To examine patterns of human immunodeficiency virus (HIV) cause-specific deaths, risk factors, and the effect of interactions on mortality, we conducted a retrospective cohort study in Zhejiang, China, from 2006 to 2013. All data were downloaded from the acquired immune deficiency syndrome (AIDS) Prevention and Control Information System. The Cox proportional hazards model was used to assess predictors of cause-specific death. The relative excess risk due to interaction and ratio of hazard ratios (RHR) were calculated for correlations between HAART, late diagnosis, and age. A total of 13,812 HIV/AIDS patients were enrolled with 31,553 person-years (PY) of follow-up. The leading causes of death of HIV patients were accidental death and suicide (21.5%), and the leading cause of death for those with AIDS was AIDS-defining disease (76.4%). Both additive and multiplicative scale correlations were found between receiving HAART and late diagnosis, with RERI of 5.624 (95% CI: 1.766–9.482) and RHR of 2.024 (95% CI: 1.167–2.882). The effects of HAART on AIDS-related mortalities were affected by late diagnosis. Early detection of HIV infection and increased uptake of HAART are important for greater benefits in terms of lives saved. It has been estimated that by the end of 2011, there was a cumulative total of 78,000 people living with HIV (PLWH) in China, with 14,293 in Zhejiang province aloneThe first case of HIV infection in Zhejiang province was reported in 1985, with escalating numbers of cases over the subsequent three decades3After the initiation of China’s national Free Antiretroviral Treatment Program in 2002, Zhejiang province launched its program in 2003 and scaled up this program over time. Over 90% of HIV/AIDS patients needing treatment accepted highly active anti-retroviral therapy (HAART) by December 20105Consequently, with the life expectancy of HIV patients substantially improved, causes of death among HIV-infected individuals have become more diverse, and deaths from AIDS-related and non-AIDS-related diseases have continued to change over the years7911Our aim was to provide a broad and up-to-date report on risk factors and all causes of AIDS-related and non-AIDS-related mortality in Zhejiang province. We also evaluated whether there is an interaction between HAART treatment and late diagnosis on the risk of AIDS-related mortality.This study is a retrospective observational cohort study in Zhejiang province with continued enrollment of HIV-infected persons at least 15 years old who were diagnosed between 2006 and 2013.Data on PLWH were collected from the AIDS Prevention and Control Information System, which generates the national epidemiology database. The data in this system are collected compulsorily during routine tracking and clinical treatment of patients. The forms used for data collection are standardized nationwide, and include demographic, treatment, death, and laboratory information completed by health care facilities [hospital and local Centers for Disease Control and Prevention (CDC)] every 3 months for AIDS patients and every 6 months for HIV+ patients.All the data from January 1, 2006 to December 31, 2013 were downloaded on July 1, 2014. An individual was defined as eligible for this study when his/her HIV infection was confirmed by Western blotting. Date censoring occurred at: a) the date of the last visit for those lost to follow-up for at least half a year; b) the date of death (AIDS-related or non-AIDS-related); and c) the end of this study on December 31, 2013 for patients who continued to be followed up.All records in the databases underwent quality control by regular database verification with regards to diagnosis of HIV infection, AIDS progression, and cause of death.3 or at least one AIDS-defining illness as defined by the World Health Organization (WHO) stage 3 or 4 diseaseCases of AIDS were reported by the local CDC based on laboratory evidence of HIV infection and CD4+ T-cell count <200 cells/mm3 or after 2011, <350 cells/mm3  Control and Prevention (NCAIDS) reporting system and analyzed anonymously, informed consent was not necessary. In China, we need to complete all of the work designated by the China CDC for HIV prevention and control, including HIV testing, HIV infection report, CD4 testing at least once a year, HAART, routine follow-up, death report, and so on. At the same time, we collect related data and upload it online as China CDC has requested. All of these procedures are routine work. This study is based on these data from routine work. We did obtain oral informed consent before HIV testing and CD4 testing as diagnosed.2 or Fisher’s exact tests were used to compare categorical variables. Mortality rates were calculated with a life table in person-years (PY); the numerator represents the number of deaths and the denominator represents the total person-years of patients at risk of death. Predictors for mortality were assessed by univariate and multivariate Cox proportional hazards regression. Crude and adjusted hazard ratios (HR) with 95% confidence intervals (95% CI) were estimated. The likelihood ratio test was used to evaluate model fit. To determine reasons for AIDS-related death and non-AIDS-related death caused by disease, those who died from drug overdose, suicide, or accidents were excluded from the model.Statistical analyses were performed using SPSS statistical software , and STATA . χPotential additive and multiplicative interactions were also assessed for initiation of HAART and age and initiation of HAART and late diagnosis. In the additive interaction analysis, factors were recorded in which the stratum with the lowest risk was considered as the reference, while both factors were considered jointly. Relative excess risk due to interaction (RERI) and 95% CIs were used to evaluate additive interactions. If 95% CIs of RERI included 0, there was no obvious additive interaction. Multiplicative interactions were calculated by the ratio of hazard ratios (RHR) and 95% CIs. If 95% CIs of RHR included 1, there was no obvious multiplicative interaction. For unbiased HR for interaction, we adjusted for potential confounding factors including age, gender, education level, area of registry, area of residence, transmission route, last CD4+ T-cell count, and coinfection with tuberculosis (TB).p ≤ 0.05 level; all hypothesis tests were two-sided.Significance was determined at the Enrollment and follow-up of the 13,812 HIV/AIDS patients in the registry with 31,553 PY follow-up is shown schematically in Of the total 13,812 HIV/AIDS patients, 1,495 (10.5%) died between 2006 and 2013, with a yearly mean mortality rate of 4.1  per 100 PY. Of the total 5,656 AIDS patients, 741 (13.1%) died, with a yearly mean mortality rate of 4.9  per 100 PY.Between 2006 and 2013, all causes of mortality decreased from 7.58 per 100 PY (2006) to 3.76 per 100 PY (2013) among HIV/AIDS patients (reduced by 50.5%), and from 8.87 per 100 PY (2006) to 5.45 per 100 PY (2013) among AIDS patients (reduced by 38.6%)  and 3. AOf all deaths in our study, 64.9% (970/1495) were among AIDS patients, and 49.5% (260/525) and 60.2% (584/970) of deaths occurred at ages over 40 years in HIV patients and AIDS patients, respectively. Leading causes of death for HIV patients were accidental death and suicide (21.5%), non-AIDS-associated malignancies (17.5%), and lung disease (excluding cancer) (14.5%). For AIDS patients, the most frequent underlying causes of death were AIDS-associated death (76.4%) and non-AIDS-defining malignancies (4.3%). There were more suicides among HIV patients under 40 years old . For all3: HR 0.160, 95% CI 0.125–0.204; 200–499 cells/mm3: HR 0.059, 95% CI 0.045–0.079; ≥500 cells mm3: HR 0.034, 95% CI 0.017–0.068, compared to CD4+ T-cell count <100 cells/mm3) , having been diagnosed with AIDS within one year of being diagnosed with HIV, compared to diagnosed with AIDS more than one year of HIV diagnosis , and last CD4+ T-cell count (100–199 cells/mmlls/mm3) . Other rlls/mm3) .3: HR 0.353, 95% CI 0.230–0.543; 200–499 cells/mm3: HR 0.234, 95% CI 0.164–0.334; ≥500 cells/mm3: HR 0.040, 95% CI 0.102–0.175, compared to CD4 + T-cell count <100 cells/mm3). HAART uptake  was an important preventive factor , late diagnosis, compared to early diagnoses , infection by heterosexual contact, compared to infection by homosexual contact , and last CD4+ T cell count (100–199 cells/mme factor . In addie factor .CI:-0.088–2.983) and multiplicative scale . Both additive and multiplicative scale interactions were found between HAART and late diagnosis, with a RERI = 5.624 (95%CI: 1.766–9.482) and RHR = 2.024 (95%CI: 1.167–2.882).The combined influences of HAART and age and HAART and late diagnosis on AIDS-related mortality are shown in In this seven-year study of mortality trends among HIV-infected patients in Zhejiang province, we found that all-cause mortality and AIDS-related mortality continued to decrease from 2006 to 2013. Other national cohort studies in China and Switzerland also reported substantial reductions in mortality of HIV/AIDS patients after HAART became available6616The increasing diversity of causes of death in the HAART era has been recognized18192122Even though AIDS-defining diseases are the leading cause of death in HIV/AIDS patients, they have decreased in favor of other causes. The leading causes of death of HIV patients were suicide , accidents, and substance use, accounting for 26.3%. This rate was lower than that reported in many cohorts, such as 32.7% in a population-based cohort study in Spain26Our findings are consistent with those already published that indicate that HAART and delayed diagnosis play an extremely important role in reducing mortality, both for AIDS-related and non-AIDS-related deaths after adjusting for potential confounding factors282930The overall finding concurs with what is known about late diagnosis typically affecting mortality among HIV/AIDS patients. Late diagnosis of HIV-infected patients leads to late entry into HIV care, including HAART uptake. We analyzed the proportion of late diagnoses in 2006–2013 and found that it decreased slightly, accounting for almost 30%, which is lower than that in Brazil and in China11An important strength of our analysis is that it identified the combined influences of HAART treatment and late diagnosis and HAART treatment and age. The aim of enrollment of AIDS patients for interaction analyses was to diminish the impact of patients without need for HAART treatment. Based on our results, the impact of going without HAART and late diagnosis on the AIDS mortality rate is more additive than the two factors present individually. Patients who were older or diagnosed late were more likely to die and progress to AIDS than younger people and those diagnosed early3032As with all cohort studies, selection bias from loss to follow-up may exist. We compared the demographic characteristics of participants at follow-up and those lost to follow-up and found that most of the patients who were lost to follow-up were younger (80% were ≤40 years old) and female migrants. The differential selection bias may lead to overestimation of the associations. For AIDS-related mortality, only 1.5% of patients were lost to follow-up, and selection bias can be overlooked. In addition, even though potential confounding factors were considered on the basis of prior knowledge and confounding assessment, there were still some unknown and unassessed confounders generating bias. Despite potential limitations, the current study had a large sample size, and we were able to evaluate both of the main associations between HAART and other factors.In conclusion, our study shows that the decline in mortality occurred stably in the HAART era. There is strong evidence that HAART treatment and early diagnosis have substantially reduced AIDS-related and non-AIDS-related mortality. The effect of HAART on AIDS mortality was affected by age and time of diagnosis, suggesting that detection of potential HIV infection and having HAART treatment  are important and bring even larger benefits in terms of lives saved.How to cite this article: Chen, L. et al. HIV cause-specific deaths, mortality, risk factors, and the combined influence of HAART and late diagnosis in Zhejiang, China, 2006–2013. Sci. Rep.7, 42366; doi: 10.1038/srep42366 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


A highly sensitive strain sensor based on a novel fiber in line Mach-Zehnder interferometer (MZI) was demonstrated experimentally. The MZI was realized by splicing a section of photonic crystal fiber (PCF) with the same length of thin core fiber (TCF) between two single mode fibers (SMFs). The fringe visibility of MZI can reach as high as 20 dB in air. In particular, the strain sensitivity of −1.95 pm/με was achieved within a range from 0 to 4000 με. Furthermore, the strain properties of different length of MZI was investigated. It was found that the sensitivity was weekly dependent on the length of MZI. The strain sensitivities corresponding to the MZI with 35 mm PCF, 40 mm PCF and 45 mm PCF at 1550 nm band were −1.78 pm/με, −1.73 pm/με and −1.63 pm/με, respectively. Additionally, the sensor has advantages of simple fabrication, compact size and high sensitivity as well as good fringe visibility. Optical fiber sensors has been widely used to monitor some parameters in sensing fields, such as temperature , refractIn this paper, we proposed a novel strain sensor based on MZI with TCF-PCF structure, which was formed by splicing a piece of TCF with the same length of PCF between two SMFs. The fusion point was more robot than that of up-tapered joint or collapsed fusion region, which making the sensor having merits of good mechanical strength and low fusion lost. In addition, a high strain sensitivity of −1.95 pm/με with a good linearity of 0.9971 in the range of 0~4 mε was achieved. Moreover, the dependence of the stain sensitivity on the length of MZI was also investigated experimentally.As shown in When axial strain is applied, the length of MZI will be increased, the resonant wavelength shift can be expressed by differentiating the Equation (1), which can be given as ,23:(2)ΔλIt can be seen that the resonant wavelength shift is a linear function of applied strain ε. Meanwhile, the strain sensitivity is mainly dependent on the change of The transmission spectra of the TCF-PCF structure with 35 mm, 40 mm and 45 mm length of PCF are shown in 2 of 0.9978, 0.9982, 0.9986 and 0.9971 for dip1, dip2, dip3 and dip4 were obtained, respectively. From the investigation, it is found that the strain sensitivity of the MZI is closed to the resonant wavelength of cladding modes and the wavelength with higher order cladding modes tend to be more sensitive to the applied strain. This is because the 2 laser (0.63 pm/με) [Additionally, the strain characteristics of sensor with different length were also investigated experimentally. In order to compare the strain characteristics of low order cladding mode and high order cladding modes, we chosen two dips around 1550 nm and 1580 nm to compare and analysis. From 3 pm/με) , Bragg g3 pm/με)  and some3 pm/με) , polariz3 pm/με) , twin-co3 pm/με) , tapered3 pm/με) , PM-PCF 3 pm/με)  and PCF 3 pm/με)  and modi3 pm/με) . However−4 dBm/με with a linearity of 0.9668 and −10.3 × 10−4 dBm/με with a linearity of 0.9945, respectively. From the results, it can be seen that the transmission loss change depends on the cladding mode and the length of MZI.The transmission loss change of the MZI with different length of PCF was also illustrated in In conclusion, we have achieved a strain sensor based on TCF-PCF structure MZI, which is fabricated by splicing a section of TCF and PCF between two SMFs. The novel structure MZI has shown better fringe visibility than that of normal PCF-MZI and a strain sensitivity as high as −1.95 pm/με was achieved in the range of 0~4000 με, which is larger than that of MZI based on tapered PCF and PM-PCF. In addition, the proposed sensor is easy to fabricate and exhibits large strain measurement range as well as low production lost, which means that the structure is attractive for the development of strain sensors.


The following information is missing from the Funding section: This research has been supported by the President’s Emergency Plan for AIDS Relief (PEPFAR) through the U.S. Centers for Disease Control and Prevention. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The following information is missing from the Competing Interests section: This research was presented in part at the 20th International AIDS Conference, Melbourne, Australia, 20–25 July, 2014. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the U.S. Centers for Disease Control and Prevention. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.


M-E/G/M-S/G-A/G/F-XG”. We also identified several hundred potential Photofrin-binding proteins using affinity purification coupled with LC-MS/MS, and confirmed the bindings of EGFR and cathepsin D with Photofrin. The enzyme activities of both proteins were significantly reduced by Photofrin-PDT. Our results shed light on the global and site-specific changes in Met-peptide oxidation among cells undergoing Photofrin-PDT-mediated oxidative stress originating from distinct subcellular sites, and suggest numerous potential Photofrin-binding proteins. These findings provide new insight into the molecular targets through which Photofrin-PDT has diverse effects on target cells.In Photofrin-mediated photodynamic therapy (PDT), cell fate can be modulated by the subcellular location of Photofrin. PDT triggers oxidative damage to target cells, including the methionine (Met) oxidation of proteins. Here, we developed a new Met-containing peptide enrichment protocol combined with SILAC-based quantitative proteomics, and used this approach to explore the global Met oxidation changes of proteins in PDT-treated epidermoid carcinoma A431 cells preloaded with Photofrin at the plasma membrane, ER/Golgi, or ubiquitously. We identified 431 Met-peptides corresponding to 302 proteins that underwent severe oxidation upon PDT and observed overrepresentation of proteins related to the cell surface, plasma membrane, ER, Golgi, and endosome under all three conditions. The most frequently oxidized Met-peptide sequence was “QAMXXM Photofrin is the most widely used photosensitizer in clinical PDT, and was the first such drug approved by the FDA for cancer treatment. In PDT, cells or tissues are exposed to a photosensitizing drug (a non-toxic dye), harmless visible light, and oxygen to produce highly reactive oxygen species(ROS) that cause tumor destruction1. More specifically, the light-stimulated photosensitizer reacts directly with biological substances and/or transfers energy to oxygen to generate singlet oxygen. This highly reactive ROS attacks many biological molecules, including lipids2, proteins3, and nucleic acids4, to cause cell death7. PDT can induce different cell fates depending on the cell type10, the utilized photosensitizer, the treated subcellular site14, and the total administered dose16. All these factors are interdependent17.Photodynamic therapy (PDT) has been approved by the U.S. Food and Drug Administration (FDA) and other health agencies in many countries for the clinical management of various cancers15. We further showed that PDT with plasma-membrane-targeted Photofrin induces necrosis-like cell death, whereas that with ER/Golgi-localized Photofrin triggers the formation of perinuclear vacuoles via SERCA dysfunction and is highly correlated with the location of the ROS generated by the treated cells16. Thus, distinct signaling events appear to be triggered when different parts of the cell are subjected to the oxidative stress elicited by Photofrin-mediated PDT. Protein oxidation exerts diverse biological effects and is a major molecular consequence of the PDT-induced generation of intracellular ROS. Therefore, PDT of human tumor cells preloaded with Photofrin at different subcellular sites appears to offer an excellent opportunity to study the protein oxidation events that occur in different subcellular locations subjected to oxidative stress. However, no previous study has examined the global or site-specific protein oxidation of PDT-exposed cells in which the photosensitizer has been dispensed to distinct subcellular locations.For Photofrin-mediated PDT, the subcellular location of the agent strongly influences the cell death response. We previously showed that Photofrin is dynamically distributed in human epidermoid carcinoma A431 cells treated with the agent in the medium: it is initially localized at the plasma membrane, but prolonged incubation allows it to enter the ER/Golgi18, signal transduction and gene expression19, apoptosis20, the antimicrobial and cytotoxic actions of immune cells21, aging22, and age-related degenerative diseases23. Proteins are among the major biomolecules targeted by ROS in cells; among the constituents of a protein, the most readily oxidized amino acid is methionine (Met), which can be attacked by various ROS, including H2O2, hydroxyl radicals, singlet oxygen, etc.25. Oxidation of Met residues can alter the protein structure, leading to the loss of enzyme activity and/or protein-protein interaction properties, as seen for calmodulin26, HIV-2 protease27, and alpha-1 antitrypsin28. Another example is caspase-3, as we previously showed that Photofrin-PDT oxidizes the Met residues of procaspase-3 and impairs its activation29.Protein oxidation has both positive and negative consequences for various biological processes, including receptor activationet al.30 generated polyclonal antibodies against oxidized Met residues, but the applicability of this strategy for the proteome-wide investigation of Met oxidation has not yet been explored. Rosen et al.31 used spectral counting to determine the percentage of Met oxidation, but did not consider the differences of ionization efficiency between oxidized and reduced Met-peptides. Gevaert et al.33 reported a COFRADIC  proteomics technology for the identification and quantification of the Met oxidation of proteins. However, this approach requires numerous steps for peptide preparation and separation, and its success critically requires the skillful application of chromatographic techniques. Whereas the above-mentioned methodologies did not use enrichment protocols to analyze Met-peptides, Grunert et al.34 applied a solid-phase reagent  to enrich the reduced form of Met-peptides for subsequent analysis by MALDI-TOF/MS. The authors provided evidence that the process removed the oxidized Met-peptides, but they assessed only model proteins. The use of this technique for proteome-wide analysis has been very limited36.Since Met residues are highly susceptible to being oxidized by various types of ROS, several groups have sought to characterize and/or quantify the Met oxidation of proteins. Oien et al.37 for Cys-peptide enrichment. We applied this enrichment method in conjunction with SILAC (stable isotope labeling by amino acids in cell culture) technology and used on-line 2D-LC-MS/MS to quantify global changes of Met oxidation in human tumor cells subjected to Photofrin-PDT-induced oxidative stress at various subcellular locations. Using this method, we detected and quantified a significant number of Met-peptides that were not detected by shotgun proteomics approach without enrichment in PDT-treated cells. We observed that the distribution of the severely oxidized proteins was well correlated with that of Photofrin. We identified 803 potential Photofrin-binding proteins in human cells, and found that 94 of them were severely oxidized in PDT-treated cells. Our data reveal for the first time the profile of Met oxidation that occurs at distinct subcellular sites of living cells subjected to Photofrin-PDT-induced oxidative stress. The developed methodology can be widely and systematically applied to other oxidative-stress-related studies.In the present study, we developed an efficient Met-peptide enrichment methodology based on the selective reaction of iodoacetyl-PEG2-biotin with reduced Met-peptides under acidic conditions, as originally described by Gygi 38, whereas methionine sulfoxide (MetO)-containing peptides (the oxidized form) and Met-free peptides do not react with iodoacetate34. Therefore, the relative oxidation level of specific Met-residues can be deduced from the ratio of the reduced Met-peptide levels observed before and after oxidative stress. Here, we applied the well-established iodoacetyl-PEG2-biotin reagent, which was originally designed to capture cysteine-containing peptides, to capture the reduced Met-peptides in biological samples. We used the following protocol  or light (CH2O) formaldehyde, and the resulting heavy peptide was oxidized by H2O2 to generate oxidized heavy peptide. Both oxidized (H) and reduced (L) peptides were purified by HPLC, quantified by LC-UV, equally mixed and then captured by the iodoacetyl-PEG2-biotin-based protocol. The enriched Met-peptides were released and analyzed by LC-MS/MS. A small portion of the equally mixed peptides was also analyzed by LC-MS/MS before the capture experiment. The results from this quantitative analysis clearly showed that (i) a very small percentage (~2.3%) of the oxidized Met-peptides can be captured as compared to its reduced form, which might result from the non-specific binding to streptavidin beads; and (ii) a small portion of the reduced (L) Met-peptides, as expected, become oxidized (to generate the oxidized (L) Met-peptides) during the enrichment procedure  for enriching Met-peptides derived from the lysates of PDT-treated A431 cells. Again, we could only detect ~31% of the Met-peptides in the trypsin-digested lysates of PDT-treated A431 cells without any enrichment process  or subjected to laser irradiation (PDT group with SILAC labeled light), and proteins extracted from the paired cell groups were equally mixed and trypsin-digested. The samples were directly analyzed by 2D-LC-MS/MS (non-enriched sample) or subjected to Met-peptide enrichment followed by 2D-LC-MS/MS analysis (enriched sample). A label-swap replication of the SILAC experiment  was performed for each condition, in an effort to improve the accuracy of our quantitation. Finally, the MS data obtained from the 12 LC-MS/MS runs were processed for protein and peptide identifications/quantifications .Our previous studies showed that the distribution of Photofrin in living cells depends on the incubation protocolIn each of the six non-enriched samples, we identified more than 24,000 peptides with an average Met-peptide percentage of ~30% . In each of the six enriched samples, we identified ~12,900 peptides with an average Met-peptide percentage of 83%  Fig. . Only 3,39. This could lead to underestimation of the change in Met-peptide oxidation  corresponding to 302 proteins that showed significantly altered Met oxidation Table . AlthougFrom non-enriched samples, we selected Met-peptide pairs with both oxidized and reduced forms that showed significant changes upon PDT treatment . Collectively, these results indicate that the systematic analysis of oxidized and reduced Met-peptides can be used to estimate the proportion of Met-peptide oxidation and the cellular oxidative status.Next, we analyzed the global distributions of the PDT/Ctrl ratios for all of the quantified peptides and proteins Fig. , and fur40. We then grouped the quantified Met-peptides by their related cellular structures (organelles), and quantitatively analyzed the Met oxidation of proteins according to the median ratios and 25th to 75th percentiles in the different GO categories  analysis41, using the non-oxidized Met-peptides as a reference set. This analysis revealed that “QAMXXMM-E/G/M-S/G-A/G/F-XG” (the bold M indicates the oxidized Met residue) is the consensus sequence for the oxidized Met-peptides  Two to three additional Met residues are located near the oxidized Met residue, potentially offering an antioxidant effect and acts as an oxidant scavenger for proteins to respond to oxidative stress43.To search for a consensus sequence for the Photofrin-PDT-induced Met oxidation of proteins, we calculated the frequency of various amino acid residues around the oxidized Met residues in the 431 Met-peptides that showed significant oxidization under Photofrin-PDT Table . For thides Fig. . This se44. This, together with our previous finding that Photofrin binds procaspase-3 and mediates its PDT-triggered Met oxidation and inactivation29, prompted us to search for additional Photofrin-binding proteins in human cells. We hypothesized that such proteins might be more susceptible to oxidative damage during Photofrin-mediated PDT. We coupled Photofrin onto carboxyl-linked agarose beads and used them to pull down proteins from A431 cell lysates. As shown in Fig. It is well known that PDT mainly attacks biomolecules near the photosensitizer, due to the very short lifetime of ROS in biological systems (<0.04 ms) and the small action radius of singlet oxygen (<0.02 mm)45, was almost completely blocked by Photofrin, regardless of PDT  and cathepsin D  for further validation. As expected, Western blot analysis confirmed that Photofrin-coupled beads could pull down both EGFR and cathepsin D from A431 cell lysates Fig. . InteresPDT Fig. . Thus, o46. Most of the studies on how the redox status can alter protein functions have focused on Cys oxidation. However, Met oxidation has gained recent attention, as it has been shown to play important roles in mediating conformational changes and regulating protein functions, such as by modulating actin assembly47, altering calcium signals26, and preventing phosphorylation49. Several studies have sought to systematically identify oxidized Met residues in proteins under oxidative stress using quantitative redox proteomics approaches. Various separation strategies and/or enrichment protocols have been used to reduce sample complexity and enhance the identification of Met-peptides in biological samples. Previously, the COFRADIC procedure developed by Gevaert et al.33 appeared to the most successful in identifying and quantifying Met oxidation on a proteome-wide scale. Here, we used the previously reported chemistry38 and a commercially available iodoacetyl-PEG2-biotin reagent37 to develop a protocol that selectively captures reduced Met-peptides in biological samples. Starting from 200 μg cell lysates, this simple protocol enabled us to identify >10,000 Met-peptides on average, with less than 17% non-Met-peptide contamination  can bind and partition into lipid bilayer membranes52, and our data showed that the proteins which reside in membrane-bound cellular structures  are among the most severely oxidized cellular proteins in all three PDT conditions -mediated PDT. Of them, 66 were also found as severely Met-oxidized proteins in our current study, including GRP78, annexin A1, EGFR, heat shock proteins, etc. 58. There are 98 proteins sensitive to other oxidative stresses; of them, 53 were also identified in this study, including heat shock cognate 71 kDa protein, cathepsin D, protein disulfide-isomerase, myosin-9, moesin, elongation factor 2, alpha-actinin-1, T-complex protein 1 subunit alpha, etc.  are ER, Golgi, or mitochondrial proteins , prion (KPKTN109MKH112MAGAA and GLGGY129MLGSA134MSRPII), actin (HQGV44MVG47MGQKDS), the sodium channel, Nav3.1 (LLFAL1476M1477MSLPAL), and caspase-3 (DNSYK39MDYPE44MGLCII)29. In this study, calmodulin showed severe oxidation (PDT/Ctrl Log2 ratio = 4.34) at Met144/145, which is located at the most C-terminal surface-exposed part of the protein. These two Met residues are reportedly important for the tertiary structure maintenance and functional regulation of this protein26. The coordination of neighboring Met residues in the 3D structure is not easy to assess. However, the identification and quantification of oxidized Met-peptides by MS plus 3D-structure analysis can provide important clues as to how spatially adjacent Met residues coordinate with each other to regulate protein functions.In the present study, we detected 302 severely oxidized proteins represented by 431 Met-peptides that contained a total of 501 Met residues Table . We analPDT Fig. . Of the PDT Fig. . Interes111MYYENSYALAVLSNYDANK and AcGADSYE318MEEDGVR  represented the most severely oxidized Met-peptides of EGFR. According to the known crystal structure of EGFR, both Met111 and Met318 are surface-exposed, while Met111 is located at the bottom of the EGF-binding pocket. Oxidation of Met111 might therefore interfere with the interaction between EGFR and EGF. Furthermore, Met766 is located near activation loop during dimerization, and it is possible that Photofrin-PDT-triggered Met766 oxidation may interfere EGFR intracellular domain dimerization and inter-molecule phosphorylation61. Regarding cathepsin D, PDT most severely oxidized Met246, which is close to the active site Asp97/295 identified in the resolved 3D structure of cathepsin D62. Thus, further detailed investigations may be warranted to elucidate how Met oxidation can regulate the activities of EGFR and cathepsin D.We identified 94 proteins as potential Photofrin-binding proteins that could also undergo severe Met oxidation upon PDT of A431 cells Table . We seleEnvironmental or drug-induced oxidative stress can cause Met oxidation of cellular proteins that participate in diverse biological processes. However, the global and quantitative analysis of protein Met oxidation remains challenged by technical limitations. This study describes a new method for Met-peptide enrichment and its combination with a SILAC-based quantitative proteomics approach, and our data provide a first insight into the oxidation status of distinct organelles in cells subjected to different Photofrin-PDT regimens, and suggest potential protein targets through which Photofrin-PDT may elicit its diverse effects on cancer cells.32. For SILAC experiments, cells were maintained in SILAC medium comprising Dulbecco’s modified Eagle medium  supplemented with10% dialyzed FBS (Invitrogen). L-lysine and L-arginine(Sigma-Aldrich) or [13C6]-L-lysine and [13C6]-L-arginine  were added at 0.1 g/L for light or heavy stable isotope labeling, respectively, and cells were cultured for at least 10 doubling times to achieve 95% incorporation64.Human epidermoid carcinoma A431 cells were cultured as previously described15. For condition I, cells were incubated with 28μg/ml Photofrin (2.5 mg/ml stock solution in 5% dextrose) in culture medium for 3 h in the dark. For condition II, cells were incubated with 28 μg/ml Photofrin for 3 h, washed with fresh medium, and incubated with Photofrin-free medium for an additional 21 h. For condition III, cells were incubated with 28 μg/ml Photofrin for 24 h. After Photofrin loading, the cells were washed once with fresh medium and irradiated with a 632.8 nm He–Ne laser (Coherent) at a fluence rate of 15 mW to yield a total energy of 10 J/cm2. Cells treated under each condition and untreated paired control cells were washed with cold phosphate-buffered saline (PBS) and lysed in 0.1% SDS. The cell lysates were vortexed at room temperature for 15 min, sonicated in an ice bath, and centrifuged at 10,000× rpm for 10 min to remove insoluble debris. The protein concentration of each lysate was determined using a BCA protein assay kit (Thermo Scientific).A431 cells were loaded with Photofrin (Pinnacle Biologics)in the dark at different subcellular sites using the previously described protocols2-biotin (Thermo Scientific) were dissolved in 6 μl dimethyl sulfoxide /18 μl acetonitrile /0.15 μl acetic acid (Sigma-Aldrich) and purified on Source 15 S resin (GE Life Sciences) to remove contaminants. A431 SILAC cell lysates (200 μg) were reduced, alkylated, digested, desalted, and vacuum dried. The dried peptide mixtures were mixed with pre-cleaned iodoacetyl-PEG2-biotin reagent . The ACN was removed with a speed vacuum, and the mixture was incubated at 50 °C for 6 h in the dark. After labeling, the excess iodoacetyl-PEG2-biotin reagent was removed using Source 15S resin, and the samples were eluted with 1 M sodium chloride/100 mM Tris pH 8.5. The eluted peptides were diluted 5-fold with water and shaken gently with 100 μl of streptavidin sepharose beads (GE Life Sciences) for 30 min at room temperature. After affinity capture, the sample was centrifuged at 1000 × g for 3 min, and the beads were washed three times with 0.2 M sodium chloride/20 mM Tris, pH 8.5. After the washing step, the beads were resuspended in 1 M Tris, pH 8.8, and 2 M DTT at 50 °C for 5 h to release the peptides. The released peptides were desalted using Source 15RPC resin, eluted by stepwise ACN  and identified by LC-MS/MS.Three hundred micrograms of iodoacetyl-PEGTwo hundred micrograms of cell lysates were digested by trypsin and reacted with bromoacetyl-glass beads under acetic condition as in the manufacturer’s instruction . After capture, the glass beads were washed and released by 1 M ammonium bicarbonate and 14% beta-mercaptoethanol. Then the samples were cleaned up by the reverse phase silica in the kit as described and analyzed by LC-MS/MS.The identified/quantified peptides were classified according to the cellular distributions of their parent proteins, as assessed by GO using the Proteome Discoverer software (Thermo Scientific). The ratios of oxidized and reduced Met-peptides classified into each organelle/subcellular site were analyzed with SigmaPlot .15. Briefly, proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with monoclonal anti-EGFR (Santa Cruz Biotechnology), anti-cathepsin D, (Santa Cruz Biotechnology), or anti-phosphotyrosine (Millipore).Immunoblotting was carried out essentially as described previouslyN-hydroxy-succinimide; Thermo Scientific) and 10 μl CarboxyLinkTM coupling gel (Thermo Scientific) at room temperature for 3 h. The beads were then washed six times with PBS and used as Photofrin-coupled beads. Photofrin-coupled or control beads were incubated with 1 mg A431 cell lysate at 4 °C for 3 h, supernatants were collected, and the beads were washed three times with 1 M NaCl and three times with PBS. The washed beads were divided into two portions. The first was subjected to SDS-PAGE. The second was subjected to reduction/alkylation by DTT/IAA and eluted by 6 M guanidine hydrochloride . The eluted proteins were diluted 20-fold with 25 mM ammonium bicarbonate to a final concentration of 0.2 M GndCl, and then digested overnight with trypsin (enzyme: substrate = 1:50). The resulting tryptic peptides were desalted with C18 Z tips (Millipore) and subjected to dimethyl labeling for quantitative analysis (see below).Photofrin (100 μg) was mixed and incubated with 2.5 μl EDC  120 mg/ml in 0.1 M MES buffer, pH 4.7, 0.45 mg NHS (65. Dimethyl-labeled peptides from control and Photofrin-coupled beads (heavy/light and swapping) were mixed and subjected to 2D-LC-MS/MS analysis. The acquired data were processed using the Proteome Discoverer software (Thermo Scientific).Dimethyl labeling was performed according to the previously described protocol2). After treatment, 20 μl of assay buffer  was added, and the autophosphorylation reaction mixture was incubated at 30 °C for 10 min. The reaction products were resolved by 7.5% SDS-PAGE, transferred to PVDF membranes, and probed with anti-phosphotyrosine (Millipore).Purified EGFR (50 ng) in 10 μl of buffer A  was treated with or without Photofrin-PDT -PDT. The untreated/treated cathepsin D (10 ng cathepsin D in a volume of 10 μl) proteins were mixed with a 5 μl reaction mixture containing 0.5 μg proinsulin (R&D Systems), and then incubated at 37 °C for 10 or 30 min. The reaction was stopped by the addition of SDS-sample buffer, and the mixture was boiled for 5 min and analyzed by SDS-PAGE/silver staining.Recombinant human cathepsin D  was dissolved and activated in 25 μl assay buffer  at 37 °C for 30 minThe peptide ratios are transformed to log2 scale, and all statistical data are expressed as the mean ± SD. Peptides ratios were considered significantly changed when the values were beyond mean ± 2 SD. Correlation analysis between two ranked variables was performed using SPSS (IBM Corporation) to calculate Spearman’s rank correlation coefficient. The quantile plots were generated using SigmaPlot.Supplementary Information


Staphylococcus aureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determine whether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measured after incubation of Staphylococcus epidermidis, group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis with the antibiotic. All bacteria released phospholipids in response to daptomycin, which resulted in at least partial inactivation of the antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shown previously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. In conclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids, which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We showed previously that  Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant enterococci (VRE) . By. By16]. S. gordonii and the GAS strain completely inactivated daptomycin at 5 µg ml−1, but GBS strains only partially inactivated the antibiotic at this concentration. At 10 µg ml−1 daptomycin, only the staphylococci, S. gordonii and the enterococci showed significant inactivation of the antibiotic, while at a concentration of 20 µg ml−1 daptomycin, only staphylococci and enterococci inactivated the antibiotic to any significant degree, with a loss of 30–60 % of antibiotic activity. However, despite triggering phospholipid release, at 40 µg ml−1 daptomycin there was relatively little (<20 %) inactivation of the antibiotic by any of the bacteria tested. Therefore, phospholipid release is finite and can be overcome with a sufficiently high dose of daptomycin.To determine whether phospholipid release resulted in the inactivation of daptomycin, the activity of the antibiotic in the culture supernatants was measured using a previously described zone of inhibition assay  Fig. 1q. DaptomyThe predominant phospholipid in the membrane of Gram-positive bacteria is phosphatidylglycerol, with much smaller quantities of cardiolipin and/or lysyl-phosphatidylglycerol also present . Our preS. aureus releases phosphatidylglycerol in response to daptomycin, and that this results in inactivation of the antibiotic by revealing a very similar phenotype for S. epidermidis. Further, these findings also support the previous observation that E. faecalis releases phospholipids in response to daptomycin [S. gordonii, also released phospholipids and inactivated daptomycin, albeit less efficiently than E. faecalis. Therefore, daptomycin-induced phospholipid release appears to be a conserved mechanism across Gram-positive pathogens.These data extend our previous finding that ptomycin , and shoS. aureus. Therefore, we undertook further experiments with E. faecalis, which was the most efficient of the enterococci and streptococci at releasing phospholipids and inactivating daptomycin, and S. aureus, in which daptomycin-triggered phospholipid release has been well characterized [Next, we wanted to explore whether the mechanism of phospholipid release and daptomycin inactivation by enterococci and streptococci was similar to that of cterized .S. aureus, daptomycin-triggered phospholipid release is an active process that requires energy, as well as protein and lipid biosynthesis [E. faecalis exposed to daptomycin was occurring via an active process, or simply as a consequence of membrane damage caused by the antibiotic, bacteria were exposed to the antibiotic in the presence or absence of a sub-inhibitory concentration of the phospholipid biosynthesis inhibitor, platensimycin [S. aureus to daptomycin (10 µg ml−1) resulted in increased phospholipid in the supernatant, but this was significantly reduced in the presence of platensimycin at half the MIC (0.25 µg ml−1) , but this was blocked when platensimycin was present at half the MIC (0.5 µg ml−1)  . Similarµg ml−1) . The preptomycin  and signptomycin . This coE. facealis indicated that the labelled antibiotic had slightly reduced bactericidal activity relative to unlabelled daptomycin [E. faecalis or S. aureus  as described previously . As repoptomycin   is higher than that of the S. aureus strain examined (1 µg ml−1), and why daptomycin triggers greater phospholipid release from staphylococci than enterococci.After 8 h incubation with BODIPY–daptomycin, the bacterial cells were pelleted and the fluorescence of both the cells and the supernatants was measured separately using a Tecan microplate reader with excitation at 502 nm and emission at 510 nm. Antibiotic attachment to the to cells . Howeverto cells . By compernatant . This diE. faecalis cultures was not due to binding of the antibiotic to the bacterial surface or the plastic vessels used in the assays. However, as a final confirmation that phospholipids released from E. faecalis inactivated daptomycin, we exposed the bacterium to daptomycin (5 µg ml−1) to trigger phospholipid release, collected the cell-free culture supernatant and added a second dose of the antibiotic (5 µg ml−1). The culture supernatant containing the released phospholipids significantly reduced the activity of the second dose of daptomycin  or the patient has a β-lactam allergy . Our finnsidered .In this work, we focussed on daptomycin because it is a last-resort antibiotic and is associated with high rates of treatment failure. However, whilst daptomycin use is increasing, it is very unlikely to have provided the selection pressure for the evolution of the phospholipid release defence mechanism described here and previously . Since cEscherichia coli can protect against membrane-acting antimicrobials such as polymixin E and colistin [Burkholderia can sequester several different antibiotics [S. aureus and E. faecalis [The discovery of phospholipid release in several Gram-positive pathogens has expanded our growing appreciation of the broad-spectrum extracellular defence mechanisms that protect bacteria against antibiotics or host defences. For example, previous work has shown that the production of outer-membrane vesicles by colistin , whilst ibiotics . These ffaecalis . Althougfaecalis , other ifaecalis . TherefoE. faecalis releases phospholipids in response to daptomycin via an active mechanism requiring de novo lipid biosynthesis and that these phospholipids inactivate daptomycin. Pathogenic streptococci also appear to be capable of inactivating daptomycin by releasing phospholipids, indicating that this mechanism is conserved amongst Gram-positive pathogens.In summary, we have demonstrated that


Describing the moment rotation (M/θ) behavior of reinforced concrete (RC) hinges is essential in predicting the behavior of RC structures under severe loadings, such as under cyclic earthquake motions and blast loading. The behavior of RC hinges is defined by localized slip or partial interaction (PI) behaviors in both the tension and compression region. In the tension region, slip between the reinforcement and the concrete defines crack spacing, crack opening and closing, and tension stiffening. While in the compression region, slip along concrete to concrete interfaces defines the formation and failure of concrete softening wedges. Being strain-based, commonly-applied analysis techniques, such as the moment curvature approach, cannot directly simulate these PI behaviors because they are localized and displacement based. Therefore, strain-based approaches must resort to empirical factors to define behaviors, such as tension stiffening and concrete softening hinge lengths. In this paper, a displacement-based segmental moment rotation approach, which directly simulates the partial interaction behaviors in both compression and tension, is developed for predicting the M/θ response of an RC beam hinge under cyclic loading. Significantly, in order to develop the segmental approach, a partial interaction model to predict the tension stiffening load slip relationship between the reinforcement and the concrete is developed. The ability of a reinforced concrete (RC) member to maintain load and rotate under increasing deflections—that is, member ductility—is vital to its ability to absorb energy inputs, such as those from seismic and blast loads. The importance of defining member ductility has made it a significant area of research since the 1960s and a substantial amount of experimental research to describe the hysteretic moment rotation (M/θ) behavior of RC members at all load levels has been performed. From this experimental research, it has been shown that the loss of stiffness associated with the cyclic loading of RC members arises due to the Baushinger effect, which describes the softening behavior of steel following reversal of load; concrete cracking and splitting along the reinforcement; cyclic deterioration of bond between the reinforcement and the concrete; and shear crushing and sliding of the concrete [In the tension region, this PI behavior of slip along the concrete to reinforcement interface is defined by the bond properties between the reinforcement and the concrete ,3,4 and In order to simulate the behavior seen in practice it is, therefore, necessary to simulate these localized partial interaction behaviors. Typical strain-based moment curvature approaches cannot directly simulate localized PI displacements and, hence, must rely heavily on empiricisms, such as the use of effective flexural rigidities to simulate tension stiffening ,20,21,22The use of standard strain-based moment curvature analysis procedures is, therefore, generally limited, in that the behavior seen in practice—that is, the formation, widening and closing of cracks in the tension region and the formation and failure of concrete softening wedges in the compression region—is not directly simulated. Moreover, the empirical factors applied to simulate the localized partial interaction behaviors are limited in use to within the bounds from which they were derived and correlation outside these bounds is often poor . In respFor members with “weak” bond—that is, for members where debonding of the reinforcement defines failure such as fiber reinforced polymer (FRP) plated members—the M/θ approach has been developed and applied to simulate both monotonic ,31 and cThis paper introduces a new tension stiffening model which allows for the formation and simulation of multiple cracks along the entire span of RC beams under cyclic loading. The segmental M/θ approach also includes the incorporation of a size-dependent stress-strain relationship for concrete under cyclic loading, which is used to model the concrete softening of RC hinges when under cyclic loading. The proposed segmental M/θ approach is then validated against experimental results.Consider the segment shown in rb in cr in rb both during crack opening and closing, as well as tension stiffening. In the compression region, slip along a concrete to concrete PI sliding plane in The rotation θ at the segment ends in def = Scr/2 is required due to symmetry around the center line C–C.To describe how the segmental approach directly incorporates the simulation of those PI mechanisms described in the previous paragraph, let us consider one half of the segment now shown in def. Prior to any strain localizations, and from the distribution of strain in NA can be adjusted until, for a given rotation θ, longitudinal equilibrium is achieved. It can be noted that this analysis prior to strain localization provides identical results to a strain-based moment curvature analysis, as it is identical apart from the starting point of a displacement profile.In rb is no longer a function of the linear strain profile in rb in rb; this can be determined through the application of partial interaction theory, which will here be extended to the cyclic load case to allow for tension stiffening. Importantly, the relationship between Δrb and Prb, as well as the strains, stresses and forces developed in the uncracked region in def = Scr/2 and, hence, it is necessary to first establish crack spacing Scr.Let us now consider incorporation of the PI mechanism associated with concrete cracking ,15,32,33r and moduli Er is centrally located in a concrete prism of area Ac and moduli Ec such that when a load Pr is applied no moment is induced.Consider the PI tension stiffening region in r in On initial axial loading P of the prism in r)n on the element, which at the first element is equal to Δr, the bond stress τ in r and is a material property which can be defined by testing. For example a schematic of the well-known model of Eligehausen et al. [perdx, where Lper is the perimeter of the reinforcing bar, the bond force B for the element in For analysis ,14,32,33r for the load to cause slip Δr in r is applied to the lefthand side of the first element in r-B. Similarly, taking the force in the concrete to be zero at a crack face, the force in the concrete at the righthand side is B. From material constitutive relationships and now knowing the average force in the reinforcement and the concrete in the element, the average strain in the reinforcement εr in c in r − εc, where the change in slip over the element is Δδn = (dδ/dx)dx and, hence, the slip in subsequent elements can be determined. A shooting technique is then applied to determine the load Pr at which full interaction is achieved for a given slip Δr and this is obtained when the boundary condition δ = dδ/dx = 0 at the same location along the prism is met, as shown in An initial guess Pcr is as shown below. The tension stiffening prism is first divided into small segments of length dx. A flowchart on the steps required is given in r.Area of steel reinforcement, Ac.Area of adjacent concrete, Aper.Perimeter of steel reinforcement, Lc.Concrete compressive strength, fc.Concrete elastic modulus, Et.Concrete tensile strength, fcr = ft/Ec.Concrete cracking strain, εy.Yield strength of steel reinforcement, σf.Ultimate strength of steel reinforcement, σy.Elastic modulus of steel reinforcement, Eh.Strain hardening modulus of steel reinforcement, EThe required input data are inserted: r = δ(1) = 0.005 mm.The slip at the crack face, Δc(1) = 0 as the concrete-concrete interfaces are not touching at the crack face.Load applied on the adjacent concrete, Pr(1) is assumed to be 1 N.Load applied on steel reinforcement, PThe first element is located at the crack face. Several boundary conditions are used: The dummy variable “i” is used to determine the number of the segment being solved. The initial value where i = 1 is used to determine the location of crack face, and larger values of i is the distance from the crack face. The length of one segment, dx = 0.1 mm.Bond stress, τ(i) is determined using the bond-slip relationship from Comité euro-international du béton-Fédération Internationale de la Précontrainte (CEB-FIP) model code.per. Strain of steel reinforcement is determined as εr = Pr(i)Ar/Er. The change in slip is then determined as ∆δ = (εr − εc)dx.The bond force, which must be known to know how much load is transferred from the steel reinforcement to the adjacent concrete, is determined as B(i) = τ(i)Lδ(i + 1) = δ(i) + ∆δr(i + 1) = Pr(i) − B(i)Pc(i + 1) = Pc(i) + B(i)Pc = Pc(i + 1)Ac/EcεWith the value of B(i) and ∆δ determined, the values of boundary conditions for the next beam segment can be calculated. Note that the value of B(i) and ∆δ must be known for these to be calculated. In practice it is not possible to reduce the slip completely to zero. As such the condition for full-interaction used is the reduction of slip such that δ(i + 1)/δ(1) < 0.01, which represents a 99% reduction from the original slip value at the crack face.r(i + 1) < 0.If the previous condition is not met, the location of full-interaction is still not met and another condition is checked, which is PIf the previous condition is also not met, the analysis will be repeated for the next beam segment; the dummy variable i increased by 1 and Procedures 2–7 are repeated.r(1) is too low as the Pr(i + 1) < 0 occurs prior to the full-interaction condition of δ(i + 1)/δ(1) < 0.01 is met. Procedures 2–7 are, thus, repeated with a higher value of assumed Pr(1).If the condition in Procedure 8 is met, the assumed value of applied load Pc > εcr.If Condition 7 is met, another condition is checked: εr, will be set. The procedure 2–10 will then be repeated.If Condition 11 is not met, the initial slip is too small to cause a primary crack and a larger value for the initial slip at the crack face, Δcr = dxi.If Condition 11 is met, the primary crack has occurred. The required output from the analysis is the primary crack spacing which is determined as SThe numerical process to determine the primary crack spacing, Scr/2 from the crack face. This is the boundary condition for the symmetrically-loaded prism in Once primary cracks have formed, the PI mechanism changes from that in r and the slip Δr shown in r.Area of steel reinforcement, Ac.Area of adjacent concrete, Aper.Perimeter of steel reinforcement, Lc.Concrete compressive strength, fc.Concrete elastic modulus, Ey.Yield strength of steel reinforcement (if applicable), σf.Ultimate strength of steel reinforcement, σr_max = ArσfUltimate load of steel reinforcement, Py.Elastic modulus of steel reinforcement, Eh.Strain hardening modulus of steel, Edef = Scr/2.Length of deformation, Lmax = Ldef/dx.Number of elements, iThe required input data are inserted: r = δ(1) = 0.01 mm.The slip at the crack face, Δc(1) = 0 as the concrete-concrete interfaces are not touching at the crack face.Load applied on the adjacent concrete, Pr(1) is assumed to be 1 N.Load applied on steel reinforcement, PThe boundary conditions are used are: The variable i = 1 is used to determine the location of crack face, and larger values of i is the distance from the crack face. The length of one segment, dx = 0.1 mm.et al. [Bond stress, τ(i) is determined using the cyclic bond-slip relationship from Eligehausen et al. .per. Strain of the reinforcement bar is determined as εr = Pr(i)Ar/Er. The change in slip is then determined as ∆δ = (εr − εc)dx.The bond force is determined as B(i) = τ(i)Lδ(i + 1) = δ(i) + ∆δr(i + 1) = Pr(i) − B(i)Pc(i + 1) = Pc(i) + B(i)Pc = Pc(i + 1)Ac/EcεWith the value of B(i) and ∆δ determined, the values of boundary conditions for the next beam segment can be calculated:The condition for full-interaction used is the reduction of slip such that δ(i + 1)/δ(1) < 0.01, which represents a 99% reduction from the original slip value at the crack face.r(1) is correct. The slip δ(1) and the corresponding Pr(1) is then recorded and a larger/smaller value of δ(1) is set, depending on whether the beam is being loaded or unloaded.If condition in Procedure 7 is met, the assumed value of applied load Pr(i + 1) < 0.If the condition in Procedure 7 is not met, the location of full-interaction is still not met and another condition is checked, which is PIf the condition in Procedure 9 is also not met, the analysis will be repeated for the next beam segment; the dummy variable i increased by 1.max since the formation of primary cracks have limited the beam sections that are under partial interaction to half the crack spacing, also known as the length of deformation Ldef.Another condition is then checked, which is i < ir(1) is too low as the Pr(i + 1) < 0 occurs prior to the full-interaction condition of δ(i + 1)/δ(1) < 0.01 is met. A higher value of Pr(1) is thus assumed.If condition in procedure 9 is met, the assumed value of applied load Pr(1) is checked whether it reaches or exceeds the ultimate load Pr_max. If the condition is not met, procedures 4–13 is repeated.The Pr and Pr(1) are then plotted to obtain the Pr/Δr relationship.If the condition in Procedure 13 is met, the steel reinforcement has fractured. The recoded values of ΔIt is now a matter of determining the relationship between the load PThe tension stiffening analysis presented in this research is considered to be an improvement to the previous research by the authors  which for at the loaded end in cr/2, as shown in r, the corresponding load Pr, the distribution in slip in During initial loading along path OAB in r/Δr relationship is strongly dependent on the load history of the local τ/δ bond properties, as well as the σ/ε relationship of the reinforcement. This dependency is a result of the inconsistencies in the signs of the τ/δ and σ/ε relationships which arise due to cyclic loading. By this it is meant that it is possible to incur increases in shear stress associated with a reduction in slip; that is, the negative friction branch F–G in the second quadrant of Unique to the initial loading case, the material constitutive relationships are restricted to the monotonic behavior; that is, the bond τ/δ relationship as defined by path O’ABCD in The first phase of unloading which takes place along path B–C in a on path B–C to a’ on path B’–C’ results in a reduction in strain. Importantly, as the strain in the reinforcement is an extending strain, the slip strain dδ/dx = εr − εc is an extending strain and, therefore, the change in slip across the element δΔ = (dδ/dx)dx results in a reduction in slip.Consider an element from Zone 1 in The behavior characterized in Zone 1 in The second phase of unloading that is along branch CD in An element from Zone 3 in i.e., in the third quadrant of Similar to phase I of unloading, in Zone 3 in r − εc has a contracting sense and, therefore, acts over an element length dx to increase the slip Δδ = (dδ/dx)dx towards zero. As shown in cr/2.An element from Zone 4 is shown in It should also be noted here that the reversal of slips in Zones 3 and 4 may result in the bar being pushed locally to a slip not previously achieved. That is, slip may result in bond stresses along the negative loading path GHIJK in The behavior discussed here is significantly different from the previous work . The newr/Δr relationship in During reloading along path DE in r/Δr relationship, these can be directly incorporated into the segmental analysis, described in Having now described the derivation of the reinforcement PFollowing the commencement of unloading, along path CD in r/Δr relationship developed for the bottom reinforcement still applies and a new Pr/Δr relationship is required for the top reinforcement.The full depth crack results in the mechanism shown in r on the layer of reinforcement which is unloading; in the case depicted in rb in rb = Prt in rt, the corresponding slip of the top layer of reinforcement can be determined from path OAB in When cracked to full depth, the analysis proceeds by imposing a slip ΔThis analysis can be continued until the slip of the bottom reinforcement reduces to zero. At this stage, the crack is taken to be closed and full interaction is assumed between the reinforcement and the concrete at the closed crack. The analysis can then continue in an identical way to that depicted in The segmental analysis presented has to this point been concerned with the incorporation of the PI mechanism associated with tension stiffening and crack opening and, hence, considered the behavior between two adjacent cracks. Let us now consider the PI wedge sliding behavior commonly associated with the formation of a plastic hinge ,15,32,33def)h and undergo total rotations of θh, and two occur in the sagging region and are of length (Ldef)s and undergo total rotations of θs.Consider the continuous beam with span length L shown in def for the purpose of quantifying rotational behavior following the commencement of concrete softening. It should also be noted that these lengths Ldef are also the plastic hinge lengths which are commonly used to quantify the ultimate deflection and moment redistribution of RC members and are usually quantified in an empirical manner [These concentrations of rotation in l manner ,26,27,28def in def encompasses the total deformation of the PI softening wedge as shown in def can be determined from the depth of the concrete softening region and as in 0 in To undertake a segmental analysis where concrete softening is taking place, the half segment length Lrb occurs at each crack face resulting in a total slip of Δrb-t = 2nΔrb, where n is the number of cracks encompassed by the softening wedge. The load developed in the reinforcement in r/Δr relationship in rb in rb. In the compression region, the analysis is identical to that applied between two cracks. However to allow for the formation and failure of the concrete softening wedge, a size-dependent stress-strain relationship derived from the mechanics of shear friction theory by Chen et al. [def-analysis where Chen et al. [def-test to that of a specimen with a size 2Ldef-analysis. Ldef-analysis is the strain require for analysis converted from the strain εLdef-test extracted from a test on a specimen of total length 2Ldef-test when a load σa is applied and Ec is the secant modulus of the concrete.For analysis, a total displacement from A–A to B–B in n et al.  is applin et al.  has propThe ability to simulate multiple cracks within one concrete wedge, as presented here, is mainly due to the new tension stiffening analysis used and is considered an improvement over the previous work  which coet al. [et al. [As an example of the application of the segmental M/θ approach, it has been used to predict the moment rotation response of beams tested by Ma et al.  and Browet al.  in Figur [et al.  in Figur [et al.  in FigurFrom et al. [It should be noted that several generic material models were used in the analysis presented in et al.  all inclThe behaviour of reinforced concrete under cyclic loading at all load levels is governed by the local partial interaction mechanisms along reinforcement-to-concrete and concrete-to-concrete sliding planes. These partial interaction behaviors define the formation and widening of cracks in the tension region, as well as tension stiffening in the tension region, and in the compression region, the formation and failure of concrete softening wedges. In this paper, a new segmental moment rotation approach for the cyclic loading of beams has been developed which directly simulates these partial interaction behaviors. For the development of this moment rotation approach, the mechanics of the load slip behavior of reinforcement embedded in concrete under cyclic loading has been derived.


The present study concerns a development of calcium aluminate cement (CAC) concrete to enhance the durability against an externally chemically aggressive environment, in particular, chloride-induced corrosion. To evaluate the inhibition effect and concrete properties, CAC was partially mixed with ordinary Portland cement (OPC), ranging from 5% to 15%, as a binder. As a result, it was found that an increase in the CAC in binder resulted in a dramatic decrease in the setting time of fresh concrete. However, the compressive strength was lower, ranging about 20 MPa, while OPC indicated about 30–35 MPa at an equivalent age. When it comes to chloride transport, there was only marginal variation in the diffusivity of chloride ions. The corrosion resistance of CAC mixture was significantly enhanced: its chloride threshold level for corrosion initiation exceeded 3.0% by weight of binder, whilst OPC and CAC concrete indicated about 0.5%–1.0%. The hydration process of CAC concrete is sensitively governed by curing temperature, thereby leading to a rapid development of strength even at an early age, often accounting for 40–50 MPa in a few days. Notwithstanding, its use for structural concrete has been limited due to a reduction of strength in a relatively hot environment, of which the risk has been, however, challenged. A very rapid development of the strength may compensate for the strength reduction at 20–60 days, since the strength gained after the reduction period is usually still higher than for ordinary Portland cement (OPC) concrete [Calcium aluminate cement (CAC) was initially developed to protect from external aggressive chemicals by reducing the hydration of Ca(OH)concrete .i.e., C12A7) is resistive against reacting with external chemicals [2O3 in oxides of CAC has led to a possibility of inhibition of steel concrete in concrete, as C3A in hydration consisting of Al2O3 and CaO binds chlorides in the pore solution then to remove from the corrosion process. Likewise, a current study showed that CAC concrete had a slightly higher resistance to the onset of corrosion, when immersed in an NaCl solution [Moreover, the CAC has benefits in raising the durability of concrete against a chemically corrosive environment, as the CA hydration i.e., C1A7 is ressolution . In a prsolution .in-situ. The ratio of CAC to OPC for the flash set is often from 3.0: 7.0 to 7.0: 3.0 [Alternatively, a mixture of CAC and OPC was suggested to mitigate the expense rise, and simultaneously to sustain the benefit of CAC concrete. The mixture has, however, a limitation in mixing and casting of fresh concrete, due to a potential risk of flash set. Unlike the setting of Portland cement, the CAC mixture with OPC may set in a few minutes after mixing, as being less practical in 7.0: 3.0 , as bein7.0: 3.0 . Also, a7.0: 3.0 . HoweverIn the present study, to identify the inhibition of CAC concrete, corrosion test including corrosion potential, chloride binding capacity and chloride transport were measured together with concrete properties (strength and setting time). In particular, a CAC mixture with OPC was intensively tested. The ratio of CAC to OPC is ranged from 5% to 15% to total binder. For the corrosion potential measurement, a mortar specimen with centrally located steel bar was cast, while the chloride binding was measured using paste. Chloride transport was evaluated by the diffusivity of chloride ions in concrete, when exposed to a salt solution. In the curing and storing process, the temperature was kept at 20 °C to avoid the influence of temperature.2.To investigate the inhibitive effect of CAC concrete, corrosion resistance was quantitatively measured by the electrochemical techniques, together with the rate of chloride transport and chloride binding capacity. Simultaneously, mixtures of CAC and OPC were fabricated to assess their inhibition effect, within the range of 5%, 10% and 15% of CAC to the total binder content. The oxide composition of CAC and OPC is given in Mix proportion for paste, mortar and concrete is given in 2.1.The compressive strength of concrete was measured with cylindrical specimens (Ø100 × 200 mm), at 7, 28, 56 and 91 days. Concrete specimens were demolded 24 h after casting and then cured in a 95% humid chamber at 20 ± 2 °C. The setting time was determined by the penetration resistance of fresh concrete. A mortar specimen was obtained by sieving fresh concrete with the 5.0 mm sieve to remove gravel immediately after the completion of concrete mixing. Then the mortar was placed in a cylindrical mold (Ø300 × 400 mm), followed by a subsequent vibration to remove air bubbles in concrete. The penetration resistance was measured at a given time interval. The initial set was defined as the time for the penetration resistance to reach 3.43 MPa, and the final set to 27.46 MPa.2.2.In the testing of chloride binding, cement paste was cast, as aggregates do not affect the chemistry between cement paste and aggregate. Six levels of chlorides were admixed in mixing water as NaCl: 0%, 0.5%, 1.0%, 1.5%, 2.0% and 3.0% by weight of binder. After casting the paste, the specimen was rotated at 6.0 rpm for 24 h to avid segregation of chloride ions, and then cured for 56 days by wrapping in a polythene film at 20 2 °C to avoid a leaching-out of ions in the cement matrix. Prior to measuring the concentration of chloride ions in the cement paste, the specimen was dried in an oven at 104 °C for 24 h and then crushed/ground to obtain dust sample, which was then sieved into the 300 μm sieve in fineness. The dust sample was stirred for 5.0 min in 50 °C distilled water to extract the water-soluble chloride [2.3.SC) was determined by the best fitting and the apparent chloride diffusion coefficient (D) was fitted to the error function solution to the Fick’s second law, for non-steady state diffusion in a semi-infinite medium, given by The rate of chloride transport was determined by the chloride diffusivity in concrete exposed to saturated salt solution. After 28 days curing of concrete in a humid chamber at 20 ± 2 °C, the specimen was cut to 50 mm in the thickness, followed by coating the specimen with epoxy resin, except one surface for chlorides to penetrate the concrete in one direction. Then, the specimen was immersed in the 4 M NaCl solution for 150–180 days before chloride profiling. The chloride profiles were obtained from samples collected by grinding the concrete surface in 2.0 mm depth increments. The acid soluble chloride concentration at all depths was measured by potentiometric titration against silver nitrite. The concentration of the surface chloride , which could accelerate the hydration process at an early age. A reduction of the compressive strength seems presumably due to the conversion process in hydration from the hexagonal CAH10 phase to the cubic C3AH6 phase, accompanying an increase in the porosity in the cement matrix [A development of the compressive strength for concrete containing the CAC was measured at 7, 28, 56 and 91 days, as given in in-situ. In the present study, a study for chemical retarders was not dealt with.The penetration resistance of fresh concrete with time is given in 3.2.The presence of chlorides in the cement matrix was evaluated by chloride binding. After 56 days of curing, to fully hydrate cement paste, the concentration of water soluble chloride in the cement paste was determined at a given total chloride concentration, then to derive the binding capacity . It is evident that a mixture of CAC and OPC had an even higher binding capacity, compared to OPC paste. Except for CAC paste, the binding capacity was reduced with an increase in the total chloride concentration in the cement matrix and then converged to about 0.68–0.72 with chloride concentration. The binding capacity for CAC paste was lower, accounting for about 0.60 at 1.5%–3.0% of chlorides. Additionally, it was seen that CAC less bound chlorides at the lower chloride concentration, ranging 0.5%–1.0%, at which other binders showed the highest binding capacity.3.3.−12 m2/s, whilst the OPC produced the diffusivity accounting for 63 × 10−12 m2/s. For the mixture of CAC and OPC, the diffusivity was in the similar range of OPC and CAC concrete, ranging from 3.32 × 10−12 to 8.00 × 10−12 m2/s. For the surface chloride concentration, OPC and CAC indicated 2.09% and 2.00% by weight of binder, while the CAC mixture produced slightly higher surface chloride, ranging from 1.88% to 2.75%.The diffusivity of chloride ions was calculated for concrete specimens immersed in a 4.0 M NaCl solution for 150–180 days. The diffusivity and surface chloride concentration were calculated using the chloride profiles with 2.0 mm, as given in 3.4.i.e., −275 mV vs. SCE), when chlorides in cast exceeded 1.0% by weight of binder. This was again observed for CAC mortar, implying that these two binders may have about 0.5%–1.0% of the critical chloride concentration for the onset of corrosion. For the mixture of CAC and OPC, however, the corrosion potential did not indicate any sign of corrosion initiation at all levels of chlorides for all duration, except at 3.0% of chlorides for 10% CAC mortar.The corrosion behavior of steel in CAC and OPC mortar containing the variation in chloride concentration was measured by the half-cell potential method. To accelerate the corrosion process, the specimens were subjected to a repetitive wet and dry cyclic condition for 20 cycles, and the corrosion rate and potential were measured every cycle after the completion of wet duration. As seen in 4.4.1.i.e., buffering to a pH fall).In the present study, a mixture of OPC and CAC had an even higher resistance to corrosion of steel, whilst CAC alone had a marginal change in the corrosion behavior in spite of a rapid development of concrete strength. The inhibition effect of CAC mixture may arise from an increase in the chloride binding capacity; chloride binding mitigates the mobility of chloride ions in the pore solution, and thus the binding capacity has been taken as an inhibition parameter. Once chlorides are bound in the cement matrix in terms of immobilization, chloride ions bound in hydration products are in fact removed from the corrosion process. As seen in In Portland cement concrete, bound chlorides may be mostly released into free by a fall in the pH of the pore solution in the vicinity of steel  and turni.e., strength development).In a previous study on the semi-quantitative analysis of XRD, no Friedel’s salt was observed for OPC paste, whereas CAC paste still contained bound chlorides at a large portion even subjected to acidification . Substani.e., < 10−12 m2/s).However, a mixture of CAC and OPC showed a prominent higher chloride threshold level for all specimens, exceeding 3.0% by weight of binder. It may suggest that CAC mixture is definitely free from the risk of chloride-induced corrosion, considering that the surface chloride concentration is usually ranged from 2.0% to 2.5% for concretes directly exposed to seawater. When it comes to the Fick’s in calculating the time to corrosion, 1.5%–2.0% of the chloride threshold level, in fact, imposes several hundreds years of the corrosion-free life of concrete structures as long as the rate of chloride transport is assumed to OPC concrete . The hydration of CA drives the highest rate of strength development: at the low temperature (< 15 °C), the hexagonal CAH10 phase is formed by hydration, while the cubic phase of C3AH6 is produced in the hydration process at the high (>60 °C) temperature. The hexagonal CAH10 phase, a dense matrix against porosity, is transformed to the cubic C3AH6 phase and alumina gel then to form the stable crystal structure. In this conversion process, the volume of the phase is decreased, which in turn forms further porosity in the matrix, and thus the strength of CAC concrete may significantly decrease. However, it does not seem that the conversion process may be problematic in strengthening CAC concrete. Arya [CAC was initially developed to avoid gypsum-bearing water on Portland cement, consisting of monocalcium aluminate 2 hydrations may minimize the risk of sulfate attack, which must accompany Ca(OH)2 in the cement matrix to react with sulfate ions to produce expansive ettringite [Notwithstanding, a CAC mixture is still beneficial for structural concrete, it is very resistive against chemical attack. As already mentioned, a higher corrosion resistance is attributed to binding of chloride ions, which could increase the chloride threshold level presumably beyond 3.0% by weight of binder. Moreover, CA-hydrations in the CAC matrix may accelerate adorption of Ca(OH)tringite ,18. The 5.In this study, CAC concrete was mainly dealt with, in particular, its strength development, setting time and chloride-induced corrosion of steel. Simultaneously, a mixture of CAC with OPC, as a binder, was tested. The conclusion derived from the present experimental study is as follows:(1)A mixture of CAC and OPC ranked a very rapid setting of fresh concrete depending on the CAC portion. However, its strength development was always lower than for OPC and CAC alone, due to an extremely rapid setting of concrete, of which cause for the lower strength was not clearly identified in the present study. It is notable that CAC solely mixed concrete ranked the highest strength at all ages in spite of a reduction of the strength after 28 days.(2)−12 to 8.00 × 10−12 m2/s, while the surface chloride was in the range of 1.88%–2.75% by weight of binder.The rate of chloride transport in terms of chloride diffusivity was not affected by CAC binder: in fact, the diffusivity of chloride ions and surface chloride concentration were not much different with binder type. The diffusivity of chlorides in concrete ranged from 2.41 × 10(3)Chloride binding capacity was strongly dependent on binder type and the amount of CAC in binder. An increase in the CAC content resulted in an increase in the chloride binding capacity, but CAC solely mixed paste produced the lowest chloride binding. Increased binding capacity may enhance the corrosion resistance of CAC mixture(4)A mixture of CAC has a very high resistance to chloride-induced corrosion of steel, compared to OPC and CAC solely mixed specimens. For the CAC mixture, no corrosion symptom was observed by monitoring of the half-cell potential, whilst OPC and CAC indicated the onset of corrosion at 0.5–1.0 of chlorides in cast. This may arise from increased chloride binding capacity and presumably buffering to a pH fall of the pore solution. The chloride threshold level for the CAC mixture exceeded 3.0% by weight of binder.


Immunohistochemistry and in situ hybridization data from rodents revealed UCN3-containing neurons in discrete regions of the central nervous system (CNS), such as the medial preoptic nucleus, the rostral perifornical area (PFA), the medial nucleus of the amygdala and the superior paraolivary nucleus. UCN3-immunoreactive (UCN3-ir) terminals are distributed throughout regions that mostly overlap with regions of CRF2 messenger RNA (mRNA) expression. Currently, no similar mapping exists for non-human primates. To better understand the role of this neuropeptide, we aimed to study the UCN3 distribution in the brains of New World monkeys of the Sapajus genus. To this end, we analyzed the gene and peptide sequences in these animals and performed immunohistochemistry and in situ hybridization to identify UCN3 synthesis sites and to determine the distribution of UCN3-ir terminals. The sequencing of the Sapajus spp. UCN3-coding gene revealed 88% and 65% identity to the human and rat counterparts, respectively. Additionally, using a probe generated from monkey cDNA and an antiserum raised against human UCN3, we found that labeled cells are mainly located in the hypothalamic and limbic regions. UCN3-ir axons and terminals are primarily distributed in the ventromedial hypothalamic nucleus (VMH) and the lateral septal nucleus (LS). Our results demonstrate that UCN3-producing neurons in the CNS of monkeys are phylogenetically conserved compared to those of the rodent brain, that the distribution of fibers agrees with the distribution of CRF2 in other primates and that there is anatomical evidence for the participation of UCN3 in neuroendocrine control in primates.Urocortin 3 (UCN3) is a neuropeptide member of the corticotropin-releasing factor (CRF) peptide family that acts as a selective endogenous ligand for the CRF, subtype 2 (CRF UCN3, tr et al. . This per et al.  and becar et al. . Pharmacstria terminalis (BSTp) and the medial amygdaloid nucleus  encoding the UCN3 precursor molecule (ppUCN3) was detected in several peripheral organs and a few cortical and subcortical areas  are sympatric in some areas of South America and were previously designated Cebus apella. They belong to the New World monkeys, and they are among the Neotropical primate species that are commonly used for biomedical research  cell bodies, fibers and terminals, and UCN3 mRNA expression are non-existent for primates. Also, Sapajus spp.), weighing 2–3 kg, were available for use in the present study and were group housed in a vivarium with water and food available ad libitum. The monkeys were provided by the Tufted Capuchin Monkey Procreation of the Araçatuba Dentistry School at São Paulo State University (UNESP) in Brazil and this study was carried out in accordance with the recommendations of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) regulations and the Institute of Biomedical Sciences Ethical Committee for Animal Research guidelines (approved protocol #40/2009).Three young male tufted capuchin monkeys  of sodium pentobarbital (30 mg/kg) and perfused transcardially (±1 liter/10 min) with 1 liter of 0.9% saline. This step was followed by 2 liters of cold 4% formaldehyde in 0.1 M sodium acetate buffer (pH 6.5) and 2 liters of cold 4% formaldehyde in 0.1 M sodium tetraborate fixative (pH 9.5). Next, the brain, spinal cord and hypophysis were dissected and cryoprotected in two solutions: (1) 10% glycerol and 2% DMSO with 0.1 M sodium tetraborate buffer (pH 9.5) for 3 days at 4°C; and (2) 20% glycerol and 2% DMSO in 0.1 M sodium tetraborate (pH 9.5) for 4 days at 4°C. We followed a formerly described protocol by Bittencourt et al. , what reThe hypophysis was processed for paraffin embedding and 5 μm thick histological sections were obtained in a rotary microtome  and collected the slices on positively charged glass slides . The histological sections were dried at room temperature, kept at 37°C for 2 days in a laboratory incubator and then at 57°C for 1 h. The sections were deparaffinized in xylene and then dehydrated in descending grades of alcohol to distilled water. For cytoarchitectonic purposes, one series of slices from each animal undergone Nissl staining (0.025% thionin). We followed the parcellation described in “A Stereotaxic Atlas of the Brain of the Cebus Monkey” by Manocha et al. , with ad260/280).The DNA extraction procedure was modified from Blin and Stafford . GenomicUCN3 (mUCN3) gene, two kits of oligonucleotides were used, which were based on the human UCN3 sequence. Due to the elevated level of conserved sequence in the mammalian UCN genes, the primers were designed to hybridize with flanking untranslated regions of the UCN3 gene. For the amplification reactions, the nest and semi-nested PCR methods were performed to reduce the non-specific binding, which consisted in a first amplification of the long DNA sequence by using specific primers and then the amplified DNA was used for a second amplification reaction. The PCR occurred in two reactions with a total volume of 50 μl (per reaction) containing 29 μl of sterile water (RNAse and DNA free), 5 μl of PCR buffer supplied with (NH4)2SO4 and 1.0 mM MgCl2 , 2 μl of each 10 mM dNTP , 120 ng of DNA, four units of Taq DNA polymerase  and 2 μl of each primer . Primer sequences were as follows: F1 (5′-atgctgatgccggtccacttc-3′) and R1 (5′-tcctccaatttgcgcatc-3′). The thermal cycling program consisted of in an initial denaturation phase for 3 min at 94°C, and for an amplification sequence of 35 1-min cycles at 94°C, 60°C, and 72°C. The program concluded with an extension phase at 72°C for 5 min. For the second PCR, 3 μl of F1/R1 were used as the template, the pairs R1 and F2 (5′-cgcaccaagttcaccctgtccctcga-3′) or R2 (5′- cctgctgagcaagaggagcttccac-3′) and F1 were carried out using the same method described previously. The PCR procedures were performed in an Eppendorf Mastercycler thermal cycler .To determine the correct sequence that amplified the translated region of the monkey ®2.1-TOPO® plasmid vector  and subcloned using TOP10 cells. Plasmid DNA sequences containing the cloned inserts were isolated using an Eppendorf Perfectprep plasmid miniprep kit . The cloned fragment sizes were confirmed by restriction digestion with EcoR1. All cloned fragments were sequenced commercially . The subcloned sequences were analyzed by searching for nucleotide and protein sequence similarity using FASTA and NCBI BLAST + software (EMBL-EBI).All the PCR-generated amplification products were separately ligated into the pCRmUCN3 mRNA, we employed 35S-labeled antisense cRNA probes in the in situ hybridization protocol, which was adapted from Simmons et al. . The plasmid was designed by subcloning a PCR fragment encoding the part of the coding region of mUCN3 into the EcoRI sites of pCR2.1-TOPO . Labeled antisense mUCN3 probes were produced by linearizing the plasmid with XbaI enzyme. Next, the appropriate RNA polymerase (T7 for anti-sense) was used to synthesize the mUCN3 probe (323 pb). Please, see Supplementary Figure S1 for probe details. The UCN3 probe fragment length was adjusted to 200 nucleotides to reduce alkaline hydrolysis, following the protocol established by Cox et al. , 0.01% tRNA, 1 mM EDTA pH 8.0, 1× Denhardt’s solution, and 10% dextran sulfate. Next, this hybridization solution (200 μl per section) was applied to monkey tissues and incubated at 56–58°C, overnight. Monkey sections were treated with 0.002% RNase A (20 μg/ml) in a solution containing 0.5 M NaCl and 1 mM EDTA (pH 8.0) at 37°C water bath for 30 min. After, the sections were submitted to gradually de-salt (stringency washes) with 1 mM DTT: 2× SSC at 50°C water bath for 1 h, 0.2× SSC at 55°C water bath for 1 h and, 0.2× SSC at 60°C water bath. Sections were dehydrated in a solution of 70% ethanol with 0.02% 2× SSC and 0.02% 5 M DTT for 10 min, air dried at room temperature. The sections were dipped in Kodak NTB-2 liquid autoradiographic emulsion, dried at 37°C for 3 h and then exposed for an average of 4 weeks at 4°C. The slides were developed with Kodak D-19 and counterstained with 0.25% thionin for reference purposes.The probes were used at concentrations of approximately 10All antisera used in this study have been characterized in other studies, and they specifically label the expected group of cells  and mice that had the UCN3 gene deleted . These mice were kindly provided by Dr Kuo-Fen Lee from the Peptide Biology Laboratory at The Salk Institute for Biological Studies in La Jolla, CA, USA. The second control employed was a competitive adsorption test, where the antisera were preincubated (overnight at 4°C) with 0–250 μM synthetic UCN3  or related peptides belonging to the CRF family, such as CRF itself  and UCN1  and the staining patterns and intensities were analyzed. Peptides were generously provided by Dr Jean Rivier of the Laboratories for Peptide Biology at the Salk Institute. Please, see Supplementary Figures S3–S5 for more details.To ensure the specificity of the antibodies, we used two sets of controls. In the first set, the same immunohistochemistry procedure used to identify UCN3-ir in monkey brain was employed in tissue from both WT mice  diluted in 0.02 M potassium phosphate buffer . The tissue was rinsed in KPBS and then was incubated for 48 h at 4°C in the primary anti-hUCN3 antiserum at 1:7000  as chromogen and nickel ammonium sulfate  as a reaction amplifier. Standard procedures were followed. However, some previously described modifications that had been shown to improve UCN3-ir cell and fiber labeling  diluted in the same blocking solution, for 2 h at room temperature. After KPBS washes, sections were mounted onto gelatin-coated slides and coverslipped with buffered glycerol medium. For CRF localization, adjacent sections from the same three different animals previously described were rinsed in KPBS, were incubated in blocking solution for 1 h, and then were incubated in anti-r/hCRF (rat/human CRF) antiserum  diluted in the same blocking solution, for 2 h at room temperature. After KPBS washes, sections were mounted onto gelatin-coated slides and coverslipped with buffered glycerol medium.Besides, we used the indirect immunofluorescence technique to study the localization of UCN3 and CRF in the paraventricular nucleus of the hypothalamus (Pa). For UCN3 localization, sections from three different animals were rinsed in KPBS and were incubated for 1 h in KPBS solution containing 0.03% Triton X-100, 1% bovine serum albumin, 5 mg/ml heparin (blocking solution), followed by incubation over 48 h at 4°C in anti-hUCN 3 antiserum  under heat and humid pressure  at 95°C for 5 min. The sections were cooled to room temperature and subjected to non-specific blocking using 5% non-fat milk, followed by secondary blocking using 3% BSA in PBS/0.3% Triton X-100 and incubated with anti-hUCN3 antiserum  coupled to a Leica microscope  using the image capture software NIS—Elements BR 3.14 . For the analysis of UCN3 immunofluorescence, the slides were examined under a Leica EL6000 microscope equipped with epifluorescence. The digital photomontages were obtained with a Nikon ECLIPSE 80i microscope connected to a Dell computer that was fitted with the Neurolucida Suite .Single UCN3-ir and CRF-ir cells in the Pa were analyzed using a confocal laser scanning microscope Zeiss LSM 780 NLO  located at the Centro de Facilidades de Apoio à Pesquisa (CEFAP/USP) at the University of São Paulo.UCN3 mRNA and UCN3-ir cell bodies and fibers were individually evaluated through semiquantitative analysis among the positively labeled regions according to a previously defined rating scale  are substitutions relative to human ppUCN3 and 61 residues are substitutions (50.83%) relative to rat ppUCN3  proved to be specific, based on the results of the controls studies performed. Table In all monkeys examined, we observed UCN3-ir cells were restricted to a few regions in the CNS, mainly in the hypothalamic and amygdaloid regions Figure ; Table 3In extrahypothalamic areas, UCN3-ir cells were predominantly found in amygdaloid areas, specifically in the medial amygdaloid nucleus , Me and anterior amygdaloid area (AA) , centrolateral thalamic nucleus (CL), paracentral thalamic nucleus (PC), granule cell layer of the dentate gyrus (GrDG) and the anterior (STMA) and ventral (STMV) regions of the medial division of the bed nucleus of stria terminalis (ST). There was no detectable UCN3 mRNA expression using the UCN3 cRNA sense probe  Figure . UCN3 mRe Figure .Visualization of fibers and terminal fields occurred predominantly in hypothalamic and limbic regions. The main sites of UCN3-ir fibers were the septal nuclei, such as the intermediate (LSI) and ventral parts (LSV) of the LS. The LSI was the main site of intense UCN3-ir Figures . In the We found moderate to high densities of UCN3-ir fibers in the JPLH, and this was associated with the presence of labeled neurons in this area. A considerable amount of UCN3-ir fibers and varicosities was observed in the following divisions of the Pa: PaMD, PaPD and the ventral division of the parvicellular part of the Pa (PaPV). We also found UCN3-ir fibers and varicosities in the preoptic hypothalamus, specifically in the anteromedial preoptic nucleus (AMPO), medial preoptic area (MPA) and lateral preoptic area , UCN3-ir fibers were observed in moderate intensity in the external layer (EL), but also in the arcuate hypothalamic nucleus (Arc), where these fibers were transiting to the ME, a pattern that is more evident at rostral levels. Moderately dense UCN3-ir fibers were restricted to the posterior lobe of the hypophysis  and the amygdalohippocampal area, parvicellular part (AHiPC). The STMA contains moderately dense UCN3-ir fibers. Few UCN3-ir fibers and varicosities were observed in the medial habenular nucleus (MHb).In extrahypothalamic areas, we found low to moderate UCN3-ir fiber densities in the Me, where it is located lateral to the In the mesencephalon, some fibers were found in the lateral periaqueductal gray (LPAG) and the ventrolateral periaqueductal gray (VLPAG). In the brainstem, there were few fibers and terminal fields in the solitary nucleus (Sol) and the caudal part of the spinal trigeminal nucleus (Sp5C). Terminal fields were also found in the centrointermediate part of the dorsal motor nucleus of vagus (CeI). There were no UCN3-ir fibers in the entire rostrocaudal axis of the spinal cord.ko/UCN3 mice, further demonstrating their specificity.Two UCN3 antisera were employed in this study. The first antiserum, PBL#6570, was used for all experiments, including cell and fiber mapping. The second antiserum, PBL#7218, was utilized for the image acquisition in some immunohistochemical experiments, but it displayed less specificity when compared to PBL#6570 and was not used for mapping. Although PBL#6570 is not species-specific, the adsorption tests demonstrated that it recognized the antigen with high specificity. Additionally, this antiserum labeled the UCN3-ir in the Pa of brains of other mammals  is more comparable in size, sulcal pattern and habits to Old World monkeys of the Macaca genus than to New World monkeys  and amygdala, these nuclei are implicated in the modulation of learning and behavior, concerning autonomic regulation. These findings confirmed that the expression of UCN3 in the MeA is sensitive to stress  of the Rhesus monkey (Sánchez et al., Sapajus spp. agrees with the CRF2 mRNA distribution in rats found by Van Pett et al. (2 system between New and Old World monkeys (Table Regarding the regions of mismatch, Sánchez et al.  describet et al. , which cys Table .2CRF mRNA expression in primates has been similarly reported in rodents by Li et al. (2 mRNA distribution (Chalmers et al., 2 in other animal models (Lewis et al., 2 in non-human primates.This partial overlap between UCN3-ir fibers and i et al. . Conversi et al.  also des2 (Lovenberg et al., Since UCN3 is the only member of the CRF family which is a highly selective ligand for CRFSapajus spp. further strengthen this notion, indicating that the UCN3 peptidergic system is optimally located to mediate and modulate stress responses in non-human primates.Venihaki et al.  reportedSapajus spp. are the paraventricular nucleus of the hypothalamus, the PFA and the juxtaparaventricular lateral nucleus, which are similar to those described for rodents. The presence of UCN3-ir fibers mainly in the ventromedial nucleus of the hypothalamus, paraventricular nucleus of the hypothalamus and in the LSI is coincidental to the CRF2 distribution in the Rhesus monkey. When taken together, these data suggest that UCN3 plays a role in stress responses and neuroendocrinological control.The main sites of UCN3-producing neurons in In this study, all authors had full access to the data and take responsibility for the data integrity and the accuracy of the data analysis. JCB: study concepts and design; study supervision. DSB: acquisition of the data. DSB and JCB: analysis and interpretation of the data; obtained funding. DSB, JCB, GBD: drafting of the manuscript. JCB, CFPL, DAL, LVS: critical revision of the manuscript for important intellectual content. PLC, JAO, CAC, KRTS, JMS, ARO: administrative, technical and material support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.