Mungbean yellow mosaic India virus (MYMIV) is a bipartite Geminivirus, which causes severe yield loss in soybean (Glycine max). Considering this, the present study was conducted to develop large-scale genome-wide single nucleotide polymorphism (SNP) markers and identify potential markers linked with known disease resistance loci for their effective use in genomics-assisted breeding to impart durable MYMIV tolerance. The whole-genome re-sequencing of MYMIV resistant cultivar ‘UPSM-534’ and susceptible Indian cultivar ‘JS-335’ was performed to identify high-quality SNPs and InDels (insertion and deletions). Approximately 234 and 255 million of 100-bp paired-end reads were generated from UPSM-534 and JS-335, respectively, which provided ~98% coverage of reference soybean genome. A total of 3083987 SNPs (1559556 in UPSM-534 and 1524431 in JS-335) and 562858 InDels (281958 in UPSM-534 and 280900 in JS-335) were identified. Of these, 1514 SNPs were found to be present in 564 candidate disease resistance genes. Among these, 829 non-synonymous and 671 synonymous SNPs were detected in 266 and 286 defence-related genes, respectively. Noteworthy, a non-synonymous SNP  at the 149th base-pair of LEUCINE-RICH REPEAT RECEPTOR-LIKE PROTEIN KINASE gene responsible for a G/C transversion [proline (CCC) to alanine(GCC)] was identified and validated in a set of 12 soybean cultivars. Taken together, the present study generated a large-scale genomic resource such as, SNPs and InDels at a genome-wide scale that will facilitate the dissection of various complex traits through construction of high-density linkage maps and fine mapping. In the present scenario, these markers can be effectively used to design high-density SNP arrays for their large-scale validation and high-throughput genotyping in diverse natural and mapping populations, which could accelerate genomics-assisted MYMIV disease resistance breeding in soybean. Mungbean Yellow Mosaic Virus is one of the most destructive as well as widely distributed plant pathogenic viruses belonging to the family Geminiviridae. This virus causes yellow mosaic disease in legumes and particularly in India, the yields of several legume crops including blackgram (Vigna mungo), mungbean (Vigna radiata), cowpea (Vigna unguiculata) and soybean (Glycine max) are challenged by Mungbean Yellow Mosaic India Virus (MYMIV). Among legumes, soybean is an economically important crop in which MYMIV causes devastating yield losses  belonging to disease resistant QTLs were retrieved from PRG database . The conserved domains in the proteins were predicted by searching against Pfam database using HMMscan v3.0  by Sanger sequencing. Approximately 500 bp flanking sequences of the selected SNPs were extracted using in-house perl programming and then flanking forward and reverse primers were designed. The sequences were amplified by PCR using genomic DNA of UPSM-534 and JS-335 along with 10 cultivars. The PCR amplification reactions were performed in a 20 μl reaction volume containing 100 ng of genomic DNA, 1x Taq buffer, 2 mM of MgClThe present study has direct relevance to the current scenario of identifying prominent breeding strategies for MYMIV resistance. Despite the efforts of soybean breeders, no major breakthrough has been achieved for imparting MYMIV resistance in soybean. Hence, molecular marker-based approaches will offer innovative avenues to breeders, geneticists and researchers to investigate the MYMIV resistance in soybean. Further, this will enable the improvement of horizontal resistances that are polygenetically inherited and for which screening and selection is cumbersome. Whole genome re-sequencing is widely practised for large-scale identification of molecular markers and their utilization in mapping of disease resistant loci. This would serve as a valuable resource for fine mapping the disease resistant loci in crop plants, including soybean. Further, high-throughput DNA sequencing and generation of large-scale expression data will provide the opportunity to explore the genetic and genomic variations at resistance genes/QTLs and pave way for conducting further downstream functional assays to clarify their mechanisms of action.MYMIV infected soybean seedlings initially showed chlorosis of leaves, which is the characteristic symptom of the disease . The disG. max var. Williams 82). Approximately 97% of the reads were mapped to the reference genome with a coverage of 95% in both the cultivars  and transversion (Tv) type of SNPs were 1042619 and 527093, respectively, with a Ts/Tv ratio of 1.9781 for JS-335 . SimilarGlycine max var. Williams 82 genome with 950068807 bases contained in twenty chromosomes was used as reference. The distribution of DNA polymorphisms detected between JS-335 and UPSM-534 was analysed with the reference genome. The frequency of SNPs and InDels identified in both the cultivars varied across twenty chromosomes  and chromosome 14 of UPSM-534 (606.68 SNPs per Mb). Similarly, higher InDel density was observed in chromosome 18 of JS-335 (441.89 InDels per Mb) and chromosome 16 of UPSM-534 (447.79 SNPs per Mb), whereas lower InDels density was detected in chromosome 4 of JS-335 (158.28 InDels per Mb) and chromosome 14 of UPSM-534 (157.58 InDels per Mb) . FurtherThe annotation of soybean reference genome was used to reveal the distribution of SNPs and InDels within various genomic regions such as intergenic and intragenic. Approximately 50% of SNPs were identified in intergenic regions in both the cultivars. Overall, a similar distribution pattern of SNPs and InDels was observed in the entire chromosome for both the cultivars. Only 10–12% of total SNPs were detected in genic regions, whereas upstream (promoter) and downstream regulatory regions had significant number of SNPs (18–22%). Within the genic region, more than 6% of SNPs were present in the introns . The 3′ Amino acid substitution was analysed to investigate the effect of SNPs in coding sequences. In JS-335, 28267 and 25533 SNPs showed non-synonymous and synonymous type modifications, respectively. Similarly, 29012 non-synonymous and 26195 synonymous changes were observed in UPSM-534. The ratio of non-synonymous to synonymous SNPs was about 1.11 for JS-335 and 1.10 for UPSM-534 . FunctioFunctional annotation of the genes showing SNP variations revealed the participation of these genes in defense response. A total of 1514 SNPs were detected in 564 defence response-related genes in both the soybean cultivars  Tables aAlthough the use of next-generation sequencing and high-throughput analysis platforms in re-sequencing of soybean cultivars and identification of QTLs related to biotic stress tolerance have been demonstrated –27, no aLEUCINE-RICH REPEAT RECEPTOR-LIKE PROTEIN KINASE (LRR-RP) gene (Glyma18g02850)  . The G tIn the present study, SNPs and InDels were generated in large-scale by re-sequencing two soybean cultivars with contrasting tolerance characteristics to MYMIV infection. In addition to the usefulness of these resources in genomics-assisted breeding for MYMIV resistance, the present study will facilitate the dissection of complex traits through construction of high-density linkage map and fine mapping. Further, these markers would be useful for high-density SNP array designing and high-throughput genotyping for variety identification, gene tagging, molecular mapping of genes/QTLs, construction of linkage map, association mapping and marker-assisted selection. Of note, the SNP identified in chromosome 18 (18–1861613) with G/C transversion at 149 bp could be used in marker-assisted breeding for MYMIV resistance after large-scale validation.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe SNP 18–1861613 is highlighted in red. Non-synonymous SNPs are indicated in bold, synonymous SNPs are underlined.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Plasmodium vivax (Pv) infection remains largely unknown. Here, we demonstrated that activated as well as “atypical” memory B cells (MBCs) are expanded in peripheral blood of Pv-exposed individuals, but their frequencies were not associated with acute infection. Aiming to investigate the association between peripheral B cells subsets and Pv-specific antibodies, we further followed-up 34 individuals exposed to P. vivax in the Brazilian Amazon area, an area of markedly unstable malaria transmission; after three cross-sectional survey , ELISA-detected specific IgG  allowed the classification of those individuals as non-responder (NR), temporary (TR) or persistent responder (PR). For AMA-1 and MSP1-19 serological groups, the frequencies of MBCs  and plasma cells (PCs) were similar among the groups. For DBPII group, we found a trend toward decreases classical MBCs according to the antibody response (NR>TR>PR). On the other hand, the frequencies of atypical MBCs increased according to the presence and persistence of DBPII antibody response (PR>TR>NR). Altogether, these results showed that atypical MBCs are expanded in Pv-exposed individuals (infected and non-infected), and it seems to be associated with the persistence of DBPII antibody response. Although preliminary, these results suggest that atypical MBCs contribute in generation of malarial antibody responses and provide insight into the role of atypical MBCs in P. vivax malaria immunity.Antibody responses generated during malaria infection represent an important component of acquired clinical immunity. Despite that, B cell subpopulations induced by the


Synthetic polymers are widely used in daily life. Due to increasing environmental concerns related to global warming and the depletion of oil reserves, the development of microbial-based fermentation processes for the production of polymer building block chemicals from renewable resources is desirable to replace current petroleum-based methods. To this end, strains that efficiently produce the target chemicals at high yields and productivity are needed. Recent advances in metabolic engineering have enabled the biosynthesis of polymer compounds at high yield and productivities by governing the carbon flux towards the target chemicals. Using these methods, microbial strains have been engineered to produce monomer chemicals for replacing traditional petroleum-derived aliphatic polymers. These developments also raise the possibility of microbial production of aromatic chemicals for synthesizing high-performance polymers with desirable properties, such as ultraviolet absorbance, high thermal resistance, and mechanical strength. In the present review, we summarize recent progress in metabolic engineering approaches to optimize microbial strains for producing building blocks to synthesize aliphatic and high-performance aromatic polymers.The online version of this article (doi:10.1186/s12934-016-0411-0) contains supplementary material, which is available to authorized users. Since the discovery and commercialization of synthetic polymers, these materials have become essential for everyday life . Currentl-lactic acid (PLLA) is to date perhaps the only good example of successful industrialization of a 100 % bio-based polymer  involved in by-product formation were deleted to minimize by-product accumulation. The gene-deleted strain was further engineered by overexpression of the pck gene (encoding phosphoenolpyruvate carboxykinase) from A. succinogenes and the pyc gene (encoding pyruvate carboxylase) from C. glutamicum, providing production of 11.2 g/L succinic acid at pH 5.7  were replaced with stronger promoters, and argR  and rpoS (encoding a stress-responsive RNA polymerase sigma factor) also were deleted  and argR was effective for increasing putrescine production due to an increase in the ornithine supply; however, argF deletion resulted in arginine auxotrophy. This problem was circumvented by the fine-tuning of argF expression through modifications of the promoter, translational start codon, and ribosome-binding site, resulting in a 60 % increase in putrescine production . T. TStreptum juice .E. coli strain [Aromatic polyimides are alternative building blocks for high-performance bio-based polymers due to their excellent thermo-mechanical performance, high chemical stability, and low coefficient of thermal expansion. A phenylpropanoid derivative of 4-aminocinnamic acid was produced by the bioconversion of the non-standard amino acid 4-aminophenylalanine using a recombinant i strain . Bio-basi strain . The 4-ai strain , suggestCompared to current aliphatic polymers, emerging bio-based aromatic polymers are value-added molecules with high thermal and mechanical properties; these polymers therefore might serve as engineering plastics. Further developments to increase the compatibility of aromatic compounds for bioprocessing will be needed to achieve high productivity of aromatic bio-monomers from renewable feedstocks.l-lactic acid production through microbial fermentation, several bio-based approaches for succinic acid synthesis recently have been commercialized [The present review aimed to provide a broad view of metabolic engineering strategies for producing building block chemicals for use in generating aliphatic polymers. We further described the current state of knowledge for the production of building block chemicals of next-generation, high-performance aromatic polymers. As described above, advances in metabolic engineering have markedly improved the productivities and yields of microbially produced polymer building blocks. Following the success of industrial cialized . However


Adipose tissue dysfunction is a prime risk factor for the development of metabolic disease. Bone morphogenetic proteins (BMPs) have previously been implicated in adipocyte formation. Here, we investigate the role of BMP signalling in adipose tissue health and systemic glucose homeostasis.Cre/loxP system to generate mouse models with conditional ablation of BMP receptor 1A in differentiating and mature adipocytes, as well as tissue-resident myeloid cells. Metabolic variables were assessed by glucose and insulin tolerance testing, insulin-stimulated glucose uptake and gene expression analysis.We employed the Bmpr1a using the aP2 -Cre strain resulted in a complex phenotype. Knockout mice were clearly resistant to age-related impairment of insulin sensitivity during normal and high-fat-diet feeding and showed significantly improved insulin-stimulated glucose uptake in brown adipose tissue and skeletal muscle. Moreover, knockouts displayed significant reduction of variables of adipose tissue inflammation. Deletion of Bmpr1a in myeloid cells had no impact on insulin sensitivity, while ablation of Bmpr1a in mature adipocytes partially recapitulated the initial phenotype from aP2-Cre driven deletion. Co-cultivation of macrophages with pre-adipocytes lacking Bmpr1a markedly reduced expression of proinflammatory genes.Conditional deletion of Bmpr1a specifically in pre-adipocytes and maturing adipocytes rather than an immune cell-autonomous effect. Mechanistically, we provide evidence for a BMP-mediated direct crosstalk between pre-adipocytes and macrophages.Our findings show that altered BMP signalling in adipose tissue affects the tissue’s metabolic properties and systemic insulin resistance by altering the pattern of immune cell infiltration. The phenotype is due to ablation of The online version of this article (doi:10.1007/s00125-016-3990-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users. Obesity is recognised as a significant risk factor for several of our most common medical conditions, such as type 2 diabetes mellitus and diseases associated with cardiovascular complications –3. The mNormal adipose tissue displays a low-grade inflammation, which is presumably due to removal of apoptotic adipocytes. In obese individuals, WAT becomes a significant source of proinflammatory cytokines, which are known to promote systemic insulin resistance . SpecifiBmpr1a) using the Myf5-Cre driver led to a specific atrophy of interscapular BAT and compensatory browning of WATs, altogether establishing the metabolic equivalence of brite/beige adipose tissue and classical BAT ..27].Analysis of gene expression on the protein level was performed as described previously . AntibodTotal RNA isolation and gene expression analysis was conducted as described previously . Primer Adipocytes were analysed using ImageJ software  .ATMs were analysed using FACS of freshly isolated stromal-vascular fractions of WAT as described previously .Body composition, activity levels and energy expenditure were assessed as described previously .Pre-adipocytes were cultured as described previously . Macrophp < 0.05 and determined by Student’s t test or two-way ANOVA when comparing multiple groups. In cases of unequal variance and non-normal distribution, non-parametric testing was conducted (Mann–Whitney U test).The data are presented as means ± SEM. Statistical significance was defined as aP2 promoter to drive adipose-specific expression of Cre recombinase to generate a tissue-specific deletion of Bmpr1a in mouse adipose tissues (aP2-Bmpr1a-KO) . ExaP2 is also expressed in cell types other than adipocytes. Specifically, it is also expressed in macrophages, where aP2 plays a role in foam cell formation [aP2-Cre may also result in gene deletion in macrophages infiltrating the adipose tissue. While this possibility is still valid, a recent study using the same aP2-Cre strain as us showed no Cre-mediated recombination in adipose tissue macrophages [Bmpr1a mRNA levels were not changed in macrophages sorted from WAT of aP2-Bmpr1a-KO mice when compared with WAT from control mice -Cre mouse strain [LysM-Bmpr1a-KO mice . Unlike aP2-Cre, which also causes recombination in adipogenic progenitor cells [Adipoq-Cre is expressed exclusively in mature adipocytes, thus targeting a more restricted population of cells within WAT [Adipoq-Bmpr1a-KO mice, decreased Bmpr1a mRNA levels were observed in WAT  was added to the co-culture to activate expression of inflammatory cytokines from macrophages  and LPS-induced cytokines  was significantly reduced in co-cultures of knockout pre-adipocytes and macrophages . Thus, alterations of signalling through BMPR1A during the earlier stages of white adipocyte differentiation, rather than in fully mature adipocytes, could be critical to the reduction of proinflammatory signals and improved insulin sensitivity.In a previous study it was reported that l muscle . Muscle-l muscle , 43. ThiaP2-Bmpr1a-KO mouse, where overall macrophage infiltration into adipose tissue is reduced, could retain a healthier metabolic profile due to a general lack of infiltrating immune cells.‘Inflamm-ageing’ is a concept encompassing age-related deterioration of the innate immune system response, low-grade chronic inflammation and the onset of age-related pathologies such as insulin resistance . In a weIn summary, our study provides new insight into the role of BMP signalling in maturing white adipocytes. In brown adipocytes, BMPs are critical for formation and thermogenic activity, whereas in white adipocytes, BMP signalling appears to regulate the endocrine interaction between cells of the adipose lineage and immune cells. A better understanding of these processes could help decipher the intricate crosstalk between adipocytes and other adipose tissue-resident cell types and this could provide novel avenues to counter the progression and pathology of insulin resistance.ESM(PDF 584 kb)Below is the link to the electronic supplementary material.


Control samples obtained from patients with irritable bowel syndrome, or tumor-matched normal colon mucosa were analyzed (n=50). MiR-182 expression increased progressively and significantly along with the colorectal carcinogenesis cascade, and in CRC liver metastases. The inverse relation between miR-182 and the expression of its target gene ENTPD5 was investigated by immunohistochemical analysis. We observed that normal colocytes featured a strong ENTPD5 cytoplasmic expression whereas a significantly and progressively lower expression was present along with dedifferentiation of the histologic phenotype. Plasma samples from 51 CRC patients and controls were tested for miR-182 expression. Plasma miR-182 concentrations were significantly higher in CRC patients than in healthy controls or patients with colon polyps at endoscopy. Moreover, miR-182 plasma levels were significantly reduced in post-operative samples after radical hepatic metastasectomy compared to preoperative samples. Our results strengthen the hypothesis of a central role of miR-182 dysregulation in colon mucosa transformation, demonstrate the concomitant progressive down-regulation of ENTPD5 levels during colon carcinogenesis, and indicate the potential of circulating miR-182 as blood based biomarker for screening and monitoring CRC during the follow-up.MiR-182 expression was evaluated by qRT-PCR and  Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer death in men and women in the United States . MetastaMicroRNAs (miRNAs) are non-coding RNAs that control gene expression at the post-transcriptional level , 7. NumeIn their seminal article, Sarver and colleagues reported an up-regulation of miR-182 in a small series of CRCs by using miRNA microarray expression analysis , 20. MorIn the present study, we further investigated the involvement of miR-182 in the transformation of the colon mucosa and CRC progression, and also extended the analysis of the relationship among miR-182 expression and its target gene ENTPD5. Moreover, we explored the diagnostic and prognostic value of circulating miR-182 as a potential biomarker for CRC patients monitoring.t-test, p=0.006), tubular adenomas with HG-IEN , and in CRCs  .To extend our previous findings on high miR-182 expression in CRC , we inve) Figure  in compat-test, p<0.0001), and CRC liver metastases  compared to normal tissues. Metastatic samples showed a higher, but not significant, miR-182 expression in comparison to primary tumors.To study miR-182 dysregulation in CRC liver metastases, we investigated miR-182 expression by qRT-PCR in a series of 20 stage IV CRCs Figure . A signiWe also investigated miR-182 expression by ISH in 5 cases of stage IV CRCs. A consistently significant overexpression in paired primary tumors and CRC liver metastasis in comparison to normal colon mucosa was observed in all the tested samples Figure . miR-182n=393). Interestingly, miR-182 expression was significantly higher in CRCs presenting lymph node  or liver metastases  at diagnosis. In univariate analysis, and considering the median miR-182 value as a cut-off limit, miR-182 expression levels negatively correlated with the overall survival of patients  (data not shown).To further strengthen these results, we evaluated the prognostic impact of miR-182 expression on a large number of CRCs in The Cancer Genome Atlas (TCGA) CRC series (By means of luciferase mutagenesis reporter assay we confirmed the relationship between miR-182 and its target gene ENTPD5. Luciferase activity was reduced 2.0 fold by miR-182 expression in HEK293T cells transfected with wild-type ENTPD5-reporter. Mutation of the predicted MRE (miRNA response-element) completely restored luciferase expression, thus demonstrating a direct interaction between miR-182 and the 3′ UTR of ENTPD5 transcript Figure .p<0.001; Figure To support this finding at protein level, we investigated by IHC the expression of ENTPD5 during colorectal carcinogenesis in a series of 20 normal colic mucosa samples, 40 tubular adenomas (LG-IEN and HG-IEN), and 20 early primary stages CRCs. Normal colocytes featured strong ENTPD5 cytoplasmic immunostaining whereas a significant and progressive lower expression was observed along with the dedifferentiation of the histologic phenotype . By using qRT-PCR and IHC, we demonstrated at mRNA and protein level a significant down-regulation of ENTPD5 from normal colon mucosa, through primary CRC tumor to liver metastases  and 10 late stages (stages III and IV) CRC patients.t-test, p=0.008) or patients with colic polyps at endoscopy   Figure . Considet-test, p=0.020; Figure We also evaluated miR-182 plasma levels before and 30 days after radical liver metastasectomy. Plasma miR-182 concentration was analyzed in paired pre- and post-operative samples from 11 CRC patients who underwent curative liver metastasectomy. We observed that miR-182 plasma levels were significantly reduced one month after surgery (paired The results of the present study can be summarized as follows: i) miR-182 is significantly up-regulated during colon carcinogenesis, and is associated to CRC patients prognosis; ii) miR-182 exerts a suppressive regulation on ENTPD5, suggesting novel molecular pathways in colon carcinogenesis; iii) miR-182 up-regulation can be detected in CRC plasma samples, and is therefore eligible as a novel diagnostic and non-invasive follow-up marker.Reports on miR-182 involvement in human cancer have been discordant. For instance, miR-182 aberrant expression has been associated to tumor progression in melanoma, endometrial cancer and prostate carcinoma , 25, 26;More recently, miR-182 enhanced expression has been associated to adverse CRC clinical characteristics and poor prognosis , 22. miRet al. who showed that both ENTPD5 mRNA and protein levels progressively decrease during the transition from normal colon mucosa, through adenoma, to adenocarcinoma . Information about TCGA and the investigators and institutions that constitute the TCGA research network can be found at “Biopsy and tissue samples were manually microdissected to ensure that each sample contained at least 80% of tumor cells. The percentage of the target lesion as obtained by manual microdissection was further validated on an adjunctive hematoxylin and eosin histology section. Total RNA was extracted using the RecoverAll kit , according to the manufacturer's instructions.C. elegans miRNAs cel-miR-39, cel-miR-54, and cel-miR-238 (synthetic RNA oligonucleotides synthesized by Qiagen) were added (as a mixture of 7 pg/pl of each oligonucleotide) to each denatured sample  with the exception of cel-miR-238, which was added after cDNA assembly. RNA was isolated using the mirVana PARIS kit following the manufacturer's protocol for liquid samples (Ambion). RNA was eluted with 50 μl of RNase-free H2O.In plasma samples, 500 μl of human plasma was thawed on ice and lysed with an equal volume of 2X Denaturing Solution (Ambion). To allow for normalization of sample-to-sample variation in RNA isolation, synthetic RNA extraction and quality controls were performed as previously described .Total RNA (1 μg) was used for first-strand cDNA synthesis using the SuperScript™ II Reverse Transcriptase kit and Taqman Assay  to detect and quantify ENTPD5 mRNA. To study mature hsa-miR-182 expression, the TaqMan MicroRNA Reverse Transcription Kit (Invitrogen) was used according to the manufacturer's instructions .−ΔΔCt.All reactions were run in triplicate, including no template controls, in a LightCycler 480 Real-Time System . Normalized expression was calculated using the comparative Ct method, and the fold change was expressed as 2C. elegans miRNAs and for hsa-miR-16 were averaged to generate SpikeIn_Average_Ct values. The median of the SpikeIn_Average_Ct values obtained from all of the samples to be compared was next calculated . The hsa-miR-182 raw Ct in a given sample was adjusted as follows: Normalized_Ct value for the miRNA in the sample = Raw_Ct value - [ - ]. All reactions were run in triplicate, including no-template controls.For plasma samples, normalization was reached using a median normalization procedure, as previously described with minor modifications [36]. For each sample, the Ct values obtained for the three spiked-in Locked nucleic acid (LNA) probes with complementarity to 21-bp sections of miR-182 were labeled with 5′-digoxigenin and synthesized by Exiqon . Tissue sections were digested with ISH protease 1  and ISH performed as described, with minor modifications [37]. Positive  and negative scrambled LNA probes were used as controls.HEK293T cells transfection was carried out as previously described . The pMiDifferences in miR-182 expression were evaluated by t-test, paired t-test and ANOVA, as appropriate. IHC data were evaluated by Kruskal-Wallis test for trend. Survival analysis on TCGA data was carried out by applying the Log-rank Mantel-Cox test. The statistical analysis was performed using STATA software .


Since the establishment of tumor angiogenesis as a therapeutic target, an excitement in developing the anti-angiogenic agents was resulted in tailoring a humanized monoclonal antibody (Bevacizumab) against vascular endothelial growth factor (VEGF): a key factor in recruiting angiogenesis. The past three decades' research in the area of angiogenesis also invented a series of novel and effective anti-angiogenic agents targeting the VEGF signaling axis. Despite the demonstrable clinical benefits of anti-angiogenic therapy, the preclinical and clinical data of the current therapeutic settings clearly indicate the transient efficacy, restoration of tumor progression and aggressive recurrence of tumor invasion after the withdrawal of anti-angiogenic therapy. Therefore, the impact of this therapeutic regime on improving overall survival of patients has been disappointing in clinic. The recent advances in pathophysiology of tumor angiogenesis and related molecular and cellular underpinnings attributed the conspiracy of compensatory angiogenic pathways in conferring evasive and intrinsic tumor resistance to anti-angiogenic agents. The understandings of how these pathways functionally cross-talk for sustaining tumor angiogenesis during VEGF blockade is essential and perhaps may act as a basic prerequisite for designing novel therapeutic strategies to combat the growing arrogance of tumors toward anti-angiogenic agents. The present review offers a discourse on major compensatory angiogenic pathways operating at cellular and molecular levels and their attributes with resistance to anti-angiogenic agents along with strategic opinions on future setting in targeting tumor angiogenesis. The sprouting of new blood vessel initiates with dissolution of vascular basal membrane, increase in vascular permeability and degradation of extracellular matrix, followed by endothelial cell (EC) migration, invasion, proliferation and tube formation. The angiogenic switch refers to a consortium of several regulatory factors, which regulates angiogenesis by maintaining a strict balance between activators and inhibitors in normal physiological angiogenesis. The pathological angiogenesis is mostly a hallmark of cancer, wherein the developed vasculature is more complex, abnormal, leaky and torturous.2Angiogenesis is a physiological process of formation of new capillaries on pre-existing vessels. The recent literature in the area of development of blood vessels is impressive in understanding the dynamics and complexities of vasculogenesis/angiogenesis.2 Till date, 10 anti-angiogenic agents have been approved by the US Food and Drug Administration: bevacizumab and ziv-aflibercept as anti-vascular endothelial growth factor (VEGF) agents, whereas sorafenib, sunitinib, pazopanib, axitinib, cabozantinib and regorafenib are approved as small-molecule RTK (receptor tyrosine kinase) inhibitors.3In 1971, Judah Folkman, a pioneer researcher in tumor angiogenesis, first highlighted the significance of vasculature for the growth and proliferation of solid tumors. He demonstrated that if a tumor is deprived from generating its own blood supply, it would not grow more than 1–2 mm in size or it may wither and die. Since the opening of this research window, the preclinical and clinical data started accumulating in a logarithmic manner, with a clear intention of inhibiting the tumor angiogenesis.4 the patients' benefit from the treatment is not satisfactory and is rather disappointing because of transient and modest performance of the anti-angiogenic agents in the clinic, off-target toxicities and intrinsic refractoriness.2 Perhaps more serious is the aggressive invasion and expedite metastasis of tumors after withdrawal of anti-angiogenic drugs.5 Besides numerous clinical trials in progress and over dozens of molecules being engineered against VEGF/VEGF receptor (VEGFR) and non-VEGF pathways,6 the entire situation warrants the understanding of molecular underpinnings and loopholes supporting the bypass angiogenic mechanisms ensuring tumor progression and metastasis even after the treatment with effective anti-angiogenic agents.Besides several appreciation reports describing the efficacy of anti-angiogenic agents in extending the survival of cancer patients by few months,In a broader sense, the multiple compensatory angiogenic factors/signaling pathways that tumors employ during anti-VEGF stress  and 2, c7 Ziv-aflibercept  is an antibody linked to IgG1 backbone and possesses VEGFR1 and VEGFR2 as extracellular domains. Its binding preference is not only with VEGF-A but also with VEGF-B and PlGF. The role of VEGF-C and VEGF-D is primarily implicated in lympangiogenesis.8 It has been reported that a VEGF-C fragment generated through proteolytic cleavage has binding affinity with VEGFR-3 and perhaps could be an alternative bypass mechanism and a potential cause of resistance to anti-VEGF-A modalities.9 NRP-1 (VEGF receptor) not only binds to VEGF-A but also binds to PlGF. Moreover, NRP-2 interacts with VEGFR-2 and may enhance angiogenic signaling. Apart from VEGF-A, NRP-2 also binds with VEGF-C and PlGF, thereby having a significant role in angiogenesis.10 Clinical evidences also strengthen the possibility of involvement of PlGF in angiogenic rescue task, as upregulated levels of PlGF were detected in patients treated with anti-VEGFR therapy.11The VEGF axis-dependent alteration pathways are initiated by the subtypes of VEGF such as VEGF-B, VEGF-C, VEGF-D and placenta growth factor (PIGF) . VEGF-B 12 hepatocyte growth factor/cMet pathway,13 angiopoietins,14 Delta4–Notch signaling pathway,15, platelet-derived growth factor (PDGF)-C,16 interleukins,17 Ephrins,18 ALK1 signaling19 and Wnt signaling.20 FGF signaling has been considered as one of the major culprit for adaptive tumor resistance to VEGF-targeted inhibitors.21 In fact, binding of FGF receptors to FGF ligands causes activation of several signaling pathways such as phospholipase Cγ–protein kinase C, phosphatidylinositol 3-kinase (PI3K-AKT-mTOR), JAK/STAT  and MAPK (mitogen-activated protein kinase: RAS/RAF/MAPK and RAS/MAPK/ERK).22 The signaling and cross-talk of these pathways , a VEGFR inhibitor. Moreover, inhibition of FGF/VEGF using brivanib was found to be effective in treating mouse pancreatic neuroendocrine tumors demonstrating adaptive/evasive resistance to VEGF inhibition.24 Plethora of preclinical and clinical findings has highlighted the significance of Delta4–Notch signalling as one of the pathways mediating the tumor refractoriness to anti-VEGF therapy.26 Inhibition of Delta4 leads to deregulation of angiogenesis, thereby resulting in excessive but non-functional vasculature, which can be conveniently used as an effective strategy for paralyzing the tumor growth that are relatively resistant to anti-VEGF therapeutic regime and for improving the therapeutic index of anti-VEGF agents by combining it with Delta4 inhibitors.26Clinical and experimental settings have identified several angiogenic growth factors having a key role in anti-VEGF escape mechanisms . Some ofpathways  are invo27 In clinical trial studies, in hepatocellular carcinoma (HCC) patients who were treated with VEGFR inhibitor, sorafenib, the progression-free survival was strongly correlated with low serum HGF levels as compared with patients with high serum HGF levels having progressive development of the disease.28 In recent cell coculture studies, a stromal cell (HSC-LX2) derived from the microenvironment of HCC was able to induce sorafenib resistance in HCC cells (Huh7) through multiple pathways including HGF/c-Met/Akt and Jak2/Stat3 .In an investigation designed to understand the modes of tumor resistance to sunitinib, the HGF/c-Met pathway was identified as a culprit for anti-VEGF rescue mechanism.30 In case of MMTV-PyMT mammary carcinomas, the higher levels of ANG2 were associated with worse response to anti-angiogenic therapy containing bevacizumab. Nevertheless, there was lack of synergistic anti-angiogenic or antitumor functions arising out of ANG2/VEGFR2 blockade.31 During VEGF arrest, administration of BowANG1  to secrete PDGF-C, which in turn triggered tumor angiogenesis.16The vessel normalization hypothesis claims that anti-VEGF agents, especially VEGFR2 blockers, normalize the vessels by proper covering of pericytes over the leaky vessels. This is a kind of vessel improvement and perhaps may act as an angiogenic escape mechanism. This proposed mechanism appears to be mediated by ANG1/Tie2 signaling.6 and, more recently, IL-17  have been implicated in tumor refractoriness to anti-VEGF agents. A clear involvement of IL-8 has been reported in anti-VEGF tumor resistance in sunitinib-treated renal cell carcinoma.17 Chung et al.33 established a new link of IL-17-driven and stromal cell-mediated signaling that confers resistance to anti-VEGF agents. Chung et al.33 also showed that IL-17 signaling cascade mobilizes the granulocyte colony-stimulating factor (CSF)-dependent recruitment of CD11b+Gr1+ immature myeloid cells, which acts as major driving force for anti-VEGF tumor refractoriness.34 Mostly, the role of EphA2/EphrinA1 signaling has been attributed with compensatory angiogenesis and concerned tumor resistance. When the functions of VEGFR1 and R2 were blocked using antibodies, the resistance toward VEGFR2 blockade was observed in the form of revascularization and regrowth of tumors. The resistance to VEGF blockade was strongly correlated with hypoxia-induced upregulation of Ephrin A1 and other compensatory pro-angiogenic factors.35 In pancreatic tumors, overexpression of Eph A2 and Ephrin A1 was observed in VEGF-treated tumors and furthermore the elevated levels were correlated with tumor refractoriness.36Several members of interleukins (ILs) such as IL-1, IL-8, IL-12 (Vasudev and Reynolds)et al.37 have demonstrated the pro-angiogenic effects of ALK1/BMP9 signaling, which includes proliferation of ECs, promotion of tumor angiogenesis in matrigel plug vascularization in a xenograft model of pancreatic cancer. In melanoma xenografts with upregulated human VEGF-A and resistance to RTK inhibitor, the combined treatment of bevacizumab in conjunction with PF-03446962  significantly improved the efficacy of VEGF/VEGFR targeting agents, thus giving a clue of involvement of ALK1 signaling in bevacizumab resistance and thereby in promoting angiogenesis under VEGF blockade.38 The combination therapy of VEGFR tyrosine kinase inhibitor and ALK1 inhibitor (ALK1-Fc) has also been assessed in VHL-deficient RCC murine xenograft models, where a clear evidence of resistance towards anti-VEGFR agents was observed.19 In zebrafish model studies, Rspo1/Wnt-mediated signaling promotes angiogenesis via VEGF-C/VEGF-R3, but not involving VEGF-A. The authors hope that the novel Rspo-Wnt-VegfC-Vegfr3 pathway may have an important role as bypass angiogenic pathway during tumor angiogenesis.20Suzuki 39 The most prominent cell populations of the BMDCs contributing to the tumor angiogenesis and progression  expressing CD11b+F4/80+, Tie2-expressing monocytes, (PDGFR)+ pericyte progenitors, a population of CD45+CD11b+ myeloid cells, tumor-infiltrated mast cells and neutrophils, and vascular endothelial-cadherin+ CD45+ vascular leukocytes and so on.40 Besides these prominent pro-angiogenic BMDCs, several other lineages of myeloid cells involved in tumor progression and metastasis are extensively reviewed elsewhere.42 An investigation probing the mechanisms of tumor resistance toward the anti-angiogenic agents, a bone marrow-derived (BMD) CD11b+Gr1+ lineage encountered in anti-VEGF rescue mechanism, has been well documented.43 The CD11b+Gr1+-driven angiogenesis was kind of co-ordinated consequence of a granulocyte–macrophage colony-stimulating factor, SDF-1α, placenta growth factor, granulocyte CSF and the granulocyte CSF-induced Bv8 (Bombina variagata peptide 8)-secreted protein.43 In brief, the CD11b+Gr1+-mediated angiogenesis is partly driven by the Bv8-dependent pathway, which is upregulated by granulocyte CSF that escapes VEGF and renders tumors more resistant to anti-VEGF agents.44Bone marrow-derived cells (BMDCs) constitute a major reservoir of vascular progenitor and vascular modulating cells, which infiltrate into stroma of tumors and have an important role in tumor progression including promotion of tumor angiogenesis independent of VEGF.gression  includes45 TAM-mediated regulation of angiogenesis has been extensively investigated in animal tumor model studies.47 PLGF was overexpressed on anti-VEGFR treatment; PLGF-mediated recruitment of pro-angiogenic and resistance-conferring TAMs might be a plausible mechanism for compensatory angiogenesis and tumor resistance.48 Nevertheless, the treatment of sorafenib in HCC tumors was also associated with elevated levels of CSF-1, SDF-1α and VEGF, which per se are chemokines for inviting macrophages.46 The finding of the HCC tumor model studies pinpoints the involvement of TAMs in tumor angiogenesis and progression under the treatment of sorafenib, which inhibits the VEGFR2, PDGF receptor and Raf kinases.BMD TAMs have been considered as a primary cause of poor prognosis and resistance to anti-VEGF agents.50 The stroma and the invasive front of a tumor are usually occupied by clad of TAFs in different types of cancers such as lung, prostrate, breast, pancreas and colon.49 In particular, TAF-mediated conspiracy of driving angiogenesis under VEGF blockade is considered as a prominent cause of anti-VEGF tumor refractoriness . Crawford et al.16 demonstrated that TAFs derived from resistant tumors sustained tumor growth and angiogenesis even after arresting the functions of VEGF. Further, it was found that TAF generated PDGF-C was a key factor in sustaining angiogenesis and tumor growth under anti-VEGF environment. The involvement of TAF-generated PDGF-C in tumor refractoriness was confirmed by arresting its functions using neutralizing antibody, which subsequently ameliorated the TAF-resistant induced angiogenesis and drastically delayed the growth of resistant tumors.16 di Tomaso et al.52 also demonstrated that the stroma-generated PDGF-C overexpression was associated with resistance to anti-VEGF treatment in glioblastoma tumor model studies.52The current state-of-the-art literature cited elsewhere focus on several dimensions of TAF-mediated cues in tumor invasion, progression, angiogenesis and metastasis.peri: around; cyte: cell) are the contractile perivascular cells that wrap around the newly formed blood capillaries. It is now a well-established fact that pericytes have a significant role in pro-angiogenic signaling, especially in vascular morphogenesis, and have direct regulatory control over EC proliferation or quiescence.53 Under hypoxic environment, the upregulated expression of VEGF-A in mature PCs recruit them to newly formed vessels.5 Pericytes also protect ECs through upregulation of Bcl-w protein, which protects ECs from apoptosis.54. Of note, anti-angiogenic agents not only prune the neovasculature but also significantly induce apoptosis in ECs.54 Autocrine VEGF-A signaling in ECs in association with PCs offers an understanding of how PCs protects ECs under anti-angiogenic environment and recur angiogenesis after the relapse of anti-angiogenic therapy. Upregulation of PDGF-B in tumor cells has profound effect on increasing the PC coverage55 and the reports state that PCs can protect ECs from VEGF withdrawal by activating compensatory pro-angiogenic signaling, especially PDGF receptor-medited angiogenic pathway in anti-VEGF therapy.55 In the current mainstream of tumor resistance, now it has been widely accepted that the anti-angiogenic drugs mainly prune ECs lacking PC coverage, while demonstrating limited efficacy on the PCs embarrassed mature vessels.56 Interestingly, low-to-moderate doses and transient treatment duration of anti-angiogenic agents have been shown to improve the efficacy of anti-angiogenic agents by a process of vessel normalization.3. Although the pro-angiogenic role of PCs is evolving, interestinlgy PCs derived from infantile hemangioma tumor of human demonstrated impressive pro-angiogenic activities and, of note, pro-angiogenic activity of these PCs was many fold higher than the retinal PCs.56Pericytes (57 Cancer stem cells (CSCs) represent a heterogenous subpopulation of cancer cells that retains the potential of self-renewal, which leads to malignant progeny. Nevertheless, CSCs have also been identified as an emerging major driving force that governs tumor recurrence and confer resistance to anti-cancer agents.58 Putative CSCs have been reported in several tumor types including brain, melanoma, colorectal, breast, hepatic, head and neck, and prostate cancer.59 Several dimensions of angiogenic functions of CSCs have been recently reviewed elsewhere.60 The reason for suspecting CSCs in alternate angiogenesis and tumor resistance lies in their capabilities to produce much higher levels of VEGF in both anoxic and hypoxic environment than non-CSC population,60 which perhaps might be boosting strong angiogenic cues after the relapse of VEGF blockade. The report describes that the treatment of sunitinib and bevacizumab increases the number of breast CSCs by upregulation of hypoxia-inducible factor (HIF)-1α and through the activation of Wnt pathway via Akt/β-catenin signaling.61In the mainstream of cancer treatment, tumor heterogeneity has seeded several enquiries and imposed challenges in relation to its causes and consequences, concern with improving the drug efficacy and challenge of ameliorating the emerging drug resistance.62 It has been described that anti-angiogenic agents induce ‘vascular regression,' which leads to increase in intratumor hypoxia and selection of more invasive metastatic clones of the cancer cells that are resistant to therapy including anti-angiogenic agents.63 Nevertheless, targeting hypoxia is being prioritized for overcoming the disappointing performance of anti-angiogenic agents in the clinic.62Tumor angiogenesis and hypoxia have been identified as hallmarks of solid tumors. HIF-1α mediates the transcription of genes involved in angiogenesis, oxygen consumption, migration and invasion of cancer cells.64. The most dramatic effect of hypoxia has been shown in recruiting BMDCs in TME, which later have an important role in compensatory angiogenesis.65 Blocking the functions of HIF impaired the mobilization of BMD pro-angiogenic cells such as VEGFR2+CD34+, VEGFR2+CD117+ and CXCR4+ Sca1+ into the circulation and inhibition of tumor angiogenesis, thereby adversely affecting the tumor growth.66 Knockdown of Phd2 (a negative regulator of HIF-1α) in human colon cancer resulted in increasing the number of pro-angiogenic CD11b+ tumor-associated myeloid cells and promotion of vascularization.67 TAMs have been shown to preferentially accumulate in the hypoxic and necrotic regions in a variety of solid tumor cancers of humans such as breast, endometrium, ovary, bladder, colon and the oral cavity (reviewed in Quail and Joyce42). CSCs preferentially reside in hypoxic niches; moreover, they have upregulated levels of HIFs.68 The hypoxia induced by anti-angiogenic agents also mobilizes and increases the number of CSCs in breast cancer.61 Hu et al.69 have demonstrated that hypoxia-induced autophagy triggers tumor cell survival and adaptive resistance to anti-angiogenic therapy in the glioblastoma. Targeting the hypoxic microenvironment might serve effective anti-angiogenic combination therapies for combating the emerging resistance towards anti-angiogenic therapy.64A vast body of literature describes the role of hypoxia in activating and upregulating the compensatory angiogenic factors/pathways and infiltrating different BMD pro-angiogenic cells including circulating endothelial progenitors and CSCs in tumor micro-environment.70 In vessel cooption, the tumor cells embrace the local blood vessels of the host during the invasion of tumors in surrounding host tissue and migrate along the vessels of the host organs.71 Vessel cooption is more frequently observed in cancers of densely vascularized organs such as the brain, lung and liver, wherein the primary tumor cells including metastases coopt with the adjacent existing quiescent blood vessels of the host tissue.72 Holash et al.70 proved that the implantation of C6 glioma cells in rat brain leads to formation of small vascularized tumors independent of angiogenesis. Kunkel et al.73 demonstrated that the systemic treatment with DC101, monoclonal antibody against VEGFR-2, increased cooption of quiescent cerebral vessels with residual tumors, which was further observed to have central cores of coopted vessels. The treatment of anti-angiogenic ZD6474 agent with brain metastases of cerebral melanoma was found to be associated with marked increase in vessel cooption.72 Interestingly, the coopted vessels respond differentially toward anti-angiogenic agents among different tumors. For example, liver metastases from breast cancer are more depended on cooption of the liver vasculature than that of colorectal origin.74The requirement of classic angiogenesis was previously thought to be a basic prerequisite for tumor progession and metastasis. Perhaps this assumption turned out as an illusion, as now sufficient evidence states that tumors can sustain their growth through various angiogenesis-independent mechanisms such as vessel cooption, vessel remodeling through intussusception, vascular mimicry  and othe75 IMG, which happens to be an adaptive response to stress and hypoxia, is observed in several human tumors such as melanoma, colon, mammary carcinomas, B-cell non-Hodgkin's lymphoma and glioblastoma . IMG has been identified as an important mechanism of rapid vascular remodeling in colon adenocarcinoma xenograft that contributes for intermittent blood flow in tumors. 76 Treatment of mammary carcinoma allograft with VEGF tyrosine kinase inhibitor (PTK787/ZK222854) resulted in the development of extensive IMG in post-relapse period.77 In Lewis lung carcinoma and RIP-Tag 2 model studies, clear evidence of IMG and rapid revascularization was observed in tumors recovered after the treatment with VEGFR inhibitors.78 Perhaps, tumors might prefer IMG during anti-angiogenic exposure, as it is faster, and thermodynamically and metabolically more feasible as compared with sprouting angiogenesis.78The intussusceptive microvascular growth (IMG) also called splitting angiogenesis is a novel mode of vessel generation and vascular remodeling that might be acting as an anti-VEGF rescue meditor. IMG involves formation of two new vessels through fission of the pre-existing capillary plexus without sprouting.et al.79 unraveled a tumor supporting crucial mechanism of generation of endothelium-independent vascular network called vasculogenic mimicry (VM). The authors demonstrated that the aggressive human melanoma cells mimic the functions of ECs and generate vascular tunnel-like phenotype that is foolproof in carrying red blood cells and plasma without involvement of ECs.79 VM has been reported in different human malignant tumours such as breast, melanoma, bladder, kidney, gliomas, glioblastomas, prostrate, ovarian, lung, sarcomas, cell renal cell carcinoma and astrocytoma.80 The imbroglio of VM is an amalgamation of three distinct elements, the primary one is the plasticity of aggressive tumor cells, followed by remodeling of the ECM and finally establishing the connections of the VM channels with the existing network of host microvessel system.81 VM is critically regulated by several signaling pathways associated with embryonic/stem cell , vascular  and hypoxia-related HIF and Twist1 . Clinical evidence of occurrence of VM in patients is strongly correlated with high risk of metastasis, poor prognosis, cancer recurrence and worse survival for patients of variety of cancers.81 Previous report entails the resistance of VM to anti-angiogenic inhibitors such as endostatin and TPN-470 in B16F10 murine melanoma model, as well as in melanoma tumor cells.83 It is interesting to note that hypoxia induces VM and, of note, CSCs, which possess tumor recurrence abilities, have a crucial role in VM.84Maniotis 85 Moreover, the systems pharmacology approaches integrating the biology of anti-VEGF resistance with the prognostic, predictive, pharmacodynamic and surrogate biomarkers coupled with computational imaging techniques such as dynamic contrast-enhanced magnetic resonance imaging and other imaging tools monitoring the vessel permeability, integrity and tumor perfusion might be helpful in ameliorating the drug resistance and optimizing the performance of anti-angiogenic drugs 86.Before we endeavor for designing novel and effective strategies in anti-angiogenic therapy, it is important to prioritize the compensatory angiogenic mechanisms as targets for improving the therapeutic index of anti-angiogenic regime. Agents counteracting the redundant functioning of pro-angiogenic pathways and growth factors, patrolling the conspiracy of BMD tumor and stromal cells including pericytes and CSCs, antagonizing the angiogenesis-independent alternative mechanisms of vascular remodeling and overcoming the rebounds of invasiveness and metastasis after drug holidays should be an integral part of a judiciously formulated combinatorial therapeutic approach of future anti-angiogenic settings.2 However, this turned out to be an illusion with growing resistance toward anti-angiogenic agents. Sizable literature has accumulated in the recent past describing the fact that tumors employ multiple mechanisms of vascularization that compensates the treatment of the currently used anti-angiogenic agents. In the midst of emerging resistance to the currently available anti-angiogenic agents, future settings should capitalize more on developing novel anti-angiogenic agents targeting compensatory angiogenic mechanisms, which may be used in combination with currently available anti-angiogenic agents. In the current situation, combination therapeutic regimes seems to be a possible approach for amelioration of tumor resistance, as several preclinical and clinical studies have demonstrated significant impact of combination therapy in improving the clinical benefits to the patients.Besides the emerging challenges, anti-angiogenic therapy has benefited not only for malignant diseases but also for non-malignant human ailments as well. Before launching of the anti-angiogenic therapy from the cradle to its action port, it was described as a therapy of ‘resistant to resistance,' owing to the genetic stability of ECs, as anti-angigenic drugs mostly target ECs.


Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. Here, we report that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation.In the  Drosophila oogenesis begins with the asymmetric division of the germline stem cells (GSCs) at the anterior tip of the germarium in the ovary bag-of-marbles (bam) in vivo working platforms for addressing how adult stem cell fate and stem cell-derived cell lineage commitment are regulated In the oogenesis, GSC cell fate is maintained by both extrinsic signals from the niche and intrinsic regulatory machineries. Cap cells (CpCs) in the niche produce BMP-like signal molecule Dpp for activating BMP signaling pathway in GSCs. Active BMP signaling maintains GSC fate by repressing differentiation via transcriptional silence of the differentiation promoting gene, Drosophila SWI/SNF chromatin-remodeling complex. There exists two subtypes of the SWI/SNF complexes in Drosophila: the Brahma (Brm)-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex polybromo/bap180, rather than osa, caused a defect in GSC maintenance reminiscent of the brm mutant phenotype. We further showed a genetic interaction of brm with polybromo/bap180 in sustaining the GSC population. Thus, we propose that Brm acts in the form of the PBAP complex to control GSC self-renewal in the Drosophila oogenesis.A growing number of evidences have indicated that GSC fate regulation can also occur at epigenetic level. We and others have identified a number of epigenetic factors involving histone modification or chromatin remodeling as regulators for GSC maintenance and germ cell differentiation Drosophila strains were maintained and crossed at 25°C unless otherwise stated. The following fly stocks were used in this study:All 1118w strain was used as wild type.Canton S (CS) and T362brmK804RUAS-brmΔ86bap1802brm, 308osa, 2osa, hsFlp;FRT80B,ubi-GFP , yw122;FRT82B,ubi-GFP (gift from Zhao-hui Wang).Mutant alleles and transgene: UAS-brm-RNAi-B35211, UAS-brm-RNAi-B34520, UAS-bap180-RNAi-B32840, UAS-bap170-RNAi-B26308 (BDSC), UAS-bap180-RNAi-V108618 . The on-target effects of the above RNAi transgenes were molecularly validated based on the RT-PCR quantative assay , c587-Gal4Gal4/UAS: brm or bap180, hsFLP; FRT80B ubiGFP was crossed to FRT80B, T362FRT80B brm, hsFLP; FRT82B ubiGFP was crossed to FRT82B, Δ86FRT82B bap180. Two-day-old female adult progenies with appropriate genotype  were heat-shocked at 37°C twice a day on three consecutive days for one hour each time. Meanwhile, the progenies from the cross of hsFLP; FRT80B ubiGFP and FRT80B or hsFLP; FRT82B ubiGFP and FRT82B were used as FRT controls respectively. Ovaries were then dissected at day 2, 7,14 and 21 after the last heat-shock treatment for the clonal analysis. The FRT clones were identified by the absence of GFP expression.Mosaic clones were generated by mitotic recombination using FLP/FRT system. To generate GSC or germline cyst clones mutant for tstubP-Gal80 and bab1-Gal4. The females were raised at 18°C until 2 days after eclosion and then shifted to 29°C for a number of days.RNAi-based knockdown experiments were performed by Gal4/UAS binary system Antibody staining was carried out as described previously Confocal images were captured on Leica TCS SP5 laser confocal microscope.GSCs were identified by the presence of a spectrosome anchored to the CpC contact site. The spectrosome-containing single germ cells in the germarium which are located away from terminal filaments and cap cells were classified as UGCs.Student's t test and Mann–Whitney test were chosen to calculate p-values.Drosophila SWI/SNF chromatin-remodeling complexes, regulates stem cell lineage commitment and stem cell proliferation in the adult Drosophila intestine Drosophila SWI/SNF complexes function in regulating the GSC fate in the ovary. For this purpose, we first examined whether Brm, the ATPase subunit of the Drosophila SWI/SNF complexes, has a role in GSC maintenance. The immunofluorescence assay using anti-Brm serum revealed that brm is ubiquitously expressed in almost all cell types in the wild type germaria including GSCs, the niche and follicle cells   or K804RUAS-brm, a dominant negative form of brm transgene under the control of nos-Gal4 driver. As depicted in brm in the germline caused a marked decrease in GSC number per germarium during three weeks after fly eclosion. Combined with the clonal analysis above, the Gal4/UAS binary system-based assay indicates that Brm is required intrinsically for maintaining GSCs.Recent studies have shown that the BAP complex, one of the le cells . To detegermaria . Statiston (ACI) , suggest2osa/308osa can be collected for an assay, we examined if loss of osa function impairs GSC self-renewal by directly counting GSC number at different time points after fly eclosion. Clearly, 2osa/308osa females had a relatively constant number of GSCs per germarium with a 14 day test period, similar to the wild type counterparts   . All dateriments , brm2/+  and osa . Thus, tbrm mutant GSCs (data not shown). Given that the lost brm mutant GSCs are able to develop into differentiated germline cysts  or K804RUAS-brm and bab1-Gal4, a GSC niche-specific driver, and performed a temperature shift assay. In the experiments, the females were raised at 18°C until 2 days after eclosion and then shifted to 29°C for a number of days. Cell number counting at different time points revealed that reduced expression of brm in the niche cells only at adulthood causes a gradual GSC loss  or bap170 (B26308 from the BDSC) in the niche cells elicited a significant decrease in GSC number per germarium in a 14 day time course, albeit reduced GSC number at eclosion was observed in the case of bap170 knock down. These data not only indicate extrinsic roles of polybromo and bap170 in GSC maintenance, but also provide more evidence that the PBAP complex controls GSC self-renewal at multiple levels.To verify if Brm in the niche acts in the form of the PBAP complex to control GSC self-renewal, we next tested polybromo and bap170, another specific subunit of the PBAP complex, for the non-cell autonomous roles in sustaining the GSC population. As depicted in UAS-brm RNAi (B35211 from the BDSC) in the niche cells and then examined the expression levels of pMad in GSCs located in the knockdown germaria. As evident in supplementary brm knockdown impairs BMP signaling output, presumably eliciting a defective GSC maintenance phenotype.CpCs in the niche produce BMP signals for keeping GSCs in an undifferentiated and self-renewing state. To determine whether knocking down the PBAP complex perturbs the niche signaling output, we expressed T362brm allele using the FRT/FLP technique and analyzed how differentiation of GSCs and their derivatives occurs in brm mutant germaria at both molecular and morphological levels. As shown in brm appear normal with respective to the cyst development  was expressed under the control of c587-Gal4, restricting brm down-regulation to ECs and early follicle cells in adults. Same as the control group, reduced expression of brm in the somatic cells did not block GSC differentiation, as indicated by the absence of accumulation of the undifferentiated germ cells (UGCs) (Escort cells (ECs) in the germarium have recently been defined as the germline stem cell differentiation niche. Considering that s (UGCs) . Thus, tDrosophila SWI/SNF complexes, Brm, Mor and Snr1, are required in the germline for oogenesis polybromo/bap180, rather than osa, cause a similar phenotype to that in brm, and brm genetically interacts with bap180 in controlling GSC self-renewal. These genetic data lead us to propose that Brm acts in the form of the PBAP complex to sustain the GSC population. Given that requirements for the core Brm complex in oogenesis are independent of either BAP-specific subunit Osa or PBAP-specific subunits Bap180/Bap170 Drosophila SWI/SNF complex subtype in the specific developmental context.Previous studies have shown that the core subunits of the bam expression. This observation rules out the possibility that the canonical BMP/Bam pathway is implicated in Brm-controlled GSC maintenance. On the contrary, knocking down brm in the niche cells impairs BMP signaling output, probably eliciting a non-cell autonomous defect in GSC maintenance. Emerging evidence indicates that the chromatin-remodeling complexes execute unique developmental roles through interacting with a variety of transcription factors and/or other co-factors such as histone-modifying enzymes in a cell-type specific or developmental-stage specific manner Like other epigenetic factors Figure S1brm knock down in the niche, rather than loss of brm function in GSCs perturbs BMP signaling. (A–D) Germaria with the control (A) or T362brm homozygous (B) GSC clone (broken circles) labeled by the absence of the nuclear GFP, or expressing bab1-gal4 alone (C) or brm-RNAi with bab1-gal4 (D), stained for pMad  or pMad and α-spectrin . Clearly, high levels of pMad expression are evident in both marked wild type control and brm mutant GSC (broken circles in A and B). By contrast, pMad expression in GSCs is remarkably reduced in the brm knock down germarium (arrowhead in D), compared with that in the control (arrow in C).(TIF)Click here for additional data file.Figure S2Molecular validation of on-targeting effects of the RNAi transgenic strains.  RT-PCR analysis shows that actin-gal4 or hs-gal4 induced expression of the UAS-brm-RNAi (A) or UAS-bap180-RNAi (B) transgene in the 3rd instar larvae leads to a reduction in the expression of endogenous brm (A) or bap180 (B) at mRNA levels. The presented gels are representative of three independent experiments.(TIF)Click here for additional data file.


Surfaces of medical implants can be enhanced with the favorable properties of titanium-nitride (TiN). In a review of English medical literature, the effects of TiN-coating on orthopaedic implant material in preclinical studies were identified and the influence of these effects on the clinical outcome of TiN-coated orthopaedic implants was explored. The TiN-coating has a positive effect on the biocompatibility and tribological properties of implant surfaces; however, there are several reports of third body wear due to delamination, increased ultrahigh molecular weight polyethylene wear, and cohesive failure of the TiN-coating. This might be due to the coating process. The TiN-coating process should be optimized and standardized for titanium alloy articulating surfaces. The clinical benefit of TiN-coating of CoCrMo knee implant surfaces should be further investigated. The purThis study focuseson preclinical and clinical studies using TiN-coated orthopaedic implants and implant material. Inclusion criteria were clinical studies on TiN-coated orthopaedic implants and preclinical studies on wear and biocompatibility of TiN-coated implant material. To identify all relevant studies on TiN-coated orthopaedic implants and implant material in English scientific literature, the following databases were searched: Medline (1947 to January 2015), Embase Classic and Embase (1947 to January 2015). No restrictions were made to the type of studies. Papers outside the English language, abstracts from scientific meetings, and unpublished reports were excluded. References of retrieved publications were used to add studies meeting the inclusion criteria that were missed by the electronic search. The Medline search is defined in Appendix. The query was checked for inconsistencies using PubMed Query Editor 0.1. Selection of studies was first performed by screening titles and abstracts. In case of insufficient information in the title or abstract, full text copies were retrieved to make a decision for the article selection. References of retrieved publications were used to add studies meeting the inclusion criteria that were missed by the electronic search.n = 32), research in medical fields other than orthopaedic , or other reasons (n = 157). No additional studies were identified from reference lists of the identified articles.The results of the combined Medline and Embase search are shown in μm3 per cell [A high variety of cell types were used for cell culture on TiN-coated implant materials: human bone marrow stem cells –17, humaper cell .2-coated cp titanium [TiN-coated materials were implanted in the femurs of dogs , rats 33, and ratitanium .P < 0.05) over 24 hours of bacteria cultures S. pyogenes and S. sanquinis on TiN-coated titanium plasma sprayed surfaces (TPS) compared with uncoated TPS [Results of three independent experiments showed a lower adhesion and proliferation  with a 26-month follow-up [Clinical studies on survival of TiN-coated orthopaedic implants are presented in ollow-up .In a postmortem retrieval analysis of a cementless TiN-coated Ti6Al4V THA one year after index surgery, the TiN-coated Ti6A14V femoral head showed circular voids without TiN-coating and voids filled with circular droplets of pure titanium . AdheredFailures of a cementless TiN-coated titanium alloy-on-UHMWPE resurfacing THA were suggested to be due to the use of conventional UHMWPE instead of highly cross-linked UHMWPE . In a caIn the radiological follow-up  of 330 hips with a press-fit polyethylene cup with TiN-coated stainless steel mesh  for the bone-implant interface, there was one case showing a radiolucent line around the cup; however, this was without clinical problems . There wIn a retrospective study of mainly cementless TiN-coated CoCrMo mobile bearing TKAs, revision surgery was performed in 4.9% . Prosthen = 51) and a CoCrMo TKA (n = 50) [In a recently published RCT, no differences in postoperative pain, KSS, revision surgery, knee flexion and knee flexion contracture, knee circumference, and knee skin temperature were observed between the TiN-coated CoCrMo TKA ((n = 50) . In both(n = 50) .In preclinical studies, TiN-coating of implant materials showed to be biocompatible with mainly favorable tribological properties. Several cohort studies of TiN-coated implants showed an overall survival exceeding 90% with a follow-up of 15 to 77 months and good clinical results , 62–64. In a pins-and-plate test of polished PVD TiN-coated CoCrMo a higher surface roughness, catastrophic cohesive failure within the layers of the TiN-coating and a fourfold increase in wear was found . Higher A high incidence of aseptic loosening was found with a new design of cemented THA using CoCrMo and TiN-coated titanium heads on UHMWPE liner . There wProsthetic fractures of the medial flange of size 5 right-sided TiN-coated CoCrMo femoral component of a TKA were found , 59. It Noteworthy is that of the 7 clinical studies, case reports not included, 4 studies , 62–64 oTitanium alloys used for articulating surfaces require surface treatment to increase hardness and reduce wear. TiN-coating has a favorable effect on the biocompatibility and tribological properties of implant surfaces. However, there are reports of third body wear due to delamination of the PVD TiN-coating on Ti6Al4V, increased UHMWPE wear and cohesive failure of the PVD TiN-coating on CoCrMo of hip implants in preclinical studies, and TiN-coating breakthrough and fretting in a retrieval study of TiN-coated Ti6Al4V femoral heads. These adverse effects might be related to the various coating processes of titanium alloys. The TiN-coating process of titanium alloy articulating surfaces should be optimized and standardized. There were no reports of adverse effects related to TiN-coating of CoCrMo knee implants. Clinical benefit of TiN-coating of CoCrMo knee implant articulating surfaces should be investigated further.


In addition, the authors used XAV939 – an inhibitor of the Wnt–β-catenin signalling pathway, which is involved in neural development – and found that XAV939-nano-stroma promoted the survival and differentiation of human fetal DA precursors. This study shows that stromal nanoparticles can be used to successfully deliver pro-survival cargos to single cells. Such an approach could improve the efficacy of cell-based transplantation therapies for human neurodegenerative diseases, including PD. 1193Page Cell-replacement approaches represent a promising strategy to substitute degenerating dopaminergic (DA) neurons in Parkinson’s disease (PD). However, long-term survival of transplanted cells is still limited by the lack of both adequate growth factor supply and stromal support. To overcome this, Su Metcalfe and colleagues developed a surrogate nano-stroma using biodegradable nanoparticles, which were preloaded with leukaemia inhibitory factor (LIF), a proneural and reparative factor. When targeted to human fetal DA neurons, LIF-nano-stroma improved the survival of these cells both


Genotype-phenotype correlations in patients with small supernumerary marker chromosomes (sSMC) are still difficult to asses.http://www.fish.uniklinikum-jena.de/sSMC.html.The presently known influence of chromosomal imbalance induced by sSMC size and origin, mosaicism of sSMC in different cells of the body and uniparental disomy (UPD) of sSMC’s sister chromosomes on the clinical outcome is summarized according to data on ~5,000 sSMC cases summarized on Two third of sSMC carriers are clinically normal. In the remainder 1/3 of sSMC patients, clinical symptoms may vary between slightly up to severely affected, including intrauterine death. Besides the known sSMC related syndromes Pallister-Killian-, isochromosome-15q12-, isochromosome-18p-, cat-eye- and Emanuel-syndrome there are numerous other yet unnamed and unidentified “sSMC-syndromes”. Recently, derivative-8- and derivative-13/21 syndromes in complex sSMC were reported.The influence of chromosomal imbalance induced by sSMC size and its origin seems to have the largest impact on the phenotype of sSMC-patients. Besides UPD of sSMC’s sister chromosomes and mosaicism of sSMC may be important for the clinical outcome. The latter is especially important to be predicted in prenatal cases.


Crescentic glomerulonephritis (GN) is the most severe form of GN and is associated with significant morbidity and mortality despite aggressive immunotherapy with steroids, cytotoxic drugs, and plasmapheresis. We examined the therapeutic efficacy of the green tea polyphenol (−)-epigallocatechin-3-gallate , a potent anti-inflammatory and anti-oxidant agent, on experimental crescentic GN induced in 129/svJ mice by administration of rabbit anti-mouse glomerular basement membrane sera. Routine histology and key molecules involved in inflammatory and redox signaling were studied. EGCG treatment significantly reduced mortality, decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. The improvements in renal function and histology were accompanied by the restoration of Nrf2 signaling (which was impaired in vehicle-treated mice) as shown by increased nuclear translocation of Nrf2 and cytoplasmic glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione peroxidase. EGCG-treated mice also showed reduction in p-Akt, p-JNK, p-ERK1/2 and p-P38 as well as restoration of PPARγ and SIRT1 levels. Lower dose of EGCG (25 mg/kg BW/day x2 weeks) treatment also significantly decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. Thus, our data illustrate the efficacy of EGCG in reversing the progression of crescentic GN in mice by targeting multiple signaling and inflammatory pathways as well as countering oxidative stress. Crescentic glomerulonephritis (GN) includes a variety of conditions characterized by glomerular fibrinoid necrosis and accumulation of cells in Bowman’s space. It can be classified into three categories: pauci-immune, immune complex-mediated, and anti-glomerular basement membrane (GBM) antibody-induced crescentic GN (anti-GBM-GN) ,2. Anti-Oxidative stress and inflammation play major roles in the pathogenesis and progression of acute and chronic kidney diseases. Overproduction of reactive oxygen species (ROS), reactive nitrogen species, and reactive chlorine species by inflammatory cells can cause tissue damage, intensify inflammation, promote apoptosis, and accelerate progression of many diseases including anti-GBM-GN . NuclearThe green tea catechins, particularly (-)-epigallocatechin-3-gallate (EGCG), are potent anti-inflammatory and anti-oxidant agents shown to inhibit leukocyte chemotaxis, quench free radicals, chelate transition metals, and interrupt lipid peroxidation chain reaction . It has All studies were reviewed and approved by the institutional review committee at UT Southwestern. Anti-GBM serum was generated by Lampire Laboratories  , 19. PreTo explore the therapeutic effect of EGCG (Sigma-Aldrich), the anti-GBM antibody-injected mice were allowed to develop full-blown nephritis for 7 days before intervention –19. On dAll urine samples were centrifuged at 14,000 rpm for 5 min. Clear supernatant was used for urine protein assay . The serum creatinine levels were measured as previously described .Mouse antibodies to rabbit immunoglobulin were assayed by ELISA as described previously . BrieflyThree-micrometer sections of formalin-fixed and paraffin-embedded (FFPE) kidney tissues were cut and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) reaction. These sections were examined in a blinded fashion. Glomerulonephritis was graded on 0–4 scale and tubulointerstitial injury was graded on a 0–5 scale, as detailed previously , 19, 22.Lipoperoxides in serum, urine, and renal tissue were determined by measurement of malondialdehyde-thiobarbituric acid with a TBARS assay kit , according to the manufacturer’s protocol. Total glutathione (GSH) content of the renal tissue was determined using a Cayman GSH assay kit . The assay uses a carefully optimized enzymatic recycling method using GSH reductase whereby the sulfhydryl group of GSH reacts with 5,5′-dithio-bis-2-nitrobenzoic acid and Ellman's reagent producing a yellow colored 5-thio-2-nitrobenzoic acid (TNB). The mixed disulfide, GSTNB (between GSH and TNB), which is concomitantly produced, is reduced by GSH reductase to recycle GSH and produce more TNB. The rate of TNB production is directly proportional to this recycling reaction, which is in turn directly proportional to the concentration of GSH in the sample. Thus, measurement of TNB at 405 or 412 nm provides an accurate estimate of GSH present in the sample. It should be noted that oxidized GSH is converted to GSH by GSH reductase in this system, which consequently measures total GSH.http://rsb.info.nih.gov/nih-image/).The renal tissue was homogenized and total protein was prepared using RIPA Lysis and Extraction Buffer . The nuclear extract was prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Scientific). Protein content was quantified using BCA Protein Assay Reagent (Thermo Scientific). For measurement of protein expression, western blotting was carried out as previously described , 23. BriTo examine the effects of low dose EGCG on renal function and pathology, subgroups of anti-GBM antibody-injected mice were allowed to develop full-blown nephritis for 7 days as described previously. On day 7 following anti-GBM antibody administration, the mice were randomized into either EGCG- or vehicle-treated group. The former group  received 25 mg/kg BW/day of EGCG orally (gavage) for 2 weeks (day 7 to day 21) until sacrifice on day 22. Normal saline was given to the vehicle group  with the same protocol. A group of mice  without anti-GBM antibody or EGCG administration was included as normal control. Sera and 24-hour urine samples (at baseline and on day 21) were collected from all mice using metabolic cages with free access to drinking water. All animals were sacrificed on day 22. Renal function and histopathology were evaluated as described previously.Data are presented as mean ± SEM. Analysis of variance (ANOVA), Fisher exact test, and Student’s t-test were used in statistical evaluation of the data as appropriate. P-values less than or equal to 0.05 were considered significant.At the end of week 1 following anti-GBM serum administration, the mice exhibited renal insufficiency and proteinuria. At the end of the 4-week observation period, the vehicle-treated group suffered a mortality rate of 27% compared with 11% in the EGCG-treated group. The vehicle-treated mice with anti-GBM-GN developed renal insufficiency and proteinuria. The EGCG-treated mice showed significantly less proteinuria and lower serum creatinine than the vehicle-treated mice . These fThe vehicle-treated mice showed moderate to severe renal injury characterized by crescent formation with significant intracapillary hypercellularity, obliterated capillary lumens, and thickened capillary walls. Tubular atrophy and dilation with hyaline casts and interstitial fibrosis were also noted. In comparison, the EGCG-treated mice exhibited milder renal injury with only occasional crescent formation and focal tubulointerstitial injury Figs.  and 2. NWe asked if the reduced renal disease in EGCG treated mice may be due to a decreased xenogenic immune response to the injected rabbit Ig. To answer this question, we compared the levels of IgG mouse anti-rabbit antibodies on day 28 using ELISA. As depicted in Renal tissue and urine malondialdehyde (MDA) levels were modestly higher in the vehicle-treated mice than the normal control group. EGCG treatment normalized the MDA levels in both renal tissue and urine. Serum MDA levels were not significantly different among the three groups . Renal tCompared with the normal control group, the vehicle-treated anti-GBM-GN mice showed a significant reduction in nuclear Nrf2 abundance, pointing to impaired Nrf2 activation. This was associated with significant reduction of GCLc, GCLm, and GPx-1 that are important products of Nrf2-regulated genes . SurprisThe vehicle-treated anti-GBM-GN mice exhibited activation of the PI3k/Akt and MAPK signaling pathways in the kidneys as evidenced by significant increase in the p-Akt, p-JNK, p-ERK1/2, and p-P38 levels . These pCompared with the normal control group, the vehicle treated anti-GBM-GN mice showed significant reduction of PPARγ and SIRT1 in the kidney tissue. EGCG administration reversed these changes . Thus, bAt the end of the 3-week observation period (2 weeks post EGCG therapy), two mice were dead in the vehicle-treated group compared with 1 fatality in the EGCG-treated group. The vehicle-treated mice with anti-GBM-GN developed marked renal insufficiency and proteinuria. The EGCG-treated mice showed significantly less proteinuria and lower serum creatinine than the vehicle-treated mice . In addiAs expected, the untreated mice with anti-GBM-GN exhibited severe renal injury and dysfunction. Treatment with EGCG in mice with established anti-GBM-GN resulted in a dramatic and unequivocal improvement in the biochemical and histological abnormalities as evidenced by significant reductions in proteinuria and serum creatinine, and improvement in histological findings including decreased glomerular crescents, less tubulointerstitial injury, and reduced glomerular and interstitial inflammatory cells. The observed amelioration of renal disease in EGCG-treated mice was not due to a reduction in immune response to the administered rabbit immunoglobulin because the levels of IgG mouse anti-rabbit antibodies were comparable between the vehicle- and EGCG-treated mice with anti-GBM-GN. In agreement with this finding, a recent study showed that EGCG did not change the titers of anti-dsDNA or the glomerular IgG deposition in experimental lupus nephritis even though the renal protective effect is striking .2O2, upregulation of myeloperoxidase and NADPH oxidase, downregulation of catalase and glutathione peroxidase, increased levels of nitrotyrosine, activation of NFkB, upregulation of inducible nitric oxide synthase and osteopontin expression, and decreased PPARγ expression [We have previously shown that glomerular disease in the mouse model of anti-GBM-GN is accompanied by oxidative damage as shown by increased levels of malondialdehyde and Hpression . We furtpression . The resThe current study, for the first time, demonstrates that the glomerular disease in a mouse model of anti-GBM-GN is associated with impaired Nrf2 activation (reduced nuclear content) along with low levels of GCLc, GCLm, and GPx1. This was accompanied by increased HO1 and NQO1 abundance in the renal tissue, reflecting Nrf2-independent regulation of HO1 and NOQ1 and involvement of other transcriptional regulators in response to inflammatory and oxidative injury as shown by other investigators –26. Trea+-dependent enzymes with deacetylase and/or mono-ADP-ribosyltransferase activity. They regulate DNA repair and recombination, chromosomal stability, gene transcription, and mediate the health-promoting effects of caloric restriction including the retardation of aging. Activation of sirtuins may increase resistance to metabolic, oxidative, and hypoxic stress in different tissues. Of the seven Sir2 homologs in mammals, the most extensively studied is SIRT1, which is cytoprotective in the kidney and participates in the regulation of blood pressure and sodium balance [Silent information regulator 2 (Sir2) proteins (Sirtuins) belong to an evolutionary conserved family of NAD balance . The antPPARγ belongs to a family of nuclear hormone receptors with protective role in kidney diseases . PPARγ a2O2 production and inhibit UVB-induced phosphorylation of ERK1/2, JNK, and P38 in cultured human epidermal keratinocytes [Multiple signaling pathways (such as AKT and MAPK/ERK) become activated in various renal diseases and following a wide variety of triggers including oxidative stress and inflammation , 38. EGCinocytes . Therefoinocytes , which iinocytes . Taken thttp://www.ars.usda.gov/nutrientdata). Thus, 10 cups of tea per day would provide sufficient EGCG dosage for a 70 Kg person.The EGCG dosage used in our study is relatively low (50 and 25 mg/kg BW/day) compared with other studies where the daily dosage varied from 50–120 mg/kg BW , 16, 20.In summary, our data suggest that EGCG ameliorates biochemical and histologic abnormalities in mice with crescentic GN. The salutary effects of EGCG are likely mediated by its anti-inflammatory and anti-oxidative properties with multiple targets including activating Nrf2-ARE and PPARr pathways and damping AKT/ERK/NF-kB pathways to reduce oxidative stress and inflammation as well as activating SIRT1 to facilitate tissue repair and regeneration. Clinical trials are needed to determine the potential utility of EGCG as a therapeutic agent for the treatment of immune-mediated glomerulonephritides and other immune-mediated diseases in humans.


It is well known that pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, which can be explained by both an inherently aggressive biology and a lack of effective chemotherapeutics. As a result, there is strong rationale for the development of effective preventive interventions in high risk populations. Chronic pancreatitis increases the risk of developing PDAC more than fifteen fold. Using selective activation of oncogenic Kras specifically in the murine pancreatic epithelium, multiple groups have shown that murine chronic pancreatitis, induced by cerulein, accelerates the development of pancreatic intraepithelial neoplasia (PanIN) and PDAC. An improved understanding of the cellular and soluble mediators responsible for this effect would have a significant impact on the development of preventive strategies for this disease.H17) and IL-17 producing-gamma-delta T cells are increased in the dense fibro-inflammatory stroma surrounding Kras-induced murine PanINs. The abundance of these IL-17 producing cells is also synergistically augmented in the context of associated chronic pancreatitis [H17 lineage, have been detected infiltrating human PanINs, emphasizing the translational relevance of the findings [Our group has recently reported that IL-17-producing-CD4+T cells (TH17) play an active role in chronic inflammation but their role in tumorigenesis remains controversial. On one hand, IL-17 has been associated with migration, invasion, recruitment of myeloid derived suppressor cells and the induction of angiogenic and anti-apoptotic factors. On the other hand, TH17 cells have been reported to mediate anti-tumor effects by promoting CTLs activities, MHC antigen expression, and production of IFN-γ. However, most of the anti-tumor effects reported have been described in the setting of advanced cancer.IL-17-producing T Helper cells  Consortium summit recommended that pancreatic cancer screening programs should be implemented on well-defined high risk groups, with the goal of detecting and treating resectable T1N0M0 PDAC and high-grade dysplastic precursor lesions. Recommended initial screening modalities include endoscopic ultrasonography (EUS) and/or MRI/magnetic resonance cholangiopancreatography. We would speculate that immune-prevention could be ideally implemented on patients enrolled into these screening programs.A recent large case-control study showed that a daily aspirin regimen may reduce the risk of developing pancreatic cancer in the general population, highlighting the importance of inflammation in pancreatic tumor initiation and progression. However, we would anticipate that targeted immune-preventive interventions may have superior efficacy in high risk patients. A variety of antibodies targeting the IL-17 signaling pathway have been under development for autoimmune diseases during the past decade. Ixekizumab and secukinumab, monoclonal antibodies against IL-17, and brodalumab, a human monoclonal antibody directed against the IL-17 receptor (IL-17RA), have shown efficacy in randomized phase II trials for psoriasis and are currently being tested in phase III clinical trials. In 2013, the FDA has approved the use of ustekinumab, an antibody that blocks the p40 common subunit of IL-12 and IL-23, for use in moderate-to-severe psoriatic arthritis and Crohn’s disease. Finally, monoclonal antibodies that specifically block the p19 subunit of IL-23 (not shared by IL-12), are currently under development and expected to have similar efficacy to IL-12 with even less adverse effects.Based on preclinical data documenting a requisite role for IL-17 in PanIN progression, specific targeting of the IL-17 pathway with monoclonal antibodies might represent an effective strategy for pancreatic cancer prevention. Before moving forward with such studies, however, additional work is required to better determine the influence of IL-17 on already established pancreatic cancers, as well as the role that IL-17 might play in in the context of immunotherapeutic interventions. By determining how IL-17 influences the entire spectrum of pancreatic cancer initiation and progression, the future of IL-17 targeting as a means of pancreatic cancer chemoprevention will become clear.


In vivo tau imaging allows the assessment of the regional distribution of tau deposits in a single human subject over time for determining the pathophysiology of tau accumulation in aging and neurodegenerative conditions as well as for application in drug discovery of anti-dementia drugs as surrogate markers. However, tau deposits show complicated characteristics because of different isoform composition, histopathology, and ultrastructure in various neurodegenerative conditions. In addition, since tau radiopharmaceuticals possess different chemotype classes, they may show different binding characteristics with heterogeneous tau deposits. In this review, we describe the characteristics of tau deposits and their ligands that have β-sheet binding properties, and the status of tau imaging in clinical studies.Tau deposition is one of the neuropathological hallmarks in Alzheimer’s disease as well as in other neurodegenerative disorders called tauopathies. Recent efforts to develop selective tau radiopharmaceuticals have allowed the visualization of tau deposits  Tau is a microtubule-associated protein that physically stabilizes microtubule assembly in axons, and pathologically, forms hyperphosphorylated aggregates in the brain in Alzheimer’s disease (AD) and tauopathies. AD is an irreversible and progressive neurodegenerative disease, clinically characterized by cognitive decline, and is the most common cause of dementia in the world. Tauopathies including some variants of frontotemporal lobar degeneration, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), tangle predominant senile dementia (TPSD), argyrophilic grain disease (AGD), and chronic traumatic encephalopathy (CTE) are also neurodegenerative conditions characterized by the accumulation of tau protein in the brain. CTE is associated with repetitive traumatic brain injury and prevalent among contact sports athletes such as American football players and military personnel victims to blasts in the battlefield . These n11C-PBB3, 18F-AV1451 (as known as T807), and 18F-THK arylquinoline series, for imaging tau deposits were evaluated in humans  provides regional and pathophysiological information non-invasively in living human subjects. Use of tau selective radiotracers will enable the noninvasive monitoring of tau pathology in patients with tauopathies, providing better understanding of tau aggregation in the brain. Tau PET imaging would help in the development of anti-tau therapeutics by assisting subject enrollment and evaluation of drug efficacy in clinical trials. Recently, several PET radiotracers with different chemotype classes, such as n humans . The demTau protein plays a role in cytoskeletal support and axonal transport by stabilizing microtubules, which is regulated by the phosphorylation of tau protein . NeuropaSix isoforms of tau protein are produced by alternative splicing of the tau gene and categorized based on the number of microtubule-binding domains into two functionally different groups; three repeat (3R) or four repeat (4R) ,25. Accocis-trans conformational isomerization of phosphorylated threonine 231-proline of tau protein plays a role in the accumulation of pathogenic tau in AD imidazopyrimidine derivative, otracers ,72. As ootracers ,72. Furt lesions . However18F-THK523 (18F-BF242) was developed as the first 18F-labeled arylquinoline derivative [In vitro autoradiography of AD brain sections demonstrates a laminar distribution of 18F-THK523 in the neocortex, which is consistent with Gallyas silver staining, but not with amyloid-β immunostaining or 11C-PiB autoradiography [18F-THK523 autoradiography shows a striking similarity to Gallyas silver-positive, but AT8-negative clusters of neurofibrillary tangles in the pre-α of the entorhinal cortex, reflecting late ghost tangles [18F-THK523 recognizes argyrophilic fibrillary tau filaments. On the other hand, THK523 failed to stain non-AD tau lesions such as Pick’s bodies and globose tangles in Pick’s disease, CBD, and PSP [Arylquinoline derivatives, BF158 and BF170, were initially identified as leading compounds of tau PET probe nearly a decade ago ,75. Thesrivative . In vitriography ,76. Inte tangles . These o and PSP .18F-labeled radiotracers, 18F-THK5105, 18F-THK5117, and 18F-THK5351. These compounds possess higher binding affinities for tau aggregates in AD brains than 18F-THK523, and preferable pharmacokinetics without defluorination in vivo [18F-THK523, these new tracers also demonstrate high selectivity for tau pathology in AD brains [18F-THK5117 preferentially accumulates in the CA1 of the hippocampus containing high density of late ghost tangles in AD brain sections. Microautoradiography using 3H-labeled compounds demonstrates selective binding ability of THK5117 to neurofibrillary pathology  showed elevated 11C-PBB3 retention in the basal ganglia, and the spatial pattern of PBB3 retention was consistent with that of brain atrophy [11C-PBB3 can cross the blood–brain barrier; therefore, some special technique is required for the quantitative analysis of PET data [ atrophy . OngoingPET data ,97,98.18F-AV1451 in regions known to contain high density of tau deposits in patients with AD with low white matter retention. A strong association was observed between the amount of radiotracer and the severity of dementia [18F-AV1451 is ongoing. A case report on posterior cortical atrophy  demonstrated that 18F-AV1451 binding in the posterior brain regions correspond to reduced 18F-FDG uptake and clinical symptom [18F-T807 in frequent areas of tau aggregates in those conditions [in vivo findings have not yet been fully validated by imaging-autopsy studies [Initial human PET studies successfully demonstrated high retention of dementia ,100. A l symptom . Case renditions ,103,104. studies .18F-THK523 imaging failed in visualizing the tau deposits clearly in patients with AD [18F-THK5105 and 18F-THK5117 demonstrated a robust difference between patients with AD and healthy control subjects in brain regions known to contain high density of tau deposits (18F-THK5117 showed better pharmacokinetics and higher signal-to-background ratio than 18F-THK5105 [11C-PiB in the same population. 18F-THK5117 positive voxels were closer to the white matter than 11C-PiB positive voxels, which may reflect the binding of 18F-THK5117 to tau deposits in layer V of the neocortex. Recent longitudinal studies of 18F-THK5117 demonstrated that the annual changes in tracer binding were significantly increased in the temporal cortex of patients with AD, which was closely associated with the annual changes in cognitive decline [18F-THK5351 PET studies demonstrated greater specific signals in the frequent sites of tau deposition and lower non-specific binding in the white matter than 18F-THK5117 [Although  with AD , 18F-THKdeposits . These t-THK5105 . The reg decline . Recent -THK5117 .in vitro binding characteristics of reported tau PET radiotracers, and status of clinical PET imaging (This paper described  imaging . Tau PET