AHNAK/Desmoyokin is a giant protein which has been recently linked to reorganization of the actin cytoskeleton, cellular migration and invasion. Here, we investigated the role of AHNAK in the pathophysiology of larynx carcinoma-one of the major subtypes of head and neck cancer. To this end, we analysed AHNAK expression in tumor tissues from 83 larynx carcinoma patients in relation to overall survival. We found that tumoral AHNAK overexpression significantly associated with poor survival of these patients both in univariate and multivariate analysis. In further studies, we combined the prognostic value of AHNAK with selected markers of inflammation, such as macrophage migration inhibitory factor (MIF) and tumor-infiltrating neutrophils (CD66b-positive cells). Both MIF and neutrophils have been linked to enhanced tumoral migration and poor clinical outcome in patients with orohypopharynx carcinoma-another major subtype of head and neck cancer. Interestingly, we found that synchronous high levels of AHNAK and MIF or AHNAK and neutrophils, respectively, were stronger predictors of poor survival than AHNAK alone. Synchronous high levels of all three markers were the strongest predictors of poor survival in our patient cohort. Taken together, our findings propose novel strategies for an accurate prognosis in larynx carcinoma and suggest potential mechanisms of inflammation-mediated tumor progression. Head and neck cancer (HNC) is the eighth most common type of cancer worldwide. Despite multiple and aggressive therapeutic interventions, there has been no fundamental improvement in the 5-year survival rates of the patients over the past decades 2+ and Protein Kinase C AHNAK (desmoyokin) is a protein of exceptionally large size (700 kDa) that is expressed in a variety of cell types in vitro studies indicated a direct interaction between neutrophils and head and neck cancer cells by showing that neutrophils were ‘primed’ by the tumor cells to release pro-inflammatory factors which promoted tumoral migration in a feed-back manner Presently, it is well established that solid tumors display an inflammatory microenvironment characterized by large numbers of tumor-infiltrating immune cells The exact molecular mechanisms responsible for tumor-host interactions in head and neck and other types of cancer have been only partially clarified. Selected soluble inflammatory mediators, such as cytokines, chemokines and metabolites of the arachidonic acid pathway, have been found to change the function and differentiation of immune cells This study investigated the relevance of AHNAK, MIF and tumor-infiltrating neutrophils (CD66b-positive cells) for the survival of patients with laryngeal carcinoma-a major subtype of head and neck cancer. We demonstrated that AHNAK overexpression significantly associated with poor survival in these patients. Interestingly, we found that high levels of AHNAK together with high MIF expression or high neutrophilic infiltration, respectively, were stronger associated with poor survival than AHNAK alone. Synchronous high levels of MIF and tumor-infiltrating neutrophils had stronger predictor values over the individual markers as well. Finally, patients with high levels of all three markers displayed the shortest survival in the entire patient cohort. Thus, our study proposes novel strategies for a more accurate prognosis in larynx carcinoma and suggests potential mechanisms of inflammation-mediated tumor progression.Tissue microarrays (TMAs) were obtained from 83 patients with head and neck squamous cell carcinoma of the larynx. The patients were treated at the Department of Otorhinolaryngology (University of Duisburg-Essen) between 1995 and 2002, and clinical follow-up was retrieved. Patient characteristics are summarized in Polyclonal rabbit anti-human AHNAK antibodies  were obtained from Sigma . Monoclonal mouse anti-CD66b antibodies clone 80H3  were from Immunotech . Monoclonal mouse anti-MIF antibodies  were obtained from R&D Systems .Tissue cores with a diameter of 3 mm were extracted from formalin-fixed/paraffin-embedded tumor tissue blocks using a skin biopsy punch . The tissue cores were then brought into recipient blocks and cut into 5 µM sections. Antigen retrieval was performed by HIER  in citrate buffer pH 6.0. Samples were stained with 0.6 µg/ml anti-AHNAK antibodies using an automated staining device . Secondary and tertiary immunoreactions were performed with a commercially available anti-rabbit IgG detection kit . Colorimetric reactions were developed with diamino-benzidine (DAB). Staining of TMAs with anti-MIF and anti-CD66b antibodies, respectively, has been described previously Tumors elicit an immune response, leading to the generation of antibodies specific for tumoral antigens. AMIDA technology (autoantibody-mediated identification of antigens) identifies proteins preferentially recognized by antibodies from cancer patients over those from healthy controls. Here, we used AMIDA to screen for tumor-associated antigens in 4 head and neck cancer patients versus 4 healthy donors, as previously described p≤0.05.Clinical data were analysed with the SPSS statistical software version 18.0 . Survival curves (5-years cutoff) were plotted according to the Kaplan-Meier method. Significance was initially tested in univariate analysis using the log-rank test or univariate Cox regression. Multivariate analysis was used to determine the independent prognostic value of selected variables using Cox's proportional hazards linear regression model. In all studies, the level of significance was set at weak AHNAK, IRS 6–8 as medium and IRS 9–12 represented strong AHNAK levels , while strong AHNAK represents overexpressed levels of the protein (from here on termed ‘AHNAKhigh’).Using AMIDA technology we initially screened for novel tumor-associated antigens (TAAs) in sera from head and neck cancer patients and we identified AHNAK as a potential candidate see  section.K levels . In healpression . Consequweak MIF represents basal levels of the protein (from here on termed ‘MIFlow’), while medium and strong MIF represent overexpressed levels of the protein (from here on termed ‘MIFhigh’). The levels of tumor-infiltrating neutrophils (CD66b-positive cells) were initially assessed as negative, weak, medium and strong  because they have been shown to associate with advanced disease and poor survival in orohypopharynx carcinoma patients-another major subtype of head and neck cancer d strong . For thehigh) or CD66b (CD66bhigh) had significantly shorter survival than AHNAKlow or CD66blow patients  (high) tended to have worse survival than those with MIFlow   . Patientog-rank) ; howeverlow/MIFlow, AHNAKlow/MIFhigh, AHNAKhigh/MIFlow and AHNAKhigh/MIFhigh). Similar groups were built for AHNAK/CD66b and for CD66b/MIF, respectively. We plotted Kaplan-Meier survival curves for the above-mentioned marker combinations , lymph node metastasis (N-stage), AJCC stage and histological grading . We founhigh/CD66bhigh/MIFhigh) had the shortest overall survival in our patient cohort   for the survival of larynx carcinoma patients. Interestingly, Kaplan-Meier analysis showed that the patients with synchronous high expression of all markers (AHNAKog-rank) .Increased effort has been made to identify cellular/molecular factors that could provide accurate information regarding cancer diagnosis, prognosis and response to therapy. In this study, we identified AHNAK as a novel independent prognostic factor for the overall survival of larynx carcinoma patients. Furthermore, we demonstrated that combined analysis of inflammatory markers (MIF and CD66b) with AHNAK or with one another has higher significance regarding survival than analysis of the individual markers. Thus, our study proposes novel strategies for a more accurate prognosis in larynx carcinoma and suggests potential mechanisms of tumor progression.in vitro, on cancer cell lines and/or investigated AHNAK at gene rather than at protein level The role of AHNAK in cancer is poorly characterized at present. In fact, a very recent PubMed search using the keywords 'AHNAK’ or ‘desmoyokin' and 'cancer' resulted in less than 20 hits. Most of the studies on this topic were performed high/MIFhigh) or high neutrophilic infiltration (AHNAKhigh/CD66bhigh), respectively, had significantly shorter overall survival than the other groups of patients. Furthermore, the statistical significance of these combinations was stronger than that of the highly significant AHNAK. Thus, our findings indicate that combined analysis of AHNAK with either MIF or CD66b might be a good strategy for an accurate prognosis in larynx carcinoma patients.Of particular interest and novelty are also our findings regarding combination of inflammatory markers with AHNAK. In this study we tested the relevance of tumoral MIF and of tumor-infiltrating neutrophils (CD66b-positive cells) for the survival of larynx carcinoma patients. Both MIF and neutrophils have been previously linked to poor clinical outcome in orohypopharynx carcinoma patients high/MIFhigh) were significantly associated with poor survival, although the individual markers were less so. These findings indicate that MIF and neutrophils might interact with each other in the tumor microenvironment to enhance the progression of larynx carcinoma. Recently, our group identified direct interactions between head and neck cancer (HNC)-derived MIF and neutrophils both in vitro and in situ. We showed that HNC-derived MIF enhanced neutrophil chemotaxis in vitro and that tumoral MIF levels correlated with the neutrophilic infiltration in tissues from orohypopharynx carcinoma patients high/CD66bhigh/MIFhigh) associates with the shortest overall survival in larynx carcinoma patients. These findings need, however, to be confirmed on larger cohorts of patients and (some) of the proposed interactions have yet to be addressed and proven experimentally in future studies.Additionally, these findings suggest that AHNAK might 'cooperate' with MIF and/or neutrophils to enhance progression of larynx carcinoma. A synergistic effect between cellular and molecular factors regarding tumor progression is supported by combined analysis of MIF and neutrophils in our cohort of patients. Specifically, we observed that synchronous high levels of neutrophilic infiltration and of MIF (CD66bIn summary, our study identifies novel molecular and cellular factors that might serve as prognostic biomarkers and might interact with each other to enhance progression of laryngeal cancer. Ultimately, these findings contribute to a better understanding and foster the development of improved therapeutic strategies against larynx carcinoma and, perhaps, other types of solid cancer as well.


Ongoing technological advances in genome sequencing are allowing bacterial genomes to be sequenced at ever-lower cost. However, nearly all of these new techniques concomitantly decrease genome quality, primarily due to the inability of their relatively short read lengths to bridge certain genomic regions, e.g., those containing repeats. Fragmentation of predicted open reading frames (ORFs) is one possible consequence of this decreased quality. In this study we quantify ORF fragmentation in draft microbial genomes and its effect on annotation efficacy, and we propose a solution to ameliorate this problem.Streptomyces genomes, fragmentation was especially enriched in some protein classes with repeating, multi-modular structures such as polyketide synthases, non-ribosomal peptide synthetases and serine/threonine kinases. Overall, increased genome fragmentation correlated with increased false-negative Pfam and COG annotation rates and increased false-positive KEGG annotation rates. The false-positive KEGG annotation rate could be ameliorated by linking fragmented ORFs using their orthologs in related genomes. Whereas this strategy successfully linked up to 46% of the total ORF fragments in some genomes, its sensitivity appeared to depend heavily on the depth of sampling of a particular taxon's variable genome.A survey of draft-quality genomes in GenBank revealed that fragmented ORFs comprised > 80% of the predicted ORFs in some genomes, and that increased fragmentation correlated with decreased genome assembly quality. In a more thorough analysis of 25 Draft microbial genomes contain many ORF fragments. Where these correspond to the same gene they have particular potential to confound comparative gene content analyses. Given our findings, and the rapid increase in the number of microbial draft quality genomes, we suggest that accounting for gene fragmentation and its associated biases is important when designing comparative genomic projects. These statistics do not include a myriad of sequenced viruses or projects not registered in GOLD.Beginning with the bacteriophage φX174 genome in 1977 by Sanger et al. [http://www.genomicslawreport.com/wp-content/uploads/2011/04/JP-Morgan-NGS-Report.pdf.For approximately 30 years following its original publication in 1977 , the SanNext-generation sequence assembly is an inherently challenging computational problem, stemming from the relatively low amount of data contained within each sequencing read, the huge volumes of data produced, and platform-specific error frequencies and profiles  includinConsequently, data occurring between genomic contig ends are omitted from draft-quality genomes. This affects comparative analyses using phylogenetic techniques; because the state of these missing characters cannot be otherwise estimated, their phylogenetic analysis is impossible. In contrast, analyses based on presence-absence of particular open reading frame (ORF) types, e.g., using BLAST , can be Whereas it is intuitive to expect ORF fragmentation in a draft genome due to the existence of multiple contigs, the extent of this problem is currently unknown. The majority of draft genomes in the NCBI database at the time of this study contained tens to hundreds of partial ORF fragments, although some fell both above and below this range Figure . The larStreptomyces genomes using three different annotation types, representing different annotation targets  and sensitivities . Using the percentage of orthologous full-length proteins as the metric of genome similarity yielded equivalent results (data not shown). Even more important than genome relatedness per se was the degree to which related samples sampled the variable genome of the taxon of interest [S. roseosporus NRRL15998, for which more partial ORFs were linked using Streptomyces sp. XylebKG-1 as a reference  compared with either S. roseosporus NRRL11379  or S. griseus subsp. griseus NBRC13350  could link relatively few of each other's partial ORFs despite the high relatedness of these genomes (Table S. clavuligerus ATCC27064 replicates) can differ in some respect, e.g., plasmid content [Inspection of the relationship between genetic relatedness and partial ORF linkage revealed insights into the varied rate of partial ORF linkage observed between genomes in our test dataset Figure . In someinterest . Both th content . RegardlTo determine whether our partial ORF linkage method could compensate for errors in KEGG over-annotation, we tested the correlation between the number of KEGG annotations after correction and both the mean ORF length and percentage of the total number of ORFs composed of fragments as described above Table . All corNext-generation DNA genome sequencing has irrevocably moved microbiology  towards extensive data volumes and the concomitant challenges of their analyses. These advances are not without attendant compromises, especially the economic trade-off between dataset breadth and quality which appears to be increasingly towards the former at the expense of the latter. Emerging technologies such as PacBio and 454 FLX Titanium XL+ offer enhanced read lengths, but these formats still cannot match the per-base costs of shorter-read sequencing platforms. Cheaper short read techniques therefore seem poised to dominate the sequencing market for the foreseeable future, especially given that human genome re-sequencing, the main economic driver for innovation in genome sequencing technologies, is focused on clinical personal genomics where short reads appear largely sufficient for reference assembly and SNP detection.Streptomyces and other similar organisms, for which the most biotechnologically interesting genes involved in secondary metabolism are both variable between strains [Given this technological reality, draft genomes are likely to dominate microbial genomic data for the immediate future. Here, we have attempted to address some of the limitations of this data type for comparative genomics analysis. We find that ORF fragmentation has the potential to dramatically cause false-negatives in some annotation types, especially those relying on relatively small stretches of sequence  genome contents. However, we must also be aware of the limitations of these data types. For example, if > 80% of the predicted ORFs in a given bacterial genome are likely to be fragmented, analyses that can accommodate ORF fragmentation, even if imperfectly, will be crucial. Extensively fragmented draft genomic data will be insufficient for some applications, even with downstream Draft genome quality will be the dominant form of microbial genomic data produced for the immediate and foreseeable future. This approach produces highly fragmented genomes, which concomitantly translates into abundant predicted ORF fragments. These fragmented ORFs lessen annotation efficacy, especially of repeating, multi-modular proteins. However, this problem can be somewhat ameliorated by increased sampling of a species' variable genome. Therefore, despite their great utility in many applications, there exist disadvantages to draft genome sequencing that need to be addressed by appropriate experimental design and deployment of sequencing resources to effectively answer the study questions posed.Streptomyces genomes used for more detailed analysis is given in Table S. clavuligerus ATCC27064 using derivatives of the same strain. Streptomyces genomes are also expected to be especially challenging to assemble using short-read techniques due to their substantial %GC bias [Streptomyces sp. PP-C42) had accompanying scaffold information, which allowed estimation of the false positive rate of our partial ORF linkage procedure (see below). Annotated protein sequences were downloaded from NCBI for those strains having complete genomes; otherwise, proteins were predicted from nucleotide contigs using Prodigal v2.00 [The contigs comprising all 1,510 partial genome sequences were obtained from the NCBI FTP folder "genomes/Bacteria_DRAFT" on May 10, 2011. Genes and their corresponding proteins were annotated using Prodigal v2.00  accordin%GC bias . All of al v2.00  as descr-5 using the predicted proteins in the Streptomyces dataset. The top COG and all Pfam hits for each query sequence were recovered for analysis. A Pfam annotation conducted using an expectation cut off of 1 × 10-3 yielded similar but slightly stronger statistical correlations compared with those shown in Table Streptomyces dataset were annotated using the KAAS web server using the single direction best BLAST method [The COG v1.0 and Pfam v25.0 databases were downloaded from the NCBI FTP site and queried by RPSBLAST with an expectation cut off of 1 × 10T method . Statisthttp://currielab.wisc.edu/. Note that this current implementation was designed to use pair-wise BLAST searches of entire proteomes due to the additional utility of these files for calculating AAIs. Other configurations  may be more computationally efficient depending on particular experimental needs. Pair-wise comparisons of each predicted proteome were conducted using BLASTp [Our partial ORF linkage algorithm was implemented as a Perl script, freely downloadable from the Currie lab website g BLASTp  with theg BLASTp . Pair-wig BLASTp .A flow diagram of our approach is presented in Figure JLK conceived of the study, participated in its design, performed the analyses, analyzed the results and drafted the manuscript. CRC participated in this study's design, helped analyze data and draft the manuscript. Both authors have read and approved the final manuscript.Parameterization of the fragment linkage algorithm by varying the maximum percentage identities, sequence overlap and minimum difference in the percent identities of a set protein fragments to a complete reference homolog. False and true positive linkage results were defined based on their concordance with the adjacent location of each fragment pair on the same contig. Each analysis was conducted on the entire Streptomyces test dataset except for Streptomyces sp. PP-C42, for which there was not available scaffold information.Click here for fileParameterization of the fragment linkage algorithm by varying the maximum percentage of identities and sequence overlap while holding the minimum difference in the percent identities of a set protein fragments to a complete reference homolog constant at 40%. The height of the surface represents the number of fragments matched using each parameter combination, and the color represents the percentage of true positives recovered using those parameters by reference to the available scaffold information.Click here for file


Despite known limitations of algorithm based designs (e.g. 3+3) they are favoured for use in dose finding studies due to simplicity and familiarity. Bayesian adaptive designs can overcome these limitations as they’re more efficient and substantially more accurate.EffTox is such a design which aims to determine the optimal, tolerable and efficacious dose. We implemented this design in a multicentre clinical trial of a novel TKI in combination with chemotherapy to treat chronic myeloid leukaemia. It is expected that the probability of efficacy may increase to a maximum then level off, thus higher doses of the combination may not result in greater efficacy. Given this non-monotone dose-response relationship, solely toxicity based dose-finding designs are not appropriate. In addition EffTox has demonstrated favourable properties of exposing fewer patients to potentially toxic and inefficacious doses, allocates more patients to the optimal dose and results in less dose-modifying toxicities in a subsequent phase-II/III trial.A Bayesian analysis is conducted using a bivariate binary probability model with prior probabilities elicited from clinicians. Dose recommendations are based on an efficacy-toxicity trade-off contour. Operating characteristics were explored via simulations of various scenarios using readily available EffTox software. The design has satisfactory operational characteristics and is flexible to be adapted trial specific requirements.Challenges in implementing this design included overcoming clinicians’ preconceptions, obtaining clinician consensus to enable development of the desirability trade-off contour and educating committees to secure funding. Overcoming these challenges allows design of robust dose-finding trials establishing an optimal, rather than sub-optimal dose.


Obesity is important for the development of type-2 diabetes as a result of obesity-induced insulin resistance accompanied by impaired compensation of insulin secretion from pancreatic beta cells. Here, based on a randomized pilot clinical trial, we report that intranasal oxytocin administration over an 8-week period led to effective reduction of obesity and reversal of related prediabetic changes in patients. Using mouse models, we further systematically evaluated whether oxytocin and its analogs yield therapeutic effects against prediabetic or diabetic disorders regardless of obesity. Our results showed that oxytocin and two analogs including -oxytocin or -oxytocin worked in mice to reverse insulin resistance and glucose intolerance prior to reduction of obesity. In parallel, using streptozotocin-induced diabetic mouse model, we found that treatment with oxytocin or its analogs reduced the magnitude of glucose intolerance through improving insulin secretion. The anti-diabetic effects of oxytocin and its analogs in these animal models can be produced similarly whether central or peripheral administration was used. In conclusion, oxytocin and its analogs have multi-level effects in improving weight control, insulin sensitivity and insulin secretion, and bear potentials for being developed as therapeutic peptides for obesity and diabetes. Developing peptides to treat obesity and/or diabetes is a relatively recent advance, but of importance, the clinical success of this concept has been evidenced by the usefulness of intestinal peptide glucagon-like peptide-1 (GLP-1) in controlling obesity as well as diabetes This was a pilot clinical study registered through Chinese Clinical Trial Register (ChiCTR-TRC-12002884), and conducted in accordance with the Declaration of Helsinki, Good Clinical Practice guidelines, and other relevant regulations by the People's Republic of China. Study protocol and informed consent form were approved by the Ethics Committee of the affiliated People’s Hospital of Jiangsu University. Written informed consent was given to each patient.Patients were recruited by clinicians through clinics of the People’s Hospital of Jiangsu University . EstimatUsing randomized placebo-controlled design , patientpost hoc analyses were used for comparisons involving more than two groups, and two-tailed Student’s t tests were used for only 2-group comparisons. Clinical data were presented as mean ± SD. P<0.05 was considered significant.Statistical analyses were performed by investigators who did not have access to patients and were blind to the information during the entire course of the study. Body weight, BMI, and waist and hip circumferences, blood glucose and insulin levels, blood pressure, lipid profiles, and parameters of hepatic and renal functions were compared among different treatment points and between OXT and placebo groups. Repeated measures analysis was used to adjust dependence among repeated observations on the same patients. ANOVA and appropriate C57BL/6 mice were purchased from Jackson Laboratory. All mice were housed in standard conditions. High-fat diet was purchased from Research Diets, Inc. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the Albert Einstein College of Medicine. Mice were subjected to body weight measurement on a daily basis surrounding surgical procedures or drug treatments to monitor physical recovery or the effect of drug treatment on body weight.Third ventricle cannulation procedure was previously described OXT and -OXT  and -OXT  were dissolved in the vehicle, aCSF. HFD-fed mice were twice injected with OXT or OXT analog via third-ventricle cannula (4 μg per injection) or i.p. (2 mg/kg body weight per injection), once at the beginning of overnight fasting and once in the morning prior to GTT. Injection of the vehicle was used as the control. For STZ model, adult C57BL/6 mice were daily injected with OXT or OXT analog vs. the vehicle via third-ventricle cannula (2 μg per injection) for 7 days, or i.p. (2 mg/kg body weight) vs. the vehicle for 3 days; all mice received daily i.p. injections of STZ (Sigma) at the dose of 40 mg/kg body weight for 4 days before these mice were subjected to GTT.For glucose tolerance test (GTT), overnight-fasted mice received a single i.p. injection of glucose (2 g/kg body weight), and blood glucose levels were measured using OneTouch Ultra2 . Fasting blood insulin levels were measured with Ultra Sensitive Mouse Insulin ELISA kits . Glucose-stimulated insulin secretion (GSIS) test was performed as previously described post hoc tests were used for comparisons that involved more than two groups. Data of animal studies were presented as mean ± SEM. P<0.05 was considered statistically significant.Two-tailed Student’s t tests were used for two-group comparisons. ANOVA and appropriate Using mouse models, our previous studies revealed that central administration of OXT can work to treat or prevent against dietary obesity Clinical epidemiology has established that obesity in humans is associated with pre-type 2 diabetes (T2D) changes such as glucose intolerance, insulin resistance and hyperlipidemia. Thus, although this pilot clinical study did not involve patients with diabetes, we still monitored blood glucose, insulin and lipid levels of study subjects. We found that while OXT treatment did not affect fasting blood glucose or insulin levels, it tended to reduce postprandial glucose and insulin levels towards healthier profiles within the normal ranges A and B.Our previous study using mouse models had shown that enhancing the intra-brain action of OXT can prevent against the development of obesity and obesity-related diabetic symptoms such as insulin resistance, glucose intolerance, pancreatic islet hypertrophy and steatosis against dietary overnutrition Next we studied if OXT and its peptide analogs could provide a protective effect against impaired insulin secretion which is known to underlie the development and progression of both T2D and type-1 diabetes (T1D). Our recent work showed that mice with genetically enhanced OXT function were significantly protected from overnutrition-induced prediabetic damages of pancreatic beta cells The above studies suggested that in addition to obesity control, OXT and its analogs can work in the brain to independently improve systemic insulin sensitivity and insulin secretion and thereof counteract against diabetes. However, given that drug delivery via brain injection is impractical in humans, we further evaluated whether peripheral OXT delivery could be workable. The rationale for this possibility includes that, the blood-brain barrier (BBB) does not completely block the passage of OXT OXT is a small neuropeptide produced by OXT neurons and released locally in the brain and systemically into the circulation. While the systemic action of OXT is important for female reproductive activities Peptides, especially those that naturally exist in bio-systems, are increasingly recognized for therapeutic potentials to treat human diseases in terms of effectiveness and safety; however, developing peptidyl drugs to target the central nervous system is still a pharmaceutical challenge. Based on current technology, direct intra-brain delivery is impractical for human beings, whereas peripherally delivered peptides often cannot effectively reach the central sites due to the physical and biological blockade of the BBB. As of now, successful cases of the central nervous system-targeting drugs are a few low-molecular weight (∼0.3KD) molecules that treat mental disorders or sleep disorders. OXT is a naturally existing peptide in mammals and has shown central anti-obesity and anti-diabetic effects per our rodent studies. Here, following our recent work showing the effect of peripheral OXT delivery in treating obesity in mice, the current work demonstrated that OXT nasal spray can effectively treat human obesity and the related lipid disorders. Along this line, our animal studies further revealed that peripheral administration of OXT analog can use a body weight-independent manner to improve both insulin sensitivity and insulin secretion in two diabetic/pre-diabetic models. Thus, with the recognition that intra-brain action of OXT is important for metabolic intervention, it is highly clinically relevant to see that OXT or its analogs can be delivered via peripheral administration to exert similar effects. We think the mechanism for the effects of peripheral OXT delivery is at least related to the central-peripheral crosstalk mediated by OXT, for instance, in addition to a possible fractural penetration of OXT across the BBB The therapeutic advantages of OXT and its analogs in treating obesity and diabetes can be further increased given that OXT has social-neuropsychiatric benefits which can possibly aid to control metabolic disease. Though initially only recognized for functions in inducing female uterine contractions and milk let-down, OXT has recently been found to be important for social recognition, pair bonding, love and care in mammals and for mental and emotional well-being in humans Material S1Study protocol for using OXT nasal spray to treat human obesity.(PDF)Click here for additional data file.Material S2Consort checklist for using OXT nasal spray to treat human obesity.(PDF)Click here for additional data file.


RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance.The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations   BRAF (V600E). Sensitivity to MEK inhibitors was shown to be associated with BRAF, NRAS as well as PTEN, PTPN5, SPRY2, DUSP4, DUSP6 mutations and to a lesser extent mutations at KRAS. Sensitivity to MEK inhibitors in NRAS mutant lines was associated with aryl hydrocarbon receptor (AHR) expression  -13.i.e, Sorafenib, Bayer) were initially thought to specifically inhibit Raf but have been subsequently shown to have multiple targets   were examined in a recent study , or the small molecule Akt inhibitor MK-2206, or the EGFR inhibitor erlotinib increased survival [Treatment of mice xenografted with vemurafenib-resistant survival . Combinasurvival . The effsurvival ,341. Thesurvival ,273. A csurvival .KRAS G12D mutation [Clinical trials are ongoing based on combining NVP-BEZ235 using inhibitors  and the chemotherapeutic drug (paclitaxel) and herceptin to treat advanced solid cancers and metastatic breast cancers which are difficult to treat (see below). BKM120 is a pan-PI3K inhibitor. It is being included in some clinical studies since NVP-BEZ235 does not inhibit PI3K-P110-β . Furthermutation . Thus toPalomid 529, a pan mTOR inhibitor, in some circumstances is effective as a single agent. Importantly when Palomid 529 was combined with either cisplatin or docetaxel it had a better effect on hormone-refractory prostate cancers . It alsoAs mentioned previously, a side effect of some chemotherapeutic drugs, such as paclitaxel, is the induction of the Raf/MEK/ERK pathway. Activation of this pathway, can under certain circumstances, promote proliferation and prevent apoptosis. Also the PI3K/PTEN/Akt/mTOR pathway can modulate the Raf/MEK/ERK pathway and altering MEK activity can have opposing effects on different cell types -349. ComThe cytotoxic effects of combinations of MEK inhibitors and paclitaxel may be specific for cells of certain origins and may depend on the levels of endogenous activated MEK/ERK present in those cells. In a study with NSCLC cells which constitutively-expressed activated MEK/ERK, no increase in paclitaxel-induced apoptosis was observed when the cells were treated with a MEK inhibitor . In contCisplatin-induced apoptosis was associated with increased levels of both p53 and the downstream Bax protein in a study with neuroblastoma cells . Activatin vitro [It should be noted that the combination of MEK inhibitors and chemotherapeutic drugs may not always result in a synergistic interaction leading to cell death. In some cases, combination therapy results in an antagonistic response. For example, combining MEK inhibitors with betulinic acid, a drug toxic for melanoma cells, antagonized the normal enhancing effects of betulinic acid on apoptosis in vitro . Furtherin vitro and preclinical in vivo data as well as the clinical trials conducted so far demonstrate that mTOR inhibitors are promising agents for HCC treatment, particularly in combination with conventional chemotherapeutic drug therapy.There are few effect therapeutic options for HCC. Combination of rapamycin with conventional cytostatic drugs such as doxorubicin and vinblastine enhances the antineoplastic activity of the respective monotherapeutic HCC treatment obtained with either doxorubicin or vinblastine alone ,351. TakThe effects of sorafenib on the treatment of HCC patients were examined in a clinical trial . A phaseThe effects of inhibiting Akt in combination with other signaling pathways and chemotherapy are being evaluated in numerous phase I clinical trials. These trials highlight the importance of targeting multiple molecules to suppress the growth of cancer which are resistant to most therapies. A combination clinical trial (NCT01245205) with the Akt inhibitor MK-2206 and the dual EGFR/HER2 inhibitor lapatinib is in progress with patients having advanced or metastatic solid tumors or breast cancer patients. NCT00848718 is a clinical trial with patients having advanced cancers to examine the effects of combining MK-2206 and the EGFR inhibitor erlotinib, docetaxel, or carboplatin + paclitaxel. NCT00963547 was a clinical trial with HER2+ breast cancer patients to examine the effects of combining MK2206 with trastuzumab (herceptin) and lapatinib. NCT01245205 and NCT01281163 are clinical trials examining the effects of combining MK2206 with lapatinib in cancer patients with advanced or metastatic solid tumors or breast cancer or just breast cancers, respectively. NCT01147211 is a clinical trial with NSCLC patients examining the effects of combining MK-2206 with gefitinib . NCT01344031 is a clinical trial with post menopausal metastatic breast cancer patients examining the effects of combining anastrozole, letrozole, exemestane (aromatase-inhibitors), or fulvestrant (an estrogen receptor antagonist). NCT01369849 is a clinical trial examining the effects of combining MK2206, with bendamustin  and rituximab  on CLL cancer patients who have relapsed or cancer patients with small lymphocytic lymphoma. NCT01243762 is a clinical trial combining MK-2206 and dalotuzumab , MK-0752 a (Y-secretase inhibitor which inhibits the NOTCH pathway from Merck) and dalotuzumab and MK-8669 (ridaforolimus a mTOR inhibitor from Merck) and dalotuzumab in cancer patients with advanced cancers. NCT01263145 is a clinical trial combining MK2206 and paclitaxel in cancer patients with locally advanced or metastatic solid tumors or metastatic breast cancers. The above mentioned clinical trials document the importance of targeting Akt and other signaling molecules as well as critical targets involved in cellular division. Furthermore the clinical trials document how basis research experimentation on these pathways is being translated into clinical therapy for cancer and other types of patients.Radiotherapy is a common therapeutic approach for treatment of many diverse cancers . Radiothin vitro using human cell lines and in vivo employing xenografts [in vitro and in vivo. The MEK inhibitor treatment was correlated with decreased Chk1 phosphorylation 1-2 hrs after radiation. The authors noticed the effects of the MEK inhibitor on the G2 checkpoint activation after irradiation, as the MEK inhibitor suppressed G2 checkpoint activation. Since ERK1/ERK2 activity is necessary for carcinoma cells to arrest at the G2 checkpoint, suppression of phosphorylated Chk1 was speculated to lead to the abrogated G2 checkpoint, increased mitotic catastrophe and impaired activation of cell cycle checkpoints. Chk1/Chk2 as serine/threonine kinases. Chk/Chk2 are important controlling regulators of DNA repair and cell cycle progression. DNA damage responses which signal through ATM and ATR activate the DNA damage transducers Chk1 and Chk2 [A side effect of radiotherapy in some cells is induction of the Ras/Raf/MEK/ERK cascade ,4. Varionografts . The MEKand Chk2 -383.2 checkpoint. It was also postulated in this study that the MEK inhibitor suppressed the autocrine cascade in DU145 prostate cancer cells that normally resulted from EGF secretion and EGFR activation. Suppression of this autocrine cascade by the MEK inhibitor may have served as a radiosensitizer to the radiation therapy. The other two cancer cell lines examined in this study (A549 and MiaPaCa2) had KRAS mutations and both were radiosensitized by the MEK inhibitor. Although these studies document the ability of a MEK inhibitor to radiosensitize certain cells, clearly other cancer cell lines without activating mutations in the Ras/Raf/MEK/ERK pathway or autocrine growth stimulation should be examined for radiosensitization by the MEK inhibitor as the KRAS mutation may also activate the PI3K pathway which could lead to therapy resistance.Mitotic catastrophe was increased in cancer cells receiving both the MEK inhibitor selumetinib and radiation when compared to the solo-treated cells . Suppresin vitro in cell lines and in vivo in xenografts [PI3K/Akt/mTOR inhibitors will sensitize the tumor vasculature to radiation both nografts ,385. mTOnografts ,387. Thenografts -393. mTOnografts . In cancnografts -396 Funcnografts -399.e.g., sorafenib, HCC). This particular Raf inhibitor also inhibits other receptors and kinases which may be required for the growth of the particular cancer. This promiscuous nature of sorafenib has contributed to the effectiveness of this particular Raf inhibitor for certain cancers. Raf inhibitors such as vemurafenib, dabrafenib, and GDC-0879 are promising for the treatment of melanoma, CRC, thyroid and other solid cancers and leukemias/lymphomas/myelomas which have mutations at BRAF V600E. However, problems have been identified with certain BRAF mutant allele inhibitors as they will also result in Raf-1 activation if RAS is mutated/amplified of if an exon of BRAF is deleted, or if BRAF is amplified or if there are mutations at MEK1 and other genetic mechanisms. Combination therapy with either a traditional drug/physical treatment or another inhibitor that targets a specific molecule in a different signal transduction pathway is also a key approach for improving the effectiveness and usefulness of MEK and Raf inhibitors.Inhibitors to the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways have been isolated and developed by various screening approaches and then in some cases modified by medicinal chemistry. Initially MEK and mTOR inhibitors were demonstrated to have the most specificity. However, MEK inhibitors may have limited effectiveness in treating human cancers, unless the particular cancer proliferates directly in response to the Raf/MEK/ERK pathway. A similar scenario is also true with mTOR inhibitors, they are most effective when there is a mutation which deregulates the PI3K/PTEN/Akt/mTOR pathways. Moreover, MEK inhibitors are often cytostatic as opposed to cytotoxic, thus their ability to function as effective anti-cancer agents in a monotherapeutic setting is limited, and they may be more effective when combined with chemo- or radiotherapy or an inhibitor which targets a different pathway or even an inhibitor which targets the same pathway. Rapamycin and rapalogs are being used to treat certain cancers which proliferate in response to mutations in regulatory genes which control the PI3K/PTEN/Akt/mTOR pathway. Raf inhibitors have also been developed and some are being used to treat various cancer patients . While rapalogs are effective and their toxicity profiles are well known, one inherent property is that they are not very cytotoxic when it comes to killing tumor cells. This inherent property of rapamycins, may also contribute to their low toxicity in humans. Interestingly and highly relevant, it has been observed that certain inhibitors which target “growth and metabolism” such as rapamycin and metformin may have very potent anti-cancer and anti-aging effects [Modified rapamycins, rapalogs are being used to treat various cancer patients, . The induction of these signaling pathways may counteract some of the effects of the signal transduction inhibitors.It is not always clear why a particular combination of a signal transduction inhibitor and chemotherapeutic drug works in one tumor type but not at all in a different tumor type. This has also been experience with the development of individual chemotherapeutic drugs, some work in some cancers but not others. This may result from many different complex interacting events. Some of these events could include: percentage of cells in different phases of the cell cycle, persistence of CICs, presence of multiple mutated activated oncogene or repressed tumor suppressor genes, epigenetic modifications and many other factors. Finally, chemotherapeutic drug therapy and other types of therapy  may induce certain signalling pathways  of melanoma in vivo. Interesting, increased drug transporter activity has not been reported in the limited number of B-Raf inhibitor-resistant samples investigated, where it has been observed in other cancer types treated with diverse small molecule inhibitors and/or chemotherapeutic drugs.A problem with some of the preceding studies is that most of the resistant cells were derived after culturing cells Scientists and clinicians often have an intentionally narrow view of a particular topic. For example, cancer researchers predominantly feel that Raf, MEK, PI3K, Akt and mTOR inhibitors will suppress the growth of malignant cancer cells. Yet MEK and mTOR and other inhibitors may also be useful in the treatment of autoimmune or allergic disorders where there is abnormal cellular proliferation. Recently it has been observed that the suppression of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways may prevent the induction of cellular senescence and aging. Clearly, these later two clinical topics, immune disorders and aging, greatly enhance the potential clinical uses of these targeted therapeutic drugs.


Xenopus laevis tadpoles to water conditioned by adult conspecifics. Then, we analysed tadpole growth, development and survivorship as a function of MHC class I and class II peptide-binding region amino acid sequence similarities between tadpoles and frogs that conditioned the water to which they were exposed. Tadpoles approached metamorphosis earlier and suffered greater mortality when exposed to immunogenetically dissimilar frogs. The results suggest that developmental regulatory cues, microbial assemblages or both are specific to MHC genotypes. Tadpoles may associate with conspecifics with which they share microbiota to which their genotypes are well adapted.Major histocompatibility complex (MHC) genes determine immune repertoires and social preferences of vertebrates. Immunological regulation of microbial assemblages associated with individuals influences their sociality, and should also affect their life-history traits. We exposed  Although host adaptations lag behind pathogens in this race owing to longer host generation times, hosts may be less susceptible to local pathogen repertoires to which they have had an opportunity to evolve defences . Local mThe vertebrate major histocompatibility complex (MHC) encodes cellular recognition of pathogens and influences mating and social behaviour. MHC genotypes of individuals determine immunorecognition of different pathogen repertoires . Host-spWhen associating with MHC-similar conspecifics, individuals may benefit from exposure to low-virulence micro-organisms that are adapted to their immune system . HoweverXenopus laevis tadpoles to water conditioned by adult conspecifics. Tadpoles and adults of this species share the same habitat and microbial environment. MHC loci regulate both immunity to bacteria [To determine the fitness consequences of MHC-based association, we exposed bacteria  and assobacteria . We asse2.X. laevis with known MHC class I and class II sequences . The flow-through system was fed by filtered, aerated water (21°C) sourced from a deep aquifer.We bred  g and j ,15; GenBff, gg, jj) twice in one night to obtain tadpoles of six MHC genotypes  [We mated six adult MHC-homozygous frogs  . We indufg, gj), in 60 l flow-through tanks and fed them sliced ox liver every 2 days. After 4 days, we stopped the water flow to allow chemical cues and micro-organisms to accumulate, and ceased feeding the frogs to limit fouling. To obtain an estimate of microbial density, we plated 10 µl of the water from each tank in triplicate onto tryptone soya agar , incubated the plates aerobically at 32°C, and counted the number of bacterial colonies at 24 and 48 h. We exposed tadpoles to water from frogs, standardized by dilution to the microbial concentration of the lowest number of colonies found .Subsequently, we isolated the maternal frogs, and two additional MHC heterozygous females  were exposed to water from each of ff, fg, gg, gj and jj adults. We randomly assigned the tadpoles into six blocks. We fed tadpoles every 2 days with ground nettle suspension, topped the water up to 1 l every 4 days to replenish evaporation, and moved each beaker one place each day to limit position effects. Three weeks after exposure, we recorded developmental stage [mageJ v. 1.3 .Two weeks after hatching, we separated 300 tadpoles, 50 of each genotype, individually into beakers containing 1 l of conditioned water. Ten tadpoles of every genotype  and block by binomial generalized linear models (GLM) using R v. 2.10 (www.r-project.org). We examined how tadpole development varied in response to the percentage of shared amino acids at MHC class I and II loci peptide-binding region (PBR) domains [We analysed how tadpole mortality varied with the number of shared MHC haplotypes . Three times as many tadpoles died when exposed to water conditioned by frogs with which they shared no haplotypes compared with when they shared one haplotype. No tadpoles died when reared in water conditioned by MHC-identical donor frogs  and PBR amino acid sequence similarity . With increasing PBR sequence similarity, development at first slowed to a minimum  and then increased .Tadpole development slowed in proportion to the number of shared MHC haplotypes  and the number of shared MHC haplotypes . Nor did tadpole size explain stage differences in our regression analyses . Kinship cues and dilution factor did not significantly influence development , growth  or survival .Tadpole size varied independently of developmental stage (4.More tadpoles died in water conditioned by MHC-dissimilar than MHC-similar frogs, but surviving tadpoles developed faster. Tadpoles exposed to cues from conspecifics dissimilar to themselves in MHC type suffered reduced fitness. These results demonstrate that the MHC identity of conspecifics with which individuals interact not only influences the social behaviour but also life-history traits of group members.Our study does not establish the mechanism by which the MHC affects development or survivorship. Possibly, developmental regulatory cues, such as those thought to modulate tadpole growth , specifiXenopus, more rapid metamorphosis also can be induced by exposure to predators, resulting in smaller juveniles [Tadpoles exposed to cues of immunogenetically dissimilar frogs developed faster without growing correspondingly larger. Such accelerated development causing tadpoles to approach metamorphosis at smaller sizes typically occurs in unfavourable conditions. In uveniles . In otheuveniles . Adults uveniles .Selection for a social environment with a compatible microbiota may explain the preferential association of tadpoles with others sharing the same MHC alleles . MHC-simX. laevis often live in turbid water. Little is known about their social behaviour in the wild [In sub-Saharan Africa, adult and larval the wild , althougthe wild  of waterSocial groups are dynamic; individuals are exposed to a changing array of micro-organisms that vary with group composition and shifting environmental factors. Our results suggest that immunogenetic identity is important in assessing the costs and benefits of sociality. How microbiota associated with specific MHC genotypes, spanning the full spectrum from mutualistic to pathogenic organisms, affect the fitness of hosts and their conspecifics remains fertile ground for future work.


A 39-year-old male with an open wound of the left lower extremity complicated by exposed hardware following open reduction and internal fixation of a tibial plateau fracture.How are soft tissue injuries in relation to fractures of the lower extremity classified?What factors must be taken into account when considering limb salvage?When is the optimal time for soft tissue coverage following traumatic lower extremity wounds?What are the options for soft tissue coverage in lower extremity reconstruction?,5Treatment of high-energy trauma to the lower-extremity with associated soft-tissue and bony injury poses a significant problem. An assessment of limb viability is made with careful examination of the vascular, bone, soft-tissue, and nerve injuries. The goal in treatment of a severely injured lower extremity is to return the patient to ambulatory status. The decision of limb salvage versus amputation while maintaining maximal functional length is a critical one.1,When referring to long bone fracture pattern and soft-tissue injury, the Gustilo-Anderson fracture classification system is typically employed to help describe injuries and prognosis.1,2To predict the outcome of salvage efforts for mangled extremities to help properly identify patients who would benefit from primary limb amputation versus reconstruction, and to attempt a better classification scheme, numerous scoring systems have been proposed. These include the Mangled Extremity Syndrome Index, Mangled Extremity Severity Score amongst others.1,4,1,1,2,1,2,4After bony stabilization, vascular repair, and adequate debridement, immediate soft-tissue coverage is indicated when hardware or vital structures are exposed.2,6-8Flap choice for lower extremity wound coverage is less important than wound bed quality and the flap's ability to deliver well-perfused tissue. Wounds around the knee may be coveraged by using the medial or lateral head of a gastrocnemius muscle flap. Care must be taken to avoid common peroneal nerve injury when using lateral muscle. If unavailable, vastus lateralis or anterior lateral thigh flaps using retrograde flow through the descending branch of the lateral circumflex femoral artery are other alternatives. Defects around the knee and lower thigh may also be reconstructed with a lateral genicular artery flap. Although free flaps are not typically used in this region, they remain an option when additional flaps are unavailable.2,2,6-8After bony stabilization, tibial defects may be reconstructed using a variety of flaps. In general, the tibial region is divided into thirds. Defects in the proximal third are best reconstructed using a gastrocnemius muscle flap. Defects in the middle third are typically repaired using pedicled gastrocnemius or soleus muscle flaps; however, as high-energy wounds in this region typically manifest with extensive zones of injury, local flaps may not be dependable. Free flap reconstruction would then be preferred. Options include rectus abdominis, scapula, anterior lateral thigh, gracilis, and latissimus muscle flaps. These are also preferred for wounds at the distal third of the tibia because of limited local tissue availability. For those in whom free flap reconstruction is not an option, a reversed sural flap may be considered. Foot and ankle reconstruction overlaps with reconstruction in the distal third of the leg, however, debulking of free flaps is often necessary to optimize form and function.1Our patient is a 39-year-old male status post fall from height resulting in bilateral calcaneal fractures and a left tibial plateau fracture. The patient underwent open reduction and internal fixation of his left tibial plateau fracture. He developed an open wound of the left lateral proximal third of his leg resulting in exposed hardware. After serial debridement, the limb was salvaged using a lateral gastrocnemius muscle flap and split-thickness skin autograft.


Chronic hand eczema (CHE) represents an inflammatory skin disease with a high prevalence ranging from 7-12% in Western industrialized countries. It was only starting from 2008 that the first, and until now the only, systemic treatment option, i.e. oral alitretinoin was approved in several countries for severe CHE unresponsive to potent topical corticosteroids. However, as the precise mechanism of actions (MOA) of alitretinoin in CHE are so far unknown, we undertook following investigations in order to shed some light on potential underlying immunomodulatory mechanisms.To investigate the mode of action of alitretinoin we stimulated human primary keratinocytes as well as leukocyte subsets and performed quantitative real-time PCR analyses, FACS-analyses, cell-proliferation assays as well as mixed leukocyte reactions (MLR).Alitretinoin acts on keratinocytes as well as dendritic cells. Keratinocytes show a significant reduction of chemokine expression after stimulation with alitretinoin whereas on dendritic cells alitretinoin inhibits the upregulation of the maturation marker CD83 as well as the co-stimulatory molecules CD80 and CD86. Consequently, alitretinoin-treated dendritic cells show significantly impaired T cell activating properties. Further analyses demonstrated that the immunomodulatory effect of alitretinoin results in a marked suppression of allogenic proliferation of leukocytes in MLR.Taken together, these results suggest that alitretinoin modulates innate as well as adaptive immune responses by suppression of chemokine-induced leukocyte recruitment and inhibition of dendritic cell-mediated T cell activation.


Patients diagnosed with neuroendocrine tumors (NETs) with hepatic metastases generally have a worse prognosis as compared with patients with nonmetastasized NETs. Due to tumor location and distant metastases, a surgical approach is often not possible and nonsurgical therapeutic strategies may apply.The aim of these systematic reviews is to evaluate the role of nonsurgical therapy options for patients with nonresectable liver metastases of NETs.An objective group of librarians will provide an electronic search strategy to examine the MEDLINE, EMBASE, and The Cochrane Library  databases. There will be no restriction concerning language and publication date. The qualitative and quantitative synthesis of the systematic review will be conducted with randomized controlled trials (RCT), prospective, and retrospective comparative cohort, and case-control studies. Case series will be collected in a separate database and only used for descriptive purposes.This study is ongoing and presents a protocol of four systematic reviews to assess the role of nonsurgical treatment options in patients with neuroendocrine liver metastases.These systematic reviews, performed according to this protocol, will assess the value of noninvasive therapy options for patients with nonresectable liver metastases of NETs in combination with invasive techniques, such as percutaneous liver-directed techniques and local ablation techniques.International Prospective Register of Systematic Reviews (PROSPERO): CRD42012002657; http://www.metaxis.com/PROSPERO/full_doc.asp?RecordID=2657 (Archived by WebCite at http://www.webcitation.org/6NDlYi37O);CRD42012002658; http://www.metaxis.com/PROSPERO/full_doc.asp?RecordID=2658 (Archived by WebCite at http://www.webcitation.org/6NDlfWSuD); CRD42012002659; www.metaxis.com/PROSPERO/full_doc.asp?RecordID=2659 (Arichived by Webcite at http://www.webcitation.org/6NDlmWAFM); and CRD42012002660; http://www.metaxis.com/PROSPERO/full_doc.asp?RecordID=2660 (Archived by WebCite at http://www.webcitation.org/6NDmnylzp). Neuroendocrine tumors (NET) arise from neuroendocrine cells and are a heterogeneous group of neoplasms -3. NETs Patients with NET liver metastases either complain of abdominal pain due to the mass effect of the tumor or excessive hormone production leading to the carcinoid syndrome, which consists of diarrhea, cutaneous flushing, various hemodynamic alterations, and wheezing ,7. MoreoA 5-year survival rate of 22% for patients with NET liver metastases has been described . TherefoMechanistically, local ablative techniques such as cryotherapy, radiofrequency ablation (RFA), and percutaneous alcohol injection (PEI) rely on the cytotoxic effects of chemicals and induce nonphysiologic temperatures into the hepatic tissue ,11. TherTreatment modalities involved in percutaneous liver-directed techniques, include bland embolization (BE), transarterial chemoembolization (TACE), and selective internal radiation therapy (SIRT). The principle of BE consists of inducing regional ischemia to the tumor. In TACE, chemotherapeutic substances are locally applied causing a cytotoxic effect. Due to the local embolization the intratumoral concentration of the cytostatic is as much as 20 times higher using TACE than in systemically applied chemotherapy . In addiPeptide receptor radionuclide therapy (PRRT), a combination of a somatostatin analog with a radioligand, a beta-emitter, is used to detect and treat NETs expressing somatostatin receptors. After systemic injection, the radioligand is internalized into the cells and transported to the lysosomes. The effect on tumor cell proliferation is based on the radiotoxicity of the radionuclide in the deoxyribonucleic acid of the target cell . HoweverThe role of systemic chemotherapy for NETs with liver metastases has been discussed vigorously. In a prospective study, Moertel et al  evaluateTargeted therapy includes multikinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and monoclonal antibodies, which interact with various molecular pathways . SunitinBiotherapy, using interferon-α and somatostatin analogues such as octreotide and lanreotide, prevents the synthesis of the polypeptide hormones and biogenic amines produced by functional NETs. This provides relief from endocrine symptoms associated with the carcinoid syndrome in 80% of patients -34. The The aim of these four systematic reviews is to determine evidence for the noninvasive treatment options in terms of symptom relief and tumor control in patients with nonresectable liver metastases of NETs.The following four systematic reviews dealing with the nonsurgical treatment options of neuroendocrine liver metastases attempt to address the following questions represented in Our research results will be reported in accordance with the standards of the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA).The eligibility criteria for inclusion as well as for exclusion of studies are illustrated in These studies will provide the basis for the qualitative synthesis of this systematic review. Single-cohort studies will be collected in a separate database and will only be used for descriptive purposes. No publication date or language restrictions will apply.1. When should locally ablative techniques  be used in patients with nonresectable neuroendocrine liver metastases?Do local ablation techniques  improve outcome  in patients with non–resection margin, tumor free (R0)/microscopic tumor lesions (R1) resectable NET liver metastases when compared with nonablative treatments ?Which local ablation technique  achieves the best outcome  in patients with nonresectable NET liver metastases?Do local ablation techniques  in conjunction with a systemic treatment  improve outcome  in patients with nonresectable NET liver metastases as opposed to a systemic treatment alone?What is the incidence of tumor dissemination in patients with NET liver metastases undergoing a local ablation technique? Does confirmation occur through imaging/biopsy during the follow-up?2. When should percutaneous liver-directed techniques be used in patients with nonresectable neuroendocrine liver metastases?Do percutaneous liver-directed techniques  improve outcome  in patients with nonresectable NET liver metastases as opposed to R2 liver resection?Which percutaneous liver-directed technique  achieves the best outcome  in patients with nonresectable NET liver metastases?Do percutaneous liver-directed techniques  improve outcome  in patients with nonresectable NET liver metastases in combination with a systemic treatment  when compared with a percutaneous liver-directed technique alone?What is the incidence of tumor dissemination in patients with NET liver metastases undergoing a percutaneous liver-directed technique? Does confirmation occur through imaging/biopsy during the follow-up?3. When should peptide receptor radionuclide therapy be performed in patients with nonresectable neuroendocrine liver metastases?Does a peptide receptor radionuclide therapy improve outcome  in patients with nonresectable NET liver metastases when compared with R2 liver resection?Does the outcome  for patients with nonresectable NET liver metastases undergoing a peptide receptor radionuclide therapy depend upon the size of liver metastases (>5- vs <5-cm diameter of the largest tumor) or their uptake on a diagnostic scan?Does the outcome  of a peptide receptor radionuclide therapy depend upon the percentage of liver volume involvement  for patients with nonresectable NET liver metastases?Does the outcome  of a peptide receptor radionuclide therapy, for patients with nonresectable NET liver metastases, depend upon the site of the primary tumor?Does a peptide receptor radionuclide therapy in combination with percutaneous liver-directed techniques  and/or local ablation techniques improve outcome  in patients with nonresectable NET liver metastases when compared with peptide receptor radionuclide therapy as a single technique?4. When should chemotherapy, targeted therapy, or biotherapy be used in patients with nonresectable neuroendocrine liver metastases?Does chemotherapy, targeted therapy and biotherapy improve outcome  in patients with nonresectable NET liver metastases as opposed to R2 liver resection?Does outcome  of chemotherapy, targeted therapy, and biotherapy in patients with nonresectable NET liver metastases depend upon the size of liver metastases (>5- vs <5-cm diameter of the largest tumor)?Does outcome  of chemotherapy, targeted therapy, and biotherapy in patients with nonresectable NET liver metastases depend upon the percentage of liver volume involvement ?Does outcome  of chemotherapy, targeted therapy, and biotherapy in patients with nonresectable NET liver metastases depend upon the site of the primary tumor?Does chemotherapy, targeted therapy, and biotherapy in combination with percutaneous liver-directed techniques  and/or local ablation techniques improve outcome  in patients with nonresectable NET liver metastases when compared with chemotherapy, targeted therapy, and biotherapy as a single technique?The electronic search strategy to scan the databases and detect all relevant articles was developed by the librarians of the Medical Library Careum . The search will be performed on the MEDLINE, EMBASE, and The Cochrane Library  databases. An endnote file, comprising all identified titles and, if accessible, the corresponding abstracts will be prepared for the investigators. Supplementary publications will be found by manual search or by reviewing reference lists. Two independent review group members will peruse titles and/or abstracts from studies, which were identified using the search profile. Afterwards, the entire text of these potentially eligible studies will be re-examined for eligibility. Any uncertainties will be discussed and resolved with a third member. A specially created Web-based, predetermined protocol will be used to extract data from the included studies for the study quality expertise and synthesis of medical findings.The data extraction parameters, include manuscript title, name of journal, first author's name, publication year, total number of patients, number of patients in the chemotherapy/biotherapy/targeted therapy group, number of patients in the nontreatment group, name of used substances, age , male to female ratio, progression-free survival, overall survival, quality of life (containing side effects), study design, and targeting objective 1-5. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) will be used to grade the quality (level) of evidence and the strength of recommendations .P values for each outcome.We will prepare a narrative synthesis of the findings from the included studies. A quantitative synthesis will be used if the included studies are sufficiently homogenous. We anticipate that there will be a limited scope for meta-analysis of a relatively large number of studies because of the range of outcomes measured across the small number of existing trials (such tumors are rare). Nevertheless, where studies have used the same type of intervention and comparator, with the same outcome measure, we will pool the results using a random-effects meta-analysis. We calculate a 95% CI and two-sided This study is ongoing and presents a protocol of four systematic reviews to assess the role of nonsurgical treatment options in patients with neuroendocrine liver metastases. Both noninvasive as well as invasive methods, such as percutaneous liver-directed techniques and local ablation techniques will be investigated.Several nonsurgical treatment options for neuroendocrine liver metastases have been reported. However, there is a lack of consensual data on the subject. These four systematic reviews described in this protocol aim to clarify the role of nonsurgical therapy modalities in patients with nonresectable NETs liver metastases. The systematic reviews will serve as a basis for developing clinical practice guidelines.


In the context of climate change, water availability will become an important limiting factor threatening biomass production, particularly in intensively managed forest tree plantations. The complexity of drought stress response calls for integrative approaches combining physiological, anatomical and molecular investigations at different scales ranging from particular cells to the whole plant. Such a strategy was developed for two eucalyptus clones used in industrial plantations in the Republic of Congo and known for their differences in term of productivity and water-use efficiency.E. urophylla * E. grandis (18-50), and one less productive hybrid obtained from open-pollination of E. alba (1-41). These genotypes were subjected to two watering regimes  in open field conditions. Growth parameters such height, diameter at breast height, stem, leaf and root biomasses as well as physiological parameters, such carbon discriminationand specific leaf area, were regularly measured over two growing seasons. At the end of the second dry season (i.e. 14 months-old trees), polyphenols accumulation and anatomical measurements were performed on mature leaves. RNA of shoot apices from trees representing the four conditions (i.e. 2 genotypes subjected to 2 watering treatments) were extracted and used to create non-normalized libraries. Using 454/Roche sequencing, we performed digital expression analysis to highlight differentially expressed transcripts showing genotype, treatments and/or genotype by treatment interaction effects. Expression of candidates genes revealed by this first analysis were assessed by RT-qPCR on shoot apices and on mature leaves.The two genetic units used in this study consisted in one more productive hybrid of Growth parameters and stem biomass analysis revealed that the two studied genotypes presented similar degrees of phenotypic plasticity in response to watering treatment during the dry season. Nevertheless, comparison of growth increments during wet seasons suggested that the more productive genotype had higher ability to recover from water shortage during the dry seasons and to use favorable conditions to increase its above-ground biomass. We found that the more productive genotype displayed greater plasticity of foliar traits in the non-irrigated treatment during the dry season, suggesting that it could adjust more quickly its leaf parameters when rainfalls resume.On mature leaves from trees subjected to water deficit, we identified mechanisms established to limit water losses by cuticle reinforcement and stomatal density decrease, to preserve leaf turgor by increase of collenchyma layer, to enhance hydraulic conductance with xylem vessel diameter decrease, and to preserve cells from reactive oxygen species through accumulation of hydroxycinnamates. Those adjustments were more intense for the more productive genotype, suggesting that this genotype may entailed higher cost strategies to preserve its leaf functions.On shoot apices, 454-sequencing provided a catalogue of 129,993 unigenes  from the initial sequencing of 398Mb corresponding to 1.14Mreads. Digital expression analysis identified 1,280 contigs displaying differential expression between the two genotypes, 155 contigs between treatments, and 274 contigs displaying genotype x treatment interaction effects.The more productive genotype mainly under-expressed genes related to photosynthetic activity during the dry season, activating genes to reallocate resources through major changes in primary metabolism, whereas the less productive genotype displayed lower levels of variation in gene abundance between treatments. This analysis allowed to reveal a set of candidate genes that could be related to phenotypic variation observed in mature leaves.The productivity of the two genotypes used in this study differed essentially in terms of ability to recover from water shortage. These differences could be explained by different strategies of investment in preservation of leaves. In the environmental gradient tested, it seemed that higher investments enabled genotype 18-50 to increase its leaf resistance, and subsequently to recover quickly high growth performance once environmental conditions become favorable. Genes that potentially underlie such physiological plasticity were revealed through integrative approaches. More investigations are now needed to valid their putative roles in phenotypic plasticity in response to water deficit. Forward genetic approaches could be used to screen the variability of gene expression and other more-integrated phenotypic traits, the nucleotide diversity of these candidate genes and their covariations on a larger genetic background.


We used immunohistochemistry and neural tracing to determine whether displacement of Purkinje cell zones into ectopic positions triggers defects in zonal connectivity within sensory-motor circuits. Despite the abnormal placement of more than 95% of Purkinje cells in scrambler mice, the complementary relationship between molecularly distinct Purkinje cell zones is maintained, and consequently, afferents are targeted into topographic circuits. These data suggest that although loss of disabled1 distorts the Purkinje cell map, its absence does not obstruct the formation of zonal circuits. These findings support the hypothesis that Purkinje cell zones play an essential role in establishing afferent topography.The cerebellum is organized into zonal circuits that are thought to regulate ongoing motor behavior. Recent studies suggest that neuronal birthdates, gene expression patterning, and apoptosis control zone formation. Importantly, developing Purkinje cell zones are thought to provide the framework upon which afferent circuitry is organized. Yet, it is not clear whether altering the final placement of Purkinje cells affects the assembly of circuits into topographic zones. To gain insight into this problem, we examined zonal connectivity in  The cerebellum is organized into an array of sagittal zones that control sensory-motor behavior , an adaptor protein that is essential for reelin signaling  transgenic mice . Mice carrying the Npy-Gfp allele were identified by genotyping using a standard polymerase chain reaction with primers designed to detect Gfp . scrambler:Npy-Gfp mice were genotyped for Gfp, phenotyped based on the severe postnatal ataxia observed in reelin pathway mutants  4/5 from scrambler heterozygote X heterozygote crosses, and each pup in the litter injected with tracer. Homozygous mutant pups were identified upon dissection, based on their small cerebella and well-understood lobule dysmorphology compared to littermate controls  0.5. The day of birth was designated as postnatal day 0 (P0). Adults were designated as P28 or older.All animal studies were carried out under an approved IACUC animal protocol according to the institutional guidelines at Albert Einstein College of Medicine and Baylor College of Medicine. Female and male Mice were anesthetized with avertin. Once all reflexes were abolished , the blood was flushed through the heart by perfusing with 0.1 M phosphate buffered saline . The tissue was then fixed by perfusing with 4% paraformaldehyde (4% PFA) diluted in PBS. The brains were then postfixed for 24–48 h in 4% PFA and then cryoprotected in buffered sucrose solutions (15 and 30% diluted in PBS). Serial 40 μm thick coronal sections were cut on a cryostat and collected as free-floating sections in PBS. Immunohistochemistry was carried out as described previously . Anti-Npy (Cat. # NAY-8060-V) was purchased from Peptides International  and has been shown to label zones in the rat cerebellum  or fluorescence. For the DAB reaction we used horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibodies  to bind the primary antibodies. Staining for fluorescent immunohistochemistry was carried out using Alexa 488- and 555-conjugated immunoglobulins , both diluted to 1:1500. Tissues sections were coverslipped using either Entellan mounting media  or FLUORO-GEL with Tris buffer . We tested the specificity of the secondary antibodies by processing the tissue sections in the absence of primary antibodies. No signal was detected in such control experiments indicating that the staining we observed was not due to non-specific signals from the Alexa or HRP conjugated antibodies (data not shown).n = 5 for each genotype) or adult mice (n = 5 for each genotype). After a 24 h (pups) or a 48 h (adults) survival period the mice were anesthetized as described above and then perfused with 4% PFA (described above). WGA-Alexa traced neurons are visible immediately upon cellular uptake and thus no additional tissue staining is required for labeling. Therefore, after the perfusion, the WGA-Alexa traced tissue was either cut and mounted for imaging or further processed for immunohistochemistry in order to examine the relationship between afferent projections and Purkinje cell zones. After tracing, the spinal cord was also cut in order to examine the size of the injection spot  and to ensure that only limited tissue damage was caused by the injection . The number of WGA-Alexa labeled mossy fiber terminals, which were identified as large terminal rosettes, were counted on each tissue section. The sum of the number of mossy fiber terminals for each region was computed and the mean for each region was used to calculate the standard error of the mean, SEM. The p value was acquired using an unpaired t-test (GraphPad Prism) to compare the differences in the number of terminals between wild type and scrambler mutants.In wild type mice, WGA-Alexa Fluor 555 accumulates as punctate deposits in mossy fiber terminals, which highlights the structure of the large terminal rosettes  that were doubled stained for Gfp and PLCβ4, and then measured the distance of a medial-lateral region of cortex that was occupied by zones that were stained using each antibody. The regions that were heavily stained for Gfp revealed the most obvious climbing fiber zones. We computed a region of overlap between the two markers—the mean percentage overlap in a given region was used to calculate the SEM. The p value was acquired using an unpaired t-test (GraphPad Prism) to compare the difference in percent overlap between afferent zones and Purkinje cell zones in wild type versus scrambler mutants. P < 0.05 was considered to be significant.For climbing fiber analysis, we acquired images from 4 to 5 sections through the anterior cerebellum of wild type and Photomicrographs of tissue sections were captured using Leica DFC360 FX (fluorescence) and DFC 490 (DAB reacted tissue sections) cameras mounted on a Leica DM5500 microscope. Images of tissue sections were acquired and analyzed using Leica Application Suite and Leica Application Suite FX software packages supplemented with a deconvolution module. For deconvolution, stacks of 10–20 sections in the z-axis were collected every 0.2–0.5 μm. The z stack images were deconvolved (~10 iterations) and then analyzed as compressed projections or as individual slices. All raw data was imported into Adobe Photoshop CS4 and corrected for brightness and contrast only. Schematics were created in Adobe Illustrator CS4.reeler mice, zebrinII and p-path are expressed within alternating zones of ectopic Purkinje cells  labeled terminal rosettes  within an equivalent sized region. We conclude that despite the lack of clear zonal boundaries and properly structured terminals, the topography of spinocerebellar afferent zones is not affected by the displacement of Purkinje cells that occurs after loss of disabled1 function in scrambler mutant mice.Similar to wild type mice Figures ,F, spino Figures ,G. Despi Figures ,G and th Figures . In cont Figures . Profilescrambler mutants, we wondered whether an altered developmental timetable could contribute to the formation of a distorted mossy fiber afferent map. To determine whether the initial patterning of mossy fibers into zones is altered in scrambler mutant mice, we used WGA-Alexa 555 to examine spinocerebellar tract development during the first week after birth. We previously determined that spinocerebellar mossy fibers resolve into a zonal pattern by ~P5 in mice . We first confirmed the identity of genetically labeled afferents as climbing fibers by double labeling transgenic tissue sections with GFP and VGLUT2, an established marker for climbing fiber terminals . Despite the severe ectopia of Purkinje cells in scrambler, we found that Npy-Gfp labeled climbing fibers were nevertheless topographically aligned with specific Purkinje cell clusters . In adjacent regions, we observed close to 100% overlap between afferent and Purkinje cell zones (for both wild type and mutant; Figures To further test the hypothesis that afferent fiber zones are correctly matched to Purkinje cell subtypes despite the abnormal cytoarchitecture in  Figures –D″. Doub Figures ,D″. MoreGfp driven by a neuropeptide Y gene promoter and a novel WGA-Alexa tracing approach to label climbing fibers and mossy fibers in the developing and adult cerebellum. Using the spontaneous mutant scrambler as a model for Purkinje cell mis-patterning, we demonstrate that despite the severe ectopia of Purkinje cell zones, the topography of afferent zones is established and afferent-target specificity is maintained.The cerebellum is organized into a complex array of topographic sagittal zones; zones are best defined by the molecular expression patterns in Purkinje cells (Apps and Hawkes, reeler, disabled1, Apoer2 and Vldlr mutants, Purkinje cell zonation is still established (Blatt and Eisenman, Cerebellar zones are clearly delineated by the patterned expression of several genes and proteins (Apps and Hawkes, scrambler mice (Goldowitz et al., scrambler mice, Npy-Gfp labeled climbing fibers and WGA-Alexa traced spinocerebellar mossy fibers terminate within severely ectopic positions although still in alignment with their normal targets, the PLCβ4 immunoreactive Purkinje cell clusters (Figures scrambler and weaver mutant mice, which is another mouse strain with ectopic Purkinje cells (Armstrong et al., reeler mutants, in which climbing fiber, spinocerebellar, and vestibulocerebellar afferents were shown to terminate into relatively normal anterior-posterior locations (Vig et al., The organization of parasagittal Purkinje cell zones is mirrored by the topography of mossy fiber (Gravel and Hawkes,  Figures , 6. Accoin vivo recordings in reeler mutants demonstrated that afferents do make connections in the ectopic clusters and afferent information is transferred to their ectopic Purkinje cells with some level of fidelity (Mariani et al., We also observed that within the ectopic Purkinje cell clusters, mossy fiber terminals failed to differentiate into their typical complex “grape-like” structure Figure . Surprisscrambler, which lack ~80% of their granule cells, and in other agranular mutants, spinocerebellar mossy fiber topography is established despite the absence of a normal mossy fiber-granule cell-Purkinje cell circuit (Arsenio Nunes and Sotelo, scrambler mutants, mossy fibers are targeted into their “correct” circuits, although within ectopic Purkinje cell zones. Thus, as long as Purkinje cells can resolve into different chemical phenotypes, zonal circuit connectivity proceeds with limited disruption.Spinocerebellar mossy fibers enter the cerebellar anlage at approximately E13/14 in mouse (Grishkat and Eisenman, Stacey L. Reeber, Courtney A. Loeschel, and Roy V. Sillitoe designed the experiments. Stacey L. Reeber, Amanda Franklin, and Roy V. Sillitoe performed the experiments. Stacey L. Reeber, Courtney A. Loeschel and Roy V. Sillitoe wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.