The application of VEGF signaling inhibitors have been associated with more invasive or metastatic behavior of cancers including hepatocellular carcinoma (HCC). We explored the contribution of MET pathway to the enhanced HCC invasion and metastasis by VEGF signaling inhibition, and investigated the antitumor effects of NZ001, a novel dual inhibitor of MET and VEGFR2, in HCC.Immunocompetent orthotopic mice model of hepal-6 was established to investigate the effects of either VEGF antibody alone or in combination with the selective MET inhibitor on tumor aggressiveness. The antitumor effects of NZ001 were examined in cultured HCC cells as well as in vivo models. MET gene amplification was determined by SNP 6.0 assay. MET/P-MET expression was detected by IHC.Selective VEGF signaling inhibition by VEGF antibody significantly reduced in vivo tumor growth of the orthotopic mice models, simultaneously also enhanced tumor invasion and metastasis, but inhibiting MET signaling attenuated this side-effect. Further study revealed that hypoxia caused by VEGF signaling inhibition induced HIF-1α nuclear accumulation, subsequently leading to elevated total-MET expression, and synergized with HGF in inducing invasion. NZ001, a novel dual inhibitor of MET and VEGFR2, markedly inhibited both tumor growth and metastasis of HCC, which showed obvious advantages over sorafenib in not inducing more invasive and metastatic behaviors. This effect is more pronounced in HCC with MET amplification and overexpression.The activation of MET is responsible for the metastasis-promoting effects induced by VEGF inhibition. MET and VEGFR2 dual blockade, NZ001, has advantages over sorafenib in not inducing more invasive and metastatic behaviors; MET amplification and overexpression can be used to identify the subgroup of patients most likely to get the optimal benefit from NZ001 treatment.The online version of this article (10.1186/s13046-018-0750-2) contains supplementary material, which is available to authorized users. Hepatocellular carcinoma (HCC) is the sixth most common cancer and one of the leading causes of cancer-related deaths in the world . HCC is MET, a transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF), has been observed to contribute to cancer metastasis, resistance to chemotherapeutic agents, and dismal outcomes of patients with solid cancers including HCC –10. WithIn the present study, first we identified the contribution of MET signaling induced by inhibition of VEGF signaling to promote malignancy of HCC in preclinical models. Then we tested if NZ001, a novel ATP-competitive multi-targeted kinase inhibitor that simultaneously inhibits both MET and VEGFR2, could suppress both tumor growth and metastasis. Finally, we found MET amplification and overexpression were useful in subgrouping the HCC patients that might get the optimal benefit from MET inhibitor treatment.For in vitro assays, NZ001 was obtained from Nanjing Zhongrunyuan Pharmaceutical Company . XL184, sorafenib and PF-04217903 were purchased from Selleck Chemicals . Goat anti-mouse VEGF antibody (AF-493-NA) was purchased from R&D Systems . NZ001, XL184 and PF-04217903 were prepared as a 20-mM stock solution in DMSO  for in vitro studies. For in vivo studies, NZ001 was formulated in sterile ddH2O and administered via oral gavage at 10 mg/kg or 30 mg/kg. Sorafenib was dissolved in Cremophor EL/ethanol  at 4-fold (4×) the highest dose. The final dosing solutions were prepared on the day of use by diluting to 1× with ddH2O and were administered via oral gavage at 30 mg/kg. VEGF antibody was injected intraperitoneally 3 times per week at 7.5 mg/kg. Recombinant human HGF, mouse HGF and human VEGF were obtained from R&D Systems . All information of primary antibodies used for Western blot and immunoprecipitation were shown on Additional file For prognostic analysis, frozen tumor and peritumor tissues were obtained from 109 patients who underwent hepatectomy for HCC at the authors’ institute between January 2005 and December 2006. For the evaluation of MET expression and microscopic vascular invasion(MVI) in HCC patients, formalin fixed and paraffin embedded tissue samples were collected from 122 patients who received the curative liver resection for HCC at the authors’ institute between September 2014 to December 2016. The entire area of the cut surface containing the greatest tumor dimensions and noncancerous liver tissue was submitted for the histologic examination. The clinicopathological characteristics of these patients were presented in Additional file To evaluate the MVI in HCC patients, the specimens were stained with hematoxylin and eosin. Microscopic vascular invasion(MVI) was defined as the presence of clusters of cancer cells floating in the portal vein, hepatic vein, or bile duct of the tumor and surrounding noncancerous tissues that were visible only on microscopy. All of the measurements were performed by two pathologists at Huashan Hospital, Fudan University  with more than 10 years of experience in hepatic pathology.Cell lines were described in the Additional file Western blot was performed as previously described . BrieflyEnzyme-Linked Immunosorbent Assay (ELISA) and immunofluorescence analysis (IF) were described in the Additional file RNA isolation and real-time quantitative reverse-transcription PCR were described in the Additional file Short interfering RNA (siRNA) sequences specifically targeting human HIF-1α (5’-GGGUAAAGAACAAAACACA-3′) and mouse HIF-1α (5’-CCCATTCCTCATCCGTCAAAT-3′) were purchased from shanghai GenePham . Cells were transfected with either target-specific siRNA or a scramble control siRNA using Lipofectamine RNAi MAX reagent  according to the manufacture’ instructions.DNA mutation analysis was described in the Additional file MET copy number variation analysis of 12 HCC cell lines and 16 Chinese patient-derived HCC cells were produced using the Affymetrix Genome-Wide Human SNP Array 6.0. The MET copy number variation analysis of mouse cell lines were produced using Affymetrix Mouse Diversity Genotyping Array. All raw data were processed with the PICNIC software program and are presented as the number of MET copies.Cell proliferation, colony-formation assays and capillary tube formation analysis were described in the Additional file Cell invasion assay and wound-healing assays were performed as previously described . The detThe detail of evaluation of immunohistochemical analysis and diagnostic scoring system were described in the Additional file 3) was calculated by the following formula: ab2/2 . Tumor growth inhibition (TGI%) [The detail of evaluation of in vivo tumor growth and metastasis in mice models of HCC was described in the Additional file n (TGI%)  was calct test was used to compare data between 2 groups. Categorical data were analyzed by the chi-square test or Fisher exact test. OS and cumulative recurrence rates were calculated by the Kaplan–Meier method and differences were analyzed by the log-rank test. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. A p-value < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS 15.0 for Windows . Quantitative data between groups were compared using the one-way ANOVA; Student In hepa1-6 orthotopic model, treatment with VEGF antibody significantly inhibited tumor growth. The sectional areas of tumors were 58.9% less in VEGF antibody treated group compared with control group. Though the growth was inhibited, tumors treated with VEGF antibody appeared to be more invasive as indicated by the irregularity of tumor border. The intrahepatic metastases (IHM) indicated by multiple tumor foci in liver were also significantly increased after VEGF antibody treatment Fig. . FurtherEmerging evidences suggest that the promoting effect on cancer metastasis caused by angiogenesis inhibition is due to the hypoxia , and tumTo further determine whether hypoxia had a direct effect on MET activation, we exposed HCC cell lines to hypoxic condition. We found that hypoxia induced HIF-1α nuclear accumulation and subsequently increased the total-MET level, but had no significant effect on MET tyrosine phosphorylation Fig. , b. As sTo determine the clinical significance of MET overexpression, we correlated the MET mRNA expression with the prognosis of HCC patients. We observed that High-MET expression group (gene expression level of tumor tissue/adjacent peritumor tissue≥2) had significantly worse outcomes than those with Low-MET group Fig. . UnivariBased on the above observation, we next investigated whether concurrent inhibition of MET signaling was sufficient to attenuate the promotion of HCC invasion and metastasis induced by VEGF inhibition. We applied PF-04217903, a selective MET signaling inhibitor, in the hepa1-6 orthotopic models, and found that PF-04217903 alone did not result in obvious alterations in tumor volume and metastasis compared with the controls Fig. . HoweverBecause of the favorable effects of combination therapy observed above, we further analyzed the potential action of NZ001 Fig. . We alsoWe further observed the effects of NZ001 and sorafenib treatment on HCC growth and intrahepatic metastasis in hepa1-6 orthotopic models. Compared with vehicle controls, both the NZ001 and sorafenib treatment significantly decreased the tumor size Fig. .Fig. 5ThTo further determine the effects of NZ001 on extrahepatic metastasis of HCC, MHCC-97H cells were directly injected into the tail vein of nude mice. Starting from first week, mice were treated with NZ001 for up to 3 weeks. We found that metastases in the lung and liver were reduced by 86.2% and 82.2% respectively in the NZ001 treated group , cohibitors , 23. We MET-amplified and MET/P-MET-high–expressing HCC cells in vitro, we next evaluated the potential action of NZ001 to MHCC97H (MET amplification and high MET protein expression) and Huh7 (non-MET amplification and low MET protein expression) xenograft models in vivo. The results demonstrated that treatments with 10 and 30 mg/kg of NZ001 led to more tumor growth inhibition (TGI) of MHCC-97H xenografts than Huh7 xenografts . These results suggested that the novel MET and VEGFR2 blockade, NZ001, significantly inhibited tumor growth and angiogenesis of HCC, especially in tumors with MET amplification and high MET protein expression.Having observed that MET and VEGFR2 inhibitors inhibited proliferation of fts Fig. , f. Wherfts Fig. . In addifts Fig. . Moreovefts Fig. . These rMET copy number and protein expression in primary HCC cells. Three out of 16 primary HCC cells exhibited MET gene amplification, which were also positive for elevated MET protein expression (IHC 3+) in HCC tissues, and showed higher sensitive to NZ001 treatment compared with other cells  was 22.9% (28/122) Fig. . Furtherice Fig. , f. ThesMET amplification and high MET/P-MET protein levels , which have been used as selection criteria for MET targeted therapy in clinical trials of lung carcinoma [MET exon 14 alterations, which resulted in increased MET protein levels due to disrupted ubiquitin mediated degradation and considered as a viable therapeutic target in NSCLC [Despite significant preclinical data supporting the role of MET as a potential oncogenic driver in HCC, the clinical data obtained with application of MET inhibitors in HCC was not appreciable , 28. Thearcinoma , showed in NSCLC , 21, wasin NSCLC , 31, theMET amplification and MET/P-MET overexpression. These further supported interrelation between NZ001 sensitivity and MET-amplification/MET overexpression and verified NZ001 as a simultaneous inhibitor of both MET and VEGFR2 signaling.Moreover, both in vitro and in vivo assays demonstrated that NZ001 selectively inhibited MET and VEGFR2, and their downstream effectors, such as P-STAT3, P-AKT and P-ERK, in a dose-dependent manner only to HCCs with In summary, we demonstrated that the activation of MET was responsible for the metastasis-promoting effects induced by VEGF inhibition, and MET signaling inhibition attenuated this unfavorable outcome induced by VEGF inhibition. MET and VEGFR2 dual blockade, NZ001, resulted more pronounced inhibition on tumor growth and metastasis of HCC and showed advantages over sorafenib, especially not inducing more invasive and metastatic behaviors. MET amplification and overexpression could be used to determine the subgroup of patients to obtain the most favorable outcome of NZ001 treatment.Additional file 1:Table S1. Primary Antibodies for WB, IF and IHC. Table S2. Clinicopathological characteristics of HCC patients for analyzing the clinical significance of MET mRNA expression (n = 109). Table S3. Clinicopathological characteristics of HCC patients for detecting MET protein expression by IHC staining(n = 122). Table S4. Clinicopathological characteristics of HCC Patients for isolating primary HCC cells (n = 16). Table S5. Univariate and multivariate analyses of factors associated with OS and TTR. Table S6. Pharmacokinetic characteristics of NZ001 in mouse. Table S7. The analysis of correlation between MET gene amplification and MET protein expression in the 16 HCC samples. Table S8. The analysis of correlation between MET gene amplification and P-MET protein expression in the 16 HCC samples. (DOCX 39 kb)Additional file 2:Supplementary Materials and Methods. (DOCX 30 kb)Additional file 3:Figure S1. The E-cadherin, vimentin, CD34, HIF-1α, P-MET and total-MET expression levels in hepa1–6 orthotopic tumors after treatment with vehicle and VEGF antibody. Figure S2. The expression levels of E-cadherin, N-cadherin and vimentin were detected by Western blot in HCC cells starved overnight and treated with HGF (10 ng/ml) for 24 h. Figure S3. After 2-week treatment with VEGF antibody, PF-04217903 alone, and their combination, the mice were killed and the liver tissues were obtained. Figure S4. The E-cadherin, vimentin, CD34, HIF-1α, total-MET and P-MET expression levels in hepa1–6 orthotopic tumors after treatment with VEGF antibody and PF-04217903 alone, or their combination. Figure S5. The effect of NZ001 on VEGFR2 and MET signaling in HUVECs and HCC cells. Figure S6. NZ001 suppressed the HGF-induced EMT in HCC cells. Figure S7. Effects of NZ001 on the spontaneous and chemokine-induced invasion of HCC cell under normoxia and hypoxia condition. Figure S8. After 2-week treatment with sorafenib and NZ001, the mice were killed and the liver tissues were obtained. Figure S9. The total-MET, HIF-1α, CD34 and P-MET expression levels in hepa1–6 orthotopic tumors after treatment with VEGF antibody and PF-04217903 alone, or their combination. Figure S10. Effects of NZ001 on the metastasis of MHCC97H in nude mice. Figure S11. Effects of NZ001 on colony-formation of HCC cells. Figure S12. The association of MET exon 14 mutation determined by sanger sequencing with NZ001 sensitivity of 12 HCC cell lines. Figure S13. The score of MET and P-MET definition for IHC staining. Figure S14. The MET/P-MET/HGF expression in HCC cell lines. Figure S15. After 2-week treatment with different concentrations of NZ001, the mice were sacrificed and the tumors were obtained. Figure S16. IHC assay showed that MET phosphorylation was greatly inhibited after NZ001 treatment in MHCC-97H xenograft tumors. (DOCX 5327 kb)


It has already been shown that FACBC-PET is useful for glioma imaging. However, there have been no reports evaluating the efficiency of FACBC-PET in the diagnosis of brain tumors in comparison with other PET tracers in clinical studies. The purpose of this study was to investigate the efficacy of FACBC-PET imaging in glioma diagnosis, compared to L-methyl-11C-methionine (MET)-PET.Six consecutive patients , who were clinically suspected of having high- or low-grade glioma, received both FACBC-PET and MET-PET within a two-week interval. T1-weighted, contrast-enhanced, T1-weighted, and fluid-attenuated inversion recovery magnetic resonance imaging was performed to assist with subsequent tissue resection. Visual findings and semi-quantitative analyses of FACBC and MET uptake, using standardized uptake values (SUVs) and lesion-to-contralateral normal brain tissue (LN) ratios, were evaluated to compare PET images.SUVs for FACBC were lower than those for MET in the non-lesion cerebral cortex, brain stem, and cerebellar hemisphere. There was a weak positive correlation between FACBC and MET uptake in glioma tissue, although L/N ratios for FACBC were higher than those for MET in all the cases.FACBC-PET showed higher contrast than MET-PET by both visual and semi-quantitative analyses and may therefore provide better assessment for the detection of glioma. This study was registered as clinical trial (No. JapicCTI-132289). Methionine is an essential sulfur amino acid necessary for cell proliferation, and thus PET with L-methyl-11C-methionine (MET), which is mainly taken up by system L-amino acid transporters, is frequently used for brain tumor imaging (Positron emission tomography (PET) imaging with amino acid analogs is often used for clinical applications, as it targets the increased amino acid transport of tumors and has proven more useful for detecting brain tumors than  imaging . For exa imaging , 4.High MET accumulation in primary brain tumors is considered to indicate malignant status and significantly unfavorable prognosis. Accordingly, MET-PET plays an important role in evaluating brain tumors, although it is difficult to evaluate benign tumors through this approach since they may show low MET uptake , 6.11C-MET, which has an extremely short half-life (20 min), can only be used in limited institutions that are equipped with a cyclotron for its production.Since MET-PET shows faint physiological uptake in normal brain , the bou18F-fluciclovine  is available for use in clinical PET studies  than MET, is useful for many departments with a PET scanner. However, there have been few reports on the efficacy of FACBC-PET for the diagnosis of brain tumors. Therefore, we aimed to compare the effectiveness of FACBC-PET and MET-PET imaging in the diagnosis of glioma.At present, the artificial amino acid radiotracer  studies . FACBC lFrom December 2013 to June 2014, six consecutive patients with suspected brain tumors  underwent both FACBC-PET and MET-PET within a two-week interval. Patients’ age ranged from 21 to 70 years  or MET (4 MBq/kg) by intravenous injection, followed by a flush with physiological saline. Emission scans of the brain were performed for a total of 10 min, after either 19 min of FACBC administration, or 20 min of MET administration. Axial and trans-axial PET resolutions in Biograph-16 were 5.5 and 5.9 mm , respectively. Emission scans were reconstructed to a matrix of 336×336 and attenuation and scatter correction were performed; voxel size was 1.02×1.02×2.00 mm.Following PET scanning, additional MRI images  for the purpose of performing tissue resection were acquired using the Achieva 1.5 tesla imager .PET scans were independently analyzed by two experts in the field of nuclear medicine and fused with those acquired by MRI using the Biograph-16 software for the navigation of each subject. PET data was analyzed using the same region of interest (ROI) settings as our previous study , manuallmean and SUVmax, respectively) were calculated for semi-quantitative analysis of FACBC and MET uptake by each lesion. The mean and maximum lesion-to-contralateral normal brain tissue ratios (LNmean and LNmax) were determined by dividing the tumor SUVmean and SUVmax by the SUVmean of the contralateral cortex. The SUVs and L/N ratios were used as indicators of the degree of PET accumulation with all values being expressed as mean±SD.These ROIs were then transferred to the corresponding PET image to calculate the uptake of each radiotracer. Activity counts were normalized relative to the injected dose per kilogram of patient body weight  as follows: SUV= (pixel count/pixel volume)/(injected radioisotope activity/body weight) × calibration factor. Mean and maximum SUVs (SUV6-methylguanine DNA methyltransferase (MGMT) promoter, Ki-67 (MIB-1), and mutant isocitrate dehydrogenase 1 protein (IDH1 R132H). The genetic diagnosis of the resected tissues was made based on the 2016 World Health Organization (WHO) classification.The collected tissues were stained with hematoxylin and eosin, and immunohistochemistry was performed for glial fibrillary acidic protein (GFAP), 1p and 19q copy number status, telomerase reverse transcriptase (TERT) promoter, the methylation status of the OHistopathological diagnosis was confirmed by surgical resection. From the six patients, there were three diffuse astrocytomas, one oligodendroglioma, one anaplastic astrocytoma, and one glioblastoma .By visual examination of all the patients, there appeared to be lower uptake of FACBC than MET in normal brain tissue. FACBC was also found to accumulate in glandular tissue  mucosa, and subcutaneously.mean as a measure of tracer uptake, FACBC was confirmed to be lower than MET in all parts of the brain parenchyma as presented in After calculating SUVmean and L/Nmax were higher in FACBC-PET than MET-PET , mucosa, and the bone marrow. Lower background uptake leads to greater lesion contrast and easier detection of tumors. Our data indicated that FACBC-PET performs better than MET-PET for visual assessment in glioma. The L/N Figures  and 2. Mmax and L/N ratio are not significantly different between Grade II and Grade III gliomas and that low-grade oligo-type gliomas have relatively high MET uptake . In cases with TERT mutation , the accumulation of FACBC also tends to be high. It has been found that TERT mutation is a promising predictor of prognosis in patients with glioblastoma . It is nin vitro studies on prostate cancer and glioma indicated that the alanine-serine-cysteine transporters (ASCT), especially ASCT2, and L-type amino acid transporters (LAT), especially LAT1, are involved in its uptake (Physiological uptake of MET is considered to occur via system L-amino acid transporters because MET is utilized by normal brain tissues as a substrate for protein synthesis, neurotransmitter synthesis, and energy production. In case of FACBC, s uptake -16. Thiss uptake , which mOverall, FACBC-PET provided better assessment for detecting gliomas than MET-PET. In terms of differential diagnosis between low- and high-grade astrocytoma, FACBC-PET was found to show the same diagnostic potential as MET-PET when taking into consideration SUVs and LN ratios. Limitation of this study was its small sample size. To confirm our observations, further investigations on other types of brain tumor, such as metastatic brain tumors and central nervous system lymphoma, are required.


West Nile virus (WNV) is an emerging infectious disease which is most commonly transmitted to humans through mosquito, and is considered a major public-health problem worldwide. The aim of the current study is to bibliometrically analyze the quantity and quality of publications indexed in Scopus from different countries to reveal the characteristics of global research output regarding WNV.This study is a bibliometric analysis based on the Scopus database. This study focused on identifying WNV publication trends with regard to publication year, publication type, prolific countries, language of publication, as well as, prolific journals, citations, and collaboration patterns.n = 519 publications; 11%). All publications related to WNV achieved h-index of 140 and were cited 124,222 times. The median [interquartile range] number of citations per article thus amounts to 9 [2-28]. The USA had the highest h-index of 131. Emerging Infectious Diseases is the most productive journal with 227 articles, followed by Journal of Virology with 162 publications. The result designated that Centers for Disease Control and Prevention was ranked the first in terms of publication output, followed by National Center for Emerging and Zoonotic Infectious Diseases.A total of 4729 publications were considered in this study, which were published between 1943 and 2016. The annual quantity of literature published before 2000 followed a low rate of research growth; while the quantity of publications after 2000 were published in a stage of rapid development. The country with the greatest number of publications in WNV research field was the USA with 2304 (48.7%) publications, followed by France with 224 (4.7%) publications, and Canada with 222 (4.7%) publications. Contributions from low- and middle-income countries (LMIC) were considerably small, that is, (Centers for Disease Control and Prevention obtained the leading position of the institutions in terms of publication output. In general, this study not only presents a full view of global WNV research, but also can contribute for future further research in this field.There is an obvious trend of WNV research after 2000, and countries with high income have more contributions in WNV research field. The research output is low among LMIC. The USA produced the largest number of publications. The  West Nile virus (WNV) is a mosquito-borne infection that is transmitted to humans by mosquito , and it Recently, bibliometric tools have been widely used to investigate the worldwide contributions in many infectious diseases related research including Ebola , dengue Findings from this study will provide a holistic picture on WNV-related research which serves as a useful reference for future studies. Furthermore, it gives a picture for authors and editorial journals about future research direction.The database used in this bibliometric study comprises Scopus® . Scopus is more comprehensive and easier to be used in biomedical field compared to any other tool for literature research, it shows all author ‘s affiliations, and it is considered as the world’s largest database for abstract and citation information , 30, thah-index. IF is a useful indicator to assess the quality of journals [h-index is used to measure the productivity and impact of published works from different countries [The methodology applied in this study was comparable to recent bibliometric studies , 31–35. journals , 37; andountries . The IF ountries . Internaountries .Statistical analysis tests were performed using the Statistical Package for the Social Sciences (SPSS) version 15.0. The frequency in count and percentage, summation, and average were used for descriptive statistics. Microsoft Excel 2003 was used for plotting the charts. In addition, to evaluate the growth pattern of research output, linear and exponential regression fitting were compared for the trend in publication. The retrieved publications were divided into three phases according to research trends over time. In this study, frequently used terms were mapped using the VOSviewer software to identify the co-occurrence of title and abstract terms. The co-occurrence networks for the most frequently used terms in the titles and abstracts of the publications related to WNV were studied over the time interval. Each term is demonstrated by a circle, where its diameter and the size of its label illustrate the frequency of the term, and its color reflects most frequently encountered topics in this field .r = 0.712 after linear adjustment). A moderate positive significant correlation was found between the years and number of scientific publications (P < 0.001). This research trend in the field of WNV can be roughly divided into the following three stages. The first stage was from 1943 to 1999, in which the annual quantity of the number of scientific publications related to WNV published was fluctuated, but was mainly in low rate of research growth. Statistical analysis revealed a low positive significant correlation between the years during 1943 to 1999 and number of scientific publications . The second period was between 2000 and 2006, in which the quantity of publications published was in a rapid increase; with the peak of publications regarding this topic was in 2003 during which the number of scientific publications reached 345 publications. According to the analyzed data there is a moderate positive significant correlation between the years during 2000 to 2006 and number of scientific publications .Thirteen document types were identified in a total of 4729 publications. Articles (3658) were the most commonly used document type which accounted for 77.4% of the total publications. They were followed by reviews , short surveys 140; 3.0%), notes  and letters . For language analysis, 22 languages were used in these publications. English is the dominant language with 4384 (92.7%) publications, followed by French , Russian  and Spanish . The total publications per year are presented in Fig. 40; 3.0%,r = −0.394, P = 0.265).In addition, the third stage was from year 2007 to 2016, during which the annual quantity of the number of scientific publications published was fluctuated, indicating a steady-state growth in the field of WNV with a slightly reduction in research rate. Statistical analysis revealed no correlation in the stage between year 2007 and 2016 and number of scientific publications . Table Furthermore, a total of 106 countries contributed to the scientific output in the field of WNV research from 1943 to 2016. Contributions from LMIC were considerably small, that is,  among these 12 journals. The most common study design of the list of the most cited publications related to WNV was “laboratory-based research” (11 publications out of 20). On the other hand, Table Centers for Disease Control and Prevention was ranked the first in terms of publication output, followed by National Center for Emerging and Zoonotic Infectious Diseases. Among the top 10 institutions, 8 were in the USA, and one each in France and Australia.Table IF value 9.558 amoThis study sheds light on how the status of research regarding WNV has developed during the past 73 years. The study also recognized the main research topics related to WNV over time. The bibliometric analysis of this study shows that the number of scientific publications concerning WNV increased in the last 73 years. These results on WNV are consistent with those obtained from similar previous analysis of the literature related to other infectious diseases –24. Thish-index, which demonstrated that WNV publications originating from the USA had the highest quality.During this 73-year period, the USA had ranked at the top regarding the quality and quantity of published publications. Although it should come as no surprise that the USA lead the world in research production in WNV, which has been found in numerous previous biomedical studies , 67–69, To the best of knowledge, this study is the first of its kind that assessed research productivity in the field of WNV during the period between 1943 and 2016 at global level. However, there were some limitations for this study and most of them were similar to previous bibliometric studies , 68, 69.US Centers for Disease Control and Prevention obtained the leading position among the institutions in terms of publication output. Furthermore, “phylogenetic evolution”, “transmission”, “signs and symptoms” and “ecology and epidemiology” may be the latest hot spots in the field of WNV, and related researches may be pioneers to direct this field in the next few years. In general, this study not only presents a full view of global WNV research, but also can contribute for future further research in this field.Although bibliometric analyses have been performed across several fields of infectious, this study may be believed to be the first study regarding WNV. In conclusion, bibliometric analyses were performed to provide estimates of research productivity related to WNV at global level. There is an obvious trend of WNV research after 2000, and countries with high income tend to have more contributions in the field of the WNV research. The research output is low among LMIC. The USA, France, Canada, Italy, Australia, the UK, Germany, Israel, Spain, and China had high productivity in publications related to WNV. The USA produced the largest number of scientific publications. The


The calcium leaching behavior of cement paste and silica fume modified calcium hydroxide paste, exposed to hydrochloric acid solution, is reported in this paper. The kinetic of degradation was assessed by the changes of pH of hydrochloric acid solution with time. The changes of compressive strength of specimens in hydrochloric acid with time were tested. Hydration products of leached specimens were also analyzed by X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric (TG), and atomic force microscope (AFM). Tests results show that there is a dynamic equilibrium in the supply and consumption of calcium hydroxide in hydrochloric acid solution, which govern the stability of hydration products such as calcium silicate hydrate (C-S-H). The decrease of compressive strength indicates that C-S-H are decomposed due to the lower concentration of calcium hydroxide in the pore solution than the equilibrium concentration of the hydration products. Furthermore, the hydration of unhydrated clinker delayed the decomposition of C-S-H in hydrochloric acid solution due to the increase of calcium hydroxide in pore solution of cementitious materials. Still, its physical–chemical behavior is not yet fully understood.Calcium leaching, which is a degradation mechanism consisting in a progressive dissolution of the cement hydrates as a consequence of the migration of the calcium ions to the pore solution, may severely damage structures like dams, nuclear waste containment structures, pipes, water storage tanks, and tunnels. The kinetics of calcium leaching of cementitious materials is very slow but can be a risk at long term. Predicting the long-term behaviour of cementitious materials in aggressive environment requires a sound knowledge of the various deterioration mechanisms that will affect the structures over their lifetimes. The mechanisms of calcium leaching have recently been the subject of extensive research6, which is only stable when the calcium concentration in the pore solution is higher than 20 mol/m3. The continuous hydrolysis of portlandite exposes the cementitious constituents of the hardened cement paste to chemical decomposition. The dissolution of hydration products from hardened cement paste produces several deleterious effects, the most obvious of which are an increase in porosity and subsequent loss in strength9. Decalcification also induces dimensional changes of cement paste10. Berner suggested that C-S-H is stable when the calcium concentration in the pore solution is 2 to 20 mol/m3, such equilibrium depends on its C/S ratio (the higher the C/S ratio of the gel the higher the equilibrium)11. C-S-H are decalcified, reducing their C/S ratio, when the calcium concentrations below the equilibrium concentration12. Mineral additions to cement, such as silica fume (SF), play a significant role in the improvement of mechanical behavior and resistance to leaching13. Lin et al. reported that the resistance increased with an increase in amount of mineral additions14. These mineral additions decrease portandite content and form denser and more stable C-S-H than Portland C-S-H by pozzolanic effect15. The denser C-S-H gel decelerates the leaching process of calcium ions and slows the deterioration of strength of specimens with silica fume16. Also, the use of mineral additions such as silica fume is reported to result in a significant reduction of the transport propertied of the mixtures17. Furthermore, the calcium leaching decreases with increasing silica fume content4, which refine the pore structure through pozzolanic reaction, thus provide resistance to the leaching process from porosity generation16.Amorphous calcium silicate hydrate, calcium hydroxide (portlandite), aluminates, ettringite, and unhydrated cement (clinker) etc. are the main constituents of hydrated Portland cements. Their chemical stability will strongly influence the durability of concrete in aggressive environment. The cement paste matrix is basically a porous material. These pores are generally filled with a highly basic solution. Portlandite is the most susceptible to hydrolysis because of its relatively high solubility in deionized water19. Calcium leaching due to ammonium nitrate solution is quicker than those obtained with the deionized water20. It is reported that 6 mol/L ammonium nitrate solution can accelerate the leaching speed up to 100 times while still get the same end products. Also, ammonium nitrate used is close to the condition immersed in deionized water. Therefore, ammonium nitrate was intensively used to experimentally investigate calcium leaching behavior.Leaching by deionized water is a very slow process, accelerated experimental procedures are needed. Hence, Carde introduced ammonium nitrate solution to accelerate leaching process based on the kinetics of the calcium leaching21 investigated the process of hydrochloric acid attack on cement mortar. Tests results showed that hydrochloric acid attacks the clinker materials of Portland cement forming some soluble salts, mostly with calcium, which are subsequently leached out. This way the porosity of cement mortar is increased. De Ceukelaire22 studied the effect of hydrochloric acid attack on three types of mortar. It is showed that the hydrochloric acid resistance of a mortar is related best to the cement content and the formation of layers of ferric hydroxide possibly can influence the leaching processes. The hydrochloric acid also achieves an acceleration of the leaching process due to the increased calcium concentration gradient in the attacking fluid phase. Thus, the object of this paper is to investigate the extents of chemical degradation by the action of hydrochloric acid on cement paste and silica fume modified calcium hydroxide paste (SF/CH system).Chandra2/kg, and the water requirement of normal consistency is 26.8%. Calcium hydroxide and hydrochloric acid are both commercial products. It can be observed from Fig. Materials. The main raw materials for experimental research are moderate heat Portland cement (MHC), silica fume (SF), calcium hydroxide (CH), hydrochloric acid (HCl) and deionized water. The chemical composition and mineralogical composition of the clinker are shown in Table To study the deterioration mechanism of hardened cementitious matrix in hydrochloric acid solution, two cementitious materials were used, one was Portland cement paste and the other was silica fume modified calcium hydroxide paste (SF/CH system). It is reported that silica fume decrease portandite content and form lower C/S ratio C-S-H gel than Portland C-S-H by pozzolanic effect, thus decelerates the leaching process of calcium ions. Therefore, it is very necessary to investigate the calcium leaching behavior of SF/CH system without cement. Mix proportions of the two cementitous material were shown in Table Step I: After 24 h, test the pH of the solution;Step II: Add hydrochloric acid slowly and stir with a glass rod until the pH of the solution equals 1;Step III: Repeat steps I and II. When the pH of the solution is less than 5, sample in the middle of the specimens, and placed in anhydrous ethanol to stop hydration. Hydration products such as CH and C-S-H were analyzed by X-ray diffraction (XRD), differential scanning caloriemetry (DSC), and thermogravimetric analysis (TG).The morphology, size and distribution of C-S-H were observed by atomic force microscopy (AFM). The atomic force microscope used in this study was a scanning probe microscope  with a silicon tip. The microscope was operated in tapping mode to provide topographic maps of the cement paste using AFM techniques. During scanning the specimen was at room conditions. Different scan ranges of 1000 nm and 800 nm were used in order to better understand the surface structure and particle shapes. The results shown in this paper consist of typical results obtained from these scans. The regions used were selected after first using the optical microscope to find the most interesting regions.Relative compressive strength of cement mortar can reflect the deterioration process of cementitious materials. Mix proportion of the cement mortar is also included in Table The data generated during the current study are available from the corresponding author on reasonable request.When hydrated cementitious materials, the pore solution of which is about 13, are in contact with hydrochloric acid, hydroxyl ions diffuse towards aggressive solution under gradient of concentration. Figure Stage I: the ΔpH of the solution of sample C was 8 (the pH of the solution was 9) and the △pH of the solution of sample SF/CH was 6 (the pH of the solution was 7). This shows that the dissolution rate of calcium hydroxide in the first stage is relatively large, which can maintain the equilibrium concentration in the pore solution of hydrated cementitious materials.Stage II: the ΔpH of the solution of sample C decreases with time, indicating the decrease of dissolution rate of calcium hydroxide. This is because calcium hydroxide must be dissolved from the inside of the sample.− dissolved from the hydrated cementitious materials was just enough to neutralize H+ in the hydrochloric acid solution. That is, supply and consumption of calcium hydroxide reached a dynamic equilibrium.Stage III: the ΔpH of sample C was kept at 5, and the ΔpH of sample SF/CH was kept at 6, indicating that OHStage IV: the equilibrium was broken and the ΔpH of the solution of the two samples declined, indicating an increased degree of calcium leaching.Stage V: the ΔpH of the solution declined to 4 (the pH declined to 5), indicating the second equilibrium.Stage VI: the ΔpH of solution declined to 3 (the pH declined to 4), indicating the decreased supply of calcium hydroxide and the increased degradation of hydrated cementitious materials.Figure − to neutralize H+. Then, the pH in the pore solution at point A began to decrease with time. The hydration products begin to decompose when the pH reduced to the ultimate lime concentration of hydration products, resulting in an increase in porosity. On the other hand, the decrease of pH in the pore solution and the increase of porosity lead to the continuous hydration of clinker, resulting in the increase of concentration of calcium hydroxide in the pore solution again. That is, supply and consumption of calcium hydroxide is a dynamic equilibrium process, as shown in equation , as shown in Fig. AFM techniques was used to provide topographic maps of the selected regions, focusing on the hydration products. The color scale for the AFM images represents differences in topography, with darker colors corresponding to lower surface height and a lighter color corresponding to a higher surface height28. The gap among C-S-H clusters in sample C-leached is larger than that of control sample (C-unleached) at 10 days. This indicates that some of the C-S-H gel in hydrated cement paste is decomposed due to the erosion of hydrochloric acid. Study shows that cohesion in cement paste results of the interactions between C-S-H surfaces in an interstitial ionic solution29. The interlayer calcium ions are responsible for creating attractive interparticle forces. The loss of calcium ions would also account for the continuous decrease in specific surface area of C-S-H. Thus, the loss of calcium ions of C-S-H contributes to the marked loss of compressive strength.Figure There is a dynamic equilibrium for the supply and consumption of calcium hydroxide in hydrochloric acid solution. This equilibrium moves from outside to the inside of the sample with time, thus, leading to the degradation of hydrated cementitious materials gradually.The hydration of unhydrated clinker in hydrochloric acid solution can improve the concentration of the calcium hydroxide in pore solution, which delay the decomposition of hydration products such as C-S-H and C-A-H.The ΔpH of the hydrochloric acid solution decreased with time, indicating the concentration of calcium hydroxide in the pore solution is lower than the equilibrium concentration, resulting the decomposition of hydration products such as C-S-H and C-A-H. The marked decrease of compressive strength observed when the pore solution of hydrated cementitious materials lower than equilibrium concentration of hydration products.According to the strength development and microstructure of hydrated cementitious materials in hydrochloric acid, the following conclusions can be drawn:


Clostridium difficile, without any impact on mortality, in real-world settings. So, although using PCT-guided antibiotic stewardship is promising, after more than a decade of randomized controlled trials on this topic the evidence in its favor is still less than compelling due to limitations in trial design, not taking into consideration fundamental aspects of PCT biology, and the absence of evidence-based antimicrobial duration in intervention and control groups. In this commentary we highlight some questions and limitations of primary PCT study data that might impact interpretation and clinical use of PCT at the bedside.Using biomarkers as a guide to tailor the duration of antibiotic treatment in respiratory infections is an attractive hypothesis assessed in several studies. Recent work aiming to summarize the evidence assessed the effect of a procalcitonin (PCT)-guided antibiotic treatment on outcomes in acute lower respiratory tract infections (LRTI), suggesting that significant reductions in antibiotic duration occur when using a PCT-guided algorithm. However, controversial evidence also suggested PCT-guided algorithms were associated with increased antibiotic duration and increased incidence of  Clostridium difficile, without any impact on mortality, in real-world settings in the US. In this commentary, we highlight some questions and limitations of primary PCT study data that might impact interpretation and clinical use of PCT at the bedside.Using biomarkers as a guide to tailor the duration of antibiotic treatment in respiratory infections is an attractive hypothesis assessed in several studies. A recent meta-analysis aiming to summarize the evidence assessed the effect of a procalcitonin (PCT)-guided antibiotic treatment on outcomes in acute lower respiratory tract infections (LRTI) suggested significant reductions in antibiotic duration occur when using a PCT-guided algorithm . HoweverA major concern in PCT-guided trials is antibiotic use in the control group. According to the World Medical Association’s Helsinki declaration, “the benefits, risks, burdens, and effectiveness of a new intervention must be tested against those of the best current proven intervention,” but defining “best current proven intervention” is difficult . HeterogFor instance, usual care is sometimes far from the best available care or what should be standard of care. Trials using a protocolized rather than an unrestricted standard care control group will likely have enhanced validity as long as the protocolized care control group is representative of standard care practices . In PCT Also, PCT algorithms consider that a PCT < 0.1 μg/L bacterial infection is very unlikely and antibiotics should not be prescribed or should be withheld. It is well known that in several bacterial infections, for example, VAP , 9, PCT In addition, another important limitation of PCT use is related to the lack of information on specific conditions and populations where its value is inadequate because of intrinsic constraints. In critically ill patients the presence of acute kidney injury or the use of renal replacement therapies has a profound effect on PCT concentrations –14. AddiClinical use of biomarker-guided antibiotic stewardship such as PCT is promising, but after more than a decade of RCTs on the topic the evidence in its favor is still less than compelling due to limitations in trial design. These limitations include not taking into consideration fundamental aspects of PCT biology and the absence of evidence-based antimicrobial duration in intervention and control groups, namely in VAP. A double-trigger criteria, in which antibiotics are stopped according to the clinical course and either decreases in biomarker levels, according to an algorithm, or the completion of 5–7 days of treatment, whichever comes first , might b


Concrete-filled fiber-reinforced polymer tubes (CFFTs) have attracted interest for their structural applications in corrosive environments. However, a weak interfacial strength between the fiber-reinforced polymer (FRP) tube and the concrete infill may develop due to concrete shrinkage and inadequate concrete compaction during concrete casting, which will destroy the confinement effect and thereby reduce the load bearing capacity of a CFFT. In this paper, the lead zirconate titanate (PZT)-based ultrasonic time-of-flight (TOF) method was adopted to assess the concrete infill condition of CFFTs. The basic idea of this method is that the velocity of the ultrasonic wave propagation in the FRP material is about half of that in concrete material. Any voids or debonding created along the interface between the FRP tube and the concrete will delay the arrival time between the pairs of PZT transducers. A comparison of the arrival times of the PZT pairs between the intact and the defected CFFT was made to assess the severity of the voids or the debonding. The feasibility of the methodology was analyzed using a finite-difference time-domain-based numerical simulation. Experiments were setup to validate the numerical results, which showed good agreement with the numerical findings. The results showed that the ultrasonic time-of-flight method is able to detect the concrete infill condition of CFFTs. Concrete-filled fiber-reinforced polymer tubes (CFFTs), as a type of hybrid compression member, are attractive for their use as composite materials in several special structural applications including piles, columns, bridge piers, poles, and highway overhead sign structures . Owing tSeveral NDT methods, such as the ultrasonic pulse velocity method ,4, acousIn this study, PZT patches were adopted as transmitters and receivers of ultrasonic waves to monitor the concrete infill for concrete-filled fiber-reinforced polymer tubes by using the ultrasonic TOF. The PZT patches were bonded on the outer surface of the CFFT. The existence of voids inside the CFFT alters the ultrasonic wave propagation path between the PZT transmitter and the PZT receiver. The proposed method is developed to monitor the concrete infill condition for concrete-filled FRP tubes. The basic principle of ultrasonic TOF to detect the defects in CFFTs is that the velocity of the ultrasonic wave propagation in FRP material is about half of that in concrete material. Any voids along the interface between the concrete and the wall of the FRP tube will drive the ultrasonic wave travel along the wall of the FRP tube. By investigating the arrival time of the equally spaced PZT receivers on the FRP tube, the concrete infill condition and the location of the defects can be identified. The methodology was first analyzed using a numerical simulation approach based on the finite-difference time-domain method. The numerical analysis was then validated through the experimental investigations, which involved four different infill conditions—an empty concrete infill, a 1/3 concrete infill, a 2/3 concrete infill, and a 100% or complete concrete infill. The proposed method can be adopted to new structures for concrete infill monitoring during concrete casting and to existing structures for void detection and determination.The CFFT under analysis consisted of an FRP tube and a concrete infill. The filling condition of the concrete directly affects the load bearing strength of the CFFT. Therefore, different filling conditions of the concrete inside the FRP tube were simulated numerically, and the wave traveling characteristics were analyzed. The geometry of the numerical model is shown in The finite-difference time-domain (FDTD) method was adopted to simulate propagation characteristics of the ultrasonic waves in the CFFT model. In order to improve the computing efficiency and reduce the wave distortion, the staggered-grid technique was used, and a perfectly matched layer (PML)-absorbing boundary condition was also applied during the numerical simulation. The Richer wavelet with a frequency of 20 kHz was chosen as the excitation wave. The time step Furthermore, As can be seen from the above analysis, the full and empty concrete infill conditions can be easily differentiated by analyzing the ultrasonic time of flight. However, in practical engineering, the concrete is normally partially absent inside the FRP tube due to inadequate compaction, creating voids and debonding defects. To qualitatively determine the concrete infill condition and locate the defects, a cross-excitation method was adopted. Two other different concrete infill conditions, i.e., the 1/3 and 2/3 concrete infill conditions, were examined using R3 and R4 as excitation sources, respectively. The waveforms of cross-excitation for the 1/3 and 2/3 concrete infill conditions are shown in For the case of the 1/3 concrete infill condition, when R3 is used as the excitation source, as shown in The experimental setup of the concrete infill detection of the CFFT using PZT-based TOF is shown in In this experimental study, the arrival time of the different receivers for the different concrete infill conditions is shown in In this paper, the feasibility of using the ultrasonic TOF method to detect the concrete infill condition of CFFTs was examined both numerically and experimentally. By investigating the snapshots of different concrete infill conditions in CFFTs in a numerical simulation, it was found that the energy of an ultrasonic wave is concentrated in the concrete material. The wave propagates faster in concrete than in the FRP material. The arrival time of the wave amplitude of the received waveforms contains critical information about the concrete infill condition inside the FRP tube. In a full concrete infill condition, the wave travels with the velocity in the concrete material, and the time of flight between the transmitter and any receiver is at a minimum value. If a void is created in between the transmitter and any receiver, such as the 1/3 and 2/3 concrete infill conditions, the wave propagation path will be directed to the FRP tube before it reaches the receiver, thus increasing the ultrasonic TOF. The ultrasonic TOF is at a maximum when there is no concrete infill in the CFFT.These results show that the PZT-based time-of-flight method is able to detect and locate the concrete infill condition of CFFTs. The proposed method can be adopted for new structures for concrete infill monitoring during concrete casting and to existing structures for void detection and determination. This method benefits from an easy operation, high flexibility, a low cost, and remote sensing, and is therefore a promising candidate for the concrete infill monitoring of concrete-filled FRP tubes.


Mutations in BLM in Bloom Syndrome patients predispose them to multiple types of cancers. Here we report that BLM is recruited in a biphasic manner to annotated DSBs. BLM recruitment is dependent on the presence of NBS1, MRE11 and ATM. While ATM activity is essential for BLM recruitment in early phase, it is dispensable in late phase when MRE11 exonuclease activity and RNF8-mediated ubiquitylation of BLM are the key determinants. Interaction between polyubiquitylated BLM and NBS1 is essential for the helicase to be retained at the DSBs. The helicase activity of BLM is required for the recruitment of HR and c-NHEJ factors onto the chromatin in S- and G1-phase, respectively. During the repair phase, BLM inhibits HR in S-phase and c-NHEJ in G1-phase. Consequently, inhibition of helicase activity of BLM enhances the rate of DNA alterations. Thus BLM utilizes its pro- and anti-repair functions to maintain genome stability. Bloom helicase is recruited to double strand breaks in an ATM dependent manner. Here the authors show that Bloom helicase is recruited to double strand breaks in an ATM and MRN dependent manner with HR and NHEJ regulated by the helicase depending on the phase of the cell cycle. In contrast to stalled replication forks that are recognized by the ATR/Chk1 axis, DSBs are known to be recognized by MRE11–RAD50–NBS1 (MRN) complex, which recruits ATM and allows its optimal activation. ATM, in turn phosphorylates NBS1 leading to the stabilization of MRN complex and subsequently the sequential accumulation of other DDR proteins, thereby allowing the progression of the DDR  and RNA helicase DEAD Box 1 (DDX1)11. ATM-mediated phosphorylation of CtIP also promotes the recruitment of BLM to laser-induced DSBs12.Mutations in tumor suppressor BLM helicase cause Bloom Syndrome (BS). BS patients are characterized by predisposition to multiple types of cancers13. For example, BLM is instrumental in the dissolution of both RAD51 nucleoprotein filaments16 and double Holliday junctions17. BLM has also been implicated in the negative regulation of error-prone microhomology-mediated end joining (MMEJ). Hence, in cells lacking BLM, the rate of MMEJ-mediated genomic rearrangements was enhanced, indicating BLM prevents this inaccurate pathway of DSB repair20.Once recruited to the site of damage, BLM is involved in its repair. The multiple mechanisms by which BLM functions during homologous recombination (HR), particularly the requirement of its post-translation modifications, have been well documented21.In recent past, site-specific cleavage in the human genome has been achieved by fusing the restriction enzyme AsiSI to a modified estrogen receptor (ER) hormone-binding domain, which only binds to 4-hydroxy tamoxifen (4-OHT), an active metabolite of tamoxifen. This has led to the generation of the U2OS–AsiSI–ER system, which has been used previously to obtain the high-resolution profiling of γH2AX around DSBs in the human genome. It was inferred that γH2AX distribution was dependent on gene transcriptionUsing this U2OS–AsiSI–ER system we have now deciphered the key determinants essential for the recruitment of BLM to the DSBs. We provide evidence that the recruitment of BLM occurs in a biphasic manner extending from 80 bp from the DSBs to 9 kb from the DSBs. While BLM recruitment does not require on its own helicase activity, it is dependent on the kinase activity of ATM and the exonuclease activity of MRE11. Another key requirement is the interaction of RNF8 ubiquitylated BLM with a second MRN complex member, Nbs1. The retention of BLM at the DSBs coincides with its co-recruitment with HR factors in S-phase and c-NHEJ components in G1-phase. The helicase activity of BLM is critical for the phase-specific recruitment of the repair factors to the DSBs. In this phase, BLM recruitment is independent of ATM kinase activity but still depends on the MRE11 exonuclease activity. Post recruitment during the repair phase, BLM not only inhibits HR in S-phase, but also c-NHEJ in G1-phase. Further c-NHEJ is inhibited in the S-phase as a backup mechanism. Thus BLM both enhances and inhibits multiple repair mechanism in a cell cycle phase-dependent manner and thereby maintains genome integrity.In an effort to understand the mechanism of BLM recruitment at the DSBs, we treated U2OS–AsiSI–ER cells with 4-OHT for different time intervals upto 4 h  with anti-BLM antibody in U2OS–AsiSI–ER cells treated with 4-OHT for 4 h. BLM recruitment to both proximal and distal regions to the DSBs on multiple chromosomes  and H3K9me3 (associated with heterochromatin), were analyzed utilizing the ChIP-seq data for U2OS cells from the ENCODE database. Analysis of the chromatin landscape on either side of multiple AsiSi sites (by superimposing the primers used for ChIP-qPCR with the ChIP-seq data) revealed that BLM recruitment occurred irrespective of the compactness of the surrounding chromatin  and the extent of recruitment of BLM was determined at different distances (80 bp to 9 kb) from the DSBs for two randomly picked AsiSI sites  and late  phase post-DSB generation. To characterize the early recruitment in more detail, we carried out parallel ChIPs with anti-BLM, anti-NBS1, anti-XRCC4, and anti-RAD51 antibodies after exposing the cells to 0.5 h of 4-OHT treatment. It was observed that after 0.5 h of 4-OHT treatment both BLM and NBS1 are co-recruited to the DSBs. The extent of BLM and NBS1 recruitment was higher than RAD51 and XRCC4. This indicated that at this early time point, the DSBs were recognized by the DNA damage sensor proteins like NBS1. At this time window, DNA repair processes have not been fully initiated Fig. .Since BLM recruitment did not depend on its own helicase activity Fig. , we wantFurther, to determine whether BLM recruitment at the DSBs depended on the enzymatic activities of MRE11 or ATM, BLM ChIP was carried out after DSB induction, in the absence or presence of either MRE11 exonuclease activity inhibitor (Mirin) or ATM kinase inhibitor (KU 55933). Treatment with either Mirin or KU 55933 prevented the phosphorylation of multiple known ATM substrates  in S- and G1-phase of the cell cycle. ChIPs using BLM, RAD51, and XRCC4 antibodies were carried out in U2OS–AsiSI–ER cells synchronized either in S- or G1-phase of the cell cycle by double thymidine block and subsequently released 24  were sequenced and the extent of different types of nucleotide changes quantitated  it is present both in the immediate vicinity and far from the DSBs , BLM is present at the DSBs along with DNA repair proteins like XRCC4 and RAD51  and ATM protein are the essential prerequisites for the recruitment process , the co-recruitment of BLM with DNA repair factors XRCC4 and RAD51 28, mCherry2-C1 , NHEJ substrate pJS296 and I-SceI expression vector pJS20 (gifts from Jemery M. Stark). All siRNAs were purchased from Dharmacon and listed in Supplementary Table The antibodies used are described in Supplementary Table 21 (a gift of Gaelle Legube) and HCT116 BLM (−/−)42 (a gift of Bert Vogelstein) cells were maintained as described. HEK293T cells were maintained in DMEM medium supplemented with 10% FCS. All cells used were tested free from mycoplasma contamination. Synchronization of U2OS–AsiSi–ER cells in S- and G1-phase by double thymidine block was carried out as published32. Supplementary Figure 43. For cell cycle analysis, the propidium iodide stained DNA content of the cells was sorted in a BD FACS Calibur and analyzed using the FlowJo software.U2OS–AsiSi–ER5 was carried out using 250 ng of the recombinant soluble substrate or 2 μl of in vitro transcribed and translated substrate (Promega). Assays were set up at 37 °C for 3 h in a total volume of 25 μl in 50 mM Tris-HCl (pH 8.0) and 1 mM DTT. Recombinant His-RNF8 (0.2 μM) was added to 0.4 μM of the indicated E2 enzymes Ubc13, 0.0125 μM E1 and 16 μM ubiquitin. Reactions were initiated by the addition of ATP (2 mM) and MgCl2 (5 mM). The reactions were stopped by boiling with 2× SDS loading dye and samples were run on Nu-PAGE 4–12% gels in 1× MOPS buffer (Invitrogen), transferred onto a nitrocellulose membrane and probed with the indicated antibodies. All lysates were made in RIPA buffer. Myc-tagged NBS1 and MRE11 were immunoprecipitated using 200 μg of RIPA extract and 1 μg of the antibody for 4 h. Interactions between immunoprecipitated myc-tagged NBS1 and MRE11 and BLM were carried out in 1× PBS + 0.1%NP40 buffer overnight at 4 °C. In vivo interactions between endogenous proteins were carried out by standard protocols using 500 μg of lysates and 2 μg of the antibody for 4 h. The protein–protein interactions with ChIP elutes was carried out after extracting the complex twice in elution buffer  for 15 min at 37 °C. The eluted fractions were pooled and processed for immunoprecipitations, as described above. For in vitro interactions, recombinant GST-BLM or GST (1 μg) was interacted with 5 μl of in vitro translated NBS1 carried out in presence of 20μCi S35 methionine. The interaction was carried out at 4 °C for 4 h. Immunofluorescence followed by confocal microscopy was carried out using antibodies and their specific dilutions described in Supplementary Table 4. All incubations with primary antibodies were for 2 hrs while the corresponding secondary antibodies were incubated for 1 hr. Confocal imaging was carried out in LSM510 Meta System  using 63x/1.4 oil immersion objective. The laser lines used were Argon 458/477/488/514 nm (For FITC) and DPSS 561 nm (for Texas Red). For immunofluorescence quantitation, a minimum of 200 cells across four biological replicates were analyzed. Data presented (mean ± standard deviation) were analyzed by unpaired two-tailed Student’s t-test.RNF8-mediated ubiquitylation of BLM21. For this purpose, 250 μg of formaldehyde crosslinked chromatin was immunoprecipitated using 2 μg of the respective antibodies used for all the ChIP assays. After washing, the immunoprecipitated complexes were re-suspended and crosslinking reversed overnight. DNA was purified by phenol/chloroform, precipitated, and analyzed by ChIP-qPCR. Alternatively, ChIP was also carried out using the ChIP assay kit  as per the manufacturer’s protocol. The extent of recruitment of the proteins at the AsiSI-generated cleavage sites was determined by ChIP-qPCR according to the percent input method. All the depicted values (mean ± standard deviation) were obtained from four independent experiments. Data were analyzed by unpaired two-tailed Student’s t-test. The primers for ChIP-qPCR analysis are described in Supplementary Table ChIP assays using anti-BLM, anti-NBS1, anti-RAD51, and anti-XRCC4 antibodies were carried in U2OS–AsiSi–ER44 in a hypotonic lysis buffer , followed by lysis by homogenization (20 strokes) in presence of protease cocktail inhibitors (Sigma) supplemented with individual components. The samples were kept on ice for 20 min, 0.5 volume of high salt buffer  added. The lysates were ultracentrifuged for 3 h at 211,422.6g in a Beckman SW50.1 rotor. Post centrifugation, the sample was dialyzed for 3 h against dialysis buffer  and frozen in deep freezer. In vitro HR assays were carried46 using (500 ng) of the substrates (pTO231 and pTO223 both of which have ampicillin resistance). Reciprocal recombination and gene conversion between the two substrates lead to the generation of a functional neomycin gene that conferred kanamycin resistance. In the assays, the substrates were incubated with 5 μg of the cell extracts prepared under different conditions. The reaction was carried at 37 °C for 30 min in an HR reaction buffer . DNA obtained after Proteinase K treatment, phenol–chloroform extraction, and ethanol precipitation was dissolved in TE buffer and plated in 1:10 ratio between Luria Broth plates containing either ampicillin or kanamycin. Colonies counted after electroporation was used to calculate fold change in recombination frequency. The entire experiment was done three times. Statistical analysis was done by Student’s t-test. Standard methods of SCE determination were followed35. Forty metaphase spreads were analyzed for each condition. Three independent experiments were carried out. The data are presented as mean ± standard deviation and the p-values were obtained by Student’s t-test. The conditions are two-tailed, unpaired data with unequal variance.Lysates for HR and NHEJ assays were prepared47 with certain modifications. NHEJ substrate pJS296 (1 μg) and I-SceI expression vector pJS20 (2 μg) were transfected in asynchronously growing U2OS–AsiSI–ER cells (−4-OHT) in the absence or presence of ML216. mCherry2-C1 plasmid (0.5 μg) was transfected in each case to determine the transfection efficiency. Thirty-six hours post transfection, the cells were collected in PBS and 50 mM EDTA, pelleted and fixed with 2% paraformaldehyde for 20 min. The percentage of mCherry and GFP-expressing cells was scored by FACS analysis in BD FACS AriaIII. The experiment was done six times, data are presented as mean ± standard deviation and the statistical analysis was done by Student’s t-test. From parallel plates, lysates were made and transfection efficiency was also determined by western blot analysis using an anti I-SceI antibody.NHEJ assays with intrachromosomal substrates were carried out six times24 with [γ-32P] ATP end-labeled double-stranded oligonucleotides containing 5′ compatible ends . The data presented are from three biological replicates and are represented as mean ± standard deviation. Data were analyzed by Student’s t-test.In vitro c-NHEJ assays were carriedt-test. The raw sequences from which the data were extracted to determine the nucleotide alterations at or near to the AsiSI sites have been submitted in FigShare: https://figshare.com/articles/Chr6_AsiIS_90404906_pdf/5852853.The genomic DNA around AsiSI sites was cloned in pUC18 and subjected to Sanger sequencing. In both −ML216 and +ML216 condition, 140 individual clones, spanning across four AsiSI sites obtained from three independent experiments were sequenced. Fifty to one hundred nucleotides on both sides of the AsiSI sites were considered to determine the accuracy of the end joining. Data were obtained from three independent experiments and represented as mean ± standard deviation, and were analyzed by unpaired two-tailed Student’s https://figshare.com/articles/Source_File_for_Images/5852940. All other relevant data are available from the authors.The individual source files of all the western blots and autoradiograms, presented as TIFF images, are attached as supplementary datasets. The combined source file for all the data are presented in Supplementary Figure Supplementary InformationPeer Review File


Pneumocystis pneumonia by 162 500, disseminated histoplasmosis by 48 000 and chronic pulmonary aspergillosis by 33 500, with approximately 60% coverage of diagnostics and antifungal agents; a total of >1 000 000 lives saved over 5 years. If factored in with the 90–90–90 campaign rollout and its effect, AIDS deaths could fall to 426 000 annually by 2020, with further reductions possible with increased coverage. Action could and should be taken by donors, national and international public health agencies, NGOs and governments to achieve the UNAIDS mortality reduction target, by scaling up capability to detect and treat fungal disease in AIDS.Deaths from AIDS (1 500 000 in 2013) have been falling more slowly than anticipated with improved access to antiretroviral therapy. Opportunistic infections account for most AIDS-related mortality, with a median age of death in the mid-30s. About 360 000 (24%) of AIDS deaths are attributed to tuberculosis. Fungal infections deaths in AIDS were estimated at more than 700 000 deaths (47%) annually. Rapid diagnostic tools and antifungal agents are available for these diseases and would likely have a major impact in reducing deaths. Scenarios for reduction of avoidable deaths were constructed based on published outcomes of the real-life impact of diagnostics and generic antifungal drugs to 2020. Annual deaths could fall for cryptococcal disease by 70 000, This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’. The majority of these deaths are attributable to opportunistic infections. As an example, across the world, HIV infection is second only to traffic accidents as a cause of death in adolescents . Dying fIn 2010, UNAIDS issued the aspirational target of zero AIDS deaths by 2015 , a reducPneumocystis pneumonia (PCP), disseminated histoplasmosis (DH) and, probably, aspergillosis (chronic and invasive). Diagnostic tools and therapies for these entities are available but are either not available or not used in many high burden countries, Nigeria being one prominent example [A major focus on efforts to reduce deaths from fungal diseases complicating HIV infection could reduce AIDS deaths by more than 30%. The major fungal causes of death in AIDS are cryptococcal meningitis,  example .1 To ensure that the figures presented are conservative, it is assumed that ART retention on therapy is excellent and resistance is minimal, whereas over 20% of patients do not meet these assumptions in low- and middle-income countries (LMICs) [A straightforward, linear scenario prediction exercise from 2016 to 2020 was undertaken to estimate the relative impact of addressing fungal diseases to reduce mortality. In summary, the current slow downward trajectory of AIDS deaths was compared with additional, gradually increasing efforts to diagnose and treat cryptococcal disease, PCP, DH and chronic pulmonary aspergillosis (CPA) after TB, as well as the benefits of increased antiretroviral therapy (ART) coverage as per the 90–90–90 campaign programme. (LMICs) –11  allows individual country, region and continent analyses to be done, with alternative assumptions.3.6 l−1. This decline to less than 200 × 106 l−1 cells occurs within 6–10.5 years from seroconversion in patients without therapy [Of the estimated 36.9 million people with HIV infection, 22 million are not currently receiving ART . The ris therapy –17. Assucurrent rates of progress in rolling out ART, 940 000 AIDS deaths are expected in 2020.Prior to the institution of the 90–90–90 target, the number of AIDS deaths was falling at about 80 000 annually, reaching approximately 1 500 000 in 2013. With the 4.Gradual movement towards the 90–90–90 ART target will likely see a further reduction in deaths . Many of5.The most conservative estimate for cases of AIDS-related cryptococcal meningitis worldwide is 372 000 , calcula6 l−1 reduces mortality from greater than 70% to 15–50% at 10 weeks, as long as meningitis is actively diagnosed and treated [There are broadly two approaches to reducing deaths from cryptococcal meningitis in AIDS. The first is based on detecting asymptomatic antigenaemia and oral treatment with fluconazole, after excluding meningitis . Screen and treat in those with CD4 counts of less than 100 × 10 treated –22. Here treated ,23. The  treated . The sec treated . Impleme treated –29. The  treated ,31.Screen and treat programmes are operational in Rwanda and being rolled out in South Africa and several other African countries. If we assume 5% coverage currently, rising to 60% by 2020, then the number of identified early cases of cryptococcal antigenaemia would rise from 13 900 to 166 950 by 2020, all amenable to treatment. Deaths would fall from 232 756 to 166 394 cases, an annual saving in 2020 of 66 363 lives and a cumulative saving over the 5 years of 192 410 lives .With screen and treat, we can also assume improved diagnostic coverage for late presenting cases, as the same simple diagnostic test can be used. Thus, a direct benefit of rolling out the screen and treat programme is improved rapid diagnosis (estimated at 80% coverage by 2020) and a markedly reduced number of unscreened, undiagnosed patients who die—from 139 125 in 2015 to 55 650 patients in 2020. These figures are included with the screen and treat totals above.For those patients who are not identified early by screen and treat, but present with meningitis, implementation of the WHO recommended treatment with amphotericin B and flucytosine should increase survival to approximately 70% at 10 weeks and 60% at 6 and 12 months . If screThe combination of both screen and treat (60% coverage) and optimal therapy (60% coverage) provides the maximal benefit, with annual deaths falling from an estimated 232 756 in 2015 to 162 876 in 2020, a cumulative gain of 300 554 lives .Table 3These estimates make the assumption that the incidence of asymptomatic cryptococcaemia and cryptococcal meningitis are constant over this 5-year period, which is unlikely. The incidence of cryptococcal infection is likely to fall as 90–90–90 rolls out. Some countries use extensive fluconazole prophylaxis, as this strategy antedated screen and treat, and this is also effective in preventing cryptococcal meningitis and deaths . However(a)6 l−1 CD4 cells [6 l−1. Those who default from ART or develop antiretroviral resistance are also at risk, but it is difficult to estimate this caseload, so 450 000 is probably a substantial underestimate.It is not known how many patients with AIDS develop PCP. The incidence or point prevalence varies substantially, increasing as gross domestic product increases , from raD4 cells –36, an ed glucan is almost always detectable in the serum of patients with PCP, so can be used as a ‘rule-out’ test, but is non-specific if elevated [All patients with PCP and AIDS who are not treated die. Diagnosis is usually clinical, but the presentation is often atypical, late and indistinguishable from bacterial pneumonia and pulmonary TB . Moleculelevated . DiagnosAssuming that 60% of patients are treated and 70% of them survive, as is the case in most LMICs ,48, the Additionally, there is a strong case for improving the diagnosis of PCP because use of high-dose cotrimoxazole carries much toxicity. Adverse reactions of high-dose cotrimoxazole include nausea and vomiting (90%), reduced oral intake in already malnourished patients, neutropenia (50%), pruritic rashes (33%), elevated liver function tests (33%) and significant anaemia (20%) ,55. A ne(b)The number of DH cases in AIDS has been estimated to be between 100 000 and 300 000 ,57. VeryHistoplasma capsulatum, a slow growing, small, intracellular yeast that enters the body via inhalation. Bat guano is its primary ecological niche, and there is substantial seasonal variation in case numbers. The clinical presentation of DH is subtly different from TB in patients with AIDS, with more gastrointestinal and fewer respiratory features, pyrexia and usually some degree of pancytopenia, also seen as part of HIV infection itself. Most patients with DH in the context of AIDS are in their 30s, and death without therapy usually occurs in 10–14 days. The fungus typically takes two weeks to grow on mycological media and does not grow on media used for bacterial culture. Patients with CD4 counts < 200 × 106 l−1 are at most risk [The fungus that causes DH is ost risk . Rapid dost risk  and moleost risk ,65.The antifungal agents of choice for DH are amphotericin B and itraconazole; fluconazole is not effective for DH in AIDS. Improved outcomes with early diagnosis and appropriate therapy can achieve 85% survival ,67.Taking the most conservative estimate of annual incidence, of 100 000 cases and assuming that the diagnosis is only made in 40% of patients currently with a 50% survival in those treated, including relapse deaths ,69, we wIf antigen and/or PCR were made available in all high incidence communities in the Americas , and both itraconazole and amphotericin B were also used, we would anticipate a reduction of approximately 60% of DH deaths by 2020. This translates into a survival gain of 48 000 lives annually by 2020 , and cum(c)Aspergillus fumigatus IgG is the key diagnostic test, [CPA is a progressive and usually fatal complication of multiple pulmonary disorders, notably TB, chronic obstructive pulmonary disease and those with non-tuberculous mycobacterial infections . Its radic test,  but is bic test, ,73. ART Aspergillus IgG antibodies and 40% of these patients died within two months [Aspergillus IgG antibody detection method used in this Kampala study had a 96% sensitivity and 98% specificity for CPA [About 935 000 patients developed pulmonary TB in the context of HIV infection in 2013 . Of theso months . In HIV- for CPA . We haveAspergillus antibodies were found in 34% of post-TB patients in UK in the 1960s, and 30% of those during treatment for pulmonary TB in Iran [In addition, there were 255 850 HIV-infected survivors of ‘smear-negative’ TB in 2013. Another prospective study showed that 2–7 years after TB treatment, 8.5% of Ugandan patients had CPA, whether HIV infected or not . This is in Iran ,80. Assu in Iran . These tAspergillus IgG testing, and availability of itraconazole and amphotericin B treatments, for 60% of patients, and is 60% effective , then mortality should fall to 20 600 (If the diagnosis and treatment of CPA are enabled by provision of o 20 600 . If the (d)Early diagnosis and treatment of the most common fungal diseases are important steps in preventing AIDS-related deaths. The actions described in The 2013 WHO cryptococcal disease guidelines  recommenwww.microfungi.net). Many molecular diagnostic tests are available but require a frozen transport chain and reliable electricity and are also expensive. Diagnosis with the β 1,3-d glucan test as a rule-out test will not be feasible for many settings owing to high costs of this assay. A low-cost diagnostic test is required for LMICs. While the best therapy, co-trimoxazole, is almost universally available, and the WHO has issued guidelines on its use for prophylaxis, there are no WHO guidelines on treatment of PCP, although the American Thoracic Society has issued management recommendations [PCP is a major AIDS-defining illness. Diagnosis is usually empirical and based on severity and sometimes obvious, but late clinical features. Concurrent bacterial infection with PCP is common ,85, and ndations . There iThe annual incidence of histoplasmosis is not known in many countries in which it is endemic. This is a barrier to adoption of the commercially available rapid ELISA-based antigen assays now commercially available, and simple low-cost lateral flow assay that will be available soon. Despite its importance in the treatment of histoplasmosis, itraconazole is not on the WHO EML, affecting availability in many countries. Similar to PCP neither the WHO nor Pan American Health Organization (PAHO), where the largest burden of disease falls, have developed guidelines for the treatment of histoplasmosis; publication and dissemination would improve both diagnosis and treatment and prevent deaths.Aspergillus antibodies are found in both groups [Aspergillus antibodies and a two-month 40% mortality were found in a group of hospitalized smear- and mycobacterial culture-negative patients in Kampala, clearly indicating that many HIV patients have pulmonary aspergillosis. Whether these cases are strictly CPA or represent invasive aspergillosis [Aspergillus IgG antibody detection. Fungal cultures for Aspergillus spp. are insensitive for CPA, not specific and may be laboratory contaminants. Recent European guidelines on the diagnosis and management of CPA are helpful [The annual incidence and 5-year point prevalence of CPA after TB were estimated using 2007 TB data and published in 2011; 372 385 incident cases per year and a prevalence of 1 173 881 (range 852 048–1 372 457) patients affected . At thath groups . Therefogillosis  is conjeTalaromyces (Penicillium) marneffei infection [These estimates of lives saved ignore other potentially fatal but treatable fungal diseases in AIDS, including oesophageal and invasive candidiasis ,90, paranfection ,95. Enhanfection .6.Scenario modelling and limited epidemiological data strongly suggest that the annual 700 000 AIDS deaths related to the four most common lethal fungal infections could be significantly reduced by the wide adoption of current diagnostic tests, antifungal agents and known systematic strategies. As the true burden of lethal fungal infections is not known, these estimates are probably conservative, although the recent (2014) UNAIDS revision downwards of AIDS deaths to 1 200 000 (from 1 500 000 in 2013) will likely alter the precise estimates, but not the thrust of the arguments.Depending on implementation and uptake, over 300 000 deaths might be saved annually, in addition to those saved by ART rollout, bringing total deaths down to 426 000, well below 500 000, by 2020 and lower subsequently, aiming to achieve the Millennium Development Goal for 2030 of ‘ending AIDS’. Preventing deaths from AIDS represents excellent value for money in a medical and broad societal sense as it prevents the loss of the most economically active participants in society. These lives can be saved in a manner that avoids duplication of efforts, by integrating fungal disease interventions and treatment guidelines into existing HIV and TB programmes and national strategies.