Laser capture microdissection (LCM) allows for the microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. According to the recent development of the LCM technologies and methodologies, the LCM has been used in various kinds of tissue specimens in dental research. For example, the real-time polymerase-chain reaction (PCR) can be performed from the formaldehyde-fixed, paraffin-embedded, and immunostained sections. Thus, the advance of immuno-LCM method allows us to improve the validity of molecular biological analysis and to get more accurate diagnosis in pathological field in contrast to conventional LCM. This paper is focused on the presentation and discussion of the existing literature that covers the fields of RNA analysis following LCM in dentistry. Several experimental techniques are available for molecular profiling studies such as DNA microarray, differential display, serial analysis of gene expression, massive parallel signature sequencing, and suppression subtractive hybridization . AlthougLCM was first introduced as a system that is able to retrieve defined cell population from human tissue samples. The original system was invented by the National Institutes of Health  to isola Analyses using LCM technology have been further developed/improved and performed in various fields. In dentistry, this technology has been utilized in different research fields such as oral embryology –10, oralOral cancer is a type of head and neck cancers developed in any part of the oral cavity or oropharynx. When oral cancer spreads (metastasizes), it usually travels through the lymphatic system and appears first in nearby lymph nodes in the neck. The new tumor at the metastatic site has the same kind of abnormal cells as the primary tumor. Although the recently gained knowledge of normal and aberrant function of oncogenes and tumor suppressor genes has provided unique opportunities to understand, and ultimately to control the processes leading to malignancy, the molecular mechanisms of this disease remain poorly understood .The recent development of hybridization-based methods utilizing cDNA arrays, provides an opportunity to identify genes expressed in normal and tumor tissues, as well as to analyze gene expression profiles in tumor progression. However, an accurate procurement of specific cell types for RNA isolation is a critical step influencing the validity of this analysis. On this point of view, LCM provides a great advantage since it enables the procurement of pure cell populations from tissue sections, a key consideration as many tumors are heterogeneous, and include areas of connective tissues, blood vessels, and even inflammatory cells that infiltrate into the tumor mass. The use of LCM to harvest cells from their native tissue environment, followed by the use of high-density oligonucleotide probe arrays to identify gene expression differences between normal and malignant oral epithelial cells, provide powerful means to decode the molecular events involved in the genesis and progression of oral cancer. In oral cancer tissues, a number of genes have been identified by either LCM/oligonucleotide microarray approach  or the Lin situ hybridization and LCM-correlated RT-PCR, the authors found that IgG heavy chain transcripts were present in the oral cancer cells and in some normal epithelial cells adjacent to the tumor. These findings suggest that tumor-derived immunoglobulin may be an important new target molecule for tumor biotherapy. LCM method can minimize possible contamination from infiltrated inflammatory cells so that cancer cells were differentially captured from the tumor tissue , cancer In clinical application, the use of LCM technologies therefore allows for scanning of gene expression patterns and search for those correlating with a disease state. Normally, clinical staging of cervical lymph nodes is carried out by clinical examination of the neck region or by ultrasound, computed tomography, and magnetic resonance imaging. However, the sensitivity of these methods is still limited due to the fact that the false negative rate is high in clinically diagnosed metastasis-negative (N0) patients. In an effort to find new biomarkers that will provide more accurate diagnosis and safer and more efficacious treatment for oral squamous cell carcinoma (OSCC), Nguyen et al. applied LCM and microarray technology to investigate the differences in gene expression profiles between primary OSCC metastasized and no metastasized to cervical lymph nodes . PrimaryWe have recently developed a methodology of immune-LCM of formaldehyde-fixed, paraffin-embedded, and coagulation factor VIII-immunostained head and neck carcinoma by using Leica LMD system . This meTooth development is the cumulative result of signaling of growth factors. Initial molecular signals in the dental epithelium induce gene expression in the adjacent dental mesenchyme. Reciprocal signaling between the epithelial and mesenchymal tissues continues throughout tooth development, resulting in the formation of a tooth of specific size and shape. Werner et al. studied the role of macrophage colony stimulating factor (CSF-1) isoforms on tooth morphogenesis by using PixCell II LCM to capture ameloblasts and odontoblast from HistoGene LCM-stained frozen tissue sections, followed by the mRNA expression analysis using RT-PCR. The results showed that CSF-1 is important for optimal cytodifferentiation and dentin matrix protein-1 (DMP-1) expression . AbsenceTooth eruption requires alveolar bone resorption as well as possible bone growth at the base of the tooth. The detailed mechanisms of tooth eruption is yet to be clarified, mainly because many issues regarding growth factors, such as types of expressing cells, temporo-spacial changes of expression patterns, and results of growth factor interaction, remain fully understood, LCM is applicable to molecular profiling of tissue specimens, permitting correlation of cellular and molecular signatures with specific cell population, especially comparison of cellular elements within the tissue microenvironment or in the variety of cell types. Thus, LCM has been applied to the studies of tooth eruption including evaluation of the tissue surrounding unerupted tooth, dental follicle microenvironment, factors involved in tooth eruption process, time expression of growth factors, and/or cytokines and type of cells releasing growth factors to conclude mechanism of tooth eruption –10, 26.The use of LCM enables collecting interested and specific cells such as stellate reticulum (SR) and dental follicle cells from the tissue surrounding unerupted tooth , 8. LCM Wise and Yao studied gene expression of bone morphogenetic protein-2 (BMP-2) and receptor activator of nuclear factor kappa B ligand (RANKL), as a marker gene for alveolar bone formation, and resorption, respectively, in the dental follicle harvested by liquid nitrogen frozen mandible of new born rats, using PALM LCM . The autThe regional differences in the expression of the bone-related genes are not the sole factors regulating bone resorption/formation during tooth eruption. Thus, cytokines and growth factors involved in bone resorption process were studied on their influence on the tooth eruption. IL-10 is an anti-inflammatory cytokine that inhibits osteoclast formation and may act to suppress the alveolar bone resorption. Liu et al. studied the dental follicle tissue harvested from cold ethanol-fixed, toluidine blue-stained sections of liquid nitrogen frozen-mandibles by using PALM LCM . They foin vivo studies . Th. Th17]. μm thickness was performed on a microtome with a new sterile disposable blade. Sample sections were mounted on poly-L-lysine coated glass foiled polyethylene naphthalate (PEN) slides for LCM . The slides were dried in an incubator at 35°C for 6 hours. Nuclear staining by hematoxylin was performed as follows: deparaffination of the slides twice in xylene for 3 minutes at room temperature, washing 3 times in 100% ethanol for 30 seconds, 90% ethanol for 30 seconds, 70% ethanol for 1 minute, and in RNase-free water for 30 seconds at 4°C, immersion in hematoxylin for 5–10 seconds at room temperature, followed by washing with RNase-free water for 30 seconds at 4°C. The slides were let to dry at 4°C and keep in freezing compartment for preservation of RNA until immune staining [Tissue preparation of hard tissue sample is important for immune-LCM, if the target tissue is surrounded by calcified tissues such as enamel/dentin and bone. In our research, we performed tissue fixation and demineralization as follows: fixation of the tissues with 10% neutral buffered formaldehyde at 4°C, and demineralization with 10% formic acid at 4°C. Paraffin embedding was performed as follows: dehydration in 70% ethanol for 30 minutes, 90% ethanol for 1 hour, 95% ethanol for 30 minutes at 4°C, and 3 times in 100% ethanol for 1 hour at room temperature, immersion 2 times in xylene for 1 hour at room temperature, 4 times in infiltrating paraffin for 30 minutes at 58°C, and embedding in paraffin. Sectioning of the paraffin embedded samples at 5 Laser capture microdissection is a useful method for obtaining microscopic samples as small as individual cells from tissues for molecular analysis. The different outcome of the various LCM studies are likely reflective of the experimental approaches and methods of analyses. First, retrieving RNA by using LCM avoids contamination of heterogeneous cellular elements. Second, sample number and the type of gene expression analysis used may be relevant to the discrepancies. Third, the stage of the tissue, source, and anatomical site of the cells, and handling methods can further result in different gene expression levels. An advantage of the LCM approach is that only a small amount of starting material is required for the extraction of a sufficient quantity of total RNA. Furthermore, the quality and integrity of the RNA make this approach suitable for use with available array technology, thus affording the possibility to define a pattern of gene expression because the combination of using LCM and applied RT-PCR protocol allows the specific isolation and characterization of selected cells . The use


Journal of the American Heart Association (JAHA),1 indicating that diabetes mellitus confers a reduced risk of thoracic aortic aneurysms and dissections. If such inverse relationship is real, this observation would have an important impact and would be revolutionary in the sense that people with diabetes mellitus, despite their chronic disease condition (as bad as it may be), would face a lower risk of suffering and dying from an acute aortic catastrophe. The expected tsunami-like global epidemic of newly diagnosed cases of diabetes mellitus in both Western societies and developing countries5 would eventually wash ashore some treasures, such as a lower attrition rate of thoracic aortic disease. How wonderful—but is this real?Are people with diabetes mellitus at reduced risk of acute aortic dissection? It would be a sweet surprise, but the provocative question relates to the findings of a nationwide case–control study by the Center for Clinical Research and Evidence-Based Medicine at the University of Texas in Houston, published in this issue of the 1 The authors used the Nationwide Inpatient Sample  from 2006 and 2007, which is supposed to approximate a 20% sample of all nonfederal, short-term general and specialty hospitals in the United States, in an attempt to extract discharge-level information on primary and secondary diagnoses and procedures from the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes and Current Procedural Terminology labels.1 Although the NIS may be considered a highly reliable source of data, its data quality is heavily dependent on human factors in coding and weighing diagnoses. The reported inverse relation of diabetes mellitus and acute aortic diseases was derived from cross-tabulation of ICD-9-CM codes with NIS diagnoses. In fact, on the basis of just 5425 case records with the primary diagnosis of thoracic aortic aneurysm and dissection (TAAD) , the authors claim that the prevalence of diabetes mellitus revealed the most consistent disparity between TAAD patients and controls: 9.7 cases of TAAD per 10 000 patients with diabetes mellitus versus 15.6 cases of TAAD per 10 000 discharged patients overall. In other words, patients with TAAD (both aneurysm and dissection) are 40% less likely to have type 2 diabetes mellitus!Crossing medicine and epidemiology obviously produces unexpected results. But let's not get cynical; rather, let's dig deeper into the details of the work of Prakash et al.6 if the primary diagnosis was an acute aortic problem that dominated the hospitalization, discharge, and aftercare? In such a scenario, a chronic, underlying diabetic condition may not even be mentioned as a secondary diagnosis. Conversely, because the calculations are derived from only hospitalized inpatients (rather than from a population-based sample), patients with diabetes mellitus are likely to be overrepresented in the sample. The magnitude of both effects is, of course, unknown.Some issues need to be discussed in this context. First, how reliable is a statistical exercise in extracting data from an incomplete data set such as the NIS? Although the authors successfully reproduced their findings in various subgroup association statistics, the source of the input data remained the same. Second, is it fair to use a relatively small sample from NIS data to draw such a far-reaching conclusion as the inverse association of diabetes mellitus and aortic diseases? Remember, the rates were 9.7 per 10 000 in patients with diabetes mellitus and 15.6 per 10 000 in patients without diabetes mellitus. Third, what are the chances that diabetes mellitus was underreported5 and whereas rates of diabetes mellitus continue to rise particularly in younger patients, aortic diseases typically become clinically apparent later in life, beyond the age of 60 years. Again, such disparity in the onset of 2 different disease conditions may contribute to the apparent underrepresentation of diabetes mellitus in the older set of patients with aortic diseases.5Fourth, is it valid and helpful to use hospital discharge information for cross-sectional association studies on the prevalence of certain disease conditions to derive a serious hypothesis? Such administrative input information was never intended to serve any scientific purpose and may not withstand the scrutiny of science. Moreover, no other reliable population-based or hospitalization-derived study documented any kind of association between type 2 diabetes mellitus and aortic diseases. In fact, clear evidence indicates that the incidence of both diabetes mellitus and aortic disease has been rising in recent years in both sexes,8 Or—even better—to find a common denominator for both conditions? Macrophages, which produce proinflammatory cytokines, and matrix-digesting metalloproteinases (MMPs) may play a key role in such an interaction because in aneurysmatic disease those cells operate in all layers of the aorta11 and promote vessel remodeling and expansion by depositing extracellular matrix degradation products and collagen.12 With enhanced glycation (as in diabetes mellitus), more glycated monomeric collagen may be formed, with effects on MMP secretion and marked reduction of MMP-9, MMP-2, and interleukin-6 secretion by activated monocytes, thereby inducing cross-links likely to increase matrix stiffness.14 The additional observation that glycated extracellular matrix inhibits monocyte MMP production may conceptually explain the potential protection of diabetes mellitus against aortic growth and aneurysm formation.15 Nonetheless, mural degradation and expansion seem to work differently in the thoracic and abdominal aorta, and their typical genetic susceptibility loci do not overlap; thus, a mitigating role of hyperglycemia is complex and not fully understood. In fact, the authors quote recent clinical evidence for decreased secretion of metalloproteinase from inflammatory cells in the aortic wall, a process that theoretically inhibits progression of TAAD by stabilizing the extracellular matrix.16 Given the assumption that such interaction works in humans, the association with a lower rate of admission or discharge for TAAD is not necessarily a logical consequence. Similarly, single cases of mutations in the smooth muscle cell–specific myosin heavy chain gene (MYH11) with upregulation of insulin-like growth factor (and antidiabetic action) are unlikely to explain any association based on hospitalization codes.17 Surprisingly, in a study from the same group, Guo et al18 recently reported an association of mutations in the smooth muscle α-actin (ACTA2) gene with thoracic aortic disease, stroke, and coronary artery disease, with the latter being clearly and causally related to type 2 diabetes mellitus.Finally, wouldn't it be more convincing to back any daring technical association study with pathophysiology or with a plausible interaction to explain any suggested association of diseases?1 tell us? Well, the hypothetical association presented here is certainly intriguing but should be viewed as a hypothesis-generating observation that suggests the need for further effort to either confirm or refute a causal relationship between diabetes mellitus and mitigated evolution of aortic aneurysm formation or prevention of rupture.What does the study by Prakash et al


The relationship between obesity, diabetes, hyperlipidemia, hypertension, kidney disease and cardiovascular disease (CVD) is established when looked at from a clinical, epidemiological or pathophysiological perspective. Yet, when viewed from a genetic perspective, there is comparatively little data synthesis that these conditions have an underlying relationship. We sought to investigate the overlap of genetic variants independently associated with each of these commonly co-existing conditions from the NHGRI genome-wide association study (GWAS) catalog, in an attempt to replicate the established notion of shared pathophysiology and risk. We used pathway-based analyses to detect subsets of pleiotropic genes involved in similar biological processes. We identified 107 eligible GWAS studies related to CVD and its established comorbidities and risk factors and assigned genes that correspond to the associated signals based on their position. We found 44 positional genes shared across at least two CVD-related phenotypes that independently recreated the established relationship between the six phenotypes, but only if studies representing non-European populations were included. Seven genes revealed pleiotropy across three or more phenotypes, mostly related to lipid transport and metabolism. Yet, many genes had no relationship to each other or to genes with established functional connection. Whilst we successfully reproduced established relationships between CVD risk factors using GWAS findings, interpretation of biological pathways involved in the observed pleiotropy was limited. Further studies linking genetic variation to gene expression, as well as describing novel biological pathways will be needed to take full advantage of GWAS results. Cardiovascular events are frequently the final common endpoint of obesity, hypertension, hyperlipidemia, diabetes and kidney disease and modification of these traits remains the Standard of Care in the primary prevention of cardiovascular disease. The clinical management of Coronary Artery Disease (CAD), hyperlipidemia, hypertension, Type 2 Diabetes (T2D) and Chronic Kidney Disease (CKD) assumes an intrinsic interplay between these conditions and in particular, shared etiological and risk factors and is strengthened by their frequent co-existence in general patient populations. Looked at from an epidemiological perspective, the clinical picture is supported by extensive evidence. Both obesity and T2D have independently and in combination, been linked with increased risk of cardiovascular disease and death The apparent coexistence of these common conditions led to efforts to categorize these composite phenotypes, characterized by constellations of atherosclerotic risk factors, with obesity and hyperglycemia at their core. However there has been a lack of evidence to support the concept that these syndromes represent a distinct phenotype and that the risk conferred by a diagnosis of ‘Metabolic syndrome’ is any greater than the risk conferred by the sum of its' parts Although large-scale GWAS have identified numerous significant SNP-trait associations, in the majority of cases the underlying pathophysiological mechanism has not been determined and in general, all known risk-variants combined explain only a small fraction of the observed heritability of these conditions Genetic pleiotropy refers to the phenomenon that single genes or variants may have an effect on multiple phenotypes The relationship between obesity, diabetes, hyperlipidemia, hypertension, kidney disease and cardiovascular disease is established and indisputable when looked at from a clinical, epidemiological or pathophysiological perspective as illustrated in The goal of this study was to investigate the overlap of genetic variants that have been associated independently with each of these commonly co-existing conditions and intermediate risk factor phenotypes in an attempt to replicate the established notion of shared pathophysiology and risk through genetic pleiotropy. We conducted an analysis to evaluate the immediate interpretability of GWAS findings in this area of research using crude GWAS-derived genomic regions without processing or filtering results with regard to directionality of the reported associations or effect sizes.Table S1 for search terms).We mined the online National Human Genome Research Institute's GWAS catalog −5) derived from GWAS publications. In order to control data quality, the catalog mandates standards for inclusion of published studies and is thus an excellent resource to study pleiotropy among GWAS-derived genetic variants The NHGRI GWAS Catalog is a curated resource for statistically significant SNP-trait associations  if the SNP falls within a gene, that gene was assigned and b) if the SNP is intergenic, both the left-flanking and right-flanking genes were assigned irrespective of distance. In all instances where multiple SNPs were mapped to the same gene, only one gene per trait was chosen from each study. We also recorded author-reported genes.We assigned genes to the associated SNPs by using the GWAS catalog's definition of We excluded human leukocyte antigen (HLA) loci that belong to the major histocompatibility complex (MHC) and contain a large number of genes related to immune system function in humans. The large extent of variability in HLA genes poses significant challenges in investigating the role of HLA genetic variations in diseases.−7. Significance of the extent of pleiotropy was calculated with two methods. We first estimated the probability of genes associated with different phenotypes overlapping by chance alone by using the hypergeometric distribution with a pool of 4,105 genes, the number of non-HLA positional genes in the full GWAS catalog (as of June ′11), and positional gene lists of size as specified in We identified all combinations of genes shared between 2 or more phenotypes using the assigned genes, for all studies combined and then for studies conducted in populations of European and African origin separately. To test for the robustness of the detected pleiotropy, ethnicity-pooled analyses were repeated using a SNP-trait associations that met a more stringent cutoff of P<1×10Figures S1, S2, S3, S4) and to have a comparison to the relationships presented in We generated ‘bubble charts’ to visually represent the pairwise overlaps of genes associated with phenotypes  was used To accurately assess the statistical significance of the GRAIL connections, we conducted simulations in which we selected 100 sets of 38 positional genes  from both the unweighted and weighted list of all genes in the GWAS catalog  that were also in the GRAIL database. Each of those 100 sets was scored with GRAIL to test for the likelihood of observing the same number or more significant connections at random., Table S2). We extracted a total of 708 associated SNPs, with 37 SNPs (5%) in coding regions, 311 SNPs (44%) in intergenic, 26 (4%) in near gene, 22 (3%) in UTR regions, and 312 SNPs (44%) in introns. These SNPs were assigned to 737 genes, with 101 genes associated with CAD and related traits, 137 with diabetes and related traits, 219 with obesity and related traits, 141 with lipids, 85 with blood pressure and related traits, and 105 with CKD and related traits . The largest number of genes was shared between CAD and lipids . There were 9 genes shared between T2D and related traits and obesity-related traits. Using only positional genes, the extent of the CAD-lipid overlap reached statistical significance (P<0.001 by hypergeometric and 0.002 by weighted permutation test), whereas other 2-way overlaps did not (Table S3).We detected 44 positional pleiotropic genes shared between at least 2 phenotypes . We also found additional suggestive 3-way overlaps of: 1) CAD, CKD-related traits, and lipids with KLHL29 and APOB (P = 0.003 by hypergeometric and 0.082 by weighted permutation test) and 2) obesity, CKD-related traits, and T2D-related traits with C6orf223 and VEGFA (P = 0.015 by hypergeometric and 0.251 by weighted permutation test). Three out of seven genes  remained significant when only studies of European populations were considered.Seven positional genes showed pleiotropy across at least 3 CVD-related phenotypes in ethnicity-pooled analysis Table 2.Figure S1). When exclusively African-ancestry studies were considered, we found that only 3 connections, between the blood pressure, lipids and chronic kidney disease phenotypes, were reproduced by one genomic region (APOB/KLHL29) .Using pair-wise phenotype overlaps, we attempted to recreate all connections from the bubble chart, −7 and found that, as expected, many overlaps disappeared, especially those with the blood pressure phenotypes, or decreased in number. Importantly, the overlaps that did not change were between the chronic kidney disease phenotypes and CAD, lipids and obesity, in addition to type 2 diabetes and lipids . It is worth noting that less pleiotropic connections were also detected if only author-reported genes were used compared to positional genes .Next, we limited the list of positional genes to reflect SNP-trait associations detected at a more stringent P<1×10CDKN2BAS, the most replicated CAD locus (chromosome 9p21), C6orf223, RPL12P33, EIF3FP3, UBA52P6 and KLHL29 that showed the greatest level of pleiotropy in this study. We re-ran GRAIL using alternative data sources, such as GO and Gene Expression Atlas, and found matches for the 2 target genes . Nevertheless, no connections were established for these genes, whereas many other connections previously obtained through the literature search were lost. We screened interaction databases KLHL29 and CDKN2BAS, supporting the notion that our knowledge of biological pathways is far from complete.To identify the key pathways suggested by the GWAS signals, we used GRAIL on positional pleiotropic genetic regions shared across at least two phenotypes. Based on abstracts published up to 2006, six out of 44 regions were dropped from the analysis as they were not found in the GRAIL database either due to the lack of sufficient literature or inconsistent mapping to Human Genomes (hg) 18. The lost genes were Figure S5).The 5 of 7 genes that showed pleiotropy across three or four phenotypes and were mapped by GRAIL did not reveal any inter-connectivity or connection to genes with an established pathway affiliation. When the analysis was repeated with abstracts published up to 2011 and inevitably informed by studies included in this analysis, we found that many of the 3+-way overlaps remained missing despite a dramatic increase in the amount of new information as expressed by the density of connections overall . The fact that only 24% of the eligible studies were conducted in non-Europeans emphasizes the value of genetic studies in diverse ethnic/racial groups. When only studies of African ancestry cohorts were included in the analyses, we found that only 3 connections, between the blood pressure, lipids and chronic kidney disease phenotypes, were reproduced .We found that genes within or flanking the reported SNPs independently replicated the clinical, epidemiological and pathophysiological notion of cardiovascular risks in the ethnicity-pooled dataset. However, when only studies of European populations were included, some of the relationships were lost (APOB) and kelch-like protein 29 (KLHL29), revealed the most extensive pleiotropy across multiple phenotypes, such as blood pressure, lipids, CKD and CAD. While APOB is well known for coding the main apolipoprotein of chylomicrons and low density lipoproteins, KLHL29's function is widely unknown. Consistent signals pointing to that genomic area included a KLHL29 intronic SNP and several intergenic SNPs positioned 19–2,323 bp from either gene, both imputed and typed, suggesting that both genes can be involved. Notably, KLHL29 was only mapped to this genomic region in studies of African ancestry. This observation could suggest ethnic/racial differences in LD in the region or an ethnic-specific variant associated with multiple risks. Recently, there have been reports of variants associated with risk in specific ancestral populations, particularly in African-ancestry populations for diabetes Several genes showed an overlap between at least three CVD-related traits Table 2.PCSK9, LDLR, LPL, APOB, APOC1  family implicated in a variety of functions in vertebrates, especially angiogenesis. Defects in VEGFA have been shown to be associated with diabetic retinopathy, diabetic nephropathy leading to end-stage renal disease and diabetic neuropathy. These genes were also connected to the lipid genes by GRAIL.We used pathway-based analysis as implemented in GRAIL to identify subsets of positional pleiotropic genes, shared across at least 2 phenotypes, involved in similar biological processes. GRAIL uses abstracts from the entirety of the published scientific literature to look for relatedness among genes within disease regions that may represent key pathways. We undertook this approach to capture both clearly established close gene relationships and potentially undocumented or distant ones. We found that the strongest connections were between genes involved in lipid transport and metabolism, such as B, APOC1 Figure 3It is of interest that many pleiotropic GWAS loci had no relationship to each other or to genes with established functional connection regardless of how current the reference data. An incomplete gene function annotation and limited knowledge of biological pathways could potentially explain this finding. Despite considerable advances in expression quantitative trait loci (eQTL) research, there are questions about the completeness of the eQTL databases. Most of the human eQTL studies to date have analyzed cell types in blood, because these are the most readily available tissues, only recently moving to a wider variety of tissues such as cortical, adipose and liver tissues −7) or based on author-reported genes instead of positional genes.We sought to analyze the data in as unbiased a way as possible. To this effect, we did not include metabolic syndrome as a CVD-related phenotype, as its definition encompasses two or more of the included phenotypes and would thus positively bias any overlapping gene lists. We also excluded catalog genes that were reported by investigators who may have had pre-existing notions about disease causality, and relied only on positional information provided by the catalog. Of note, the complete replication of the established relationships was not reproduced when the analysis was limited to associations that reached genome-wide significance Click here for additional data file.Table S2List of included studies.(XLSX)Click here for additional data file.Table S3−7.List of GWAS positional genes associated with at least two CVD-related phenotypes. In bold are genes that showed overlaps in studies where only GWAS studies of cohorts of European ancestry were included. Underlined are genes that showed overlaps under the more stringent GWAS threshold of P<10(DOCX)Click here for additional data file.Figure S1Bubble Chart representing the positional GWAS genes intersection in cohorts of European Ancestry only. The size of the phenotype is representative of the percentage of genes studied attributed to that phenotype. Line thickness is representative of the number of intersecting genes between two phenotypes.(TIF)Click here for additional data file.Figure S2Bubble Chart representing the positional GWAS genes Intersection in studies in cohorts of African Ancestry only. The size of the phenotype is representative of the percentage of genes studied attributed to that phenotype. Line thickness is representative of the number of intersecting genes between two phenotypes.(TIF)Click here for additional data file.Figure S3−7. The size of the phenotype is representative of the percentage of genes studied attributed to that phenotype. Line thickness is representative of the number of intersecting genes between two phenotypes.Bubble Chart representing the positional GWAS genes intersection in the ethnicity-pooled analysis with more stringent GWAS P-values<10(TIF)Click here for additional data file.Figure S4Bubble Chart representing the GWAS author-reported genes intersection in the ethnicity-pooled analysis. The size of the phenotype is representative of the percentage of genes studied attributed to that phenotype. Line thickness is representative of the number of intersecting genes between two phenotypes.(TIF)Click here for additional data file.Figure S5A plot of 38 genes that overlapped between at least two CVD-related phenotypes based on the PubMed abstracts published up to 2011. Plotted using VIZ-GRAIL. For each plot, phenotypic overlap is arranged along the outer circle; blue indicates three-way or four-way overlaps. Inner circle represents individual genes; genes in bold belong to one of the connecting lines in the middle of circle. The redness and thickness of lines connecting pairs of genes represent the strength of the connections.(TIF)Click here for additional data file.


Microsatellite instability (MSI) is the molecular hallmark of DNA mismatch repair deficiency. Since its initial description in colorectal cancer (CRC) in 1993 and its association with Lynch syndrome, the most common inherited cancer predisposition world-wide, accumulating evidence suggests that MSI status may also be of concrete prognostic and predictive value in the management of sporadic CRC. This mini review aims at providing a concise survey of the molecular basis and the multifaceted role(s) of MSI status in today’s clinical practice. Saccharomyces cerevisiae, and eventually opened up a new field in cancer research  several research groups had made an intriguing observation: widespread somatic alterations at short, repetitive DNA sequences, referred to as replication error (RER+) phenotype or, more specifically, microsatellite instability (MSI) , consist of a few to several thousands of tandemly repeated motifs made up of one (mono) up to six (hexa) nucleotides and are thought to account for approximately 3% of the human genome . Given tDue to their repetitive sequence structure, it is not surprising that microsatellites exhibit a particularly high mutation rate compared to non-repetitive, unique DNA stretches. During replication of the nuclear genome DNA polymerases, due to slippage, often fail to correctly duplicate such microsatellite tracts. The resulting insertion/deletion loops can consequently lead to insertions or deletions of repeats, thereby altering the length of the respective microsatellite . In eukaryotes these errors are corrected by the DNA MMR system: its heterodimers MSH2/MSH6 and MSH2/MSH3 detect the replication error (licensing step) and recruit the MLH1/PMS2 complex through which degradation of the mutated stretch and resynthesis are initiated . As origSince MSI analysis of colorectal tumors provided a straight-forward, though indirect, means to identify LS patients great attention was given to the development of selection criteria and microsatellite markers to be used for testing. In 1996 and 2002 the National Cancer Institute in Bethesda, MD, held workshops in which the Bethesda guidelines for the identification of LS patients were defined , 8. MSI To assess the presence of MSI in a given tumor the NCI workshop recommended to analyze a panel of five microsatellites including two mono-  and three di-nucleotide markers ; depending on the mutational pattern, a secondary panel including additional mononucleotide  and/or complex microsatellites  should be tested . AlternaThe molecular dissection of CRCs into MSI-H and MSS tumors allowed to delineate two major, virtually exclusive pathways of genetic instability: chromosomal instability, which results in aneuploidy and is present in about 85% of CRC, and MSI. Whether MSI-L CRC constitute a pathogenetic class of their own is still a matter of debate: with regard to clinical, biological, and morphological parameters they closely resemble those of MSS CRC. Since the analytical sensitivity to detect MSI-L is dependent on the number of microsatellite markers analyzed, result interpretation, and comparison between different studies investigating MSI-L and MSS tumors are heavily compromised; furthermore, extensive genotyping efforts have failed to demonstrate fundamental differences , 11. Giva priori chance of 50% of having inherited the pathogenic MMR mutation.The discovery of MSI in the majority of LS-related CRC led by analogy to a similar biochemical defect in yeast to the identification of the underlying MMR germ line mutations in MLH1, MSH2, MSH6, and PMS2. Heterozygous carriers of a MMR gene alteration are at a greatly increased lifetime risk to develop cancers of the LS tumor spectrum, mainly CRC (25–70%) and endometrial cancer (30–70%) . KnowledIn contrast to their sporadic MSS CRC LS patients’ tumors typically have a comparatively favorable prognosis and absence of distant organ metastasis; together with the observation that LS cancers are accompanied by an intense immune response with dense lymphocytic infiltrates points to a possible protective effect by the immune system .per se remains underdiagnosed , to 5-FU treatment  based chemotherapy. In the context of MSI and an underlying (hereditary or sporadic) defect in MMR the use of 5-FU in MSI-H CRC merits particular attention: as demonstrated in numerous reatment , 20. In reatment , 22. TheAs exemplified by the rapidly growing list of cancer genomes analyzed by means of ever more complete as well as cost-effective high-throughput (“next generation”) sequencing methods, a novel era of truly personalized medicine seems to be at hand . It is tThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The primary goal of palliative cancer care is typically to relieve suffering and improve quality of life. Most approaches to diet in this setting have focused only on eating as many calories as possible to avoid cachexia. However, as the concept of palliative care has evolved to include all aspects of cancer survivorship and not just end of life care, there is an increasing need to thoughtfully consider diet and nutrition approaches that can impact not only quality of life but overall health outcomes and perhaps even positively affect cancer recurrence and progression. In this regard, there has been a recent emphasis in the literature on nutrition and cancer as an important factor in both quality of life and in the pathophysiology of cancer. Hence, the primary purpose of this paper is to review the current data on diet and nutrition as it pertains to a wide range of cancer patients in the palliative care setting. In cancer care models, the primary goals of palliative care are to relieve suffering and improve quality of life across the cancer illness trajectory. As the concept of palliative care has evolved to include all aspects of cancer survivorship and not just end of life care, there is an increasing need to thoughtfully consider lifestyle behaviors that can positively affect health outcomes and cancer progression for those who have responded to oncological interventions. In that regard, there has been a recent emphasis in the literature on nutrition and cancer as an important factor in both quality of life and in the pathophysiology of cancer. Hence, the primary purpose of this paper is to review the current data on diet and nutrition as it pertains to a wide range of patients with cancer in the palliative care setting.  At one time, palliative care referred to “end of life” only, in which case patients were encouraged to eat whatever they felt like. If the patient deteriorated, the standard nutritional strategy became more focused on quality of life (QOL) and relief of symptoms, rather than active nutritional interventions aimed at improving outcome. At this stage of the cancer illness, the expectation was short survival time, and the major goal was to minimize cachexia. Thus, any calories were deemed to be “good” calories. As a broader number and range of patients come under the purview of this expanded palliative model, recent research has suggested that it may be more appropriate to develop a specific nutritional strategy which can help improve the overall health and well-being of cancer patients, including those in the traditionally defined palliative care setting. In fact, when making decisions about nutritional support, one must consider diagnosis, prognosis, current status and symptoms, pathways of disease progression, treatment and possible side effects, patient comfort and peer and social support, socioeconomic status, cultural and religious views, and ethical and legal issues. ensuring an adequate amount of calories,reducing foods and dietary habits that have been specifically linked to modifiable risk factors for cancer reoccurrence,creating a diet that minimizes inflammation, insulin resistance, and oxidative stress,ensuring an adequate amount of specific nutrients and selective nutritional supplementation linked to cancer prevention/recurrence. Hence, the primary purpose of this paper is to introduce the concept of diet as a potential survivorship intervention as opposed to sustenance only. Our goal is to synthesize the known knowledge in a way that might assist clinicians while making nutritional recommendations toUsing these strategies and based upon the most current data available, appropriate, individualized approaches to palliative care diet and nutrition can be established for a wide spectrum of patients with cancer. The diagnosis of cancer often powerfully motivates survivors to alter their lifestyle habits, so healthcare providers need to be aware of what changes patients are likely and willing to make in order to determine the most appropriate recommendations . This reIt should be stated at the outset that this paper pertains broadly to all types of cancer. However, we also recognize that there may be differences in the nutritional approach to patients with different types of cancers. Thus, patients with lymphoma may have different requirements or benefit differently from various supplements than patients with sarcoma. At the present moment, we do not feel that there is sufficient data to help with these distinctions. In addition, this paper pertains primarily to adult patients rather than children. While some of the concepts below would certainly pertain to pediatric patients and an overall analysis of nutrition in children would be extremely valuable, space does not permit an additional review and analysis of this area of palliative care.Nutrition should be a priority early in palliative care. Food has a primary role in life for everyone including people with advanced illness. Adequate nutrition is essential, not only to meet physiological requirements, but also because it has additional psychological, spiritual, social, and cultural benefits for patients and caregivers. Strategically addressing these additional aspects is of high importance in the palliative setting.  The hypothesis that increased energy expenditure leads to weight loss experienced by patients with cancer has been supported in the literature , 3. HoweThe American Cancer Society (ACS) provides some important guidelines for nutritional support in patients with cancer. The ACS guidelines acknowledge that “many persons with advanced cancer may need to adapt food choices and eating patterns to meet nutritional needs and to manage symptoms and adverse effects such as pain, constipation, and loss of appetite” .  The specific dietary guidelines from the ACS, however, are relatively general and include choosing foods and drinks in amounts that help patients get to and maintain a healthy weight; limiting the amount of processed and red meat eaten; eating at least 2(1/2) cups of vegetables and fruits each day; and choosing whole grains instead of refined grain products. These guidelines mirror others that emphasize high-calorie, high-energy meals in small portions , and suc The notion of palliative care traditionally evoked a cachectic picture. However, many patients are overweight at the time of diagnosis or become so after treatment . Women d Hence, optimal body mass index and body metrics should be pursued in an appropriate manner based upon the status of the patient, whether cachectic or overweight. The health care team should be intimately involved in addressing the specific caloric needs of each cancer patient throughout the clinical course, both in terms of the number of calories required and the quality of those calories.Eating foods that contain high amounts of excess sugars is an unhealthy diet even in the palliative care setting. Such energy-dense, nutrient-poor foods include refined sugars, candies, fried foods, and so-called “junk food.” The literature contains ample consensus that eating such foods favors the onset of obesity and diabetes, both factors in cancer development. For example, a high intake of refined carbohydrates has been associated with cancer in general  and more Red and processed meat consumption has consistently gained a reputation as a contributor to disease, including cancer. Data is emerging that red and processed meats may influence disease recurrence and mortality as well, for example, for colorectal cancer survivors. Evidence shows that consumption of red meat can activate cancer genes in the colon  such as  The data also suggests that red meat, in particular, is pro-inflammatory and procarcinogenic. For example, the European Prospective Investigation into Cancer and Nutrition-Potsdam study of 2,198 men and women found that the consumption of red meat was significantly associated with higher levels of the inflammatory markers GGT and hs-CRP when adjusted for potential confounding factors related to lifestyle and diet . Another A mouse study demonstrated that a high animal fat diet could actually induce tumor growth and progression, including epithelial-mesenchymal transition (EMT) and inflammation . This stP < 0.05), with a stronger association for processed red meat. In this study, the intake of fruits or vegetables had no correlation with bladder cancer, but higher levels of vitamin B12 intake were found to be protective .  A dietary analysis of 1171 patients with bladder cancer compared a variety of dietary factors to those of 1,418 non-cancer patients . The res In an analysis of patients with early stage breast cancer from the Life After Cancer Epidemiology Study  research Overall, the cumulative data suggests that refined carbohydrate and red meat consumption should be avoided or significantly reduced in the diet of patients with cancer as there are many other healthier sources of caloric intake. Achievement and maintenance of a healthy body composition via a plant-based diet high in fruits, vegetables, and whole grains and low in saturated fats and red or processed meats should be the guidelines imparted to patients from their health care providers. As it has become increasingly apparent that cancers use inflammatory pathways for growth and metastases, the concept of utilizing anti-inflammatory diets has received more attention. The literature indeed shows more studies exploring the link between cancer and inflammation . InflammκB), STAT3, and eIF4E activity [κB and STAT3 activation pathways have the potential to prevent and/or treat cancer and to be of use in the palliative care setting. For instance, compounds called triterpenes are particularly adept at suppressing inflammatory pathways linked to cancer. The mechanism of action is most likely related to the suppression of different cytokines, in addition to prostaglandin E2, particularly from activated macrophages [Platycodon, pristimerin, ursolic acid, and withanolide. These can be obtained from botanical medicines and diet [ Mounting evidence suggests that chronic inflammation mediates most chronic diseases, including cancer, and causes a great deal of morbidity and ultimately mortality. Hence, good targets for anticancer strategies include major regulators of inflammation, cellular transformation, tumor cell survival, proliferation, invasion, angiogenesis, and metastasis such as transcription factors nuclear factor-kappa B . Symbiotic intestinal microbiota generate responses that help reduce inflammation and enhance the health of the gut. Dysbiotic segmented filamentous bacteria induce Th17 cells and promote inflammation, which is a factor in cancer development. Unfavorable microbiota and their toxic components have been shown to act on both Nod1 and Nod2 receptors, and their defective signaling may account for the development of inflammatory bowel disease (IBD). IBD increases the risk of colorectal cancer . Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus delbrueckii spp. bulgaricus) recovered faster from the acute inflammatory phase of IBD and had lower disease severity in the chronic phase. Adenoma and adenocarcinoma formation was also diminished by both treatments. VSL#3 increased the mRNA expression of TNF-α, angiostatin and PPARγ, whereas CLA decreased COX-2 levels. Both CLA and VSL#3 suppressed colon carcinogenesis, although VSL#3 showed greater anti-carcinogenic and anti-inflammatory activities than CLA. CLA modulated expression of COX-2 levels in the colonic mucosa, whereas VSL#3 targeted regulatory mucosal CD4+ T cell responses [ Beneficial intestinal microbiota due to their regulatory function of gut immune response can prevent or retard neoplastic growth. Probiotic bacteria can produce metabolites such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid that has anticarcinogenic effects. In one study, mice treated with CLA or a probiotic called VSL#3  has been more frequently suggested to influence various processes associated with an increased cancer risk. So far, mechanisms that could explain the preventive action of probiotics against colorectal cancer onset may include (a) binding and degradation of potential carcinogens; (b) quantitative, qualitative, and metabolic alterations of the intestinal microflora; (c) production of anti-tumorigenic or antimutagenic compounds; (d) competition with putrefactive and pathogenic microbiota; (e) enhancement of the host's immune response; (f) direct effects on cell proliferation; (g) improvement of the host's immune response; (h) antiproliferative effects via regulation of apoptosis and cell differentiation; (i) fermentation of undigested food; and (j) inhibition of tyrosine kinase signaling pathways , 54. Lactobacillus casei DN-114 001 in decreasing the incidence and grade of RT-induced diarrhea [Lactobacillus GG supplementation may reduce the frequency of severe diarrhea and abdominal discomfort related to 5-FU-based chemotherapy [ Particularly meaningful to patients in treatment regimens that include radiation therapy (RT) to the abdominal region for cervical, ovarian, prostate, sigmoid, or colorectal cancer is the potential therapeutic benefit from probiotics. RT can upset the colonization resistance of the indigenous gut flora, causing RT-induced diarrhea, enteritis, and colitis sometimes in more than 80% of patients. Randomized trials have demonstrated efficacy of probiotics such as VSL#3 and diarrhea . Probiotdiarrhea , 57. Simdiarrhea . Lactobaotherapy .  Symbiotics, the combination of probiotics and prebiotics, have been studied in patients with advanced colorectal cancer, who may have associated comorbidities such as reduction of immunity, increased rate of infections, impaired cicatrisation of wounds, and muscle weakness. The role of chronic inflammation is known in many of these complications. Investigators are in pursuit of knowledge in this field. The Human Microbiome Project holds promise to help us gain factual and useable knowledge to help patients with cancer . This NIOverview of Probiotics. Probiotic type/benefits/dose.L. acidophilus and B. delbrueckii subspecies bulgaricus: antibiotic-associated diarrhea 2 × 109 daily for 5–10 days.L. acidophilus and B. longum: antibiotic-associated diarrhea 5 × 109 daily for 7 days.L. rhamnosus GR-1 and L. fermentum RC-14: vulvovaginal candidiasis 109 bacteria in skim milk, twice daily  for 14 days.11 daily for 8 weeks.VSL#3 irritable bowel syndrome 9 × 1012 twice daily for 6 weeks plus conventional therapy.Ulcerative colitis active flares: 1.8 × 1012 bacteria twice daily.Pouchitis 1.8 × 10It has been noted that “despite the positive results and plethora of agents, bacterial combinations and concentrations, the inconsistency in administration, the inhomogeneity of comparison groups and lack of stringent clinical endpoints remain obstacles in the effort to establish a definitive clinical strategy at this time. Further work is warranted to gain a keen understanding of their clinical value in CRC patients” . It shou However, it is not yet possible to make generalized recommendations on when and when not to take specific prebiotics and probiotics at this time. Based on the available evidence, it is reasonable that physicians and patients discuss these issues together and come up with personal strategies using available products, sound clinical judgment, and the best current research evidence.ω3-PUFAs) such as alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid are associated with a decreased incidence and severity of several chronic diseases including cardiovascular disease (CVD) and cancer” [ω3-PUFAs such as eicosapentaenoic acid (EPA) and polyphenols such as curcumin and resveratrol have been demonstrated to have anticolorectal cancer activity in preclinical models. The EPA in the free fatty acid (FFA) form has been shown to reduce adenomatous polyp number and size in patients with familial adenomatous polyposis [There is accumulating evidence that the use of omega-3 fatty acids has the potential as anticancer compounds. How they interact with the cancer process is becoming clearer as investigations unfold. “Diets rich in omega-3 polyunsaturated fatty acids  and studied the mechanisms of action. The results showed that DHA induces apoptosis in TNBC cells via activation of caspase-8 and -9 dependent proapoptotic events [ω-3 PUFA diet that blocks microRNA-21, thereby increasing the tumor suppressor gene PTEN levels which may help prevent expression of CSF-1 in breast cancer [Health, Eating, Activity, and Lifestyle Study had subjects complete a food frequency/dietary supplement questionnaire and provide blood samples to assess diet and inflammatory markers [ω-6 relative to ω-3 PUFAs were associated with greater C-reactive protein levels and greater odds of having fatigue (OR of 2.6 for the highest versus the lowest tertile of intake). The results suggest that reducing ω-6 while increasing ω-3 fatty acid intake might be beneficial for reducing fatigue in patients with cancer.  Exogenous PUFAs have been shown to attenuate breast cancer cell proliferation and migration, suggesting a mechanism in which PUFAs restrain breast cancer growth partly via their inhibition of transient receptor potential channels (TRPC) . DHA hasc events . Expresst cancer . A study markers . The resin vivo study, a diet with high levels of EPA and DHA strongly suppressed the growth of human pancreatic cancer xenografts in athymic nude mice, by inducing oxidative stress and cell death [ω-3 PUFAs have been shown to preserve muscle mass and function in cancer patients even during active treatment. During chemotherapy, omega-3 fatty acids may contribute to a reduced inflammatory response, and some small studies showed that omega-3 fatty acids increase the response rate to chemotherapy [ Some studies suggest that dietary intake of EPA and DHA in foods and supplements may have protective associations against the development of endometrial cancer . EPA andll death . In addiotherapy . The relationship between specific fatty acids and prostate cancer survival remains a field with need for more research. Dietary intake of 14 fatty acids was analyzed in a population-based cohort of 525 Swedish men with prostate cancer. Among all men, those with the highest omega-3 DHA and total marine fatty acid intakes were 40% less likely to die from prostate cancer.ω-3 PUFAs. Axonal sensory peripheral neuropathy is a major concern in paclitaxel therapy. Omega-3 fatty acids have beneficial effects on neural damage from their effects on neuronal cells and participate in inhibition of the formation of proinflammatory cytokines involved in peripheral neuropathy. Patients with breast cancer have a longer disease free survival rate with the newer therapeutic agents. Finding a way to mitigate the disabling effects of iatrogenic peripheral neuropathy may significantly improve the patients' quality of life [ω-3 PUFAs supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery [ω-3 PUFAs in patients with cancer is a desirable practice.  Complications of treatment such as neuropathy may potentially be helped by  of life . In more surgery . In viewIn the past few years, there has been a growing emphasis on the importance of maintaining adequate levels of vitamin D to help maintain overall health and immune function. Similarly, maintaining adequate levels of vitamin D in advanced cancer and palliative care patients would seem an easy and logical strategy. The data below strongly supports the importance of achieving adequate blood levels of vitamin D in such patients.  From a physiological perspective, several studies support the importance of vitamin D to help with cancer management. The active form of vitamin D interacts with the vitamin D receptor (VDR) to induce antiproliferative, anti-invasive, proapoptotic, and prodifferentiation activities in prostate cancer cells . Via sev Clinical studies have also suggested an important role for vitamin D in cancer. To begin, several studies have shown that patients with cancer often have low levels of vitamin D. For example, a study of 195 patients presenting to a radiation oncology clinic with advanced stages of cancer showed that approximately 75% had vitamin D levels that were either deficient (<20 ng/mL) or suboptimal (20–30 ng/mL) . The aut Studies have reported an inverse relationship between vitamin D intake and the risk of breast cancer. Studies have also suggested that there are improvements in cancer survival after a diagnosis of breast cancer in women when they have higher levels of vitamin D . Vitaminβ-carotene supplement users. Another study of 658 patients with various types of cancer showed that patients with 25(OH)D levels below 46 nmol/L at diagnosis experienced shorter survival . In addi3 was lower than that found in a noncancer reference group [3 concentrations over 10 ng/mL  compared to women with concentrations below 10 ng/mL . Another study of 1800 patients with breast cancer showed that serum 25OHD (>30 ng/mL) at initial diagnosis of breast cancer significantly correlated with smaller tumor size, improved overall survival, and disease specific survival, especially in postmenopausal patients [ In patients with ovarian cancer, serum concentration of 25(OH)Dce group . In addipatients .P < 0.05) after 3 months which also corresponded to improved serum levels of vitamin D [ Of course, while the association between low vitamin D levels and cancer is supported by a number of the mentioned studies, perhaps the more important question is whether vitamin D supplementation will actually be beneficial in the management of cancer patients, and for this paper, whether such treatment might be useful and advisable in the palliative care population. One study showed that when 69 vitamin D deficient patients were given 2,000 u vitamin D per day, there was a marked improvement from baseline in fatigue  in 60 women with breast cancer and aromatase inhibitor-induced musculoskeletal symptoms showed that, at 2 and 6 months, measures of pain such as the FIQ pain, BPI worst-pain, BPI average-pain, BPI pain-severity, and BPI pain interference scores were significantly better in those patients receiving vitamin D compared to placebo . 3 between 3,000 and 5,000 IU daily which generally achieves adequate levels in most treated patients with a target of approximately 50 ng/mL. Consideration should also be given to include supplementation of vitamin D cofactors such as calcium and phosphate in the appropriate setting, especially when deficient. Few studies have actually been performed in palliative care patients, but given the above data, it would seem reasonable to ensure that serum vitamin D levels are maintained. Vitamin D supplementation would likely be beneficial both with regard to the cancer itself and perhaps more importantly for the palliative care patient, helping to maintain quality of life. In addition, there appears to be a very little problem with providing vitamin D supplementation in terms of adverse effects or interference with medications. Thus, adequate vitamin D supplementation to maintain a serum level of at least 50 ng/mL would seem to be an appropriate nutritional goal in palliative care patients. This could be done with either daily supplementation or weekly typically with doses of vitamin DEven multivitamins could potentially have an emerging role in the management of cancer patients in general and in palliative care patients specifically. Kwan et al.  evaluate Another controversy regards the use of antioxidant supplements during chemotherapy treatment. Although traditionally this has not been considered good practice, there is mounting evidence that antioxidants may be helpful. A systematic analysis of 19 randomized, controlled clinical trials of antioxidant concurrent use in chemotherapy patients revealed that in most studies, antioxidant supplementation resulted in either increased survival times, increased tumor responses, or both, as well as fewer toxicities than controls . The sup Another controversial area is antioxidant supplements during radiation. Many acute and chronic effects of ionizing radiation are mediated by reactive oxygen species and reactive nitrogen species which deplete antioxidant stores, leading to cellular apoptosis, stem cell depletion, and accelerated aging. Epperly et al.  have fou In recent years, a large number of studies have attributed a protective effect to polyphenols and foods containing these compounds  against cancer development and propagation, when used as chemopreventive agents. The mechanism of action of these polyphenols is most likely based upon antioxidant activity . HoweverDeveloping a healthier diet with the addition of specific supplements designed to reduce oxidative stress and inflammation should be an important part of an overall palliative care plan. It is interesting to note that the cancer diagnosis is frequently a motivator for improving diet and nutrition. A study of 1,560 patients with breast cancer [ The majority of cancer fighting nutrients should be obtained via a wholesome diet. Nutrient-dense foods contain substantial amounts of key nutrients in relation to the dietary energy they provide . Nutrien In conclusion, it is important to consider the diet and nutritional planning for patients in the palliative care setting, focusing on foods and supplements that provide cancer fighting nutrients, reduce oxidative stress and inflammation can help overall quality of life, and have a beneficial effect on their cancer and other medical conditions. Anything done by the patients on their own to take an active role in improving health and cancer outcomes is desirable on multiple levels. This is an era of increasing current and future global cancer burden, coupled with concomitantly shrinking health care budgets and resources. Hopefully, this paper serves as stimulus for further research and activity in exploring the concept of dietary intervention in palliative care patients with cancer as a possible treatment plan costrategy for changing the “host” in ways which favor physiological function of the whole body and contemporaneously oppose cancer biology, while addressing caloric, macro, and micronutritional requirements.With the projected global future cancer burden and attendant health care costs, every opportunity should be taken to teach patients and, therefore, their close contacts behaviors which could positively influence the health of the population at large.


Lactobacillus and Enterococcus. In addition, hierarchal analysis revealed a significant increase in the relative abundance of the phylum Actinobacteria, the order Actinomycetales, and several novel genera under the family Bifidobacteriaceae and Coriobacteriaceae, in the LWB group. Functional annotation of the shotgun sequences suggested that approximately 9% of the 4709 Kyoto Encyclopaedia of Gene and Genome (KEGG) hits identified were impacted by the LWB-diet. Open Reading Frames (ORFs) assigned to KEGG category xenobiotics biodegradation and metabolism were significantly greater in the LWB-enriched diet compared to the control and included the pathway for benzoate degradation [PATH:ko00362] and glycosaminoglycan degradation [PATH:ko00531]. Moreover, the number of ORFs assigned to the bacterial invasion of epithelial cells [PATH:ko05100] pathway was approximately 8 fold lower in the LWB group compared to controls. This study demonstrated that LWBs have the potential to promote gut health and can aid in the development of optimal diets.The gastrointestinal tract is populated by an array of microbial species that play an important role in metabolic and immune functions. The composition of microorganisms is influenced by the components of the host’s diet and can impact health. In the present study, dietary enrichment of lowbush wild blueberries (LWB) was examined to determine their effect on colon microbial composition and their potential in promoting gut health. The microbial composition and functional potential of the colon microbiota from Sprague Dawley rats fed control diets (AIN93) and LWB-enriched diets (AIN93+8% LWB powder substituting for dextrose) for 6 weeks were assessed using Illumina shotgun sequencing and bioinformatics tools. Our analysis revealed an alteration in the relative abundance of 3 phyla and 22 genera as representing approximately 14 and 8% of all phyla and genera identified, respectively. The LWB-enriched diet resulted in a significant reduction in the relative abundance of the genera  Vaccinium angustifolium) (LWB) are excellent sources of fiber, manganese, and polyphenols, such as anthocyanins Dietary enrichment of blueberries demonstrated impacts on gut microbial population dynamics and gastro-intestinal tract (GI) health 9−1012 microorganisms, and may contribute up to a 10% increase in caloric absorption Microbial catabolic reactions can yield products that are more biologically active than the parent compounds, and therefore may be more beneficial to the host Bifidobacteria more than two fold Enterobacteriaceae counts Enterobacteriaceae family and different Enterococcus species have been documented to increase with a concomitant decrease in BifidobacteriaBifidobacteria and other beneficial species. Recent developments in molecular biology allow for the simultaneous analysis of genes and have revealed insights into the molecular basis of the native gut microbiota in response to dietary changes. Advances in DNA sequencing technology have dramatically changed the way scientists investigate the microbial communities that populate the gut Lowbush wild blueberries have demonstrated a positive effect on native GI microbiota through the increase of beneficial microorganisms There is a lack of knowledge with respect to how LWB affect the function and cellular mechanics of gut microbes and how this interaction could potentially impart health benefits to the host. The objectives of this research were to i) characterize the function of the gut microbial community of the Sprague Dawley (SD) rats using metagenomic techniques and ii) use this model to detect compositional and functional changes in a LWB enriched diet. Investigating changes in gut microbiota in response to LWB enrichment may provide insights into how LWBs impart health benefits to their host.ad libitum and diet consumption was measured daily. Animals consumed 20±4 g of feed per day, an amount equivalent to 24.0±5.2 mg of anthocyanins and 4.5 g of fiber per day in the LWB diet 2/5% O2 inhalation for 3 min and samples from the large intestine were taken as follows.The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the Institutional Animal Care and Use Committees of the University of Maine (Protocol #A2011-01-03). Nine male, three-week old Sprague-Dawley (SD) rats, approximately 90 g in weight, were obtained from Charles River Laboratories . They were randomly divided into 2 groups: the control group (N = 4), which was fed a control diet (AIN93), and the treatment group (N = 5), which was fed a blueberry-enriched diet, (AIN93+8% w/w LWB powder substituting for dextrose) et al., 2011 Animals were dissected in a laminar flow hood to ensure sterility. Once the rat cavity was fully exposed, the intestines were unwound. Samples for metagenomic analysis were taken from the proximal colon, approximately one inch downstream of the cecum. The colon contents (0.37±0.13 g) were collected by elevating one end of the large intestine and pushing contents into a sterile sample container. The samples were then snap-frozen in liquid nitrogen and stored at −80°C. A QIAamp DNA stool kit was used to extract metagenomic DNA with modifications to the protocol described by Li de novo assembled using SOAPdenovo software package (http://soap.genomics.org.cn/soapdenovo.html), and normalized to ensure that each genome was represented equally regardless of size. Quality reads were then analyzed using MetaPhyler, mapping genes against a reference genomes using BLAST t-test Approximately 1.0 µg of high-quality DNA was processed using an Illumina TruSeq DNA sample prep kit following the manufacturer’s instruction . Final individual libraries were validated, pooled based on their respective 6-bp adaptors, and sequenced at 100 bp/sequence read using an Illumina HiSeq 2000 sequencer. All Illumina raw sequences were deposited into MG-RAST databases (MG-RAST Accession # from 4470921.3 to 4471270.3). The mean numbers of whole genome shotgun (WGS) sequence reads used in this study are listed in denovo. Open reading frames (ORFs) were predicted from contigs ≥200 bp using FragGeneScan (v1.14). The resultant ORFs were independently annotated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.Raw sequence reads were uploaded into a MG-RAST server for quantitative views of the microbial populations in the rat proximal colon based on WGS sequence data Metastats (http://metastats.cbcb.umd.edu). The method relied on two assumptions: first, that each of two treatment populations has multiple samples: and second, that the relative abundance data were available for specific features, such as relative abundance assigned to each taxon, or numbers of sequences mapped to a given biological pathway. The matrix was normalized from raw abundance measures to a fraction representing the relative contribution of each feature to each of the individuals. A modified t-test was then used to compute a two-sample t statistic. The threshold for the t statistic was chosen to minimize the number of false positives by controlling p values using the nonparametric t-test with 1000 permutations, which dealt with the underlying distributions that were non-normal. For low frequency (sparse) features, such as low abundance taxa, Fisher’s exact test was used to model the sampling process according to a hypergeometric distribution (sampling without replacement).Statistical analysis was performed using P = 0.016), Bacteroidaceae , Rikenellaceae , and Ruminococcaceae  were among the most abundant. Similarly, 306 prokaryotic genera were identified in the rat proximal colon using MetaPhyler. Among them, Lactobacillus, Bacteroides, Alistipes, Clostridium, Ruminococcus, and Akkermansia were the major representatives in the rat colon. In addition to MetaPhyler, the microbial composition of the rat colon microbiota was also characterized independently using the MG-RAST pipeline which identified 32 phyla and 752 genera. The most abundant phyla identified by MG-RAST were Bacteroides, Firmicutes, and Proteobacteria, and the most abundant genera identified were Bacteriodes, Alistipes, Clostridium, Lactobacillus, Prevotella, and Parabacteriodes  2-fold higher abundance in the LWB rats than the control. Members of the order Actinomycetales were twice as abundant and included the genera Thermonospora and Cornynebacterium, and novel genera classified under the family Nocardioidaceae, Pseudonocardiaceae, Micrococcaceae, and Actinomycetaceae , which represents almost a 5 fold reduction. In addition, the relative abundance of Enterococcus was significantly reduced from 0.06% to 0.002%, representing a 30-fold reduction, as a result of the LWB dietary treatment (ce reads . Comparereatment .P<0.05)  using the MG-RAST pipeline. Statistical comparisons revealed significant changes in ≈9% of all KO identified as a result of the dietary treatment. The five most abundant level 2 categories identified were translation, amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, and replication and repair . Among tP<0.05) . BenzoatDiet has an influence in shaping the development and composition of the gut microbiota, which is also defined by host genetics and the bacteria acquired at birth Enterococcus and Clostridium spp. translocations to the liver in SD rats Enteococcus faecalis, a commensal bacterium of humans and animals, has shown the capability to induce irritable bowel syndrome (IBS) in IL-10 gene-deficient mice, suggesting that certain enteric microbiota tend to be more opportunistic and can induce colon inflammation Enterococcus spp. in mice fed diets supplemented with blueberries Enterococcus spp. in the LWB enriched diet. The results from this study and recent research suggest that the protective anti-inflammatory effect of blueberries can be accredited to microbial metabolism, which is dependent on the composition of the microbiota.The permeability of the colonic epithelium is an important aspect of gastro-intestinal health, and increases in permeability can allow for gut-derived bacteria and toxins to infiltrate the liver via the portal circulation. Blueberries have demonstrated reduction in the degree of parenchymal infiltration and Bifidobacteria and Lactobacilli are normally present in the large intestine of healthy humans in numbers ranging from 108–1010 CFU/ml and 106–108 CFU/ml, respectively Lactobacillus is higher in the dextrose diets, reflecting their capability to competitively ferment simple sugars in the rat colon. The shifts toward members of the Actinobacteria phylum at the expense of Lactobacillus species could be a reflection of the complexity of LWB compound catabolism and that microbial digestion of LWB may require a concerted effort of multiple species In vitro human fecal batch cultures have demonstrated the enrichment of Lactobacillus and Bifidobacteria, with the addition of 1 g/L gallic acid and 200 mg/L of anthocyanins Lactobacillus and Bifidobacteriain vivo studies with humans fed freeze-dried LWB in a smoothie demonstrated significant increases of only Bifidobacteria species and no impact on Lactobacillus populations Although many microbial members of the large intestine have not been identified, it is known that some species of bacteria preferentially colonize the large intestine Thermospora, Corynebacterium, Microccaceae, and Nocardiodaceae, were detected in a higher abundance in the LWB enriched diet. In termite hind guts, members of the phylum Acntinobacteria have demonstrated their involvement in xenobiotic metabolism and these microorganisms could possibly contribute to the degradation of benzoate compounds derived from blueberries Bifidobacterium, Escherichia, and Lactobacillus isolated from human fecal samples have been reported to execute the hydrolysis reactions of phenolic acids and polyphenols Bifidobacterium longum has a large number of predicted proteins (more than 8%) related to the catabolism of nondigestible plant polymers, including enzymes involved in the degradation of complex polysaccharides and xenobiotics Lowbush wild blueberries are exposed to numerous microorganisms with extensive metabolic capacities that are lacking in the host. Previous research demonstrated that anthocyanidins glycosides are hydrolyzed extensively by the intestinal microflora, and the product of microbial digestion is often less stable and possibly more bioavailable Slackia, Bifidobacteria, and Coriobacteriaceae spp. Research using SD rats demonstrated that blueberry and blackcurrant enriched diets increased Bifidobacteria species after dietary treatment and lowered concentrations of pro-inflammatory markers Bifidobacteria fed LWB was observed Coriobacteriaceae family with respect to hepatic triglycerides, glucose/glycogen levels, and enterohepatic circulation Bifidobacterium/Coriobacteriaceae impacted plasma cholesterol levels in hamsters, with Bifidobacteria being associated with high density lipoprotein particles (HDL) and Coriobacteria being associated with non-HDL particles Bifidobacterium/Coriobacteriaceae ratio in the intestines than infants fed formula, suggesting a beneficial evolutionary adaptation for the harvest of energy from otherwise indigestible oligosaccharides Bifidobacterium/Coriobacteriaceae ratio than the control diet. Our results provide insight into microbial community’s response to LWB and should help elucidate the potential benefits in the gut and host.Lowbush wild blueberry enrichment may favor microbial species that can harvest energy from LWB components and could possibly promote beneficial microbial populations. Health properties attributed to beneficial bacteria include modulation of colonic microbiota by inhibiting a wide range of pathogens, improvement of lactose digestion, reduction of serum cholesterol, stimulation of the immune system through cytokine stimulus, reinforcement of intestinal epithelial cell tight junctions, and increased mucus secretion Although the microbiome of rats differs from humans, the murine model is a powerful tool to study population dynamics and related metabolic functions. Metagenomic studies can determine microbial community profiles, gene presence/absence and abundance, and functional repertoire; however, they can only infer an observed phenotype since a gene’s presence does not imply its expression or functionality


Fibroblast growth factor 21 (FGF21) is an emerging regulator of local and systemic metabolic homeostasis. Treatment with pharmacological levels of FGF21 alleviates obesity and associated metabolic diseases including diabetes. However, beyond anti-obesogenic effects, the normal roles and underlying mechanisms of FGF21 as an endocrine hormone remain unclear. A recent wave of studies has revealed that FGF21 is a stress-induced endocrine factor in liver, muscle, and other tissues that targets adipose tissue and adipocytes through the FGFR1-betaKlotho complex. Adipose tissues and adipocytes within diverse tissues respond with metabolites and adipokine signals that affect functions of body tissues systemically and cells within the local microenvironment adjacent to adipocytes. Normally this is to prevent impaired tissue-specific function and damage to diverse tissues secreting FGF21 in response to chronic stress. Therefore, diverse stressed tissues and the adipose tissue and adipocytes constitute a beneficial endocrine and paracrine communication network through FGF21. Here we attempt to unify these developments with beneficial pharmacological effects of FGF21 on obesity in respect to inter-organ stress communication and mechanisms. Since the debut in 2005 , fibroblBasal serum levels of FGF21 vary widely among individuals and are generally low in pure strains of laboratory mice . DeficieStudies in mice indicate that FGF21 is inducible and the liver is a major source of circulating FGF21 , 10. ParIn contrast to the liver that is a switching station for processing metabolites in support of other organs and tissues and the adipose tissue that serves as a storage depot of energy and metabolic precursor of lipids, muscle is a major fuel consumer. To meet high aerobic catabolic demands for ATP, muscle secretes myokines that call on liver and adipose tissue to supply adequate fuel. Consistent with this concept, several recent studies indicate that FGF21 is induced in skeletal, heart, and gastrocnemius muscle under conditions that cause local and systemic metabolic stress –25. ThesIn addition to liver and muscle, it has been suggested that FGF21 is inducible and secreted into the circulation in other tissues and organs under stress conditions. Brown adipose tissue (BAT) may be a source of FGF21 in response to cold stress and fetal-to-neonatal transition , 29. WhiThese studies suggests that the activation of FGF21 in liver, muscle, and other tissues is a response of these tissues and organs to stress triggered by external challenge and internal cellular pathogenesis that lead to defects in metabolic homeostasis. In support of this notion, several nuclear receptors and transcription factors that are involved in metabolic stress responses regulate FGF21 expression. These include PPARα/γ, RAR, ChREBP, SREBP1c, LXR, STAT3/5, p53, and ATF4 , 33–39. Several recent studies using direct ablation of FGFR1 or KLB in adipocytes, administration of activating antibody targeting specifically the FGFR1-KLB complex, or induction of dysfunctional adipose tissue –44 proviTaken together, these studies indicate that stress messenger FGF21 and adipose FGFR1-KLB team up to respond to stress caused by a wide range of metabolic abnormalities and pathogenesis. The stress-responsive communication mediated by extra-adipose FGF21 and adipocyte FGFR1-KLB occurs predominantly between adipose tissue and at least two tissues whose roles in metabolism are quite different, but when stressed have major consequences on the organism: (1) liver whose role is to maintain lipid, carbohydrate, and organism metabolic homeostasis through synthesis, degradation, and temporary storage; and (2) muscle which is a major fuel consumer predominantly powered by the oxidation of fats and carbohydrates (Figure As a first responder to the SOS message carried by FGF21, how do adipocytes through FGF21-mediated activation of FGFR1-KLB aid stressed tissues and in particular liver and muscle? In addition to as a reservoir of energy, adipose tissue is an endocrine organ capable of systemic signaling through its lipid metabolites and adipokines , 48. A nTwo major effects of FGF21 directly on adipose tissue stand out that may contribute to relief of metabolic stress on liver and muscle. Activation of the adipocyte FGFR1-KLB complex modulates the rate of adipocyte lipolysis and fatty acid oxidation that indirectly relieves hepatocyte steatosis or muscular lipid overload. Upregulation of expression of genes involved in fatty acid oxidation, browning of white fat and thermogenesis is also thought to collectively contribute to the anti-obesogenic effects , 40, 49 Are adipocytes via the FGFR1-KLB partnership the primary or even specific target of systemic or local FGF21 signal observed in diverse body tissues? FGF21-stimulated changes in systemic metabolites and adipokines whose receptors are distributed widely have potential to indirectly affect practically all body tissues. Moreover, adipocytes that are characterized by expression of FGFR1-KLB and present in the microenvironment of most, if not all, tissues have potential to affect functions of adjacent tissues via FGF21-controlled paracrine products in the microenvironment. A careful dissection of both FGFR isotypes and KLB and signals elicited by the partnership in parenchymal and adipocytes of diverse organs that show a response to systemic FGF21 is required to clarify whether non-adipocyte cells constitute additional direct targets for FGF21. It has been suggested that FGF21 may signal directly in hepatocytes , pancreaSince the discovery of the regulatory function of FGF21 in metabolism in 2005, studies have focused on the dramatic systemic pharmacological effects and clinical potential of FGF21 in alleviation of obesity and diabetes. These have obscured its general role as a stress hormone in organ-specific physiopathologies with adipose tissue as its tissue target and adipocyte FGFR1-KLB as its molecular target. The adipocyte FGFR1-KLB complex accounts for the weight reduction and anti-diabetic effects of pharmacological FGF21 –44. It aOne important question is whether the treatment of metabolic abnormalities accompanying obesity and diabetes and the relief of stress in diverse tissues occur through the same or overlapping adipocyte signals controlled by FGF21 Table , 46, 49.Adipokines have emerged as FGF21-induced endocrine signals that also communicate back to stressed tissues. The upregulation of adiponectin currently is in the spotlight , 50, 51 In addition to the tissue-specific responses to FGF21-stimulated adipocyte signals that alter metabolism to reduce tissue stress, the mechanisms that control the induction of FGF21 in specific tissues in crisis also requires clarification Table , 57, andThe overarching canonical role of members of the diverse FGF superfamily in the adult is coordination of tissue homeostasis through autocrine and paracrine growth-promoting signaling controlled by matrix heparan sulfate. With the loss of affinity for heparan sulfate and gain of selectivity for the FGFR1-KLB complex that predominates in mature adipocytes, FGF21 has evolved as an inter-organ stress signal to adipocytes for negative regulation of systemic and cellular metabolic stress. This concept may also apply to evolution of other endocrine FGFs referred to as the FGF19 subfamily. The inter-organ crosstalk between ileal FGF19 and liver FGFR4-KLB is a negative regulatory mechanism of postprandial bile acid levels serving to prevent stress and damage resulting from exposure to prolonged and elevated levels of bile acids . SimilarThe 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.


The tumor suppressor function of the promyelocytic leukemia (PML) protein was first identified as a result of its dysregulation in acute promyelocytic leukemia, however, its importance is now emerging far beyond hematological neoplasms, to an extensive range of malignancies, including solid tumors. In response to stress signals, PML coordinates the regulation of numerous proteins, which activate fundamental cellular processes that suppress tumorigenesis. Importantly, PML itself is the subject of specific post-translational modifications, including ubiquitination, phosphorylation, acetylation, and SUMOylation, which in turn control PML activity and stability and ultimately dictate cellular fate. Improved understanding of the regulation of this key tumor suppressor is uncovering potential opportunities for therapeutic intervention. Targeting the key negative regulators of PML in cancer cells such as casein kinase 2, big MAP kinase 1, and E6-associated protein, with specific inhibitors that are becoming available, provides unique and exciting avenues for restoring tumor suppression through the induction of apoptosis and senescence. These approaches could be combined with DNA damaging drugs and cytokines that are known to activate PML. Depending on the cellular context, reactivation or enhancement of tumor suppressive PML functions, or targeted elimination of aberrantly functioning PML, may provide clinical benefit. The resultant PML-RARα fusion oncoprotein acts in a dominant negative fashion over wild type (wt) PML, as reiterated in a mouse model  were empirically identified to provoke a profound therapeutic response against APL, before the molecular pathogenesis of the disease was established  gene was initially described in the pathogenesis of Acute Promyelocytic Leukemia (APL), where it fuses with the Retinoic Acid Receptor α (RARα) gene as a consequence of the chromosomal translocation Promyelocytic leukemia comprises multiple isoforms, which predominantly localize to the nucleus  is a physiological E3 ubiquitin ligase of PML, and showed increased PML expression in multiple tissues of E6AP KO mice. One of the functional implications of this finding was that lymphoid cells derived from E6AP deficient mice were more susceptible to DNA-damage induced apoptosis, associated with enhanced accumulation of PML-NBs . UHRF1 was demonstrated to target PML for degradation by mediating its polyubiquitination in human umbilical vein endothelial cells (HUVEC) and cancer cell lines , and promotes the proteasomal degradation of PML and PML-RARα , which is able to localize to the PML-NBs in breast cancer cells (MDA-MB-231), phosphorylate PML at two sites (S403 and S505), resulting in the recruitment of Pin1, and subsequent proteasomal degradation of PML by yet to be identified E3 ligase , was shown by the elegant work of Scaglioni et al. , to promThe acetylation of PML represents an additional post-translation mechanism regulating PML. Treatment of HeLa cells with the HDAC (histone deacetylase) inhibitor, trichostatin A (TSA) resulted in increased acetylation of PML leading to efficient induction of apoptosis  to PML is essential for PML-NB formation and maturation, and may also mark PML for ubiquitination. SUMO may either be non-covalently bound to PML through the SUMO binding domain , specific cytokines involved in anti-viral responses, immune-surveillance as well as anti-proliferative processes, which are known to be potent inducers of PML   kinase pathway, which promotes efficient SUMOylation of PML . In the background of mutant p53, PML tumor suppression cannot only be lost, but its activities can be conscripted to provide growth advantage. In fact, when PML was knocked down in these cancer cells they lapsed into growth arrest  may hold promise for therapy. However, targeting this pathway will need careful consideration as it is also involved in PML-dependent hematopoietic stem cell (HSC) maintenance   stem cells are resistant to conventional therapy including Bcr-Abl tyrosine kinase inhibitors and can be a source of relapse (Holtz et al., 2O3 is usually very effective as it specifically targets the pathogenic PML-RARα fusion protein for degradation and reactivates functional PML. However, there is a proportion of APL patient that remains resistant and therefore novel therapies are required. Clearly, the restoration of PML to inhibit cancer growth emerges as a promising targeted strategy (Wolyniec et al., Over the last decade, immense progress has been made regarding our understanding of molecular pathways regulating PML stability. Major post-translational modifications such as phosphorylation, acetylation, SUMOylation, and ubiquitination and their interactions have been extensively studied in normal and cancer cells. Although we have identified certain kinases and E3 ubiquitin ligases of PML, less is known about potential phosphatases and deubiquitinases, which are likely to play important regulatory functions. Another key limitation in our understanding of PML is the shortage of information about isoform specific effects and the potential problem with protecting and activating cancer promoting or cancer suppressing proteins. Hence, the new generation of mouse models specific for PML isoforms will be absolutely necessary to address these important issues. Treating of APL patients with AsThe 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.