trauma, arterial insufficiency and rarely vasculitis . Some patients suffer from a skin-limited form , while in others it evolves into SLE, although this process is not fully understood. A key exogenous trigger to the onset of cutaneous disease activity is exposure to UV radiation. It has been shown that photosensitive patients with cutaneous lesions express anti-Ro/SSA autoantibodies. In vivo studies have demonstrated up-regulation of antigens such as Ro52 in keratinocytes . This isFigure 4.There has been some interest in the role of excess iron stored in the skin as hemosiderin, in the pathophysiology of chronic venous disease (CVD). It is now thought that the severe skin changes (such as lipodermatosclerosis) and leg ulceration associated with CVD happen after iron overload occurs. The mechanisms underlying the deleterious effects of local cutaneous iron deposition in CVD are shown in high macrophages, continuous uptake of Hb is thought to be the cause high intracellular concentrations of heme-iron which induce an unrestrained pro-inflammatory macrophage activation. Macrophage iron can be further increased during inflammation by virtue of increased systemic or local hepcidin expression, which leads to reduction in ferroportin, an iron eﬄux protein, resulting in intracellular iron accumulation. Individuals may also be predisposed to CVD disease through a genetic inability to counteract the skin iron overload  have shown that skin iron levels reflect the liver iron overload. Interestingly, this technique has been proposed as a clinical tool to enable better informed decisions on when to initiate, change or stop phlebotomy therapy. In both CVD and hereditary hemochromatosis, parenchymal iron deposition leads to activation of metalloproteinases and subsequently fibrosis. Hereditary hemochromotosis has also been used to study the effects of iron on the aging process. Relative iron overload may also have a deleterious effect on normal skin aging, as iron chelators assist “successful” normal skin aging when applied topically .Leg ulceration represents one of the main causes of morbidity in sickle cell anemia (SCA). Known risk factors for leg ulcer development in SCA include Hb (≤6 g/dL), lower levels of fetal Hb, hemolysis, raised lactate dehydrogenase , infectiOver recent years there has been some advancement in knowledge about iron in the skin and iron deficiency in cutaneous wound healing. It is clear from studies on pathology of CVD that high iron in macrophages can induce unrestrained proinflammatory macrophage activation. Furthermore in cases of iron deficiency/anemia of inflammation, when serum hepcidin levels are elevated, hepcidin/ferroportin interaction can lead to increased iron concentration in cells particularly macrophage and this could also have a detrimental effect on wound healing. Iron deficiency without inflammation is likely to affect one of the later stages of wound healing such as remodeling. Additional in-depth scientific study of both the underlying pathophysiological mechanisms and role of local cutaneous iron in conditions associated with iron overload and iron deficiency is a priority. Iron is a potential therapeutic target in the skin by application of topical iron chelators and other novel pharmacological agents, and in delayed cutaneous wound healing by treatment of iron deficiency.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.


MCC BSP-/- cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteoprogenitors. No mineralized colonies were observed in BSP-/- cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/- culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/- cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors.Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/- mice, we studied the impact of the absence of BSP on  The SIBLING  are a fin vitro osteogenesis in cultures of calvaria cells (CC) isolated from BSP+/+ and BSP-/- six day old mice by collagenase digestion. We show in this cell culture model that BSP-/- bone cell cultures display a constitutive impairment of osteogenesis, resulting in part from a defect in osteoprogenitor numbers, and which is partly rescued by a higher cell density.OPN and BSP are highly expressed by osteoblasts, osteoclasts and hypertrophic chondrocytes, and BSP is particularly abundant in sites of primary bone formation . BSP is Ibsp gene were replaced by a PGKneo cassette that created a null allele in mouse embryonic stem (ES) cells . After ras Nägy  31] . The. Thein vctor EB1 .While it is possible that BSP would play a part in the density-dependent differentiation of osteoblasts, how exactly osteoblast differentiation and mineralization are inhibited in standard density BSP-/- MCC cultures is still an open question. MEPE ASARM is cleaved-out and freed by CatB, and captured/degraded by PHEX. In BSP-/- standard density cultures PHEX expression is lower and CatB is higher as compared to BSP+/+, which may result in higher amounts of active, inhibitory ASARM. However, neither MEPE nor DMP1 are expressed in these cultures, and the only possible source of ASARM is OPN. In high density BSP-/- cultures, DMP1 and MEPE are expressed, but PHEX expression increases and CatB decreases, suggesting reduced ASARM peptide amounts and a lesser inhibition of mineralization. Blocking CatB activity in our standard density cultures continuously or within time-windows and with different concentration of CU074, a specific inhibitor, did not affect nodule formation or mineralization in either genotype, suggesting that this protease is not involved. There is presently no evidence that CatB is the enzyme cleaving out the ASARM from OPN, and other enzymes should be sought for. In BSP-/- MCC cultures, we used a cocktail of two inhibitors targeting a wide spectrum of protease activities, including cathepsins D and B. This treatment induced an increase of the number of mineralized colonies formed in the dishes. While this suggests that proteases are at least in part involved in the inhibition mechanism, it is not established at present that the inhibitor treatment targeted an enzyme cleaving OPN by the ASARM peptide or at any other point in the sequence, and as mentioned both full-length OPN and some of its peptides appear able to block mineralization . Furtherin vitro, consistent with low levels of bone forming activity in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors, explaining the smaller size of the CFU-ALP observed in bone marrow cultures and the lower number of CFU in MCC cultures. The proteolytic processing of the OPN protein might play a part in the inhibition of osteogenesis and mineralization in the absence of BSP. These hypotheses will orient future studies aimed at clarifying the respective roles of SIBLING proteins on osteogenesis.In conclusion, the present study shows that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity


Close interpersonal contact likely drives spatial clustering of cases of cholera and diarrhea, but spatial clustering of risk factors may also drive this pattern. Few studies have focused specifically on how exposures for disease cluster at small spatial scales. Improving our understanding of the micro-scale clustering of risk factors for cholera may help to target interventions and power studies with cluster designs. We selected sets of spatially matched households (matched-sets) near cholera case households between April and October 2013 in a cholera endemic urban neighborhood of Tongi Township in Bangladesh. We collected data on exposures to suspected cholera risk factors at the household and individual level. We used intra-class correlation coefficients (ICCs) to characterize clustering of exposures within matched-sets and households, and assessed if clustering depended on the geographical extent of the matched-sets. Clustering over larger spatial scales was explored by assessing the relationship between matched-sets. We also explored whether different exposures tended to appear together in individuals, households, and matched-sets. Household level exposures, including: drinking municipal supplied water , type of latrine , and intermittent access to drinking water  exhibited strong clustering within matched-sets. As the geographic extent of matched-sets increased, the concordance of exposures within matched-sets decreased. Concordance between matched-sets of exposures related to water supply was elevated at distances of up to approximately 400 meters. Household level hygiene practices were correlated with infrastructure shown to increase cholera risk. Co-occurrence of different individual level exposures appeared to mostly reflect the differing domestic roles of study participants. Strong spatial clustering of exposures at a small spatial scale in a cholera endemic population suggests a possible role for highly targeted interventions. Studies with cluster designs in areas with strong spatial clustering of exposures should increase sample size to account for the correlation of these exposures. While clustering of cholera incidence had been previously described, the relative role of similar risk behaviors versus transmission dynamics is not well understood. We explored how risk factors for cholera clustered at the sub-community scale, and found significant more correlation in risk behaviors among spatially matched households than the community as a whole. We found clustering of single risk factors, and co-occurrence of different risk factors. Our results indicated that the distribution of risk behaviors may play a role in the clustering of cholera cases at very small  spatial scales. This had implications for spatially matched study designs, which may be overmatching on some exposures. It also may lead us to rethink targeted interventions, suggesting a role for more intensive highly targeted interventions as a supplement to more generalized campaigns. Cholera is responsible for over 100,000 deaths each year and is endemic in many countries . CholeraUnderstanding if and how risk factors cluster may help us better understand how cholera and diarrheal diseases cluster. Furthermore, cholera prevention strategies often consist of behavioral interventions aimed at encouraging communities to adopt safe water, sanitation and hygiene practices . The preHere we use data from spatially matched households enrolled as controls during a case-control study to explore whether potential risk factors for cholera and other diarrheal diseases cluster at small spatial scales. The risk factors considered include lack of access to water, poor sanitation, overcrowding, source of drinking water, and poor hygiene and food handling practices –17. Thes2 with a population density over 100,000 per km2 [V. Cholerae O1 or O139 cultured from their stool sample. Hospital surveillance was conducted at the Tongi Hospital and the icddr,b Dhaka Hospital, both frequented by Arichpur residents.The present study used data from households selected as spatially matched controls in a case-control study conducted between April and October 2013 in Arichpur in Tongi Township in Dhaka, Bangladesh. Arichpur is a cholera endemic working class neighborhood, and is approximately 1.2 km per km2 . CholeraStudy staff attempted to enroll four spatially-matched control households (the matched-set) for each primary case household. To select a control household, staff would first select a number (one for each control household) from a random number table, then attempting to enroll the household that number of doors to the right of the entrance of the primary case household. Special instructions were applied for households in multi-story dwellings and when a natural boundary (e.g. railroad or river) was encountered. Household GPS locations were collected at the front door of each household (resolution ~3-5m).During one of the two household visits to each control household, we collected data on exposure to suspected cholera risk factors at both the household and individual level. Demographic variables including household size and number of rooms were collected. We also collected data on hygienic behavior including the availability of hand soap as observed by the team, and whether the household members boiled all of their (self-reported) sources of drinking water. Exposures related to sanitation and latrine included type of latrine and number of households sharing a latrine. For access to drinking water, we measured: 1) whether the household consumed drinking water from municipal supplied water (supplied water) and/or tubewell water in the past month by self-report; 2) distance to water source from front door of the house as measured by the team; 3) whether households had intermittent access to drinking water; and 4) whether and how households stored drinking water  , sharing a latrine with other households, storing drinking water, a distance of > 10 meters to nearest drinking water source from front door, intermittent drinking water supply, not always boiling drinking water from all reported source, no hand soap available, drinking municipal supplied water, and drinking tubewell water –17. AlthWe explored the clustering of each household level exposure within matched-sets by calculating the intraclass correlation coefficient (ICC). For individual level exposures, we explored clustering within matched-sets as well as within individual households. We estimated the ICC using a two-level random effects logistic regression model with no additional covariates specified using the GLLAMM program in Stata 2012 ,23. We aTo understand whether clustering of each household and individual level exposure exhibited fine scale spatial dependence, we explored the association between the level of clustering in matched-sets and their geographic extent. We quantified the clustering within each matched-set (within matched-set concordance) as the proportion of pairs of households  within the matched-set that had the same exposure. We defined the spatial extent of matched-sets as the median distance between any two households within the matched-set. The confidence interval of the association was assessed using bootstrapping (1000 iterations) by resampling all matched-sets, households within the matched-sets, and then individuals within the households.We also explored whether clustering existed at larger spatial scales . We quantified concordance of exposures between two matched-sets (between matched-sets concordance) as the proportion of all possible pairs of households, one from each matched-set, that had the same exposure. We used non-parametric locally weighted polynomial regression models (LOESS) to visually assess how between matched-sets concordance for each exposure varied with the distance between centroids of matched-sets. We considered distances up to 780 meters (approximately ¾ of the diameter of Arichpur) as households and individuals that were separated by over 780 meters were limited. We hypothesized decreasing concordance between matched-sets for exposures that showed clustering beyond the spatial extent of the matched-sets. If concordance remained static beyond a certain distance threshold, the threshold likely corresponded to the spatial extent of the clustering. We used linear regressions to quantify the trend of decreasing concordance up to the threshold and calculated the confidence intervals using bootstrapping (1000 iterations) by resampling all matched-sets, households within the matched-sets, and then individuals within the households.To understand which household level exposures clustered with one another, we calculated the probability of co-occurrence of different household level exposures within the same household, or matched-set. We then calculated the ratio of this probability to the probability of exposures appearing together by chance if they were uncorrelated. Likewise, we calculated the co-occurrence of each pair of individual level exposures within individuals, households and matched-sets , while values less than one suggested two exposures tend to not appear together. Confidence intervals were calculated by resampling all matched-sets, households within the matched-sets, and then individuals within the households through 1000 bootstrap iterations.We obtained written informed consent from adult study participants or parents/guardian for minors. The study protocol was reviewed and approved by the ethical review committee of Johns Hopkins Bloomberg School of Public Health and icddr,b.Two hundred and thirty-nine households were recruited in 61 spatially matched-sets. Of those, 171 households (72%) in 48 matched-sets had complete household level exposure data, and were included in the present study . Eight hWe observed pronounced clustering of household level exposures within matched-sets . NeighboMore spatially compact matched-sets tended to have higher concordance in exposures than the larger ones, though the trend was not statistically significant for any exposure . AveragiVisually assessing the LOESS plots of the exposures, we found clustering beyond the spatial extent of the matched-sets in household level exposures related to drinking water sources, as manifested by the decreasing concordance between matched-sets over space. The spatial extent of the clustering was approximately 350 meters for the use of supplied water and tubewell water, and 460 meters for distance over 10 meters to the closest water source , since cHousehold level unhygienic practices were more common among households with infrastructure that was suspected to increase cholera risk. For example, households having no hand soap at home tended to tended to share a latrine with neighbors  . MoreoveIn addition, among the same matched-sets, unhygienic practices were correlated with infrastructure that was suspected to increase cholera risk. For example, households not always boiling drinking water tended to be in the same matched-set as those using a pit latrine  and those over 10m to the nearest water source  . ClusterWe found substantial clustering of single exposures and co-occurrence of different exposures suspected to modify cholera and diarrhea risk within groups of nearby households, and an overall trend of households nearer to one another being more likely to share risk factors. This suggests that even within a relatively compact neighborhood there can be substantial spatial dependence of risk for cholera and diarrheal diseases driven by spatial homogeneity in behavior or access to improved water and sanitation.Aggregating risk profiles at the neighborhood-level may be misleading and obfuscate hidden spatial heterogeneity, hence we may miss pockets of high risk. Thus, interventions targeted at micro-scale clusters of high cholera risk may be an important complement to general disease prevention or control campaigns. In cholera endemic neighborhoods, clusters of high-risk behavior could be identified prospectively through community surveys, or by canvasing the area around incident cholera cases. These groups could then be slated for more intensive water, sanitation, and hygienic interventions including education and provision of supplies. This latter course has proven effective in preventing onward spread of cholera in households, and may work equally well within clusters with high cholera risk .Some study designs must account for spatial clustering of exposures at a fine scale. In a case control study, matching controls by proximity aims to control for environmental confounders that are unknown or difficult to measure, and is an efficient and practical method for risk factor studies or vaccine effectiveness studies in low-resource settings , 26. HowThe study has several limitations. Our analysis focused only on one neighborhood in urban Bangladesh. Factors that are specific to the Arichpur neighborhood such as the network of water infrastructure affect the spatial scale of clustering. However, the result may likely be observed in other densely populated urban areas where cholera is most prevalent and similar risk profile is present. Thus, a clear understanding of risk factors present in the community is important for cholera interventions and studies. In addition, some exposures in our study are self-reported. Participants may be more likely to report low risk behavior, and may have undermined the level of clustering. However, the conclusion of microscale clustering would hold especially for the household level exposures, because they were less likely to be biased as infrastructure related to water supply and sanitation are very unlikely to change. Furthermore, 11 out of the 69 hospital-based cholera positive households did not participate in our study, and 13 out of the 31 households recruited from the pharmacy did not provide stool samples . If thesIn conclusion, our study demonstrates clustering of individual and groups of exposures modifying risk of cholera and diarrheal diseases within immediate neighbors, and an overall trend of clustering over the entire Arichpur neighborhood. Based on our findings on clustering of high-risk behaviors, we recommend incorporating community risk assessments to supplement general water, sanitation, and hygienic intervention campaigns. For clustered studies such as those designed to assess vaccine effectiveness and identify risk factors, we call for an initial risk assessment for level of risk factor clustering, and determine the required sample size accordingly.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.


Recipients of solid organ transplants (RSOT) have a highly increased risk for developing cutaneous premalignant and malignant lesions, favored by the lifelong immunosuppression. Vascularized composite tissue allografts (VCA) have been introduced recently, and relevant data are sparse. Two patients with skin cancers  have been so far reported in this patient group. Since 2000 we have been following 9 recipients of VCA  for the development of rejection and complications of the immunosuppressive treatment. Among the 9 patients, one face-grafted recipient was diagnosed with nodular-pigmented basal cell carcinoma of her own facial skin 6 years after graft, and one patient with double hand allografts developed disseminated superficial actinic porokeratosis, a potentially premalignant dermatosis, on her skin of the arm and legs. Similar to RSOT, recipients of VCA are prone to develop cutaneous premalignant and malignant lesions. Prevention should be applied through sun-protective measures, regular skin examination, and early treatment of premalignant lesions. NMSC developing in RSOT are often multiple and thereby entail significant morbidity and nonnegligible mortality in this patient group. Vascularized (composite) tissue allografts (VCA) are a more-recently introduced type of allografts aiming to replace nonvital tissues (such as face and hands), lost following severe trauma, or treatment of life-threatening or severely disfiguring tumours. VCA are immunogenic and necessitate lifelong antirejection IST, exposing their recipients to infectious and neoplastic complications of chronic immunosuppression. Up till now two VCA recipients  have reportedly developed postgraft NMSC (BCC and SCC) [Recipients of solid organ transplants (RSOT) are at increased risk for developing various types of cancer, favored by the life-long immunosuppressive treatment (IST) necessary to avoid graft rejection. Skin cancers are the most common ones . The incand SCC) , 7. We r in situ carcinoma that was treated by conization. At the 6-year anniversary of follow-up (at the age of 44 years) the patient complained of a small asymptomatic, well-demarcated, pigmented nodule of the left temple close to the outer canthus that had appeared several months prior to consultation and was very slowly enlarging , or BCC [Our second patient developed PK, a rather uncommon skin disorder that may be seen in immunosuppressed patients, including RSOT. PK is thought to develop from the expansion of a clone of abnormal keratinocytes, favored by immunosuppression. The incidence of posttransplant PK varies according to the published series, from 0.34 to 10.7%, and the delay of appearance after graft in renal transplant patients is 4.5 years . Althoug, or BCC  in 7–11%, or BCC  and deat, or BCC . Our sec, or BCC , we beliSimilar to other SOTR, VCA recipients may develop premalignant and malignant skin lesions. Their precise incidence in this patient group will be better assessed when a larger collection of cases will be available. Premalignant skin lesions and NMSC are favored by the IST that cannot be circumvented but are mainly due to sun-exposure; since this factor can be avoided, VCA recipients should be educated to protect themselves with sun-protective clothing and use of sunscreens. Regular skin self-examination, dermatological follow-up, and early detection and destruction of all premalignant lesions are strongly advisable, as in other RSOT. Secondary prevention also includes IST revision, for example, conversion to mTOR inhibitors that have been shown to decrease skin carcinogenesis in renal transplant recipients .


Polyglutamine diseases are fatal neurological disorders that affect the central nervous system. They are caused by mutations in disease genes that contain CAG trinucleotide expansions in their coding regions. These mutations are translated into expanded glutamine chains in pathological proteins. Mutant proteins induce cytotoxicity, form intranuclear aggregates and cause neuronal cell death in specific brain regions. At the moment there is no cure for these diseases and only symptomatic treatments are available. Here, we discuss novel therapeutic approaches that aim in neuronal cell replacement using induced pluripotent or adult stem cells. Additionally, we present the beneficial effect of genetically engineered mesenchymal stem cells and their use as disease models or RNAi/gene delivery vehicles. In combination with their paracrine and cell-trophic properties, such cells may prove useful for the development of novel therapies against polyglutamine diseases. Polyglutamine (polyQ) diseases are late-onset progressive neurodegenerative disorders; they include Huntington's disease (HD), Dentatorubral-pallidoluysian atrophy (DRPLA), Spinal and bulbar muscular atrophy (SBMA), and Spinocerebellar ataxia (SCA) −1, −2, −3, −6, −7, and −17. They are caused by a trinucleotide (CAG) repeat expansion within the coding region of unrelated genes that do not seem to share a common function or conformation. With the exception of SBMA, mutant genes are inherited in an autosomal dominant manner. CAG expansions encode for longer polyQ chains in the produced protein Orr, . As a reThe symptoms of the disease manifest around the fourth decade of the life depending on the length of the polyQ tract in mutant proteins. The protein degradation machinery plays a pivotal role in the cellular response to expanded polyQ chains  have recently emerged as excellent tools for cell-based assays since they can differentiate into neuronal cell populations. Using fibroblasts from polyQ-disease patients, researchers have generated iPSC-derived neurons for SCA2, SCA3, HD, and SBMA  represent a complementary alternative to iPSCs in terms of disease modeling. These adult stem cells can be easily isolated from various tissues  and expanded Would it be possible to produce a robust pathogenic polyQ phenotype in MSCs? To this end, we generated an inducible mesenchymal stem cell model for the polyQ disease SCA1 using the Sleeping Beauty transposon system . This indicates that a subpopulation of MSCs may have embryonic characteristics and multipotential differentiation properties. Recently, researchers have identified an MSC population which is resistant to severe cellular stress conditions  of the striatum are selectively lost. Attempts to replace these neurons have been made using fetal striatal tissue or striatal progenitors derived from human ES cells  in a SCA1 mouse model that displays progressive ataxia and PC loss. NPCs were stereotactically engrafted into the cerebellum of SCA1 mice which showed improved motor skills after transplantation. Transplanted NPCs were detected in close vicinity to the molecular layer of PCs suggesting that their neuroprotective effect was mediated by direct contact with host neurons  combines the advantages of cell and gene therapy. These cells have an intrinsic ability to express cell-trophic cytokines which promote neuronal growth, reduce release of free radicals and suppress inflammation  in neuronal cells. They generated MSCs expressing shRNA antisense to HTT and co-cultured them with cells expressing HTT142Q. Levels of HTT142Q were markedly reduced in neuronal cells indicating the direct communication and exchange of RNAi molecules between MSCs and HTT142Q expressing cells . Additionally, the effect of MSCs is more prominent when repeated doses of cells are administered  reach the injured area and (b) survive for a substantial period of time in order to exert their therapeutic effect. MSCs are usually attracted by chemokines released by degenerating cells and activated microglia (Aarum et al., GE-MSCs should also encode for genes that strongly suppress polyQ-induced neurodegeneration preferably at early stages of the disease. In the last years, several studies have described modifier genes that modulate cytotoxicity and aggregation of mutant proteins. Some of them, have a well-defined mode of action, suppressing the formation of toxic oligomers that are early formed in polyQ diseases and correlate with neuronal cell loss. For example, components of the ubiquitin-proteasome machinery upregulate proteasomal activity and their overexpression in damaged neurons may induce clearance of polyQ protein aggregates. At the molecular level, the interaction of polyQ ATXN1 with proteins like PUM1 or RBM17 is protective in SCA1 models (Lim et al., in vivo, GE-MSCs tend to home into injury sites and fuse with damaged neurons, delivering protective molecules. These cells have also the ability to promote neuronal survival. Potentially, co-transplantation of iPSCs and GE-MSCs may promote the engraftment of patient-specific iPSC-derived neurons and the survival of endogenous neurons in damaged brain areas. We strongly believe that stem cells hold great promise for the development of novel cell-based therapies for polyQ diseases.Stem cells are a valuable tool to face polyQ diseases. Different types of them have different modes of actions. iPSCs can give rise to patient-specific neurons; together with NPCs they can be used for cell replacement therapies. MSCs can be easily isolated and modified to overexpress polyQ proteins, serving as disease models for drug discovery. When transplanted ES, SP: wrote the manuscript, GK: edited the manuscript.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.


Aminolevulinic acid (ALA) is the first metabolite in the heme biosynthesis pathway in humans. In addition to the end product heme, this pathway also produces other porphyrin metabolites. Protoporphyrin (PpIX) is one heme precursor porphyrin with good fluorescence and photosensitizing activity. Because tumors and other proliferating cells tend to exhibit a higher level of PpIX than normal cells after ALA incubation, ALA has been used as a prodrug to enable PpIX fluorescence detection and photodynamic therapy (PDT) of lesion tissues. Extensive studies have been carried out in the past twenty years to explore why some tumors exhibit elevated ALA-mediated PpIX and how to enhance PpIX levels to achieve better tumor detection and treatment. Here we would like to summarize previous research in order to stimulate future studies on these important topics. In this review, we focus on summarizing tumor-associated alterations in heme biosynthesis enzymes, mitochondrial functions and porphyrin transporters that contribute to ALA-PpIX increase in tumors. Mechanism-based therapeutic strategies for enhancing ALA-based modalities including iron chelators, differentiation agents and PpIX transporter inhibitors are also discussed. Considered as pigments of life, porphyrins attract tremendous human curiosity about their chemistry, biosynthesis, role in homeostasis and pathogenesis, and potential as therapeutic agents. As a class of tetrapyrroles with highly conjugated heterocyclic structure, porphyrins typically have intense absorption of light in the visible range, giving the characteristic red color in animals (due to heme) and green color in plants (due to chlorophyll). It was nearly 150 years ago that hematoporphyrin (HP), a crude porphyrin extract from blood, was first shown to have fluorescence property and about 100 years ago that the fluorescence of HP was found useful for detecting tumors . These iBecause all porphyrins are biosynthesized from aminolevulinic acid (ALA), an early precursor in the heme biosynthetic pathway that is found in nearly all mammalian cells, ALA can be used to boost the production of endogenous porphyrins for many diagnostic and therapeutic uses. Following the pioneer work by Malik , KennedyThe key to the successful use of ALA as a PDT and imaging agent lies in the preferential accumulation of PpIX in target cells following ALA administration. Extensive research has been performed to determine the molecular mechanism involved in enhanced ALA-PpIX in tumor cells. In this special issue on Advances in PDT, we would like to summarize research on this topic over the past two decades. We begin with an overview of the biosynthesis and transport of PpIX, and then summarize current understanding on the mechanism involved in preferential ALA-mediated PpIX synthesis and accumulation in tumors. Next, we discuss mechanism-based therapeutic strategies for enhancing ALA-based tumor detection and PDT. Finally, we end with perspectives on areas for future studies. For readers who are interested in the clinical applications of ALA and its derivatives, there are excellent recent reviews ,11,12,13PpIX is a precursor to heme, the final product of the heme biosynthesis pathway . This paBecause free porphyrin metabolites such as PpIX and heme are prone to cause oxidative stress and cell damage, cells maintain a low level of free porphyrins through mechanisms including well-coordinated heme biosynthesis and degradation, rapid utilization of free heme for hemoprotein synthesis, and extracellular transport of excess free porphyrins . Heme biElevated PpIX fluorescence is commonly observed in a variety of tumor cells and tissues following ALA administration compared with normal counterparts , which pBecause PpIX is a metabolite produced in the heme biosynthetic pathway, it is reasonable to assume that the enhanced level of PpIX in tumor cells after ALA incubation is due to changes in the expression or activity of heme biosynthesis enzymes in tumor cells. Comparing the expression or activity of heme biosynthesis enzymes between tumor and normal cells or tissues does reveal significant differences in some cases. However, conflicting results, often dependent on tumor type, are commonly seen in the literature.ALAS gene expression was compared between micro-dissected tumor tissues from colorectal cancer patients and corresponding normal tissues by RT-PCR [ALAS expression in tumor tissues was significantly lower than in normal tissues. However, both ALAS gene expression and protein level were found increased in HCC4017 non-small-cell lung cancer (NSCLC) cells compared with normal cells, and increased ALAS protein level was shown in a panel of human lung cancer xenograft tumor samples [y RT-PCR . ALAS ex samples . It shouPBGD expression or activity is associated with cell transformation [PBGD gene expression or enzyme activity in the oncogenesis of cervical [PBGD expression between human colorectal cancer and normal tissues [PBGD does not result in increased PpIX production, suggesting a complex interplay between PBGD and other heme biosynthesis enzymes [PBGD has been suggested to play an important role in elevated ALA-PpIX production in tumor cells based on the findings that increased ormation ,20, and ormation ,22,23 anormation ,25. Thercervical , prostatcervical  and breacervical  cancers,cervical . Significervical ,30, alth tissues . Because tissues . However enzymes .UROD gene expression or enzyme activity in tumor initiation and progression. UROD was highly expressed at an early stage, but not at a late stage, in Friend virus-induced erythroleukemia in mice [UROD expression was significantly elevated in tumor biopsies from head and neck cancer patients [UROD expression were more likely to have shorter disease-free survival, suggesting the involvement of UROD in cancer progression. However, in human clear-cell renal carcinomas, there was no correlation between the UROD activity and the concentration of total porphyrins or the degree of malignancy [There is evidence showing the involvement of increased  in mice . Enhance in mice . UROD expatients . In addilignancy .FECH gene expression or enzyme activity has often been found reduced in a variety of tumor cells/tissues including liver [FECH gene expression significantly increased ALA-PpIX fluorescence in human colon [FECH reduced cell sensitivity to ALA-PDT by decreasing PpIX production [As an enzyme responsible for converting PpIX to heme, ng liver , bladderng liver , colorecng liver ,23, esopng liver  comparedng liver ,36. Silean colon , urothelan colon , glioma an colon  and breaan colon  cancer coduction ,40,41, sThe observation that PpIX is often accumulated in tumor cell mitochondria leads to a speculation that enhanced ALA-PpIX is related to mitochondrial alterations in tumor cells. Comparing mitochondrial content (as indicated by the fluorescence intensity of MitoTracker dye) and the activity of cytochrome c oxidase  with ALA-PpIX level in different tumor cell lines indicates a correlation between ALA-PpIX level and mitochondrial content, but not with cytochrome c oxidase . HoweverRecent studies suggest that alterations in energy metabolism, particularly enhanced glycolysis, are involved in enhanced ALA-PpIX in tumor cells . SimilarALA-mediated PpIX synthesis depends on active ALA uptake and effective transport of different porphyrin metabolites between the cytosol and mitochondria by porphyrin transporters . PorphyrABCB6 expression than normal brain tissues [ABCB6 expression than glioma tissues with lower PpIX fluorescence, and ABCB6 overexpression significantly increases ALA-PpIX fluorescence in glioma cell lines [versus cell membrane.One porphyrin transporter involved in PpIX synthesis is the ATP-binding cassette sub-family B member 6 (ABCB6) . Origina tissues . The notll lines . Howeverll lines  and endoll lines , and trall lines , suggestABCG2 causes PpIX accumulation, particularly in mitochondria, and results in mitochondrial damage, indicating that PpIX is an endogenous substrate of ABCG2 [As a drug efflux transporter responsible for cancer cell resistance to many anticancer agents, the ABCG2 transporter probably plays the most important role in the extracellular transport of PpIX. Knockdown of of ABCG2 ,55. The of ABCG2 . Increasof ABCG2  and cellof ABCG2 . In a reof ABCG2 .Although ALA-based modalities have been used in the clinic for detecting and targeting tumor tissues, its applications are limited by inadequate and heterogeneous PpIX production in tumor cells ,60. ThusALAS2 gene variants with increased enzymatic activity than the wild-type enzyme [CPOX-overexpressing vector [FECH [ABCG2 [It is desirable for a therapeutic agent to enhance ALA-PpIX level through multiple mechanisms. For example, differentiation agent vitamin D is able to enhance PpIX synthesis by up-regulating CPOX and reduce PpIX conversion by down-regulating FECH . It is ie enzyme , as wellg vector , signifior [FECH ,38,39 anH [ABCG2  gene expBecause ferrous iron is a substrate necessary for converting PpIX to heme by FECH, removal of ferrous iron by iron chelators prevents this conversion, resulting in PpIX accumulation. The non-specific metal ion chelator ethylenediamine tetraacetic acid (EDTA) was first used to enhance ALA-PpIX accumulation in leukemia cells . A more in vivo. It increases PpIX levels in human skin [CP94  is another iron chelator that is superior to DFO in enhancing ALA-PpIX accumulation likely due to its lower molecular weight and higher lipophilicity, resulting in better tissue penetration . CP94 isman skin ,76, bladman skin  and glioman skin  cancer cman skin , indicatCPOX over-expression alone is enough to increase PpIX fluorescence [Induction of keratinocyte differentiation by increasing calcium concentration in medium enhances ALA-PpIX production and cytotoxicity to ALA-PDT through increased ALA uptake, increased PpIX synthesis by elevating CPOX expression, and decreased PpIX efflux . Followirescence .α level, which may enhance extrinsic apoptosis induced by ALA-PDT [CPOX gene transcription [Both the active form of vitamin D   and diet ALA-PDT . The meccription .The identification of PpIX as an endogenous substrate of ABCG2 transporter leads to the use of ABCG2 transport inhibitors to enhance ALA-PpIX fluorescence and PDT effects . IncreasThe past two decades have witnessed extensive research aiming at unraveling the mystery of enhanced ALA-PpIX in tumors and seeking therapeutic approaches to boost PpIX levels in tumors with insufficient PpIX accumulation. As a result of this intensive study, considerable knowledge has been obtained about various tumor-related pathological alterations that contribute to an increased level of PpIX in tumors following ALA stimulation. This knowledge has provided the foundation for the clinical application of ALA for detecting and targeting tumors, particularly in the skin, brain and bladder. It has also stimulated the exploration of mechanism-based therapeutic approaches to enhance ALA-based modalities, which has led to many encouraging preclinical and clinical results.etc.). More importantly, we do not know the cause of these pathological alterations in tumor cells. Is enhanced ALA-PpIX directly caused by oncogene activation in tumor cells? If so, what oncogenes are more likely to enhance tumor PpIX level and how? Obviously, answers to these fundamental questions will not only help us understand the biological meaning of increased ALA-PpIX level in tumors, but also have important implications in selecting appropriate patients for ALA-based modalities.However, the molecular mechanism underlying enhanced ALA-PpIX in tumor cells remains elusive and those promising results are yet to be translated to clinical practice. To fulfill the potential of ALA-based modalities, more mechanistic basic research, well-designed clinical trials, and collaborative studies between bench scientists and clinicians are needed. In basic research, many cellular and functional alterations that contribute to an enhanced ALA-PpIX level in tumors have been identified. But we lack a comprehensive understanding of the underlying connection between these different pathological alterations and the relationship between these alterations and cell phenotypic changes (gaining survival advantage, metastatic potential, avoiding metabolic catastrophe In clinical research, it is certainly encouraging to see promising results from pilot clinical studies where ALA-PpIX is used for diagnosing tumors other than glioblastoma, and iron chelators are able to improve PDT outcome. However, these clinical studies are limited by very small sample size and sometimes patients with mixed diseases. Well-designed large-scale clinical trials that will eventually convince the medical community and regulatory agencies to accept these new ALA-based therapeutic modalities are needed. Finally, in the field of oncology where personalized medicine is taking the lead, the importance, as well as the benefit, of collaborative research between bench scientists and clinicians cannot be overemphasized. ALA is certainly not good for all types of cancer nor for all patients with a certain type of cancer. Identifying the appropriate disease and patient subsets who will benefit the most from ALA-based modalities is probably the most important task facing researchers in this field in the next decade. Through more collaborative research between basic and clinical scientists, it is hoped that we will have more mechanistic understanding of ALA-based modalities and more patients will benefit from these modalities.


According to data published by the Centers for Disease Control and Prevention, over 6 million people undergo a variety of medical procedures for the repair of articular cartilage defects in the U.S. each year. Trauma, tumor, and age-related degeneration can cause major defects in articular cartilage, which has a poor intrinsic capacity for healing. Therefore, there is substantial interest in the development of novel cartilage tissue engineering strategies to restore articular cartilage defects to a normal or prediseased state. Special attention has been paid to the expansion of chondrocytes, which produce and maintain the cartilaginous matrix in healthy cartilage. This review summarizes the current efforts to generate chondrocytes from adipose-derived stem cells (ASCs) and provides an outlook on promising future strategies. This limitation often leads to the accelerated development of osteoarthritis or the remodeling of cartilage defects with fibrous or fibrocartilaginous tissue, which has decreased mechanical potential compared with hyaline cartilage.The loss of cartilage tissue due to trauma, tumor, or age-related degeneration is generally associated with poor prognosis and symptoms that require long-term follow-up treatment and represents an ongoing clinical challenge in hand and orthopedic surgery. Due to the limited vascularization of cartilage tissue, chondrocytes in vitro over 2-3 weeks, and the reinjection of chondrocytes into the injured cartilage covered with a periosteal flap [Autologous chondrocyte transplantation (ACT) was the first chondrocyte tissue engineering technique to be applied in daily clinical practice, accomplished by Brittberg et al., in 1994 [eal flap .The use of embryonic stem cells (ESCs) has been suggested to obtain a high number of autologous chondrocytes. However, ethical concerns have limited the clinical application of ESCs. Recent advances in induced pluripotent stem cell (iPSC) research have clearly shown that differentiated somatic cells can be reprogrammed into a multipotent state. Since the first description of iPSCs in 2006 was by Yamanaka et al., this new field has grown continuously, and different experimental approaches to nuclear reprogramming, including nuclear transfer, cell fusion, and transcription-factor transduction, have been developed . DespiteThe potential of adipose tissue in regenerative medicine has been underestimated for a long time, being reduced to the simple function of energy storage. The first report on the application of fat tissue for autologous reconstruction was published by Neuber, who performed a lipofilling procedure in an infraorbital rim in 1893 . Two yea in vivo studies, the use of bone marrow-derived mesenchymal stem cells (BMSCs) has proven to be an effective new treatment strategy for the repair of damaged cartilage in several animal models. Additionally, recent studies in rabbits compared mesenchymal stem cell lines from different sources with a focus on their chondrogenic potential and showed slightly better results for BMSCs; however, ASCs were also capable of substantial cartilage remodeling [ de novo cartilage formation in vivo by injecting ASCs in combination with fibrin glue subcutaneously in nude mice [Regardingmodeling , 21. Theude mice . Since 2ude mice . in vivo studies, are of great interest in this dynamic field of tissue engineering.Therefore, reviews of the current concepts, such as the chondrogenic differentiation of ASCs via mechanical forces, as well asMesenchymal stem cells (MSCs) are multipotent mesoderm-derived progenitor cells that can be isolated from various human tissues, including bone marrow (BMSCs), umbilical cord blood (CBSCs), muscular tissue (MDSCs), and adipose tissue (ASCs). MSCs derived from human adipose tissue have been successfully differentiated into functional adult white or brown fat cells as well as neural, muscle, tendon, bone, or cartilage cells , 24, 25. in vitro and in vivo [To minimize MSC population variability, the initial population of cells and the differentiated progeny are defined by examining the expression of specific cell surface markers. In addition to different surface antigen profiles, the individual therapeutic capacities of MSC populations can also greatly differ. For instance, in the treatment of myocardial infarction, MSCs from discrete populations demonstrate different healing performances in cardiac regeneration  but poss in vivo , 27. Add in vivo . The don in vivo . The dif in vivo . TherefoCompared with other MSCs, a shift to utilizing ASCs has recently taken place because of their better bioavailability and easier harvesting by liposuction.In general, two principal techniques are used to harvest adipose tissue from the human body: plastic section and liposuction. For liposuction, different tumescent solutions have been developed in recent decades. Among the pioneers in modern liposuction, Illouz applied a technique initially introduced by Fischer, who used blunt cannulas for suction of subcutaneous fat tissue , 32. LatThe Coleman technique is still the most frequently used harvesting protocol; however, other techniques, such as microharvesting using smaller cannulas for suction, as proposed by Nguyen et al., have also gained popularity . Many stThe medical device industry also focuses on this issue by providing systems that promise increased cell viability and ASC enrichment, such as Celution, Cytori Lipobank , Adivive , and a constantly growing number of similar products.Although the Coleman technique is still the international standard for harvesting adipose tissue, a future shift to other techniques and further refinements seems likely.μm nylon mesh. The filtrate is then centrifuged to separate the stromal vascular fraction (SVF) from free lipids and mature adipocytes. After several washing steps and an optional erythrocyte lysis, the SVF cells are resuspended in proliferation medium and cultured for 24 hours to produce adherent cells.The isolation of ASCs from freshly harvested lipoaspirate relies on well-established protocols . After lThe weakness of this procedure is that there are occasionally a low number of viable cells due to excessive lysis. Therefore, many variations have been introduced into the original protocol in recent years, and most laboratories apply slightly modified protocols, which have been established based on practical experience.A variety of differentiation protocols have been published to achieve the chondrogenic differentiation of ASCs . These t in vitro can be induced by adding various supplements and growth factors to the basic medium. In this regard, factors, such as transforming growth factors beta 1 and 3 , bone morphogenetic protein 4 (BMP 4), sex determining region Y box 9 (SOX 9), and basic fibroblast growth factor (bFGF), alone or in combination, have high chondrogenic potential. Certain animal models have demonstrated the importance of FGF 2 signaling activation for the induction of cartilaginous repair in full-thickness articular cartilage defects [β 1 in human wound fluids have been demonstrated to be important in the healing process and can also be found in lipoaspirates [The chondrogenic differentiation of ASCs defects . Moreovespirates .β 1 (10 ng/mL), and insulin (6.25 μg/mL).The most commonly used protocols for the chondrogenic differentiation of ASCs involve the supplementation of basic medium  with ascorbate-2-phosphate (50 nM ASAP), TGF-The industry currently offers a large variety of ready-to-use chondrogenesis supplements for MSCs, such as OriCell , PromoCell C-28013 , and StemPro A10071-01 chondrogenesis differentiation kits .  in vitro [ in vivo.In general, cartilage and the resident chondrocytes are exposed to low oxygen tension ranging from 2 to 7% saturation . Severalin vitro , 44. Thein vitro –47. ThusIn vitro, ASCs tend to grow as a monolayer in cell culture and avoid cell-cell contact by growth inhibition. However, excessive cell accumulation, as occurring in high-density micropellets, is a fundamental prerequisite for chondrogenic differentiation. In recent years, three-dimensional (3D) constructs, such as scaffolds, various hydrogels, alginate gels, and matrices, have been developed to overcome growth inhibition [hibition . These 3hibition .The induction of stem cell differentiation by applying mechanical forces is an innovative concept in artificial tissue generation. It has been established that the cytoskeleton interprets and responds differentially to mechanical forces from the microenvironment . In thisIn vitro, different strategies can be used to apply mechanical forces to cells. The FlexCell system  is an up-to-date technique that is based on seeding the cells on a silicone membrane that can be stretched or flexed in a static or cyclic mode using vacuum pressure. Other strategies focus on applying pressure to cell cultures with static or dynamic stamps or centrifugal force.Although preliminary results show a high potential and promising future for mechanical chondrogenic differentiation in tissue engineering, the underlying molecular mechanisms have not yet been extensively studied in detail.Numerous methods have been applied in the past to monitor and evaluate the process of chondrogenic differentiation . These m in vitro and in vivo. The differentiation of these cells can be induced by various stimuli.Lipofilling is the mostly commonly used procedure for the volumetric correction of depressed scars, flap remodeling, breast reconstruction, or the treatment of contour deformities. Human ASCs have good potential to differentiate into cartilaginous tissuesTaken as a whole, ASCs represent a very promising resource for further research in cartilaginous tissue engineering due to the increased bioavailability and easier harvesting of ASCs compared with other mesenchymal stem cells. Nevertheless, the chondrogenic differentiation of human adipose-derived stem cells is still a dynamic field.An ideal technique would combine liposuction with the intraoperative isolation of ASCs and the enrichment of these cells with chondrogenic factors, followed by the direct injection of this cocktail into osteoarthritic joints. Many researchers worldwide are working on attaining these ideal conditions, but numerous challenges remain, such as clinical safety issues and the issue of immune responses to an allogeneic transplantation.


Patients with end-stage renal disease (ESRD) suffer from a number of complex neurological complications including vascular damage and cognitive dysfunction. It is of great significance to detect the neurological complications and improve the prognosis of ESRD patients. Many new noninvasive MRI techniques have been steadily used for the diagnosis of occult central nervous system complications in ESRD patients. This gives an opportunity to understand the pathophysiological mechanisms of these neurological disorders. This paper is a review that presents the MRI findings of occult brain damage in ESRD patients, outlines the applications of advanced MRI techniques, and introduces a brief perspective in this study field. End-stage renal disease is now recognized to be a growing public health problem . It has Magnetic resonance imaging (MRI) is of great value for the diagnosis of apparent cerebrovascular damage. However, conventional MRI is limited in detecting neurological abnormality in ESRD patients with and without subtle vascular damage but with cognitive impairment. In the last decades, some advanced MRI techniques, such as diffusion weighted imaging (DWI), diffusion tensor imaging (DTI), and blood oxygen level dependent functional MRI (fMRI), have been widely used in the clinical and basic scientific studies. These advanced MRI techniques have been used to detect occult vascular damage, brain blood flow changes, brain atrophy, and brain functional connection in the resting state. This uncovers potential pathophysiological mechanisms of cognitive deficits in ESRD patients. This paper reviews the pathophysiological mechanisms of cognitive impairment, clinical presentations, and the potential applications of advanced MRI techniques in ESRD patients, especially focusing on patients without overt neurological complications. Last, a study perspective in this field is presented.Though the cognitive decline is prevalent in patients with ESRD, its cause and pathophysiological mechanisms have not yet been clearly understood. More recently, researchers attributed the cognitive disorders and dementia to the disorders of the kidney-brain axis , 7, 8. TIn patients with ESRD, there is a wide range of diverse neurological complications such as white matter changes, cerebral atrophy, osmotic demyelination syndrome, dialysis encephalopathy, hypertensive encephalopathy, dementia, intracerebral hemorrhage, and opportunistic infections –14. Pati1WI, T2WI, and fluid attenuated inversion recovery (FLAIR) images, is valuable for the diagnosis of these lesions [It has been reported that ESRD patients are inclined to develop neurological disorders such as white matter changes, cerebral atrophy, cerebral infarction, intracerebral hemorrhage, posterior reversible encephalopathy syndrome, osmotic demyelinization syndrome, cerebral infection, sinus vein thrombosis, and dialysis disequilibrium syndrome. Conventional MRI, which includes T lesions , 14. Det lesions , 14.Recently, some new MRI techniques have been widely used in the metabolic brain diseases. White matter microstructure changes, mild cerebral atrophy, cerebral microbleed, and brain function alterations can be detected with the advanced MRI techniques. These advanced MRI techniques are potential measures to investigate the occult neuropathological changes from brain structural and functional aspects in patients with ESRD. The following sections will introduce the applications of these advanced MR techniques used in the evaluation of brain damage in ESRD patients.Conventional MRI has high sensitivities for obvious cerebral atrophy, whereas it is limited in the diagnosis of early stage of cerebral atrophy. Voxel-based morphometry (VBM) is a whole-brain, unbiased technique for describing regional cerebral volume and tissue concentration differences in structural magnetic resonance images . It can ESRD patients on hemodialysis show significant cerebral atrophy. This is correlated with longer hemodialysis duration and cognitive deficits. Prohovnik and his coworkers compared the global cerebral atrophy of ESRD patients on hemodialysis dialysis with healthy controls by VBM . They fo2WI sequences, T2∗WI is more sensitive in depicting small hemorrhage [Microbleeds may be a predictor of intracerebral hemorrhage, which is associated with cognitive dysfunction. It has been reported that the incidence of intracerebral hemorrhages in ESRD patients on hemodialysis has been approximately five to ten times higher than that in the general population . Comparemorrhage .2∗WI, while only two of the 53 control patients had microbleeds on T2∗WI. The total number of microbleeds in 11 hemodialysis patients was 44, whereas the control group had only two microbleeds. On T2- and T1WI only about 30% of microbleeds were shown as either hyperintensity or hypointensity, suggesting the higher sensitivity of T2∗WI. In another study by Watanabe [2∗WI. This rate (35%) was higher than that of asymptomatic or healthy elderly individuals (about five to six percent). A correlation between microbleeds and intracerebral hemorrhage was found in this study. More microbleeds are associated with higher risk for intracerebral hemorrhage and cognition dysfunction. Therefore, T2∗WI should be used to detect microbleeds in ESRD patients.The incidence of microbleeds was found to be higher in the hemodialysis patients compared with the controls. In the study by Yokoyama et al. , eleven Watanabe , 80 pati2∗WI, susceptibility-weighted imaging (SWI) can detect more microbleeds [Compared with Trobleeds . SWI is robleeds . One repAnother method, susceptibility mapping, which is sensitive to the subtle changes of iron content has been used in the studies of brain iron deposition in patients with haemodialysis. Susceptibility maps were reconstructed from original phase data. It can quantitatively calculate the susceptibility and the method has been shown to strongly correlate with iron concentration in the brain. With susceptibility mapping, Chai et al. found that the susceptibility of the bilateral caudate head, putamen, substantia nigra, red nucleus, and dentate nucleus in haemodialysis patients was higher than those in healthy controls, indicating that abnormal iron deposition did occur in patients with haemodialysis . In a laDiffusion weighted imaging (DWI) provides unique information about various pathological changes of the brain by depicting the microscopic movement of water molecules. The measurement of the water motion can be quantified absolutely by the introduction of the apparent diffusion coefficient (ADC) in DWI. The ADC mainly reflects the rate of diffusion in living systems. Diffusion tensor imaging (DTI) is an extension of DWI which could fully yield diffusion anisotropy effects, providing even more precise details on tissue microstructure . With DTDWI may be an effective indicator to predict the prognosis in the ESRD patients. Kim et al.  reportedDWI is of great value to define the nature of edema, which is useful in explaining the pathogenesis of brain edema in ESRD patients. Kim et al.  reportedDTI studies have demonstrated that ESRD patients have asymptomatic white matter damage. Hsieh et al.  found thDTI and fiber tractography have been used to investigate the pattern of brain white matter changes in patients with ESRD. Kim et al.  used dif1H-MRS) has been widely used in the diagnosis of brain diseases as well as the understanding of neurological disorders. N-Acetylaspartate (NAA), choline-containing compounds (Cho), and myoinositol (mI), and so forth are closely related to the cerebral metabolic changes in ESRD patients.Magnetic resonance spectroscopy (MRS) permits noninvasive evaluation of cerebral metabolites in vivo. It can provide specific information about the neurons, axons, the energy of cells, and the state of the membrane. Proton MRS  in the brain [The cerebral blood flow (CBF) could be measured via dynamic susceptibility contrast and arterial spin-labeled perfusion (ASL) MRI. Dynamic contrast enhanced MR perfusion imaging uses gadolinium (Gd) as contrast agent which limits its use in ESRD patients. ASL technique utilizes arterial blood water as endogenous tracer by using radiofrequency irradiation. Without using contrast agents and radioactive tracer, ASL MRI measurements of cerebral blood flow have been validated against he brain . Previouhe brain . With thASL MRI could be used as an effective means to investigate the cerebral blood flow changes in hemodialysis patients. One ASL MRI study by Prohovnik et al.  found thBlood oxygen level-dependent (BOLD) signal has been accepted as the most widely used image contrast in functional MR imaging (fMRI) of the human brain  since itIn the past years, much attention has been focused on detecting the brain activity in patients in the resting state and a few studies have been done on ESRD patients in a resting state using different analysis methods. Regional homogeneity (ReHo) is one of the algorithms to analyze the BOLD signal of the brain. It is proposed based on the hypothesis that the brain activity would more likely occur as clusters rather than as a single voxel; thus Kendall's coefficient of concordance (KCC) was used to evaluate the similarity between the time series of a given voxel and its nearest neighborhoods . In one t-test showed a typical spatial pattern in the DMN for the healthy control subjects (HCs) in the resting state consisting of the bilateral PCC and adjacent precuneus, angular gyri, anterior cingulate cortex, MPFC, and superior temporal cortex . A similar DMN spatial pattern was observed in patients with ESRD , precuneus, rostral anterior cingulate, and medial prefrontal cortex (MPFC). An abnormal DMN has been found to be related to ESRD. A resting-state fMRI study by Ni et al.  demonstrl cortex    with theIn conclusion, the advanced MRI is helpful in assessing the minimal neurological complications in patients with ESRD. With the application of multimodality MR techniques, further studies may uncover the neural mechanism underlying cognitive dysfunction in ESRD patients. Advanced MRI techniques are of great value in the early detection and diagnosis of minimal nephroencephalopathy (MMSE < 24). Thus, it would be helpful in evaluating the occult brain changes and improving both the morbidity and mortality of these ESRD patients.