Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus (MSSA), vancomycin-resistant enterococci (VRE), and vancomycin-susceptible enterococci (VSE) between spray irrigation workers using reclaimed water and office worker controls. Nasal and dermal swabs from 19 spray irrigation workers and 24 office worker controls were collected and analyzed for MRSA, MSSA, VRE, and VSE. Isolates were confirmed using standard biochemical tests and polymerase chain reaction assays. Antimicrobial susceptibility testing was performed by Sensititre® microbroth dilution. Data were analyzed by two-sample proportion, chi-square, Fisher’s exact tests, and logistic regression. No MRSA or VRE were detected in any samples. MSSA was detected in 26% and 29% of spray irrigators and controls, respectively. VSE was detected in 11% and 0% of spray irrigation workers and controls, respectively. The adjusted odds of MSSA, multidrug-resistant MSSA, and either MSSA or VSE colonization were greater among spray irrigation workers, however results were not statistically significant. Future studies with larger sample sizes are needed to further evaluate this relationship.As reclaimed water use expands, it is important to evaluate potential occupational health risks from exposure to this alternative water source. We compared odds of colonization with methicillin-resistant  Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) have both been identified in wastewater throughout the treatment process, including final effluent in the U.S. and Europe [Between 5%–6% of municipal wastewater effluent, approximately 2.22 billion gallons per day, is reclaimed and reused in the United States . Landscad Europe ,10,11,12d Europe ,9. As rad Europe , includiStaphylococcus aureus is a bacterial pathogen that colonizes multiple body sites, most commonly the nostrils, and causes a number of infections, including skin and soft tissue infections, pneumonia, and septicemia [S. aureus (MSSA) have been detected in air samples from a number of environments, including wastewater treatment plants [. Since these bacteria can be aerosolized from water and are capable of colonizing skin and soft tissues, exposure through inhalation is of concern, particularly among workers at both wastewater treatment plants and spray irrigation sites who use or come in contact with the reclaimed water.pticemia . MRSA ant plants . Since tEnterococci are opportunistic pathogens that can cause urinary tract infections, bacteremia, and endocarditis . BetweenS. aureus or enterococci survive wastewater treatment and distribution to reuse sites, spray irrigation workers using reclaimed water could be exposed to these organisms through dermal contact or inhalation [If halation . To our halation ,5. In th3/day of wastewater, with a peak capacity of 1.51 million m3/day. The incoming wastewater (influent) at Mid-Atlantic WWTP1 includes domestic and hospital wastewater, and the following treatment steps are employed at the plant: screens, primary clarifier, activated sludge reactors, secondary clarifier, sand filters, chlorination, dechlorination and discharge. At the time of the study, the chlorination dose was 2–3 mg/L, followed by dechlorination with sodium bisulfite such that the chlorine residual in the effluent was <0.1 mg/L. The reclaimed water spray irrigation site included in this study is located in the Mid-Atlantic region of the USA. The site was chosen based on the willingness of the site operator to participate in the study. This site receives treated wastewater from a tertiary wastewater treatment plant (Mid-Atlantic WWTP1) ,12 that The effluent (discharge) from Mid-Atlantic WWTP1 is sent to our spray irrigation study site through an enclosed pipe. Once the treated wastewater reaches the spray irrigation site, it passes through an aluminum screen and is then treated with 254 nanometer wavelength ultraviolet (UV) radiation bulbs that produce a minimum of 30,000 microwatt seconds per square centimeter. After UV treatment, the water is pumped into an open-air storage pond at a rate of 230,000 gallons per day with a peak capacity of 4 million gallons. Based on turf irrigation needs, water from the storage pond is pumped to spray heads throughout the site. Spray irrigation workers also carry backpack spray systems to irrigate additional areas. The spray irrigation site employs eight full-time employees and approximately 22 seasonal employees each year.This study was approved by the University of Maryland College Park, Institutional Review Board, IRB Protocol 09-0211. A total of 43 subjects were enrolled in the study; 19 spray irrigation workers from the study site who were occupationally exposed to reclaimed water, and 24 office worker controls from an academic work setting who were not exposed to reclaimed water or healthcare settings on the job. Study subjects were selected through a convenience sample based on employment status. Office worker controls were matched by age (±2 years), sex, and race to the spray irrigation workers and recruited into the study in person and over email. Individuals were excluded from participation if they reported a nosebleed three days prior to sample collection to avoid dislodging blood clots.S. aureus nasal colonization rates among healthcare workers is greater than the 20%–30% colonization rate found in the general population [Participants were asked to complete a short survey containing questions related to sociodemographics, as well as questions related to risk factors associated with MRSA colonization and previous MRSA diagnosis. The survey also asked participants about previous work in healthcare facilities and household members who work in healthcare facilities, because pulation ,25. The A total of 94 nasal (48 from spray irrigation workers and 46 from office worker controls) and 94 dermal swab samples (48 from spray irrigation workers and 46 from office worker controls) were collected between August 2009 and February 2011 when the irrigation spray heads were in use. Participants were sampled at multiple time points when possible. On average, participants were each sampled 2.19 times. Nasal swabs were collected using Liquid Stuart Medium Transport swabs . The swab was inserted approximately 1.25 cm into the participant’s right nostril and gently rotated five times on the inside wall of the nostril ,26. DermS. aureus and Enterococcosel agar for isolation of total Enterococcus spp. Baird Parker agar plates were incubated at 37 °C for 24 h, while Enterococcosel agar plates were incubated at 41 °C for 24 h. Resulting black colonies with halos on Baird Parker, and colonies with a black precipitate on Enterococcosel agar were considered presumptive S. aureus and enterococci, respectively. These colonies were purified on Brain Heart Infusion agar and archived in Brucella broth with 15% glycerol at −80 °C. S. aureus ATCC 43300 and Enterococcusfaecalis ATCC 29212 were used as positive controls and phosphate buffered saline was used as a negative control throughout the isolation process.All media was obtained from Becton, Dickinson and Company . Nasal and dermal swabs were streaked onto Baird Parker agar for isolation of total S. aureus were identified using the Gram stain, the coagulase test , and the catalase test. For confirmation of S. aureus and MRSA differentiation, the S. aureus-specific nuc gene, the MRSA-specific mecA gene, and a 16S rDNA internal control were PCR amplified in a multiplex reaction as described previously [S. aureus ATCC 43300 was used as a positive control for PCR amplification of nuc and mecA genes.eviously ,28. DNA et al. [ddl genes of Enterococcus faecalis and E. faecium, the vanC1 and vanC2/3 genes of E. gallinarum and E. casseliflavus, and a 16S rDNA internal control [E. faecalis ATCC 51299, E. faecium ATCC 51559, E. casseliflavus ATCC 25788, and E. gallinarum ATCC 49573. Colonization among study participants was defined as MRSA, MSSA, VRE, or VSE isolated and confirmed from any swab sample  during the study period.Enterococci were presumptively identified using the Gram stain, the catalase test, and by detection of pyrrolidonyl peptidase (pyr) activity . Confirmation was accomplished using a modified multiplex PCR assay previously described by Micallef et al. . Genomicet al. . Briefly control . PCR ampS. aureus (n = 97) and Enterococcus spp. (n = 20) isolates. Briefly, overnight cultures were transferred to sterile demineralized water (Trek Diagnostic Systems) to achieve a 0.5 McFarland standard. Then, 30 μL of the suspension for S. aureus and 50 μL of the suspension for enterococci was transferred to sterile cation-adjusted Mueller Hinton broth (Trek Diagnostic Systems), and 50 μL of the broth solution was then dispensed into the GPN3F MIC plates (Trek Diagnostic Systems Inc.) with the following antibiotics: erythromycin , clindamycin , quinupristin/ dalfopristin , daptomycin , vancomycin , tetracycline , ampicillin , gentamicin , levofloxacin , linezolid , ceftriaxone , streptomycin , penicillin , rifampin , gatifloxacin , ciprofloxacin , trimethoprim/sulfamethoxazole , and oxacillin + 2%NaCl . Plates were incubated at 37 °C and read after 18–24 h. Enterococcus faecalis ATCC 29212 and S. aureus ATCC 29213 were used as quality control strains. MICs were recorded as the lowest concentration of an antimicrobial that completely inhibited bacterial growth [Antimicrobial susceptibility testing was performed using the Sensititre® microbroth dilution system with GPN3F minimal inhibitory concentration (MIC) plates  in accordance with the manufacturer’s instructions on all PCR-confirmed t-test was used to compare the number of antibiotics that isolates expressed resistance against. Logistic regression models were used (1) to determine if the odds of ever being colonized with the bacteria of interest were different for spray irrigation workers compared to controls, while controlling for education, duration of job, yearly income, and current smoking status; and (2) to conduct analyses of spray irrigation worker odds of colonization. A generalized linear mixed effects model (GLMM) was used to evaluate the odds of being colonized with S. aureus over time by occupational status. In all cases, p-values of ≤0.05 were defined as statistically significant. All statistical analyses were performed using Stata/IC 10  or SAS 9.2 .Descriptive statistics were reported including the percentages of study participants that were positive for MSSA and VSE by worker classification. Differences in sociodemographic variables between spray irrigation workers and controls were compared using the chi-square or Fisher’s exact test. Statistical analyses of antibiotic resistance data were limited to MSSA (n = 32) and VSE (n = 3) isolates expressing unique antimicrobial resistance profiles; this allowed us to reduce bias that could be introduced by including possible clones. A two-sample p<0.001; p = 0.01). Spray irrigation workers reported “currently smoking” (p = 0.002) and “smoking more than 100 cigarettes in the past six months” (p < 0.001) more than the controls (The participation rate for the study was 88% (43/49). Participants ranged in age from 17 to 66 years and both spray irrigation workers and office worker controls were composed largely of Caucasian males . The meacontrols . Slightlp = 0.84).No MRSA was detected in any of the nasal or dermal swabs collected from the spray irrigation workers or controls. MSSA was recovered from 28% (12/43) of all study participants. Twenty-six percent (5/19) of spray irrigation workers had nasal swabs that were positive for MSSA during at least one sampling event . Among cvs. 0%; p = 0.19).VRE was not detected in any nasal or dermal swab samples from the spray irrigation workers or controls . A greatp = 0.30) (Thirty-three percent (14/43) of all participants were colonized with either MSSA or VSE. A greater proportion of spray irrigation workers compared to controls were colonized with at least one of the bacteria of interest ( = 0.30) .vs. 8.3%) (p = 0.40) (p = 0.13) and linezolid (p = 0.13) (p = 0.17) and ampicillin (p = 0.25) (p = 0.37). In total, 97 MSSA isolates were recovered from nasal and dermal swabs: 57 isolates from spray irrigation workers, and 40 from controls. However, statistical analyses concerning antibiotic resistance patterns among these isolates were limited to 32 isolates that could be confirmed as unique using phenotypic analyses . Isolates were resistant to a variety of the 18 antibiotics tested. A greater percentage of spray irrigation workers compared to controls were colonized with MDR MSSA (11%  = 0.40)  and a gr = 0.13) . A great = 0.25) . MSSA isIn total, 20 VSE isolates were isolated from two spray irrigation worker nasal swabs. Of the three isolates that were phenotypically unique, all three were resistant to rifampicin and all three were either resistant or intermediately resistant to quinupristin/dalfopristin.p = 0.75) (data not shown).Our unadjusted and adjusted logistic regression models indicated that the odds of being colonized with MDR MSSA or either MSSA or VSE were greater among the spray irrigation workers compared to the controls; however, the differences were not statistically significant . In the The results of our logistic regression models focused only on spray irrigation workers showed that most of the variables used in our model did not have statistically significant effects on the odds of the spray irrigation workers being colonized with MSSA, MDR MSSA, VSE, or either MSSA or VSE (data not shown). However, spray irrigation workers, who reported either personally having worked in a healthcare setting or having a household member who had worked in a healthcare setting (n = 8) tended to be more likely to be colonized with either MSSA or VSE compared to those who did not report this type of exposure but the difference was not statistically significant .S. aureus [S. aureus [et al. that found that the prevalence of MRSA colonization in the U.S. is approximately 1.5% based on the 2003–2004 NHANES data [Based on the 2003–2004 National Health and Nutrition Examination Survey (NHANES), between 27.2% and 30% of the U.S. population are colonized with . aureus . It has . aureus ,31. TwenNES data . TherefoNES data .et al. of rural U.S. hospitals, 22% of patients with positive VRE cultures had been in the hospital for under 48 h or were outpatients, prompting classification as a CA-VRE infection [Community-associated VRE (CA-VRE)  is rarely reported in the USA. ,33, howenfection . TherefoE. faecalis and E. faecium have previously been isolated in small numbers from the upper respiratory tract; however, in a large-scale hospital study of VRE and MRSA colonization by Warren et al., the majority of VRE-positive specimens were recovered from stool or rectal samples (87%) compared to respiratory (0%) and soft tissue and wound samples (2%) [et al., 13% of all hospital patients who provided an oropharyngeal  culture had VRE-positive results [les (2%) ,36. Yet, results . Rectal S. aureus or enterococci colonization between reclaimed water spray irrigation workers and office worker controls, the higher percentage of spray irrigation workers colonized with MDR MSSA and VSE raises potential public health concerns for those working with or otherwise exposed to reclaimed water. Several previous studies have analyzed the risk of different types of infections among individuals occupationally, recreationally, or residentially exposed to reclaimed water that has undergone various levels of treatment and found conflicting results [et al. in France identified that farmers exposed to wastewater treated in stabilization ponds reported more respiratory and GI symptoms compared to a non-exposed group of controls [Although we found no statistically significant differences in the odds of  results ,5. A stu results . With ad results . Durand  results . They fo results . Becausecontrols .S. aureus and enterococci among workers using reclaimed water in the United States. Although the differences between the odds of MSSA, MDR MSSA, and VSE colonization among spray irrigators vs. controls in this study were not statistically significant, the data still provide evidence of potential human health issues that should be further investigated with larger sample sizes across the USA. This is particularly important with regard to potential exposures to MRSA—a leading cause of hospital-acquired infections and a microorganism associated with a growing number of community-acquired infections—because MRSA has been detected in treated U.S. wastewater that is used in reclamation activities [Legionella spp. and Aeromonas spp. since these waterborne microorganisms have also been isolated from reclaimed water [To our knowledge, the current study is the first to evaluate occupational exposures to tivities . Our restivities ,12. Simied water ,39,40.Our findings suggest that the odds of MSSA, multidrug-resistant MSSA, and either MSSA or VSE colonization between spray irrigation workers using reclaimed water and those who are not routinely exposed to reclaimed water are not statistically significantly different. However, the lack of statistically significant findings could be an artifact of the limited number of spray irrigation workers available to participate in the study. As reclaimed water use continues to grow, additional studies with larger samples sizes are needed to further evaluate occupational exposures to human pathogens originating from this water source.


Lung cancer is currently the leading cause of cancer-related death in worldwide, non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. Surgery, platinum-based chemotherapy, molecular targeted agents and radiotherapy are the main treatment of NSCLC. With the strategies of treatment constantly improving, the prognosis of NSCLC patients is not as good as before, new sort of treatments are needed to be exploited. Programmed death 1 (PD-1) and its ligand PD-L1 play a key role in tumor immune escape and the formation of tumor microenvironment, closely related with tumor generation and development. Blockading the PD-1/PD-L1 pathway could reverse the tumor microenvironment and enhance the endogenous antitumor immune responses. Utilizing the PD-1 and/or PD-L1 inhibitors has shown benefits in clinical trials of NSCLC. In this review, we discuss the basic principle of PD-1/PD-L1 pathway and its role in the tumorigenesis and development of NSCLC. The clinical development of PD-1/PD-L1 pathway inhibitors and the main problems in the present studies and the research direction in the future will also be discussed. Lung cancer is currently the leading cause of cancer-related death in the worldwide. In China, the incidence and mortality of lung cancer is 5.357/10000, 4.557/10000 respectively, with nearly 600,000 new cases every yearDuring the last few decades, significant efforts of the interaction between immune system and immunotherapy to NSCLC have been acquired. Recent data have indicated that the lack of immunologic control is recognized as a hallmark of cancer currently. Programmed death-1 (PD-1) and its ligand PD-L1 play a key role in tumor immune escape and the formation of tumor microenvironment, closely related with tumor generation and development. Blockading the PD-1/PD-L1 pathway could reverse the tumor microenvironment and enhance the endogenous antitumor immune responses.In this review, we will discuss the PD-1/PD-L1 pathway from the following aspects: the basic principle of PD-1/PD-L1 pathway and its role in the tumorigenesis and development of NSCLC, the clinical development of several anti-PD-1 and anti-PD-L1 drugs, including efficacy, toxicity, and application as single agent, or in combination with other therapies, the main problems in the present studies and the research direction in the future.Cancer as a chronic, polygene and often inflammation-provoking disease, the mechanism of its emergence and progression is very complicated. There are many factors which impacted the development of the disease, such as: environmental factors, living habits, genetic mutations, dysfunction of the immune system and so on. At present, increasing evidence has revealed that the development and progression of tumor are accompanied by the formation of special tumor immune microenvironment. Tumor cells can escape the immune surveillance and disrupt immune checkpoint of host in several methods, therefore, to avoid the elimination from the host immune system. Human cancers contain a number of genetic and epigenetic changes, which can produce neoantigens that are potentially recognizable by the immune systemThe present research of immune checkpoint molecules are mainly focus on cytotoxic T lymphocyte-associated antigen 4 (CLTA-4), Programmed death-1 (PD-1) and its ligands PD-L1 (B7H1) and PD-L2 (B7-DC). CTLA-4 regulates T cell activity in the early stage predominantly, and PD-1 mainly limits the activity of T-cell in the tumor microenvironment at later stage of tumor growthet al. showed that PD-1 was also expressed on regulatory T cells (Treg) and able to facilitate the proliferation of Treg and restrain immune responsePD-1 is a type 1 trans-membrane protein that encoded by the PDCD1 genePD-1 has two ligands: PD-L1  and PD-L2 , that are both coinhibitory. PD-L1 is expressed on resting T cells, B cells, dendritic cells, macrophage, vascular endothelial cells and pancreatic islet cells. PD-L2 expression is seen on macrophages and dendritic cells alone and is far less prevalent than PD-L1 across tumor types. It shows much more restricted expression because of its more restricted tissue distribution. Differences in expression patterns suggest distinct functions in immune regulation across distinct cell types. The restricted expression of PD-L2, largely to antigen-presenting cells, is consistent with a role in regulating T-cell priming or polarization, whereas broad distribution of PD-L1 suggests a more general role in protecting peripheral tissues from excessive inflammation.et al.’s research also demonstrated that neuroglioma with PTEN gene deletion regulate PD-L1 expression at the translational level by activating the PI3K/AKT downstream mTOR-S6K1signal pathway and, hence increase the PD-L1 expressionPD-L1 is expressed in various types of cancers, especially in NSCLC1113141618+ T effector memory cells into CD8+ central memory cellsLike the CTLA-4, the PD-1/PD-L1 pathway down-modulates T-cell response by regulating overlapping signal proteins in the immune checkpoint pathway. However, their functions are slightly different28Researches on the mechanism of PD-1/PD-L1 pathway mediating immune escape are still ongoing, especially the mechanism of PD-L2 is still unclear. These researches provide the theoretical basis and research direction for the further immunotherapy targets research.Historically, immunotherapy has been ineffective in cases of NSCLC, which has been thought to be a type of non-immunogenic cancer; nevertheless, NSCLC can evade the immune system through various complex mechanisms which we have discussed above. Researchers had discovered that the count of peripheral and tumor lymphocytes which are capable of suppressing tumor immune surveillance reduced in patients with advanced non-small cell lung canceret al.’s research demonstrated that, PD-L1 had higher positive results in lung adenocarcinoma with higher grade differentiation and vascular invasion and positive PD-L1 expression correlated with less cancer recurrence. PD-L1 expression is correlated with better relapse-free survival (RFS), but unrelated with overall survival (OS)Some retrospective studies showed that the overexpression of PD-L1 and is closely related to the poor prognosis and high invasiveness in NSCLC patients38In addition, a study involving 583 NSCLC cases with surgically resected for single-nucleotide polymorphism (SNP) analyses has reported that: there were no significant differences in different PD-1 SNP statuses  and the ratio was extremely similar to the healthy control in a previous study. But the survival time of the patients with the GG phenotype of PD-1 was significantly shorter compared to the patients with GA or AA, and the GG phenotype patients significantly had a worse prognosis in the SCC populationThe process of tumorigenesis, progression and metastasis is a process of complex and consecutive changes. The activation of oncogenes and the inactivation of tumor suppressor genes by mutations and the imbalance of immune system are all essential components of the process. An imbalance to the regulation of the immune system changes the tumor-specific T-cell immunity in the tumor microenvironment and adjusts the tumor progression and metastasis. It has been reported that the mutations of tumor-related genes are associated with the imbalance of immune system. In the process of the development of lung cancer, there are a lot of genetic mutations, such as EGFR, ALK and so on. Studies have shown that, PD-L1 expression in EGFR mutation samples is higher than in wild type, and the mutation can promote the expression of PD-L1Researchers have shown that the PD-L1expression of tumors may be associated with the efficacy of NSCLC patients who received immunotherapy. Recently, a multicenter phase I clinical trial result of pembrolizumab used for treatment of advanced NSCLC patients was published in New England Journal of Medicine. The result suggested that the response rate for patients with PD-L1 positive (a proportion score of at least 50%) exceeded the PD-L1 negative (a score of 1 to 49% and the group with a score of less than 1%) for previously treated patients (P < 0.001) and for previously untreated patients (P = 0.01). Progression-free and overall survival were shorter among patients with a proportion score of 1 to 49% or a score of less than 1% than among those with a score of at least 50%In a multi-center clinical trail focused on specific antibody nivolumab that specifically blocking PD-1 showed that cumulative response rates  were 18% among patients with NSCLC (14 of 76 patients). Immunohistochemical analysis was performed on pretreatment tumor specimens obtained from 42 patients, objective response rate of PD-L1 positive and negative was 9 of 25 patients (36%), 0% (0/17) (P = 0.006)respectivelyCurrently, studies focused on the reasons of non-response to anti-PD-1/PD-L1 therapy for patients with NSCLC scarely have reported. However, analyzing on the basis of currently available clinical and basic studies, it can be assumed that different responses to anti-PD-1/PD-L1 therapy are not caused by single factors, but the result of the interaction of various factors .In addition, some investigators found that PD-1 expression on tumor cellsTherefore, the mechanism and the relationship during PD-1/PD-L1 and the immune therapy efficacy and the selection of patients still need further basic studies and large sample phase II or III clinical trials to be confirmed.Nivolumab  is a human monoclonal IgG4 antibody that essentially lacks detectable antibody-dependent cellular cytotoxicity (ADCC). Inhibition by monoclonal antibody of PD-1 on CD8+ TILs within lung cancers can restore cytokine secretion and T-cell proliferationIn another early phase I trial of nivolumabSingle-agent trials of nivolumab are planning or ongoing on NSCLC . In addition, there are clinical randomized trials which focus on the comparison of nivolumab and plain-based combination chemotherapy . In March 4, 2015, nivolumab was approved by the US Food and Drug Administration for treatment of patients with metastatic NSCLC (squamous cell carcinoma), when progression of their diseases during or after chemotherapy with platinum-based drugs.Pembrolizumab (MK-3475) is a highly selective, humanized monoclonal antibody with activity against PD-1 that contains a mutation at C228P designed to prevent Fc-mediated ADCC. It is now in the clinical research phases for patients with advanced solid tumors. Its safety and efficacy were evaluated in a phase I clinical trial of KEYNOTE-001. The best response according of 38 cases of patients which initially accepted pembrolizumab 10 mg/kg q3wwas 21%  and the median PFS of responder still has not reached until 62 weeks. The researchers also found that the antitumor activity of pembrolizumab was associated with the PD-L1expression44Clinical trial of pembrolizumab monotherapy is ongoing for patients with NSCLC (NCT01840579). Randomized trials comparing pembrolizumab to combination chemotherapy (NCT02142738) or single-agent docetaxel (NCT01905657) are ongoing in PD-L1 positive patients with NSCLC.Pidilizumab is a humanized IgG-1K recombinant anti-PD-1 monoclonal antibody that has demonstrated antitumor activity in mouse cancer models. In a first-in-human phase I dose-escalation study in patients with only advanced hematologic cancers, there is no clinical trials of NSCLC presentlyAnother therapeutic method based on the PD-1/PD-L1 pathway is by specific binding between antibody and PD-L1, thus preventing its activity. It was speculated that utilizing PD-L1 as therapeutic target maybe accompanied by less toxicity in part by modulating the immune response selectively in the tumor microenvironment. However, since PD-L2 expressed by tumor cells or some other tumor-associated molecules may play a role in mediating PD-1-expressing lymphocytes, it is conceivable that the magnitude of the anti-tumor immune response could also be blunted.BMS-936559/MDX1105 is a fully humanized, high affinity, IgG4 monoclonal antibody that react specifically with PD-L1, thus inhibiting the binding of PD-L1 and PD-1, CD80 . Initial results from a multicenter and dose-escalation phase I trial of 207 patients(including 75 cases of patients with NSCLC) showed durable tumor regression (objective response rate of 6%–17%) and prolonged stabilization of disease (12%–41% at 24weeks) in patients with advanced cancers, including NSCLC, melanoma and kidney cancer. In patients with NSCLC, there were five objective responses (in 4 patients with the nonsquamous subtype and 1 with the squamous subtype) at doses of 3 mg/kg and 10 mg/kg, with response rates of 8% and 16%, respectively. Six additional patients with NSCLC had stable disease lasting at least 24 weeksMPDL3280A is a human IgG1 antibody that targets PD-L1. Its Fc component has been engineered to not activate antibody-dependent cell cytotoxicity. In a recently reported phase I study, a 21% response rate was noted in patients with metastatic melanoma, RCC or NSCLCFifty-two patients were enrolled in an expansion cohort of the phase I trial of MPDL3280A, 62% of them were heavily pretreated NSCLC (≥3 lines of systemic therapy) and the ORR was 22%MEDI4736 is a human IgG1 antibody that binds specifically to PD-L1, thus preventing PD-L1 binding to PD-1 and CD80. Interim results from a phase I trial reported no colitis or pneumonitis of any grade, with several durable remissions, including NSCLC patientsOn the basis of the favorable toxicity profile and promising activity in a heavily pretreated NSCLC population, a global Phase III placebo controlled trial using the 10 mg/kg biweekly dose has been launched in Stage III patients who have not progressed following chemo-radiation (NCT02125461). The primary outcome measures are overall survival and progression-free survival.AMP-224 was a B7-DC-Fc fusion protein which can block the PD-1 receptor competitivelyA variety of approaches for combining PD-1/PD-L1 pathway inhibitors with other therapeutic methods have been explored over the past few years in an effort to offer more feasible therapeutic options for clinic to improve treatment outcomes. Approaches have included combinations with other immune checkpoint inhibitors, immunostimulatory cytokines (e.g. IFN-y) cytotoxic chemotherapy, platinum-based chemotherapy, radiotherapy, anti-angiogenic inhibitors, tumor vaccine and small-molecule molecularly targeted therapies many with promising results61Preclinical evidence exists for the complementary roles of CTLA-4 and PD-1 in regulating adaptive immunity, and this provides rationale for combining drugs targeting these pathways. In a Phase I study in 46 chemotherapy-naive patients with NSCLC, four cohorts of patients received ipilimumab (3 mg/kg) plus nivolumab for four cycles followed by nivolumab 3 mg/kg intravenously every 2 weeks. The ORR was 22% and did not correlate with PD-L1 statusIn another Phase I study, 56 patients with advanced NSCLC were assigned based on histology to four cohorts to receive nivolumab (5–10 mg/kg) intravenously every 3 weeks plus one of four concurrent standard “platinum doublet” chemotherapy regimens. No dose de-escalation was required for dose-limiting toxicity. The ORR was 33–50% across arms and the 1-year OS rates were promising at 59–87%At present, there are many other clinical trials of combination therapy are ongoing. A second stage of phase I trial testing about the combination of MEDI4736 with tremelimumab is also enrolling (NCT01975831). Other combinations regimens being tested include nivolumab in patients with NSCLC with chemotherapy, bevacizumab, erlotinib or ipilimumab (NCT01454102), and in advanced solid tumors with IL-21 (NCT01629758). Ongoing combination therapy trials with pembrolizumab include co-administration with cisplatin + pemetrexed or carboplatin + paclitaxel in NSCLC (NCT01840679). In addition, MPDL3280A is being studied in combination with cytotoxic chemotherapy in patients with NSCLC. Researches in regard to the therapy effectivity of combining PD-1/PD-L1 pathway inhibitors with IFN-g, cancer vaccine, soluble CD80 are ongoing or have got results in animal models and other tumor subtypesCurrent research on the combination therapy between PD-1/PD-L1 inhibitors and radiotherapy primarily focuses on animal experimentation (tumor-bearing mice). In a study of glioblastoma multiforme (GBM), investigators tested the combination of anti-PD-1 antibody with stereotactic radiotherapy in a mouse orthotopic GBM model. The results demonstrated that combination-therapy can significantly prolong the survival compared with either modality alone. Long-term survival was seen only in the combination-therapy group, with some mice alive more than 180 daysIn another study of preclinical melanoma and renal cell carcinoma mouse models, scientists found that PD-1 wild-type mice can recapitulate the radiotherapy-induced antitumor responses observed in PD-1 knockout mice after they received anti-PD-1 body therapy, thus prolonging their survivals. The combination of anti-PD-1 therapy and radiotherapy can also elicit the reduction in size of non-irradiated, secondary tumors outside the SABR radiation field The feasibility of combination therapy was that radiotherapy maybe had an effect on up-regulation of PD-L1 on tumor cellsThe PD-1/PD-L1 inhibitors are considered to have a good safety, little toxicity and relatively well tolerated therapeutic method. In different trials (mentioned in this review), the incidence rate of immune-related adverse events (irAEs) exist large difference, but mainly focus on grade 1, 2, such as: fatigue, rash, pruritus, diarrhea, drug-related pneumonitis, vitiligo, colitis, hepatitis, hypophysitis, thyroiditis and vomit. However, there are still some reports which pointed out that a certain proportion of grade 3 or 4 irAEs occured. In a clinical trail involving a total of 296 patients, no maximum tolerated dose was defined, Grade 3 or 4 irAEs occurred in 14% of patients; three deaths resulted from pulmonary toxicity. In a clinical trial with combination therapy, the patients were followed up at least 4 months, grade 3-4 irAEs occurred in 22 of 46 (48%) and led to discontinuation in 16patientsThe PD-L1 expression has been shown to correlate with positive responses to PD-1/PD-L1 pathway inhibition in clinical studies and with poor prognosis in many cancers including NSCLC45However, there are still some controversies for this viewpoint. PD-L1 is an inducible molecule and tumors are frequently heterogenous. Therefore, the threshold of PD-L1 expression positive or overexpression is different in different trials. In addition, even for the same patients, if we observe different region cells, the threshold is different too. For example, discordance between primary tumor and metastases for PD-L1 positivity in both directions has been observed in kidney cancer in 15% (5 of 34) of patientsSo, the role of tumor expression of PD-L1 must be further elucidated. Further clinical studies should address these associations.Although much is known about the PD-1/PD-L1 pathway in tumor immunity, many problems remain unexplored. For example: According to different patients, how to choose an appropriate therapic approach, PD-1/PD-L1 inhibitors or chemotherapy or combination therapy or others and whether there are different curative effect between PD-1/PD-L1 inhibitors and other therapies and does the differences make sense? Whether PD-1/PD-L1 inhibitors can be used for the first-line? Does blockade of the PD-1/PD-L1 pathway result in activation of only tumor-reactive T cells or also T cells that are auto-reactive to non-tumor antigens and that could cause undesirable autoimmunity? Whether there are any other factors impact research results about the biological markers, such as: PD-L2, or other gene mutation or expressed proteins, whether could predict prognosis by two factors or more? Thus, additional basic immunology studies and larger samples of clinical trials are essential.The research of cancer immunotherapy provides a new wide space for cancer treatment (including NSCLC), and compared with other therapeutic method, immunotherapy has its unique advantages, such as: relative safety, effectivity, less and low grade side effect and so on. Especially with the discovery and continued in-depth study of PD-1/PD-L1 pathway in the immune regulation mechanism, many significative conclusions were reported. Data from many clinical trails suggest that some patients with NSCLC have been benefited from the drugs of anti-PD-1 and anti-PD-L1 already. However, summarized what have been discussed above, only a small fraction of patients benefit from PD-1 or PD-L1 inhibitors treatment. But with the continuous studies on biomarker and combined treatment in PD-1/PD-L1 pathway, new research progress will be acquired as well. We will make significant progress on treatment and in control of NSCLC.How to cite this article: He, J. et al. Development of PD-1/PD-L1 Pathway in Tumor Immune Microenvironment and Treatment for Non-Small Cell Lung Cancer. Sci. Rep.5, 13110; doi: 10.1038/srep13110 (2015).


The flexible active humidity sensors were fabricated by transferring the nanofibers from silicon to a soft polymer substrate. The sensors exhibited outstanding piezoelectric energy-harvesting performance with output voltage up to 2 V during the vibration process. The output voltage generated by the NaNbO3 sensors exhibited a negative correlation with the environmental humidity varying from 5% to 80%, where the peak-to-peak value of the output voltage generated by the sensors decreased from 0.40 to 0.07 V. The sensor also exhibited a short response time, good selectively against ethanol steam, and great temperature stability. The piezoelectric active humidity sensing property could be attributed to the increased leakage current in the NaNbO3 nanofibers, which was generated due to proton hopping among the H3O+ groups in the absorbed H2O layers under the driving force of the piezoelectric potential.The development of micro-/nano-scaled energy harvesters and the self-powered sensor system has attracted great attention due to the miniaturization and integration of the micro-device. In this work, lead-free NaNbO Nowadays, the development of high-performance humidity sensors has attracted great attention because monitoring and controlling environmental humidity is important for industry, agriculture, and people’s daily lives . Thanks et al. have demonstrated self-powered pH and UV sensors with a ZnO nanowire-based piezoelectric nanogenerator as their powering sources [Since the power consumption of the micro-scaled sensors has decreased to a much lower level due to the miniaturization of sensing layers, the electrical power generated by energy harvesters such as solar cells, thermoelectric devices, and piezoelectric nanogenerators is sufficient to maintain the sensors . Among t sources . Partial sources . The sen3 nanofibers (NFs) have exhibited ultra-high sensitivity and selectivity to water molecules at room temperature. The surface-absorption of water molecules leads to a great enhancement of a NF’s conductivity [3 nanowires have also been reported to have an outstanding piezoelectric energy-harvesting performance by Jung et al. [3 nanowires for building high-performance active humidity sensors.Recently, a novel type of self-powered sensor system was demonstrated by Zhu and his co-authors, showing that the ZnO nanowires could act as both the piezoelectric energy harvester and the sensor unit ,16,17. Tuctivity . Simultag et al. . Those r3 NFs were synthesized using a far-field electrospinning method. A flexible active humidity sensor based on the NaNbO3 NFs was assembled on the polydimethylsiloxane (PDMS) substrate. The energy-harvesting behavior of the devices under various humidity conditions was investigated. The device exhibited highly sensitive active humidity sensing performance at room temperature. Moreover, the working mechanism of the active humidity sensor was also discussed in detail.In this work, the perovskite NaNbO3 NFs were firstly synthesized on the clean silicon substrates through a far-field electrospinning method. The detailed experimental procedures are as follows: First, the NaNbO3 sol-gel of 0.4 mol/L was prepared by the reflux reaction of the niobium ethoxide and sodium acetate anhydrous in the mixture of acetic acid and 2-methoxyethanol with a volume ratio of 1:3. The reaction lasted for 1 h at 95 °C and was protected in a dry N2 atmosphere to prevent hydrolysis of the niobium ethoxide. All chemical reagents were analytically pure and purchased from Alfa Aesar in China. After the reaction, a transparent yellow NaNbO3 solution was obtained. Then, the electrospinning precursor was prepared by mixing the NaNbO3 solution with the ethanol solution of poly(vinylpyrrolidone) . After stirring for 12 h at room temperature, the precursor was transferred into a 5-mL plastic syringe, which was then positioned onto the syringe pump to electrospin the NFs. The clean silicon substrate was positioned on the metal sample collector, which was placed 10 cm away from the syringe tip. The electrospinning process was then started at a voltage of 1.4 kV/cm, and the precursors ejected at a constant rate of 0.36 mL/h. Finally, the substrates were covered by white composite NFs of the NaNbO3 solution, and the PVP was dried at 80 °C for 5 h. The dried composite NFs were firstly pre-sintered at 450 °C for 1 h to combust the carbons and then annealed at 700 °C to form the perovskite NaNbO3 polycrystalline NFs. All heat treatments were done in an oxygen atmosphere. The structure and morphology of the samples was characterized by the X-ray diffractometer  and field-emission scanning electron microscopy .The NaNbO3 NFs were transferred from the silicon substrate onto the flexible PDMS substrate. Firstly, a certain amount of PDMS silicone was spin-coated on the samples and then cured at 60 °C to make the NFs stiff on the PDMS polymers. Then, the silicone was carefully lifted off from the substrate to transfer the NFs onto the soft polymer substrate. After that, the Pt interdigital electrodes (IDEs) were deposited on the NFs/PDMS surface by using a shadow mask during the sputtering process. Finally, the devices were completed after wire leading. Then, the NaNbO3 NFs were poled with a DC voltage of 20 kV/cm at room temperature. The electrodes of the devices were then shorted for 30 min to remove the stored charges. The piezoelectric energy harvesting and active humidity sensing performance of the devices were measured with a self-designed measuring system as well as a data acquisition (DAQ) card , a digital multimeter (Keithley 2000), a charge amplifier , and the WS-60A gas sensor testing system. Among them, the Keithley 2000 multimeter was used for testing the output voltage generated by the sensors when the experiment was conducted inside an air-dry oven to test the temperature stability. The DAQ USB-6210 was used to test the output voltage at room temperature.In order to fabricate the flexible nanogenerators, the prepared NaNbO3 NFs on the silicon substrate after the annealing treatment. The NFs were randomly arranged on the substrate with a good size distribution and smooth surface morphology. The average diameter of the NFs was ~78 nm according to the statistical result shown in 3 (JCPDS card No. 74-2437).3 active sensor. Because the heat treatment is necessary for the formation of perovskite NaNbO3 NFs, silicon substrates were used for the synthesis of the NFs. Due to the porous structure of the NFs on the silicon substrate, the top layer of the NFs could be packaged by the PDMS soft polymers through the spin-coating and the curing process. Thereafter, the NFs could be easily transferred onto the flexible PDMS substrate by lifting off the PDMS layer. After the IDE deposition, the flexible device based on the electrospun NaNbO3 NFs could be obtained. As shown by the photo image in 3 in dimension.3 NFs was lower than 0.2 V, which was too weak to be recognized from the strong background noise, and only 10% of the output voltage was generated by the sensors with NaNbO3 NFs  compared with the input impedance of the DAQ cards (~MΩ), the noise in the recorded voltage signals was unstable and too high to recognize the relatively lower voltages during the sampling process [It is known that the sensitivity of sensor systems mainly depends on the signal-to-noise ratio (SNR) of the signals. Because of the high-frequency sampling and much higher impedance of the NaNbO process . This li3 sensors with the voltage signal conditioned by the charge amplifier. The sensing performance was tested by changing the humidity of the environment when the NaNbO3 sensors were continuously vibrating under the knocks of the rotating stick. As shown in 3 NFs could be attributed to the electron flow in the external circuit under the driving force of the piezoelectric potential [3 NFs and compensate the piezoelectric potential. Once the piezoelectric potential was removed due to the release of strain, the accumulated electrons would flow back to the positive side along the external circuit and generate a negative voltage peak. As reported, the electrical field applied along the axial direction of the NaNbO3 NFs could lead to protons hopping among the surface-absorbed H3O+ groups [3 NF when the piezoelectric potential was generated due to bending motion. This additional charge flow would lead to leakage current inside the piezoelectric materials and thus decrease the output voltage of the devices. When the environmental humidity changed, the concentration of protons on the surface of the NFs also changed. As a result, the output voltage changed due to the variation in leakage current. Moreover, the leakage current density in the NFs was in a linear correlation with the concentration of protons and absorbed water molecules [3 sensor compared to the reported CeO2/ZnO and SnO2/ZnO sensors should be attributed to the decreased output voltage amplitude after the signal conditioning. However, it is worth noting that the structure of the NaNbO3 nanofiber-based sensors was much more stable than the sensors based on the ZnO nanorod arrays, which was assembled through the stacking of the top electrode and the nanorod arrays.otential ,27,28. T+ groups ,29. Therolecules . Therefo2 gas was introduced to and then removed from the testing chamber, respectively. The output voltage did not exhibit any change during the introducing and removing process of ethanol and H2, which confirmed the excellent selectivity of the sensor. In order to evaluate the influence of temperature on the sensing result, the energy conversion property of the sensors under 38% RH was tested when the temperature increased from 24 to 80 °C. As shown in Moreover, the sensor also exhibited fast response speed to the variation in humidity. As shown in 3 piezoelectric NFs with a uniform size distribution were synthesized using a far-field electrospinning method. By lifting off the PDMS layer coated on the NFs and the electrode deposition, a flexible active humidity sensor was obtained. The sensors could generate an impulsive output voltage with an amplitude up to 2 V during the vibration process. The energy-harvesting performance was confirmed to have originated from the piezoelectric effect of the NaNbO3 NFs. By conditioning the voltage signal with a charge amplifier, the noise in the recorded voltage signals was suppressed by half, which could be attributed to matching impedance and filtering high frequency signals. The output voltage of the NaNbO3 sensors after the signal conditioning exhibited a negative linear correlation with the environmental humidity when the humidity changed from 5% to 80% RH. The humidity-dependent output voltage provides the potential of the NaNbO3 device to be applied as an active humidity sensor with the sensitivity of 2 mV/%RH. The variation in output voltages with humidity can be attributed to the humidity-dependent leakage current inside the NaNbO3 NFs, which was induced by protons hopping among the surface-absorbed H3O+ under the driving force of piezoelectric potential. The active sensor also exhibited a fast response speed, as well as great selectivity and temperature stability. The active humidity sensor based on NaNbO3 NFs provides an effective solution for self-powered sensor systems with high sensitivity, simple structure and fabrication processes, and low production costs.In this work, monoclinic NaNbO


TAR DNA-binding protein 43 (TDP-43) is a nuclear protein, but it is redistributed in the neuronal cytoplasm in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Because small transgenic animal models often lack cytoplasmic TDP-43, how the cytoplasmic accumulation of TDP-43 contributes to these diseases remains unclear. The current study is aimed at studying the mechanism of cytoplasmic pathology of TDP-43.We established transgenic pigs expressing mutant TDP-43 (M337V). This pig model shows severe phenotypes and early death. We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain. Transgenic TDP-43 interacts with PSF, an RNA splicing factor that associates with NeuN to regulate neuronal RNA splicing. The interaction of TDP-43, PSF and NeuN causes PSF and NeuN mislocalize into the neuronal cytoplasm in transgenic pigs. Consistently, abnormal PSF-related neuronal RNA splicing is seen in TDP-43 transgenic pigs. The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains.Our findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology. TAR DNA-binding protein 43 (TDP-43) is a multifunctional, nuclear protein that binds RNA to regulate RNA processing, including alternative splicing, miRNA production, and mRNA trafficking and stabilization –6. TDP-4To date, a variety of transgenic TDP-43 animal models have been established and provided us with valuable tools to uncover gene expression alteration and aberrant RNA splicing caused by TDP-43 , 7, 15. The lack of cytoplasmic mislocalization of TDP-43 in small animals may be due to unknown species differences that regulate the subcellular localization of TDP-43. Because of the greater similarity between pigs and humans, we have used transgenic pigs to investigate the pathology caused by mutant polyglutamine proteins and SOD1 and uncovered unique pathology not seen in small animals , 27. In There are a number of mutations in human TDP-43, and transgenic expression of wild type human TDP-43 also causes neuropathology in animal models , 28. ConNewborn transgenic TDP-43 pigs appeared as normal at birth as non-transgenic pigs. However, they started to gain less body weight from 3 months after birth compared to non-transgenic pigs Fig. . The extOf the dead TDP-43 pigs, we were able to collect brain tissues from some animals for western blotting and immunohistochemical studies. Using anti-TDP-43 , which recognizes endogenous pig TDP-43 and flag-tagged transgenic TDP-43 via western blotting, we found that the levels of transgenic TDP-43 in the spinal cord and cerebellar cortex tissues in different transgenic pigs are lower than or equivalent to the endogenous pig TDP-43 Fig. . Also, tWe know that mutant TDP-43 affects the spinal cord motor neurons to cause ALS , 31, so TDP-43 is involved in gene transcription and RNA splicing via its interactions with other proteins. To identify any pig proteins that could interact with TDP-43 and are involved in TDP-43-mediated toxicity in transgenic pigs, we generated GST-TDP-43 and used it to pull down pig brain cortex proteins. The GST pull-down uncovered a band that is specific to GST-TDP-43 pull-down Fig. . Via masPSF is found to interact with NeuN to activate neural cell-specific alternative splicing . We thenAfter verifying the in vivo association of TDP-43 with PSF and NeuN, we wanted to know whether their subcellular localization is altered in TDP-43 transgenic pig brain. We performed immunocytochemical studies of the spinal cord of transgenic TDP-43 pigs and observed that TDP-43 was concentrated in the nucleus and also distributed in the cytoplasm Fig. . ImmunosThe interaction of TDP-43 with PSF/NeuN led us to focus on alternative splicing of NMHC II-B (myh 10), nonmuscle myosin heavy chain II-B whose splicing is regulated by PSF and NeuN  and is iTo prove a causal role of TDP-43 in reducing NMHC II-B levels, we transfected mutant TDP-43 in PC12 cells and then examined the half-life of endogenous NMHC II-B using cycloheximide treatment. The transfection of TDP-43 promoted the degradation of endogenous NMHC II-B in PC12 cells Fig. . BecauseWe have obtained ALS patient brain tissues that were found to contain TDP-43 aggregates. Because ALS patient brains had undergone extensive neurodegeneration, we had to examine the neurons that were not degenerated and remained clear cell body morphology in order to assess protein subcellular distribution. We found that those neurons in the ALS brains show more cytoplasmic distribution of PSF and NeuN in the cerebellar cortex and spinal cord than those in the control brains Fig. . The cytNext, we wanted to see if NMHC II-B level is also decreased in ALS patient brains. Using RT-PCR to examine alternative splicing of NMHC II-B, we found that tissues from 5 ALS patient cortexes contained more –N30 isoforms than Alzheimer’s disease (AD) and control normal individual brains Fig. . WesternSince NMHC II-B is important for neuronal intracellular trafficking of receptors like NMDA receptor –40 and sBased on the finding that mutant TDP-43 interacts with PSF, we proposed a model for the effects of cytoplasmic mislocalization of mutant TDP-43. In this model, pathogenic TDP-43 is distributed in the cytoplasm. Due to its association with PSF and NeuN, the cytoplasmic mislocalization of TDP-43 may lead to the increased levels of PSF and NeuN in the cytoplasm, thereby reducing the nuclear levels of PSF and NeuN. The reduced PSF/NeuN in the nucleus can impair the nuclear function of these proteins and lead to abnormal RNA splicing, such as greater production of –N30 isoform, which is unstable or becomes toxic. The reduced level of NMHC II-B therefore contributes to the neuropathology caused by mutant TDP-43 Fig. .TDP-43 contains a bipartite nuclear localization signal sequence and a nuclear export signal that allows nuclear-cytoplasm shuttling . TDP-43 Since transgenic TDP-43 is expressed ubiquitously under the control of the ubiquitin promoter, it is also possible that the toxicity of mutant TDP-43 in peripheral tissues contributes to the severe phenotypes of TDP-43 pigs, which will require further experiments to investigate. An interesting finding in our study is that transgenic mutant TDP-43 is also distributed in the cytoplasm, a phenomenon that is seen in patient brains. Viral vector expression of TDP-43 in cynomolgus monkey spinal cord was found to result in the cytoplasmic distribution of TDP-43 . Thus, lHow cytoplasmic distribution of TDP-43 contributes to neuropathology remains unknown. The cytoplasmic mislocalization of TDP-43 is thought to result in the loss of the nuclear TDP-43 and its sequestration of other proteins in the cytoplasm, leading to theories of loss and gain of function caused by TDP-43 . For exaStudies of gene expression in TDP-43 transgenic animal models indicate that mutant TDP-43 affects the splicing and expression of a large number of genes , 15, 49.In TDP-43 transgenic pig brains, we saw diffuse staining of TDP-43 in the cytoplasm without clear aggregates. The formation of cytoplasmic aggregates may be age-dependent or may only occur in old pig brains. The lack of obvious cytoplasmic TDP-43 aggregates supports the idea that soluble TDP-43 is toxic in our TDP-43 transgenic pigs. In fact, this idea has been suggested by the earlier finding that deleting the NLS in mutant TDP-43 could cause the cytoplasmic distribution of mutant TDP-43 and severe phenotypes in transgenic mice without aggregates . Also, aAccording to our proposed model, the mislocalization of TDP-43 in the cytoplasm and its interactions with PSF/NeuN may contribute to the cytoplasmic distribution of PSF and NeuN, thereby reducing their effect on RNA splicing in the nucleus. It is possible that the interaction of mutant TDP-43 with PSF/NeuN in the nucleus can impact the function of PSF/NeuN, as the reduced neurite outgrowth of transfected cells showing both cytoplasmic and nuclear distribution of mutant TDP-43 can be alleviated by NMHC II-B. However, TDP-43 is known to interact with a large number of proteins. The contributions of the interactions of TDP-43 with its partners to ALS pathology may depend on cell types, subcellular regions, and species-related factors, which could confer differential posttranslational modifications to modify these interactions.Using nuclear transfer technology, we established transgenic pigs expressing mutant TDP-43. Like other transgenic TDP-43 animal models, transgenic TDP-43 pigs show severe neurological phenotypes, supporting a toxic gain of function of mutant TDP-43 in large mammalian animals. We also found that mutant TDP-43 is distributed in the cytoplasm in the transgenic pig brain, a phenomenon that is seen in patient brains. By focusing on the TDP-43-PSF interaction and its related RNA splicing function, we provide new evidence for species-dependent cytoplasmic distribution of mutant TDP-43 and a new insight for how the cytoplasmic accumulation of mutant TDP-43 can contribute to abnormal gene expression. These findings should also have implications for identifying other pathological pathways and interacting proteins involved in ALS and FTLD and help uncover therapeutic targets, as well.pBudCE4.1-PSF was obtained from Dr. V.M. Kalscheuer’s lab . pEGFP-N2. After 48 h, 500 μg/ml G418  was added to the medium to select transfected cell colonies, and the plates were incubated in media containing G418 for approximately 10 d. The surviving cell colonies were selected and propagated in a fresh 48-well plate. Colonies that proliferated well were then expanded and screened for the expression of TDP-43.Animal use followed the NIH Guide for the Care and Use of Laboratory Animals. The animal use protocol was approved by the Institutional Animal Care and Use Committees (IACUC) at Guangzhou Institute of Biomedicine and Health (GIBH), Chinese Academy of Sciences. TDP-43 (M337V) cDNA  was tagg2 for 18–22 h at 39 °C. The embryos were then surgically transferred into the oviduct of a surrogate female on the first day of standing estrus. Pregnancy status was monitored using an ultrasound scanner between days 30–35 post-transplantation. Transgenic piglets were identified by PCR using primers (forward primer: 5’-TTGAACTATGCGCTCGGGGTTG-3’ and reverse primer: 5’-GTTCGGTTGTTTTCCATGGGAGAC-3’) and western blot analysis with anti-TDP-43 antibodies.The ovaries of pigs were collected from long white pigs at a local slaughterhouse. The cumulus-oocyte complexes were isolated and cultured as described previously , 27. AftThe movement capabilities of transgenic pigs were tested by treadmill running as described in our recent study . A suitaHuman cortex and spinal cord tissues were obtained and archived via an institutional review board and Health Insurance Portability and Accountability Act-compliant process at neuropathology/histochemistry core of Emory University. Autopsies were performed following death with post-mortem interval of 6–13 h. The spinal cord and cortex tissues were obtained from the postmortem brains of ALS patients  who died at 67–74 years of age and were confirmed to have TDP-43 aggregates via post-mortem histologic analysis. Non-ALS tissues were obtained from neurologically unaffected patients  and Alzheimer’s disease patients  who died at 53–92 years of age. For immunofluorescence studies, tissues were fixed in formalin and embedded by paraffin, sectioned, and stored at room temperature (16-25 °C). For Co-IP and RT-PCR analysis, tissue segments stored at −80 °C were used.GST-TDP-43 recombinant fusion proteins were produced in bacteria (BL21) and purified with glutathione-sepharose beads (Sigma). Pig cortex tissues (0.1 g/ml) in NP-40 lysis buffer  were homogenized using a Teflon homogenizer for 30 strokes. The lysates were added with protease inhibitor cocktail  plus proteasome inhibitor and kept on ice for 30 min. Lysates were sonicated for 10 s and centrifuged at 1000 × g for 2 min. The supernatants (5 μg/μl protein) were pre-cleaned by incubating with 100 μg GST protein and 30 μL glutathione-sepharose beads for 4 h at 4 °C and then subjected to GST-TDP-43 pull-down assay. Analysis of the GST-TDP-43 pull down bands was performed by AB SCIEX TripleTOF® 5600 system at Institute of Genetics and Developmental Biology in Beijing.Cultured cells or brain tissues were homogenized in NP-40,1 × PBS or RIPA buffer with 1× protease inhibitor as previously described . For immThe cytosolic fraction was obtained by centrifuging the brain lysates at a high speed 15000 × g for 15 min, the supernatant was used as cytoplasmic for Western Blot and Co-Immunoprecipitation. For nuclear fractionation, we added 1000 μl of NP-40  buffer to 0.1 g pig brain cortex tissue on ice for 10 min. The tissues were then homogenized with a glass homogenizer for 30 strokes and sonicated of 15 s. The lysates were centrifuged at 4 °C at 800 × g for 10 min. The pellets were re-suspended in 374 μl of buffer , pH 7.9) with 26 μl of 4.6 M NaCl to give the final concentration at 300 mM NaCl and then homogenized with 20 full strokes in Teflon homogenizer on ice and sonicated for 10 s. The homogenized samples were kept on ice for 20 min and then centrifuged at 24,000 × g for 20 min at 4 °C. The supernatant was used for western blotting or Immunoprecipitation.2. PC12 cells were maintained in DMEM/F12 medium with 5 % fetal bovine serum and 10 % horse serum, 100 U/ml penicillin at 37 °C in an atmosphere of 5 % CO2 and 95 % humidity. NGF was used at 100 ng/ml  to induce neurite extension for the PC12 cells.PC12 cells stably expressing flag-TDP-43-EGFP were described in our recent study . HEK293TCulture of primary neurons from mouse brain cortex was performed as described previously . BrieflySections from human or pig brains were fixed in 4 % paraformaldehyde/1 × PBS, embedded in paraffin or Tissue ® Tek O.C.T compound (SAKURA), and sectioned to 10 or 40 μm slices. The paraffin embedded sections were deparaffinized with xylene and sequential ethanol  and rehydrated with water. Rehydrated slices were heat-treated for antigen retrieval in citrate buffer. The treated slices were then stained by antibodies to TDP-43, NeuN, and PSF and detected using DAB staining. Images of micrographs were taken using a Zeiss microscope (Axiovert 200 MOT).To quantify neurons expressing transgenic TDP-43 in transgenic pig brains and ALS patient brains, we used 5–8 images obtained with 20 × N.A. 0.8 objective per section and 5 sections per group (n = 3-5 samples per group). To quantify cultured cells, we examined more than total 300 cells per group in at least three independent experiments. To measure the lengths of neurites of cultured neurons, imaged cells were selected at random, and all visible processes of selected neurons were imaged. The transfected neurons whose neurites could be clearly identified were selected to measure the lengths of their neurites using a fluorescent microscope of ZEISS Axiovrert 200 M microscope. The images were analyzed by measuring the longest branch of each neurite and the relative length was quantified as pixel using NeuronJ plugin image software, which is used for quantification of elongated structures.For analyzing histological changes in the muscle sections, a minimum of 3 random fields (corresponding to 200–300 cross-sectioned fibers) were photo micrographed for gastrocnemius muscle of non-transgenic and TDP-43 transgenic pigs; all type fibers were analyzed for their size using Scion Image software .t-test (two tailed) for comparing two different groups. A value of p < 0.05 or p < 0.01 was indicated with one asterisk (*) or two asterisk (**), which represent statistically significant.For statistical analysis, data were obtained from three or more independent experiments. Statistical analysis was performed using Microsoft Excel and Student’s Animal use followed the NIH Guide for the Care and Use of Laboratory Animals. The animal use protocol was approved by the Institutional Animal Care and Use Committees (IACUC) at Guangzhou Institute of Biomedicine and Health (GIBH), Chinese Academy of Sciences. Human cortex and spinal cord tissues were obtained and archived via an institutional review board and Health Insurance Portability and Accountability Act-compliant process at neuropathology/histochemistry core of Emory University.


The deposition and evolution of the FDTS-based seed layer is investigated by X-ray photoelectron spectroscopy, Auger electron spectroscopy, and transmission electron microscopy. Crystalline quality of graphene transferred from Cu is monitored during formation of the seed layer as well as the HfO2 growth using Raman spectroscopy. We demonstrate that FDTS-based seed layer significantly improves nucleation of HfO2 layers so that graphene can be coated in a conformal way with HfO2 layers as thin as 10 nm. Proof-of-concept experiments on 200 mm wafers presented here validate applicability of the proposed approach to wafer scale graphene device fabrication.We investigate the use of perfluorodecyltrichlorosilane-based self-assembled monolayer as seeding layer for chemical vapour deposition of HfO We adapt an established process used for surface energy modification in the fabrication of micro-electro-mechanical systems (MEMS) structures and their cavities2 on graphene. Wafer scale compatibility and maturity of the proposed pre-treatment can enable fast adoption for the fabrication of graphene-based devices on large-diameter wafer scale.In this work we investigate a pre-treatment of graphene with perfluorodecyltrichlorosilane (FDTS) prior to HfO2/Si(100) wafers before and after FDTS coating are shown in 20−1, FWHM(2D): 36 cm−1) proving that the FDTS coating process does not negatively affect the crystalline quality of graphene sheet. The observed red shift of about 8 cm−1 after SAM formation may be associated with a process induced electrostatic doping of graphene22The representative Raman spectra of a graphene layer transferred to 300 nm SiO2 substrate and weaker C signal due to the absence of graphene. The additional scans performed on the remaining area (regions 1 and 2) show reduced Si and O signals and an intense C peak due to the closed graphene layer. A fluorine signal of similar intensity is detected in all investigated regions proving that the FDTS SAM was coated homogeneously on the substrate surface. More detailed chemical analysis of the SiO2/graphene/FDTS interfaces was performed by X-ray photoelectron spectroscopy (XPS), as shown in The optical microscope image of a transferred graphene layer after the FDTS deposition process is shown 2 substrate which can be detected through the graphene layer. After the FDTS coating process, a clear F1s appears and the O1s peak and Si 2s and 2p peaks are attenuated. Based on these XPS results, we can also conclude, that no additional elements are incorporated during the FDTS deposition into the graphene layer  rises with temperature. As to be seen in the spectra, the characteristic oxidized carbon species (COOR) signals are also observed at a binding energy of 288 eV282/Si substrate is adsorbed, resulting in an increase of the signal of oxidized carbon species C-O and COOR and a peak shift of the C-O component28The intensity of the aromatic C-C signal is reduced after the annealing at 400 °C, as illustrated in As the decomposed FDTS chains could interact with graphene, a Raman analysis was performed after the annealing procedure in order to investigate the quality of the graphene layer. As illustrated in The following conclusions can be drawn from the discussions above: The FDTS seed layer covers graphene homogenously at room temperature and this process does not affect the crystalline quality of graphene. During thermal treatment, long fluorine chains partially decompose and some fluorine species desorb from the surface as it is evidenced by reduced F1s signal. After annealing the high crystalline quality of graphene remains unaffected as proved by Raman spectroscopy investigations.2 by CVD on pretreated graphene layers. As demonstrated by Lukosius et al.2 on transferred graphene is feasible without any initial pretreatment of the graphene surface with the restriction, that HfO2 films have to be thicker than 20 nm to be uniform and closed. Due to nucleation sites created by the FDTS based seed layer, the minimum HfO2 film thickness should be drastically reduced. In the first step, we chose a thickness of nominally 10 nm HfO2 to test the influence of the FDTS nucleation capability. Two graphene samples were transferred to pre-structured 75 nm TiN/Si substrates, which will be used for ongoing electrical characterization. After the transfer process one sample was coated with FDTS, while the other sample was left pristine. Finally, 10 nm HfO2 were deposited by CVD onto both samples.The next section is dedicated to the growth of HfO2 layer, transmission electron microscopy (TEM) was applied. The samples were firstly coated with an Al-layer to protect the sample and then a lamella was prepared. According to the high resolution TEM images, there is a huge difference between the two samples, as illustrated in 2 layer, the sample with FDTS coating  to remove the polymer. Target substrates in this study were 200 mm Si(100) wafers covered with either 300 nm thermal SiO2 or 75 nm CVD TiN layers.Commercially available graphene was transferred from Cu growth substrates by detaching graphene from Cu foils with poly(methyl methacrylate) (PMMA) assisted wet etching in Ammonium persulfate (80 mg/ml in water)3(CF2)7(CH2)2SiCl3) coating of graphene was carried out with a 200 mm AURIX tool . The standard FDTS coating process used in MEMS fabrication is performed at substrate temperatures of 30 °C and is divided into two parts: the first part cleans and activates the surface by exposure of the wafer to O2 plasma, the second part provides the N2 bubbled exposure to FDTS and DI water at a process pressure of 40 Torr and for a duration of 300 s. The flow rate of the two carrier gases are 20 sccm for FDTS and 100 sccm for DI water. As O2 plasma readily introduces defects in the crystalline lattice of graphene, this step was omitted in the process flow.The FDTS (CF2 growth was performed in an atomic vapor deposition tool with Hf(NMeEt)4 precursor and oxygen as the reactive gas2 deposition was set to 400 °C for a duration of 3 min. The deposited layers are polycrystalline. More details about the depositions of HfO2 by CVD have been reported elsewhereHfO−8 Torr and the spectra were measured with X-ray beam size of 100 × 100 μm. A PHI 670 Auger Nanoprobe (Perkin Elmer) was used for Auger electron spectroscopy. The spectra were measured with a beam voltage of 10 keV. Structural investigations and EDX-measurements were done by transmission electron microscopy using a FEI Tecnai Osiris instrument operated at 200 kV.A Renishaw InVia micro-Raman spectrometer equipped with a 514 nm wavelength laser, an 1800 lines/mm grating and 50x objective was used to acquire Raman spectra. XPS measurements were performed using PHI 5000 Versa Probe II (ULVAC-PHI) with monochromatic Al-Kα irradiation. The chamber pressure was ~3 × 10How to cite this article: Kitzmann, J. et al. Perfluorodecyltrichlorosilane-based seed-layer for improved chemical vapour deposition of ultrathin hafnium dioxide films on graphene. Sci. Rep.6, 29223; doi: 10.1038/srep29223 (2016).


The internal transcribed spacer (ITS) sequence was demonstrated as the optimal barcode for identifying Radix Astragali and its adulterants. This new analysis method is helpful in identifying Radix Astragali and expedites the utilization and data mining of DNA barcoding.Radix Astragali is a popular herb used in traditional Chinese medicine for its proimmune and antidiabetic properties. However, methods are needed to help distinguish Radix Astragali from its varied adulterants. DNA barcoding is a widely applicable molecular method used to identify medicinal plants. Yet, its use has been hampered by genetic distance, base variation, and limitations of the bio-NJ tree. Herein, we report the validation of an integrated analysis method for plant species identification using DNA barcoding that focuses on genetic distance, identification efficiency, inter- and intraspecific variation, and barcoding gap. We collected 478 sequences from six candidate DNA barcodes (ITS2, ITS,  Astragalus membranaceus and Astragalus mongholicus according to Chinese Pharmacopoeia (2010 edition). Radix Astragali is widely used for its antiperspirant, antidiuretic, and antidiabetic properties and as a tonic drug [Radix Astragali (Huang Qi), a commonly used Chinese medicinal material, is mainly sourced from the plants ofopoeia 200 editionnic drug . It possnic drug –6. Astragalus, Hedysarum, and Malva are often used in its stead [ matK (maturase K) and rbcL  of chloroplast genome, and coxI (cytochrome c oxidase 1) of the mitochondrial genome [ Astragalus) and 478 sequences from six barcodes were used to validate the new method for identifying Radix Astragali and adulterants and to accelerate the data utilization of DNA barcoding.Due to the high market demand for Radix Astragali, a diverse group of adulterants with similar-morphological characteristics from genuses, such asts stead . The trats stead , thin-lats stead , Fourierts stead , and higts stead , all, rets stead  or amplits stead , are unsts stead , 15. It ts stead , 17. In ts stead , 3′ untrts stead , ITS . Supernatant was removed and placed into a new tube before adding 700 μL isopropanol and blending for 15–20 minutes. The mixture was centrifuged in CB3 spin columns for 40 s at 12000 rpm. The filtrate was discarded and 500 μL GD (adding quantitative anhydrous ethanol before use) was added before centrifuging at 12000 rpm for 40 s. The filtrate was discarded and 700 μL PW (adding quantitative anhydrous ethanol before use) was used to wash the membrane before centrifuging for 40 s at 12000 rpm. This step was repeated with 500 μL PW, followed by a final centrifuge for 2 minutes at 12000 rpm to remove residual wash buffer. The spin column was dried at room temperature for 3–5 minutes and then centrifuged for 2 minutes at 12000 rpm to obtain the total DNA.The material specimens were naturally dried and 30 mg of dried plant material was used for the DNA extraction. Samples were rubbed for two minutes at a frequency of 30 r/s in a FastPrep bead mill , and total genomic DNA was isolated from the crushed material according to the manufacturer's instructions . We made the following modifications to the protocol: chloroform was diluted with isoamyl alcohol (24 : 1 in the same volume) and buffer solution GP2 with isopropanol (same volume). The powder, 700  psbA-trnH barcodes, as presented in μL reaction mixtures containing 2 μL DNA template (20–100 ng), 8.5 μL ddH2O, 12.5 μL 2× Taq PCR Master Mix , and 1/1-μL forward/reverse (F/R) primers (2.5 μM). The reaction mixtures were amplified in a 9700 GeneAmp PCR system . Amplicons were visualized by electrophoresis on 1% agarose gels. Purified PCR products were sequenced in both directions using the ABI 3730XL sequencer .General PCR reaction conditions and universal DNA barcode primers were used for the ITS, ITS2, and psbA-trnH, matK, rbcL, and COI), which were downloaded from the GenBank database . Complete ITS2 sequences were obtained using the HMMer annotation method, based on the Hidden Markov model (HMM) . All of database . Sequenc psbA-trnH and matK regions provided a higher rate of success than the other three barcodes using the BLAST1 method. However, matK, ITS, and psbA-trnH performed better than the other three barcodes, based on the nearest distance method. ITS and psbA-trnH obtained higher genetic distances, so the matK, ITS, and psbA-trnH barcodes were the preferable methods for identifying Radix Astragali and its adulterants based on superior sequencing efficiency and identification efficiency.A total of 478 sequences for six barcodes were analyzed, from which 161 sequences were obtained from Astragalus Radix and its adulterants. Sequence information and identification success rates are listed in  matK, and rbcL regions outperformed the ITS and psbA-trnH regions for gap rates. However, when we compared all of the average inter- and intraspecific distances, the ITS2, rbcL, matK, and psbA-trnH regions performed better than the ITS and COI regions. Therefore, in terms of intra- and interspecific variation, ITS2, matK, and rbcL are the preferable options for identifying Radix Astragali and its adulterants.Six parameters to analyze intraspecific variation and interspecific divergence were employed to assess the utility of six DNA barcodes . We expe rbcL, COI, ITS, and matK regions possessed less relative distribution of inter- and intraspecific variation than psbA-trnH and ITS2, although there were no nonoverlapping regions for the six barcodes. Hence, the rbcL, COI, ITS, and matK regions are more successful at identifying Radix Astragali and its adulterants, from the standpoint of barcoding gap analysis.Analysis of the DNA barcoding gap presents the divergence of inter- and intraspecies and indicates separate, nonoverlapping distribution between specimens in an ideal situation . In our  psbA-trnH successfully differentiated Radix Astragali and its adulterants. Furthermore, it produced areas of obvious separation for Radix Astragali. The remaining five barcodes also differentiated Radix Astragali and its adulterants. Each species clustered together, separate from other species. Considering the difficult amplification and sequencing and fast and accurate identification purpose of DNA barcoding, we did not add all the sequence data of ITS2 and psbA-trnH to build ML tree and subsequent analysis.Maximum likelihood (ML) is a general statistical criterion in widespread use for the inference of molecular phylogenies . An ML tRadix Astragali is reported to possess 47 bioactive compounds and has many bioactive properties –37. Vari psbA-trnH, rbcL, and matK from chloroplast genome; COI from mitochondrial genome), genetic distance and ML Tree were calculated by MEGA 6.0 software, and identification efficiency, intra- and interspecific variation, and barcoding gap were calculated using the Perl language algorithm. Results of the six indicators assessed are shown in  psbA-trnH outperformed other barcodes in terms of identification efficiency. ITS2 performed better in terms of genetic distance, gap rate, and inter- and intraspecific variation. RbcL performed better in terms of barcoding gap and inter- and intraspecific variation. Although ITS2 was part of the ITS sequence, it performed poorly in identification efficiency. Therefore, we suggest that the ITS sequence is the optimal barcode, and that the psbA-trnH region is a complementary barcode for identifying Radix Astragali and its adulterants.In this study, we validated a new analytical method for identifying Radix Astragali using DNA barcoding. Seventy-seven specimens of Radix Astragali and its adulterants were collected, and the sequences of 29 species reported in the literature were downloaded from the GenBank database. Based on the 478 sequences for six barcodes (ITS2, ITS from nuclear genome;In conclusion, we describe a new analytical method for the use of DNA barcoding in the identification of Radix Astragali. Six indicators, including average genetic distance, BLAST1 and the nearest distance method for identification efficiency, inter- and intraspecific variation, and gap rate were tested to evaluate six DNA barcodes using bioinformatics software and the Perl language algorithm. The ITS sequence was the optimal barcode for identifying Radix Astragali and its adulterants. This method provides a novel means for accurate identification of Radix Astragali and its adulterants and improves the utilization of DNA barcoding in identifying medicinal plant species.


AbstractCitharoceps Chamberlin was diagnosed by the presence of a very distinctive stridulatory apparatus composed of two patches of ridges on the sides of the cephalic region, and a stridulatory thorn on the prolateral region of the femur I. Currently, this genus is a junior synonym of Ariadna Audouin, with the assumption that the stridulatory apparatus could constitute an exclusive feature of its unique known species, Citharocepsfidicina Chamberlin, currently senior synonym of Citharocepscalifornica Chamberlin & Ivie. In the present study, Citharoceps is revalidated and redescribed based on the occurrence of the stridulatory apparatus in Citharocepsfidicina and Segestriacruzana Chamberlin & Ivie, and also on the presence of distinguishable characters, such as the length of the labium-sternum junction, ventral median spine on male metatarsi I, and strong sclerotized interpulmonary fold in females, forming a conspicuous median flap. Segestriacruzana is transfered to Citharoceps, with Citharocepscalifornica removed from the synonym of Citharocepsfidicina, and proposed as a junior synonym of Citharocepscruzana, due to the similarity between the additional material examined and the original description. Males of Citharocepsfidicina and Citharocepscruzana are described for the first time. Segestriidae currently comprises 120 species distributed in three genera: Segestria Latreille, 1804, Ariadna Audouin, 1826 and Gippsicola Hogg, 1900 , and Queensland Museum, Brisbane . The morphological examinations and descriptions follow The specimens examined are deposited in the American Museum of Natural History, New York , California Academy of Sciences, San Francisco , Collection of the Cabrillo National Monument Park, San Diego , Darrel Ubick collection, San Francisco (CDU), Segestriasenoculata : DENMARK: Zealand Island: Tisvilde, Tisvildeleje  (DMS), 1♂ 1♀ 3imm., 19-20.V.1991, C. Griswold & N. Scharff leg. (CAS 9032847). Ariadnamaxima : CHILE: Concepcion Province: Estación Escuadrón  (DMS), 2♂ 5♀ 3imm., 20.IX.1980, N. Cekalovic leg. (AMNH); Santiago Province: Santiago  (DMS), 1♀, 20.X.2009, T. H. Kawamoto leg. (IBSP 166664). Gippsicola sp.: AUSTRALIA: Queensland: Massey Range, 4km W of Centre Bellender Ker, 1250m  (DMS), 2♂, 9–11.X.1991, Monteith, Janetzi & Cook leg. (QM S91041); Bellender ker Range, Summit TV Station, 1560m  (DMS), 1♀, 1–7.XI.1981, Earthwatch/Queensland Museum leg. (QM S30617).Vouchers for comparative studies: PageBreakTaxon classificationAnimaliaAraneaeSegestriidaeChamberlin, 1924gen. reval.Citharoceps Chamberlin, 1924: 607.Citharocepsfidicina Chamberlin, 1924Citharoceps is distinguished from other segestriid genera by the presence in males, females and immatures of a conspicuous stridulatory apparatus composed of two patches of grooves on both sides of cephalic region , Baja California, Mexico, 4.VII.1921, Soiver leg., deposited in CAS 1392, examined). Chamberlin, 1924: 608 . – World Spider Catalog, 2015.California:UNITED STATES OF AMERICA. San Diego County: San Diego, La Jolla  (DMS), Eucalyptus grove, 1♀ 1imm., XII.1965, F. Ennik leg. (CAS 9039517); San Diego, Point Loma Peninsula  (DMS), 2♂, 8-12.IX.2003, D. Chan & T. Duffield leg. (CNMP); Baja California del Norte:MEXICO.  2.5mi S. of the Halfway House on Mex. Highway #1 (RB-3)  (DMS), 2♀ 1imm., 21.XI.1962, P. R. Craig & D. Dailey leg. .Citharocepsfidicina differ from those of Citharocepscruzana by the slightly shorter and thicker embolus . Total length 5.2; carapace 2.64 long, 1.76 wide. Palps light brownish orange . Total length 8.0; carapace 3.72 long, 2.36 wide. Palps orange, gradually darkening distally. Legs: robust, with long setae, and with ventral tibial and metatarsal spines of similar size : Total length 5.0–5.2; carapace 2.64–2.72 long; femur I 2.36–2.44. Female (n=3): Total length 6.08–8.0; carapace 3.0–3.72 long, 1.84–2.36 wide; femur I 1.92–2.56.South of California, USA and Baja California, Mexico Fig. .Citharocepsfidicina. In addition, that immature was collected with two females that match perfectly with the females collected in San Diego.We feel that species attribution is unproblematic. The immature specimen collected in Baja California del Norte by Craig and Dailey in 1962, matches perfectly with the type material of Taxon classificationAnimaliaAraneaeSegestriidaecomb. n.Segestriacruzana34°00'N; 119°45'W) (DMS), Santa Barbara County, California, USA, III–IV.1913, R. V. Chamberlin leg., deposited in AMNH, examined). –  Chamberlin & Ivie, 1935: 7  (DMS), California, USA, following  Chamberlin & Ivie, 1935: 8, pl. 5, figs 32–33  (DMS), 1♀, 22.II.2002, D. & S. Ubick leg. (CDU); Greenfield, Arroyo Seco Camping, Indians Road, el. 1126’  (DMS), 1♂ 2imm., 18.VI – 24.X.2004, D. & S. Ubick leg. (CDU); Coast Ridge Trail  (DMS), 0.8 mi SE Nacimiento Rd., el. 3000’, 1♀ 2imm., 1.VI.1991, D. Ubick leg. (CDU); San Luis Obispo County: Cayucos  (DMS), 300’, 1♀, 15.XI.1937, O. Bryant leg. (CAS 9039527); Santa Barbara County: gully SW of U. C. Field Station, 80-90m, Santa Cruz Island  (DMS), 1♂ 1imm., 22.IV.1994, D. H. Kavanaugh leg. .Citharocepscruzana differ from those of Citharocepsfidicina by the presence of a relatively longer and slender embolus . Total length 8.4; carapace 4.0 long, 2.76 wide. Palps light orange . Total length: 11.92; carapace 4.44 long, 2.6 wide. Palps reddish orange, gradually darkening distally. Legs reddish orange, with pairs I-II darker. Femur, patella and tibia I-II darkly marbled mainly on the ventral, prolateral and retrolateral regions : Total length 8.0–8.4; carapace 3.92–4.0 long, 2.4–2.76 wide; femur I 3.08–3.2. Female (n=3): Total length 9.32–11.92; carapace 4.24–4.44 long, 2.6–2.76 wide; femur I 2.72–2.92.Monterey County to Santa Barbara County, California, USA Fig. .PageBreakCitharoceps was found from the type locality of Segestriacruzana. After the examination of the immature holotype of Segestriacruzana, it was detected that it possessed the stridulatory apparatus exclusive for Citharoceps, not detected by Citharoceps were found from the middle coast of California that resemble those described by Citharocepscalifornica, together with a male, from the same region, that is similar to the one from Santa Cruz Island. Thus, Citharocepscalifornica is removed from its synonym with Citharocepsfidicina, due to its greater size and darker coloration, and placed as junior synonym of Citharocepscruzana.One male of Citharoceps Chamberlin, 1924 comprises only coastal regions of the state of California, USA, and Baja California, Mexico  in the carapace; and thorns (plectron) in the femur I. This type was also described by The occurrence of a stridulatory apparatus in spiders has been reported in at least 25 spider families , and alsplogynae  and Enteelegynae . LegendrPageBreakCitharoceps is unknown, but considering that it occurs in males, females and immatures, it seems unlikely that it has a courtship function. Citharocepsfidicina making a loud buzzing sound while rubbing the carapace ridges. Considering this information, the function of the stridulatory apparatus in Citharoceps could involve defensive buzzing sounds, as described by Micrathenagracilis , but future ethological studies are needed to corroborate this hypothesis.The function of the stridulatory apparatus in


The fully human monoclonal antibodies nivolumab (Nivo) and ipilimumab (Ipi) block the interaction between the immune checkpoint receptors programmed death-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4), respectively, and their cognate ligands, restoring antitumor immune response. Phase 1 studies of Nivo monotherapy  or Nivo+Ipi combination therapy  demonstrated durable clinical activity and objective response rates (ORRs) of 31% to 40%, respectively, in patients with advanced melanoma (MEL). Evaluation of PD-L1 expression using immunohistochemistry (IHC) suggested a correlation between pretreatment tumor PD-L1 expression and clinical response to Nivo monotherapy [MEL patients received Nivo monotherapy (n=107) or Nivo+Ipi combination therapy . Tumor and tumor infiltrating lymphocyte (TIL) surface programmed death ligand 1 (PD-L1) expression in formalin-fixed, paraffin-embedded (FFPE) tumor tissue was evaluated by IHC with an automated assay (Dako) using the 28-8 Ab. Tumor PD-L1 positivity (PD-L1+) was defined as ≥5% cell membrane staining of any intensity; any expression on TILs was considered positive. Absolute lymphocyte counts (ALC) were measured in serial peripheral blood samples and lymphocyte subsets were evaluated using flow cytometry.P< 0.05).Tumor PD-L1-positive expression was observed in 45% and 37% of samples from the 003 and 004 studies, respectively. In 003, inclusion of any immune cell staining increased PD-L1 positivity to 92%. A numerically higher ORR was observed in MEL patients with PD-L1+ tumors with Nivo monotherapy or with sequential but not concurrent combination therapy. Neither study demonstrated an obvious change in ALC; however, phenotypic changes in T-cell subsets, including increases in the percentage of CD4 and CD8 expressing HLA-DR, ICOS and/or Ki67, were seen with combination therapy. In both studies, responses were observed irrespective of tumor PD-L1 or ALC status. In an exploratory analysis low pretreatment myeloid derived suppressor cells (MDSC) correlated with higher ORR with combination therapy (PD-L1 positivity is associated with tumor response with Nivo monotherapy; however, some responses were observed independent of PD-L1 or ALC status. No correlation between response and PD-L1 or ALC status was seen with combination therapy. MDSC levels may correlate with response to combination therapy. Future phase 3 randomized studies will explore these markers and other phenotypic changes in immune cell populations that might predict activity of Nivo in patients with MEL and other advanced cancers.


In this perspective article, we question how well model organisms, the ones that are easy to cultivate in the laboratory and that show robust growth and biomass accumulation, reflect the dynamics and interactions of microbial communities observed in nature. Today’s -omics toolbox allows assessing the genomic potential of microbes in natural environments in a high-throughput fashion and at a strain-level resolution. However, understanding of the details of microbial activities and of the mechanistic bases of community function still requires experimental validation in simplified and fully controlled systems such as synthetic communities. We have studied methane utilization in Lake Washington sediment for a few decades and have identified a number of species genetically equipped for this activity. We have also identified co-occurring satellite species that appear to form functional communities together with the methanotrophs. Here, we compare experimental findings from manipulation of natural communities involved in metabolism of methane in this niche with findings from manipulation of synthetic communities assembled in the laboratory of species originating from the same study site, from very simple (two-species) to rather complex (50-species) synthetic communities. We observe some common trends in community dynamics between the two types of communities, toward representation of specific functional guilds. However, we also identify strong discrepancies between the dominant methane oxidizers in synthetic communities compared to natural communities, under similar incubation conditions. These findings highlight the challenges that exist in using the synthetic community approach to modeling dynamics and species interactions in natural communities. For centuries, Biology has relied on model organisms for understanding the details of metabolism, regulation, evolution trends, etc. For example, rats, mice, nematode worms and yeast are all used as models for understanding complex metabolism and regulation in humans . MicrobiMethane oxidation is an important environmental process that mitigates release of large quantities of methane, mostly generated biogenically, via degradation of complex organic matter . This prMethylomonas (gammaproteobacteria) and Methylosinus  of major players, as observed at pure culture level, compared to activities/conditions approximating environmental settings. The existence of such discrepancies would have major implications for the selection of laboratory models and the experimental conditions. It would affect not only how we interpret the data on their performance, but also how we scale up these findings to the level of predicting and modeling global elemental cycles. Ultimately, we question whether our model choices and their manipulation in simplified laboratory settings allow for realistic interpretation of microbial activities in natural environmental settings.Figure 1), to address the discrepancies between the performance of natural communities in semi-in situ conditions and laboratory performance of pure culture isolates. Even though all 50 organisms originated from Lake Washington sediment, we did not follow any specific design for community structure, assuming that species that thrive in the specific incubation conditions that we apply would increase in their abundance and species that are not fit for these conditions would decrease in their abundance. Live cultures were mixed together in similar cell counts and were incubated for several weeks, with periodic transfers and dilutions, as previously described (2), low methane/high oxygen , and high methane/high oxygen . While the first two conditions mimicked gas concentrations in the opposing parts of methane and oxygen gradients in the natural lake sediment, the third condition represented a typical laboratory setting. We addressed two main questions: (i) whether Methylomonas and Methylosinus would outperform Methylobacter and Methylosarcina, to reflect their robust performance in the laboratory, and (ii) whether species other than Methylophilaceae, Flavobacteriales or Burkholderiales would persist in methane-fed communities, also based on laboratory performance of the cultivated species. In each of the communities, rapid loss of species complexity was observed after a few days of incubation, as previously observed for natural communities of high complexity  while methanotrophs of the family Methylocystaceae were undetectable. However, significant differences compared to natural community dynamics were uncovered in terms of the dominant methanotrophs. While Methylobacter species were found as dominant under the HL condition in natural samples . The dynamics of the Methylosarcina population also differed from the ones previously observed in natural communities. While Methylosarcina was reported to outperform Methylobacter under the HH condition .We assembled synthetic communities of 10 methanotrophs, 36 non-methanotrophic methylotrophs, and 4 non-methylotrophic heterotrophs . The results from these incubations clearly demonstrate that the performance/fitness of the three model organisms, as pure cultures, did not correlate well with their performance/fitness as parts of synthetic communities, even when simple two- to three-species communities were considered . In turn, their performance/fitness as parts of synthetic communities did not correlate well with the performance/fitness of their closely related counterparts as parts of natural communities . However, performance/fitness of the simple (3-species) and the complex (50 species) synthetic communities showed some agreement . Thus, we conclude that synthetic communities, at least as applied to methane-oxidizing communities and in the conditions used, appear to behave differently from the natural communities. These observations question, more generally, our reliance on data from laboratory manipulations for global predictions as related to microbe-mediated environmental processes.We next measured growth curves for pure cultures of three methanotroph species, Methylomonas spp., hardly seen in natural communities, showing robust performance, in agreement with their documented growth characteristics  The strains that we successfully isolated in pure cultures poorly reflect strains that are active in the natural habitat. However, we do have evidence of their close relatedness at the genomic level (Microbial communities involved in methane metabolism were used here as one example of a functional microbial assemblage involved into a specific biogeochemical process. In microbial ecology, and in ecology in general, fitness and performance of individual species are often considered as predictors of success in natural communities. However, the necessity of measuring performance in natural settings has been advocated . We hereeristics . In factsediment , and we ic level , understLC and ZY designed the experiments, ZY carried out experiments, SK carried out flow cytometric counting, DB carried out iTag data clustering. ZY and DB prepared figures. LC, ZY, SK, and DB wrote the manuscript.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.