Sarcoidosis is a granulomatous lung disorder of unknown cause. The majority of individuals with sarcoidosis spontaneously achieve full remission (uncomplicated sarcoidosis), however, ~20% of sarcoidosis-affected individuals experience progressive lung disease or cardiac and nervous system involvement (complicated sarcoidosis). We investigated peripheral blood mononuclear cell (PBMC) microRNA and protein-coding gene expression data from healthy controls and patients with uncomplicated or complicated sarcoidosis. We identified 46 microRNAs and 1,559 genes that were differentially expressed across a continuum of sarcoidosis severity . A total of 19 microRNA-mRNA regulatory pairs were identified within these deregulated microRNAs and mRNAs, which consisted of 17 unique protein-coding genes yielding a 17-gene signature. Pathway analysis of the 17-gene signature revealed Jak-STAT signaling pathway as the most significantly represented pathway. A severity score was assigned to each patient based on the expression of the 17-gene signature and a significant increasing trend in the severity score was observed from healthy control, to uncomplicated sarcoidosis, and finally to complicated sarcoidosis. In addition, this microRNA-regulated gene signature differentiates sarcoidosis patients from healthy controls in independent validation cohorts. Our study suggests that PBMC gene expression is useful in diagnosis of sarcoidosis. Lung involvement is commonly manifested as bilateral hilar lymphadenopathy and pulmonary infiltration with more severe cases developing pulmonary fibrosis. Cardiac and neurologic involvement are also associated with significant morbidity and death4. This broad spectrum of clinical manifestations makes diagnosis challenging and prolonged.Sarcoidosis remains a systemic inflammatory disease of unknown etiology with an unpredictable course that affects all races and ethnicities that is characterized by the presence of non-caseating epithelioid granulomas in one or multiple organs. The disorder is extremely heterogeneous with more than 50% of sarcoidosis patients experience remission within 3 years after diagnosis, and over 66% of patients experiencing remission within 10 years6. Acute phase reactants like serum amyloid (SAA) levels are significantly higher in active disease similarly to ACE7, but likewise suffers from low specificity and sensitivity. HLA-DR allele and cytokines such as tumor necrosis factors  are overexpressed in sarcoidosis as well and correlated with fibrosis development8. No single biomarker has proven to exhibit significant sensitivity and specificity to be recommended as a monitoring and prognostic tool for standard clinical use.Clearly validated biomarkers that can accurately sub-phenotype patients with sarcoidosis are of enormous utility as they may identify subjects at increased risk of complicated sarcoidosis and allow for the delivery of targeted therapies to prevent progressive multi-organ deterioration. Unfortunately, blood, bronchoalveolar lavage fluid, exhaled breath concentrate, and cerebrospinal fluid have all been assessed as sarcoidosis biomarkers by multiple methodologies, including enzyme-linked immunosorbent assay and proteomic analysis, but with limited success. For example, angiotensin converting enzyme (ACE) is the most commonly studied biomarker but is non-specific and does not accurately correlate with severity11, particularly relevant in assessment of personal risk for developing complicated sarcoidosis10. For example, Koth et al. analyzed the transcriptomic gene expression data from whole blood of sarcoidosis patients and accordingly built a classifier using the gene expression data, which distinguished sarcoidosis patients from healthy controls in an external validation cohort with a fairly good accuracy9. Recently, we investigated genome-wide gene expression in peripheral blood mononuclear cell (PBMC) in sarcoidosis patients and identified a 20-gene signature, which distinguishes uncomplicated sarcoidosis from complicated sarcoidosis10. This gene signature also exhibited a substantial predictive power when classifying sarcoidosis patients from healthy controls in two external cohorts.As a consequence, we and others have proposed the integration of blood gene expression profiling as an opportunity to explore potential useful molecular signatures in sarcoidosisIn this study, we proposed to incorporate microRNA (miRNA) expression information with protein-coding gene expression data to identify sarcoidosis biomarkers. Firstly, we identified a list of differentially expressed miRNAs in PBMCs from patients with sarcoidosis. Secondly, we searched for a list of target protein-coding genes for the deregulated miRNAs and based on these target genes, developed a novel miRNA-regulated peripheral blood gene signature that accurately differentiates sarcoidosis patients from healthy controls and complicated sarcoidosis patients from uncomplicated ones. Pathway analysis of the gene signature revealed Jak-STAT signaling pathway as the most significantly represented pathway. A severity score, based on the expression of the gene signature, exhibited a significantly increasing trend from healthy control, to uncomplicated sarcoidosis, and finally to complicated sarcoidosis. These studies suggest that PBMC gene expression is useful in diagnosis of sarcoidosis, and more importantly, in the identification of patients with complicated sarcoidosis.P < 0.05) that were differentially expressed with severity . In total, we identified 46 miRNAs (adjusted 12 accession: GSE37912). Spearman’s rank correlation test was conducted between gene expression and sarcoidosis severity. In total, 1,559 genes showed significant correlation (adjusted P < 0.0005), among which 340 genes showed positive correlation between gene expression and severity while the expression of 1,219 genes was negatively correlated with severity  to select the top miRNA-mRNA binding pairs14. We intersected these predicted binding targets against the differentially expressed protein-coding genes revealing a total of 425 miRNA-mRNA pairs. Among these miRNA-mRNA pairs, we only retained the pairs that demonstrated a significant negative correlation (adjusted P < 0.005) between miRNA and mRNA expression. This step yielded 19 miRNA-mRNA pairs consisting of eight unique miRNAs and 17 unique protein-coding genes  was assigned to each patient based on the expression of the 8-miRNA signature, which is a linear combination of the miRNA expression values weighted by direction of differential expression (See Methods for details). Higher SmiRNA implies more severe sarcoidosis. As expected, a significant positive correlation (Spearman’s rank correlation test: ρ = 0.651 and P = 4.4 × 10−9) was identified between SmiRNA and sarcoidosis severity in our discovery cohort . The 8-miRNA signature based severity score of sarcoidosis patients is significantly higher than that of controls in the Germany cohort (t-test: P = 3.4 × 10−6)  is 1.000 in this cohort −6) Fig. . The areort Fig. , which sort Fig. .Figure 3Sgene) to each patient based on the expression of the 17-gene signature, which is a linear combination of the protein-coding gene expression values weighted by direction of differential expression (see Methods for details). Patients with more severe sarcoidosis are expected to have a higher Sgene. Not surprisingly, we found a significant positive correlation (Spearman’s rank correlation test: ρ = 0.615 and P = 5.6 × 10−9) between Sgene and sarcoidosis severity in our discovery cohort . The other dataset (GEO accession: GSE18781)18 is from Oregon Health Sciences University and includes 25 healthy controls and 12 sarcoidosis patients (Oregon cohort). The 17-gene signature-based severity score of sarcoidosis patients is significantly higher than that of controls in both the validation cohorts (t-test: P = 1.5 × 10−8 for the UCSF cohort and P = 3.8 × 10−5 for the Oregon cohort) rt) Fig. . The AUCely Fig. . Principely Fig. .Figure 4P = 1.9 × 10−2 and 2.09 × 10−5) .To understand the discriminative power of the 17-gene signature in distinguishing sarcoidosis from other lung diseases, we collected two more blood transcriptomic datasets (GEO accession: GSE42826 and GSE4257), the first consisted of 52 healthy controls, 25 sarcoidosis patients, 11 tuberculosis (TB) patients, six pneumonia patients, and eight lung cancer patients. TB, pneumonia patients, lung cancer were considered as non-sarcoidosis lung disorders. The second dataset comprised of 123 healthy controls, 87 and 47 cases with COPD and sarcoidosis respectively. Principal component analysis on the expression of the 17-gene signature in this dataset indicates that the first principal component significantly differentiates the sarcoidosis patients from the non-sarcoidosis lung disorders in both datasets   Fig. .AUC in both validation cohorts. Figure P = 0.019).We next addressed whether the classification power of the 17-gene signature is superior to the other genes that are related to sarcoidosis. To answer this question, we conducted a second resampling test. We limited the resampling pool to the genes that were differentially expressed with sarcoidosis severity  that potentially relate to sarcoidosis severity, including miR-23a, miR-23b, miR-30c, miR-185, and miR-223, now recognized to be related to pulmonary hypertension18. Jak-STAT signaling pathway is an intracellular cascade initiated in response to cytokine signaling. Several genes in Jak-STAT pathways have already been associated with sarcoidosis, such as IL1531, IL23R32, and STAT118. According to the current knowledge of the molecular mechanism leading to sarcoidosis, genes involved in T-cell receptor selection, T-cell activation and apoptosis, and cytokine regulation are also highly associated with the pathogenesis of sarcoidosis33. In our 17-gene signature, CBLB and IL6ST are known to be involved in T-cell receptor signaling and cytokine-cytokine receptor interaction, respectively, according to the definition in KEGG database. However, we failed to identify a single gene within our 17-gene signature that overlapped with the previously published sarcoidosis blood gene signatures by Koth et al.9 and Zhou et al.10, respectively.We also explored the utility of a protein-coding gene expression signature in sarcoidosis. We identified the 17-gene signature, in which the genes were potentially regulated by the miRNAs within the 8-miRNA signature. In agreement with prior reported studies, the 17-gene signature was enriched in Jak-STAT signaling pathwayet al. suggests that resampling test should be used to evaluate the predictive power of a given gene signature instead of focusing on nominal P-value, because most the published signatures are not significantly better than the randomized gene signatures of identical size20. Using resampling tests, we found that the classification power of the 17-gene signature is significantly better than that of the random gene sets selected from human genome.A published bioinformatical study by Venet et al.9 and Zhou et al.10, the 17-gene signature was developed based on miRNA expression information and predefined miRNA-gene interactions. Statistically-derived gene signatures by whole genome screening are often highly accurate in the patient data sets from which they were identified, yet most of them have not been validated as useful clinical tools. Using resampling test, we demonstrate that the miRNA-regulated 17-gene signature performs even better than the random gene sets selected from the pool of sarcoidosis related genes, which suggests that incorporation miRNA expression information into gene signature identification may significantly decrease false positive rate when developing gene signatures for complex human diseases.In contrast to the published sarcoidosis gene signatures derived from whole genome screening by Koth et al. recently published overexpression of miRNas involved in angiogenesis, miRNA -27b, miR-192 and miR-221 in BALF cells from patients with acute sarcoidosis, none of these were significantly overexpressed in PBMCs34. Crouser et al. also profiled DE-miRNA in lung and lymph tissue and observed no overlap in miRNA expression in PBMC relative to diseased lung tissue; however TGF Fβ/WNT was a commonly regulated pathway30. Jazwa et al., suggested miRNA-34 regulates SIRT1 and the expression of IFN- γ in sarcoidosis35.Despite multiple efforts to identify concordant DE-miRNA in sarcoidosis, it is recognized that patterns of miRNA expression differ between BAL cells, lung tissue, lymph nodes and PBMCs suggesting organ specific regulation. In our initial 8-miRNa signature we identified miR-23a, miR-23b, miR-30c, miR-185, and miR-223 related to sarcoidosis severity. Kiszalkiewicz In summary, we derived a molecular signature consisting of 17 protein-coding genes, which are potentially regulated by deregulated miRNA in sarcoidosis. This signature can be independently used as potential novel molecular markers for differentiating patients with sarcoidosis, especially for distinguishing the patients with risk of complicated sarcoidosis. Although gene expression variation in PBMCs may not necessarily reflect the dynamics of the tissue microenvironment in sarcoidosis patients, PBMC gene expression information is useful in diagnosis of sarcoidosis, and more importantly, in the identification of patients with complicated sarcoidosis.36, neurologic sarcoidosis (e.g. evidence of hyperdense MRI lesions)37, or severe pulmonary sarcoidosis  . The healthy controls were collected in a matching procedure for both age and gender of the patients. As for the sarcoidosis patients, we didn’t find significant difference in age (t-test: P > 0.05) and gender (χ2-test: P > 0.05) between uncomplicated and complicated cases. Patient characteristics and treatment information are summarized in Supplementary Table The study was approved by the University of Arizona Institutional Review Board with written informed consent obtained from all subjects, and was performed in accordance with the principles in the Declaration of Helsinki. We defined complicated sarcoidosis as cardiac sarcoidosis (e.g. ventricular arrhythmias)normexp with offset value 10 based on a convolution model39 and normalized using the global Lowess regression algorithm. Only the miRNAs being present in at least two third of total samples were further analyzed. The miRNA expression data of the validation cohort, the Germany cohort, were obtained from the GEO database (GEO accession: GSE34608), which were based on Agilent-019118 Human miRNA Microarray 2.0 G4470B.For the discovery cohort, we profiled the PBMC miRNA expression level for 35 healthy controls, 17 patients with uncomplicated sarcoidosis, and 13 patients with complicated sarcoidosis, using Exiqon miRCURY LNA Array v11.0 . Total RNA was isolated from PBMCs according to manufacturer’s protocol. Array hybridization was performed by Exiqon with the quantified signals background corrected using 10, which were based on Affymetrix Human Exon 1.0ST Array. Briefly, gene expression data were summarized using robust multi-array average (RMA) algorithm40 embedded in the Affymetrix Power Tools v.1.12.0. Adjustment for possible batch effect was conducted by COMBAT41. We consider a transcript cluster to be reliably expressed in these samples if the Affymetrix implemented DABG (detection above ground)42P-value was less than 0.01 in at least two third of total samples. The gene expression data of the UCSF (GEO accession: GSE19314)9 and Oregon cohorts (GEO accession: GSE18781)18 were downloaded from the GEO database, which were based on Affymetrix Human Genome U133 Plus 2.0 Array.The protein-coding gene expression data of the discovery cohort are available from the GEO database (GEO accession: GSE37912)SmiRNA) is a linear combination of expression values of the miRNAs within the 8-miRNA signature. The formula is shown below:NmiRNA is the number of miRNAs in the 8-miRNA signature; i  is a linear combination of expression values of the genes within the 17-gene signature45. The formula is shown below:Ngene is the number of protein-coding genes in the 17-gene signature; i (as shown in Table i; i across all samples, respectively; and “sgn” denotes the sign function.8-miRNA and 17-gene based severity scores were calculated for each human subject, respectively. 8-miRNA based severity score (entclass1pt{minimaSupplementary Information


Research suggests that consumers often mishandle food in the home based on survey and observation studies. There is a need for a standardized tool for researchers to objectively evaluate the prevalence and identify the nature of food safety risks in the domestic environment. An audit tool was developed to measure compliance with recommended sanitation, refrigeration and food storage conditions in the domestic kitchen. The tool was piloted by four researchers who independently completed the inspection in 22 homes. Audit tool questions were evaluated for reliability using the κ statistic. Questions that were not sufficiently reliable (κ < 0.5) or did not provide direct evidence of risk were revised or eliminated from the final tool. Piloting the audit tool found good reliability among 18 questions, 6 questions were revised and 28 eliminated, resulting in a final 24 question tool. The audit tool was able to identify potential food safety risks, including evidence of pest infestation (27%), incorrect refrigeration temperature (73%), and lack of hot water . The audit tool developed here provides an objective measure for researchers to observe and record the most prevalent food safety risks in consumer’s kitchens and potentially compare risks among consumers of different demographics. Despite numerous interventions and regulations meant to control contamination of food products, foodborne illness continues to be a major public health problem in the United States, with an estimated 9.4 million cases, 56,000 hospitalizations and 1400 deaths each year . The majStaphylococcus aureus [Escherichia coli [Campylobacter jejuni [Salmonella spp. [Listeria monocytogenes [The presence of microbial contamination in the domestic environment has been well documented. Enteric bacteria, normally eliminated by proper cooking or by adequate sanitation, have been isolated from almost every surface in the kitchen ,13,14,15s aureus ,15,16,17hia coli ,13,16,17r jejuni , Salmonella spp. ,19, and ytogenes ,20,21. TEscherichia coli, Shigella, and S. aureus. Contamination on the hands can significantly increase the spread of microorganisms around the kitchen, for pathogens picked up from handling raw meat as well as enteric pathogens from the food preparer [Cross contamination is the transfer of harmful microorganisms from raw food to utensils, surfaces, or other foods. During meal preparation up to 71% of consumers were observed touching surfaces in the kitchen directly after handling raw meat ,23. Conspreparer ,28. Almopreparer ,29, but preparer ,23.Listeria monocytogenes and Staphylococcus aureus.Temperature abuse may occur when a food is heated or cooled improperly. Many consumers report using visual or tactile methods to know when meat is done cooking, such as checking the color of the meat or feeling the meat for firmness and texture ,30. ThesAlthough most authors agree that consumers frequently mishandle food at home there is a lack of consistency between results from study to study. Self-reported data, the most common method to evaluate consumer food handling behavior, is inconsistent and reported behavior does not always represent actual behavior . When coVisual inspection to document conditions related to food safety is the traditional method used to evaluate retail and foodservice settings, including restaurants, hospitals, and schools ,38. In oThis type of visual inspection may also be useful to study consumer households. Unlike other methods, the use of an audit tool provides a standardized checklist to record conditions that are actually present at the time of inspection. The instructions and questions on the audit tool must be evaluated for reliability to ensure they are clear and objective. A reliable instrument will produce the same results after repeated use, such as use by multiple people to evaluate a given set of conditions. This type of reliability, called inter-rater reliability, measures the extent to which a group of raters agree in their responses to a question . This stSome studies have utilized this approach to evaluate specific conditions in the domestic environment, such as refrigerator cleanliness and organization ,41, presAn audit tool was developed to measure compliance with recommended sanitation, refrigeration, and food storage conditions in the domestic kitchen. The instrument is formatted as a checklist, with multiple-choice questions to be used by the researcher to record visual observations from the environment. This method is commonly used in the food industry to inspect restaurants and food service establishments, and several pre-existing audit tools served as a model for the one created in this study ,45. A liIn order to be included in the audit tool each question had to be related to at least one target behavior, directly observable, and able to be measured or determined objectively. All conditions that are either directly associated with target behaviors or necessary to control the target behavior were further evaluated. From this list only conditions that could be directly observed by the auditor, with no input from the participant, were considered. The last criterion for inclusion was measurability and objectivity of the questions. In order for an instrument to be reliable each question must be able to be measured objectively, with no chance of different interpretations between different observers using the tool. While many conditions could be easily measured in an objective way, some were inherently subjective but considered especially relevant to food safety. For these criteria a set of observation guidelines were created to accompany the audit tool. These guidelines provided a list of standards that allowed the auditor to observe a subjective quality (such as “cleanliness”) in an objective way.It should be noted that the audit tool was developed and modified based on the assumption that the consumer would practice appropriate hygiene and food handling practices. That is, if soap and towels are available they will be used for proper handwashing. Similarly, if cleaning supplies are available they will be utilized and if cutting boards are available they will be used appropriately to avoid cross contamination. Actual observations of how consumers utilized available supplies were not conducted. Similarly therefore, it was assumed that a lack of supplies for proper hygiene and food handling would make it likely that these practices were not being carried out, and therefore increase risk.i.e., Cross Contamination Hazards, Time/Temperature Abuse, etc.), and were reorganized into groups based on location in the kitchen. This format allowed auditors to complete the inspection in an efficient and systematic manner. The final instrument contained five sections, each referring to a specific area of the kitchen and bathroom: General Kitchen, Kitchen Sink, Counters/Cabinets, Refrigerator/Freezer, and Bathroom Hygiene. The initial audit tool developed for the pilot study consisted of 52 questions.Three experts with backgrounds in food safety and domestic hygiene reviewed the draft audit tool for accuracy, usability, relevance to the domestic environment and overall completeness. The questions on the audit tool were originally categorized by food safety topic (p < 0.05) based on a lowest acceptable κ of 0.6 with a goal κ of 0.8 [The audit tool was pilot tested in domestic kitchens to determine inter-rater reliability of the questions as well as to further refine the instrument. For this study the lowest level of kappa (κ) acceptable to consider a question sufficiently reliable was initially set at 0.6—the high end of moderately significant κ values . It was κ of 0.8 .A convenience sample was recruited from Drexel University in Philadelphia, PA, USA, via department-wide e-mail advertisements and through word of mouth. The email included information about the commitment required for the study (a single home visit lasting approximately 20 min), the eligibility criteria , and the compensation ($20.00 in cash). Volunteers were asked to contact the researcher if they were interested in participating in the study. Appointments were given on a first come, first serve basis in order to reach the goal of 22 participants. A total of 38 people volunteered to participate in the study. From those 38 volunteers, three responded too late (after completion of the pilot study), seven provided contact information but could not be reached, four lived too far away and two canceled their appointments and were unable to reschedule. Volunteers were screened for eligibility via telephone questionnaire prior to scheduling appointments.Four researchers conducted inspections in 22 homes in and around the city of Philadelphia, PA, USA during May and June 2012. This study was approved by the Drexel University Institutional Review Board (IRB) and all participants were asked to sign informed consent forms before participating in the study. Prior to the onset of data collection a comprehensive training session was held to familiarize the auditors with the instrument. During this time the auditors reviewed the guidelines for recording observations, clarified the meanings of important terms used throughout the audit tool, and discussed how to handle unexpected scenarios. Informal training sessions were done periodically throughout the duration of the pilot study to remind the auditors to follow certain guidelines or to address new issues.At the participant’s home, each of the four auditors conducted an independent observation of the domestic environment using the audit tool. During this time the auditors did not discuss their observations with each other and could only consult their list of guidelines to determine how to evaluate conditions. The audit tool contained five sections and a total of 52 questions. The majority of the questions were multiple choice with only two answer choices (YES or NO), with a few open-ended sub-questions. Each section was followed by a blank space for the auditor to record any additional comments or relevant observations.The General Kitchen section contained 10 questions about the overall state of the kitchen, such as whether pets were observed on or near surfaces where food is prepared, evidence of pest infestation, maintenance of trash receptacles, and the availability and storage of cleaning materials. Six questions were included in the Kitchen Sink section to record if soap was present (and what type), evaluate the overall cleanliness of the sink, drain, and sponge/dishcloths. Kitchen water temperature was measured by running water into a container with an immersed thermometer , −20 to 110 °C; Ertco; Lafayette, NJ, USA) placed in the sink. Water was run for five minutes or until reaching 43 °C, whichever came first. The Counters/Cabinets section contained 11 questions about the overall condition and cleanliness of appliances, kitchenware, counters and utensils. This section also asks the observer to record if the participant owns a meat thermometer.The fourth section, Refrigerator/Freezer, was the longest section with 22 questions. Questions from this section recorded the condition and cleanliness of the refrigerator and freezer (interior and exterior), food storage methods , adherence to expiration and use-by dates, and the presence of foods commonly associated with foodborne pathogens . The temperature of the refrigerator was also measured using a pen-style infrared thermometer  and recorded in this section. This was done by holding the thermometer approximately 2 inches away from a food container as close to the middle of the refrigerator as possible, immediately after opening the refrigerator door. The last section, Bathroom Hygiene, contained two questions about the presence of soap and hand towels near the bathroom sink only if a bathroom was present on the same level of the home in which the kitchen was located.The auditors were encouraged to record additional comments or to elaborate on their answers as often as possible to provide the most complete picture of the conditions in the home. Upon completion of the inspection each participant was compensated $20 and provided with basic food safety information.Audit tool data from home inspections was numerically coded and entered into an Excel spreadsheet. κ statistic was calculated and used to evaluate inter-rater reliability for each question on the audit tool . Observan = 11) were male, and the rest female. Participant age ranged from 21 to 75 with a mean age of 31. The purpose of this pilot study was to evaluate the reliability, functionality and appropriateness of the audit tool, rather than record individual characteristics of the participants. For this reason a convenience sample was used and minimal demographic information was obtained.All 22 participants were located in or around the city of Philadelphia, PA, USA. Half of the participants (n = 22). Missing data was encountered for 13 questions when raters unintentionally left a question blank. Any question with a missing response was omitted from the analysis and κ was calculated for the remaining number of homes (n < 22).A summary of the κ values for 24 questions which were eventually included in some format in the final audit tool is shown . In mostκ values for questions from the audit tool varied from −0.031 to 1.0, with the minimum acceptable level of agreement pre-set at κ = 0.6. Of the initial 52 questions included in the audit tool, three questions had a κ of less than zero, indicating the level of agreement between raters was less than what was expected by chance. Eight questions had slight to fair agreement (κ = 0–0.2) and twelve questions had moderate levels of agreement (κ = 0.41–0.60). Seven questions had substantial levels of agreement (κ = 0.61–0.80) and the remaining 16 questions had almost perfect agreement (κ ≥ 0.81). Many of the questions on the audit tool with moderate levels of agreement (0.41–0.60) related to the condition and cleanliness of countertops, sinks, cutting boards and refrigerators . These cMany conditions related to unsafe food handling behaviors were observed using the audit tool. These results represent only a small number of homes (22), however indicate that the audit tool is capable of detecting a range of unsafe food handling conditions in the domestic environment. Observations related to cleaning and sanitation revealed several potential risks, such as a lack of cleaning materials other than dish soap in 5% of homes audited . Food coSeveral conditions were observed that may increase risk of cross contamination in the domestic environment . Evidencn = 6) were below the recommended temperature of 5.0 °C and 9% (n = 2) had temperatures greater than 10 °C.Conditions related to time and temperature abuse of food were identified in a majority of the homes audited . Meat thThe goal of this pilot test was to create a user friendly, reliable instrument that was appropriate to audit the domestic kitchen to be used in a larger study and potentially by other researchers. Based on these results two types of modifications were required to create the final version of the audit tool—eliminating questions that were particularly cumbersome or not essential for the goals of the project, and eliminating questions with low reliability. First, audit tool questions that were related to overall sanitation but considered least likely to contribute to the presence of foodborne pathogens in the home were eliminated. Items that were considered least likely to be directly associated with the presence of pathogens included evidence of tobacco use in the kitchen, maintenance of trash cans, and cleanliness of kitchen appliances. Questions that were particularly difficult and cumbersome to observe were also eliminated. The draft audit tool contained 22 questions in the Refrigerator/Freezer section, many of which required the rater to spend a significant amount of time moving food items to look through the refrigerator. This task becomes extremely difficult with more than one rater and if the refrigerator is very full, so many items from this section were omitted in the final version.Only half of the questions from the audit tool met the standard for the minimum acceptable level of reliability determined prior to the onset of the study (κ ≥ 0.6). Ten questions with low κ scores were considered non-essential or cumbersome and eliminated right away. The remaining 13 questions were considered particularly relevant to the goals of the study and necessary to include in the final instrument. Six of these 13 questions had a κ score between 0.5 and 0.6 and were included “as is” in the final audit tool because κ of above 0.5 is still considered a moderate level of agreement . The sevIn order to improve the reliability of questions that scored low in the pilot study the audit tool guidelines and instructions were also modified. The original guidelines were several pages in length, making them difficult to refer to during inspections. Changes to the guidelines decreased the length to a single page and helped clarify subjective questions. For example, the modified guidelines list exactly what criteria are necessary to consider a sponge or dishcloth dirty. Another problem that occurred during the pilot study was the tendency of raters to overlook certain items, such as not looking in a specific drawer in the refrigerator for meat products. The audit tool instructions have been modified to clearly state if the rater should look in specific locations to answer the question .This research has resulted in the development and validation of a user friendly, objective audit tool available with instructions for use see . It is i


Anti-immune programmed death-ligand 1 (PD-L1) pathway is used by the tumor to overcome immune system and serves as immunotherapy target in various malignancies.To investigate the expression of PD-L1 in vulvar squamous cell carcinoma (vSCC) and to assess it's clinicopathological and prognostic significance.Immunohistochemical PD-L1 expression was evaluated in 84 vSCCs with previously defined status of p16 and DNA-HPV, infiltration of immune cells: CD8+, CD4+, FOXP3+, CD56+, CD68+, and GZB+ cells. PD-L1 positivity was defined as ≥5% of PD-L1-positive cells. Survival analyses included the Kaplan–Meier method, log-rank test and Cox proportional hazards model.PD-L1 expression was detected on cancer and peritumoral immune cells. PD-L1-positivity of cancer nests  was correlated with higher infiltration of CD4+ (p=0.037), CD8+ (p=0.02), FOXP3+ (p=0.007), CD68+ (p=0.021) cells, while PD-L1 positivity of peritumoral immune cells  was correlated with higher infiltration of intraepithelial FOXP3+ cells only (p=0.037).PD-L1-positivity of cancer cells but not immune cells, was more frequently observed in p16-negative tumors (p=0.004). High-risk HPV-status did not correlate with the PD-L1 status of cancer and immune cells (p=1.000) and (p=1.000) respectively). Median follow up was 89.20 months (range 1.7-189.5). PD-L1 positivity of peritumoral immune cells was found to be an independent favorable prognostic factor for OS. Conclusion: This study highlights the importance of comprehensive PD-L1 assessment in both cancer and immune cells. PD-L1 expression on peritumoral immune cells seems to be an additional prognostic factor in vSCC patients and may influence the results by anti-PD-L1 treatment. The immune microenvironment of vulvar squamous cell carcinoma (vSCC) has been studied intensively by our group –3 and thINK4a expression regardless to high-risk HPV-DNA status [INK4a-negative tumors.Recently, we have found that cancer immune surveillance as represented by tumor infiltrating lymphocytes (TILs) and tumor associated macrophages (TAMs) depends on p16A status  as varioINK4a overexpression, contributes to shaping of the tumor microenvironment and p16INK4a-status could stratify patients for separate immunotherapeutic approaches in vSCC.This suggests that not HPV infection itself, but p16Recently, there has been a breakthrough in cancer immunotherapy against various cancer types by employing immune checkpoint blockade, particularly using antibodies directed against programmed death-ligand 1 (PD-L1) pathway members .PD-L1 , which is expressed on many cancer and immune cells, plays an important part in blocking the ‘cancer immunity cycle’ .INK4a and high risk (hr) DNA-HPV status in the primary tumor.This study aimed to evaluate PD-L1 expression in vSCC tumors and to look for the correlation of this biomarker with clinical and pathological features of vSCC patients including TILs, TAMs as well as p16PD-L1 expression was detected on cancer cells (CC-stars) and tumor-infiltrating immune cells (IC-arrows)  PD-L1 positive CC were found while PD-L1 positivity in peritumoral immune cells was disclosed in 51 of 84 (60.7%) cases.Lack of PD-L1 expression (CC-PD-L1 and IC-PD-L1 negative tumors) was observed in 26 of 84 (31%) vSCC. Dual expression of PD-L1 (CC-PD-L1 and IC-PD-L1 positive tumors) was notified in 20 of 84 (23.8%) cases. PD-L1 was exclusively expressed on cancer (CC-PD-L1 positive and IC-PD-L1 negative) and immune cells (CC-PD-L1 negative and IC-PD-L1 positive) in 7/84 (8.3%) and 31/84 (36.9%) case respectively. Detected variants of PD-L1 expression are depicted on Figure CC-PD-L1-positivity was correlated with higher intraepithelial infiltration of CD4+ (p=0.037), CD8+ (p=0.02), FOXP3+ (p=0.007), CD68+ (p=0.021) immune cells  (Table CC-PD-L1-positivity was frequently observed in p16-negative vSCCs (p=0.004).CC-PD-L1-positivity was similar in (hr)HPV-DNA positive and negative vSCCs (p=1.000).Lack of difference in IC-PD-L1–positivity was observed in cases with mutually excluding p16 and (hr)HPV-DNA status .CC-PD-L1 and IC-PD-L1-positivity did not depend on pT (Fisher's exact test) (p=0.748 and p=0.158), presence of lymph node metastases (p=0.640 and p=0.656), tumor differentiation grade G1/G2,3 (p=1 and p=0.815), (Mann-Whitney U test) age (p=0.523 and p=0.072) and depth of invasion (p=0.361 and 0.502) respectively (Table CC-PD-L1-positivity did not influence prognosis of vSCC patients (p=0.164) Figure .Patients with primary tumors positive for IC-PD-L1 expression had improved OS compared to IC-PD-L1 negative ones (p= 0.017) Figure .In the next step, the cohort of 84 vSCC patients was divided into four groups based on variants of CC-PD-L1 and IC-PD-L1-positivity: CC-PD-L1(+)/IC-PD-L1(-) n=7; CC-PD-L1(+)/IC-PD-L1 (+) n= 20; CC-PD-L1(-)/IC-PD-L1(+) n=31 and CC-PD-L1(-)/IC-PD-L1(-) n=26.Survival analyses comparing all variants of PD-L1 expression indicated the group of patients with CC-PD-L1(-)/IC-PD-L1(+) tumors presented trend towards significantly better prognosis (p=0.061) Figure . When thINK4a status.Additionally, impact of PD-L1-positivity on survival was assessed in groups of patients with different p16INK4a-negative and p16INK4a-positive (p=0.3763 and p=0.1639 respectively).CC-PD-L1-positivity did not influence the overall survival in cases having tumors p16INK4a-positive tumors (p=0.0271) while it was not prognostic for patients with p16INK4a- negative tumors (p=0.0821) . Compared with the whole tumor sections, tumor microarrays (TMAs) may be less representative, especially while assessing the biomarkers expressed in the tumor infiltrating immune cells .PD-L1 positivity was defined as ≥5% of cells regardless the intensity of membranous staining. This is consistent with several publications analyzing this biomarker within other malignancies –12. An aPD-L1-positivity of immune cells was more frequently observed within vSCC than PD-L1-positivity of cancer cells (60.7% vs. 32.1%). We found only two publications dealing with PD-L1 in vulvar cancer , 14. NonINK4a-overexpression modulates immune cells infiltration regardless to (hr)HPV-DNA status [INK4a-negative tumors although more infiltrated with TILs  had worse outcome than cases with p16INK4a-positive vSCCs [INK4a-negative vSCCs suggesting that anti-immune programmed death-ligand 1 (PD-L1) pathway is at least partially responsible for worse outcome in these patients. However, CC-PD-L1 did not reveal prognostic significance either in entire cohort nor in patients with p16INK4a-negative tumors.Recently we have reported that p16A status . Patientve vSCCs , 15. HerInk4a-negative tumors which were found to be more infiltrated by TILs and TAMs [The mechanism by which CC-PD-L1 surface expression is induced is quite ambiguous and includes either immune cell and cancer cells. Besides adaptive PD-L1 up-regulation in an inflammatory cytokine milieu caused by TAMs and/or Interferon-γ –18, cancand TAMs . Lack ofInterestingly, PD-L1-positivity of immune cells was found to be independent prognostic factor for overall survival of vSCC patients. Similar findings were published for head and neck cancer patients suggesting that comprehensive assessment of PD-L1 expression could bring an information for more precise usage check point inhibitors . Recent Several previous reports have shown that PD-L1 expression is a negative prognostic factor in several cancer types, including renal, colorectal, and lung cancers –26, but INK4a-status of the primary tumor, this protective effect was observed only for p16INK4a-positive cases. (hr)HPV-DNA status did not influenced PD-L1-positivity either on cancer and immune cells which supports our previous results suggesting lack of clinicopathological significance of HPV-DNA positivity within vSCC [Notably, we have found that IC-PD-L1-positivity improves survival in entire cohort, but when the patients were subdivided into two groups based on p16hin vSCC , 15.When we separately analyzed PD-L1-positivity on CC and IC, which was categorized into four variants of vSCC cases, we found that patients with CC-PD-L1(-)/IC-PD-L1(+) tumors had significantly best outcome.Few hypotheses could explain this paradoxical favorable prognosis of PD-L1 expression on ICs. IC-PD-L1 expression could be driven by adaptive mechanisms such as exogenous inflammation mediated immune attack and then reflected pre-existing immunity , 30. IC-Here, IC-PD-L1-positivity was correlated with unfavorable intraepithelial FOXP3+ lymphocytes while CC-PD-L1-positivity was correlated with favorable CD4+, CD8+ as well as unfavorable FOXP3+and CD68+ immune cells. Thus, indication of more favorable pre-existing immunity remains unclear.INK4a and (hr)HPV-DNA status. Such correlations have never been described before.However, with the exception of PD-L1 expression all other data were retrieved from our previous studies, originality of current report is supported by completely new study project and design. Immunohistochemical PD-L1 expression has never been analyzed before within vulvar cancer tissue. By analyzing all available data for this cohort, we were able to present in single publication, not only patterns of expression and prognostic significance of this biomarker but also associations with TILs, TAMs, p16This study provides a new insight into immune surveillance on vSCC as for the first time demonstrates that although PD-L1 could mediate the occurrence of cancer immune escape, it also indicates an effective immune response.The weaknesses of the current study are the retrospective design and the small size of cohort involved. Its strength lies in the consistency of the treatment of patients under uniform standards and their long observation, revealing recurrences and hence enabling the assessment of the prognostic significance of all the analyzed biomarkers.This retrospective study was approved by the Polish Ministry of Science and Higher Education review board, who determined that further informed consent was not required as informed consent for tissue sampling was obtained from all patients prior to surgical treatment and written consent was given by the patients for their information to be stored in the hospital database and used for research.INK4a and (hr)HPV status[Our cohort of 85 patients previously studied with known p16PV status, as wellINK4a and (hr)HPV status were retrieved from our recent study [We analyzed all 84 primary tumors for PD-L1 expression. Data on TILs were retrieved from our previous studies , 5. Datant study .Mouse anti-human monoclonal antibody against PD-L1  was obtained from Dako Inc. Mouse anti-human monoclonal antibodies against CD4 (NCL-L-368), CD8 (NCL-L-295), CD56 (NCL-CD56-1B), were obtained from Novocastra, Inc. Mouse anti-human monoclonal antibody against FOXP3 (cat. No ab20034) and CD68 (cat. No ab955) were obtained from Abcam, Inc. Mouse anti-human polyclonal antibody against Granzyme B (cat. No 760-4283) was obtained from Ventana Medical Systems, Inc. Mouse anti-human monoclonal antibody against p16 (cat. No sc-56330) was obtained from Santa Cruz Biotechnology Inc., USA [Immunohistochemical staining for PD-L1 was performed as follows. Four-micron-thick serial sections were cut, placed onto slides, and deparaffinized. For epitope retrieval, slides were immersed in Target Retrieval Solution  and heated in a pressure cooker. Then, they were incubated for 90 min with primary antibody . The reaction was visualized using the EnVision FLEX (DAKO). Appropriate positive (tonsil) and negative  controls were included in each staining. Immunohistochemistry results were evaluated by two independent pathologists blind to the clinical data.PD-L1 cell expression was referred as positive if membranous staining was identified and showed a continuous honeycomb pattern. Cytoplasmic staining of PD-L1 was disregarded. Expression of PD-L1was categorized into two groups according to the percentage of PD-L1-positive cells. PD-L1 positivity was defined as ≥5% of cells regardless to intensity of membranous staining –12.INK4a status was previously described for this cohort by our group [Immunohistochemical staining for other antibodies: p16, CD8, CD4, FOXP3, CD68, CD56, GZB with evaluation and classification of TILs and p16Tissue dissection and DNA preparation as well as mucosal HPV DNA amplification and genotyping were detailed for this cohort in our other recent study .The statistical analysis was performed using the Fisher's exact probability test. The difference between numerical variables was verified by Mann-Whitney U test.p values <0.05 were regarded as significant in all analyses.Overall survival curves were estimated by the Kaplan–Meier method and compared by the two-sided Fox test. Univariate and multivariate analysis was performed using Cox regression model. All analyses were performed using the statistical softwareStatistical 13 (Stat Soft Inc.).PD-L1 expression on IC, not on CC, is independent predictor of favorable OS in surgically treated vSCC patients. Assessment of the expression of immune-related molecules in whole tissue section slides could produce evidence relevant to the appropriateness of treatment via immune checkpoint blockade.Moreover, our findings highlight the importance of comprehensive assessment of both TC and IC in vSCC and suggest that usage of antibodies directed against PD-L1-pathway seems to be favorable for subjects having tumors negative for PD-L1 peritumoral immune cells. Thus, incorporating peritumoral immune cells into the classification of PD-L1 expression seems to be necessary to select the beneficial vSCC patients for anti-PD-L1 treatment.


Linear Dirac dispersion relation is nearly preserved and the calculated small effective mass in the order of 0.05mo suggests high carrier mobility. Density of states and space charge distribution of the considered heterobilayer structure near the conduction and the valence bands show unsaturated π orbitals of stanene. This indicates that electronic carriers are expected to transport only through the stanene layer, thereby leaving the h-BN layer to be a good choice as a substrate for the heterostructure. We have also explored the modulation of the obtained band gap by changing the interlayer spacing between h-BN and Sn layer and by applying tensile biaxial strain to the heterostructure. A small increase in the band gap is observed with the increasing percentage of strain. Our results suggest that, Sn/h-BN heterostructure can be a potential candidate for Sn-based nanoelectronics and spintronic applications.The structural and electronic properties of stanene/hexagonal boron nitride (Sn/h-BN) heterobilayer with different stacking patterns are studied using first principle calculations within the framework of density functional theory. The electronic band structure of different stacking patterns shows a direct band gap of ~30 meV at Dirac point and at the Fermi energy level with a Fermi velocity of ~0.53 × 10 In fact, stanene, a buckled honeycomb structure of 2D hexagonal tin film, has drawn keen interest due to its intriguing prospect as quantum Hall insulator2, topological insulator3 and topological superconductor4. In addition, stable low buckled form of stanene7, synthesized on the Bi2Te3 (111) substrate8 is theoretically investigated to support a large-gap 2D quantum spin Hall (QSH) state at room temperature and enables superior electric conduction with zero dissipation10. However, stanene has a zero band gap without spin orbiting coupling (SOC) while ~0.1 eV SOC induced band gap has been reported in the literature11. This zero band gap feature limits the use of stanene in the high performance semiconductor based nanoelectronic devices such as field effect transistor12. In this context, Garg et al.13 has recently reported the opening of a band gap in stanene by the patterned boron-nitride (BN) doping in stanene using density functional theory (DFT). In addition, Chen et al.14 has studied graphene-stanene heterobilayer models with several stacking methods and reported a band gap of ~77 meV for the most stable configuration. Furthermore, graphene/hexagonal boron nitride (graphene/h-BN) bilayer has been reported to show a small band gap at Dirac point in contrast to the zero band gap of graphene monolayer as well as higher carrier mobility than the graphene bilayers17. In fact, chemical stability and significant band gap of the monolayer h-BN18 make it promising for heterostructure based nano-devices.Of late, group-IV two dimensional (2D) materials such as silicene, germanene and stanene have received special attention attributed to their similar electronic properties with the graphene and intriguing prospects in the nanoelectronic device applications5, we report the structural and electronic properties of stanene/hexagonal boron nitride (Sn/h-BN) heterobilayer using first-principles density functional theory (DFT) calculations. A band gap opening along with the appearance of nearly linear relation near the Dirac point is observed in the proposed heterobilayer structure. Three representative models of the hybrid structure based on different stacking are investigated in terms of their stability, electronic property such as band structure, density of states, real space charge density and effective mass as well as the modulation of band gap under tensile strain and varying the interlayer distance. Our proposed structures with a finite band gap and high carrier mobility would provide further insight and encouragement on the modeling of stanene-based nanoelectronic and spintronic devices.Hence, following the deep scientific interest in modulating the band gap and other electronic properties while preserving the Dirac cone of stanene19. The electron-ion interactions are accounted using norm conserving Troullier Martin pseudo-potentials20. To describe the electron exchange correlation energy, the generalized gradient approximation (GGA)21 with Perdew-Burke-Ernzerhof (PBE) exchange correlation functional is implemented. In order to consider the Van der Waals (vdW) inter-molecular attractive forces, we have used DFT-D222, an empirical dispersion correction proposed by Grimme23 throughout the simulations. For the structural optimization, the plane wave basis cut off is set at 550 eV (40 Ry) with a convergence threshold on force of 10−4 Ry/a.u. The first Brillouine zone of the unit cell is sampled with a 12 × 12 × 1 Monkhorst-Pack grid for the geometry optimization and a 15 × 15 × 1 grid for the subsequent calculations. The interaction between the two adjacent bilayers is eliminated by introducing a sufficient vacuum of 20 Å along the direction perpendicular to the surface.The electronic properties of the proposed heterobilayer structure are investigated using the density functional theory (DFT) with a plane-wave basis set using the Ab initio code PWSCF package of Quantum Espressom* is the particle effective mass, E(k) is the dispersion relation, k is the wave vector and ћ is the reduced Planck constant.The effective masses of electrons 24 and simulation27 studies. This is also in close agreement with the reported theoretical studies by Liu et al.11, Nissimagoudar et al.28 as well as by Peng et al.29. Again, in our study after the geometry optimization the lattice constant of h-BN is 2.504 Å, which is also in close agreement with the previously reported studies33 in the literature. The optimized lattice constant of stanene is ~47% greater than that of h-BN and therefore we have considered stanene/h-BN heterostructures composed of 4 × 4 lateral periodicity of h-BN and 2 × 2 lateral periodicity of stanene, as shown in Fig. In our study, the geometry optimized lattice constant, Sn-Sn bond length and buckling height of the stanene are 4.687 Å, 2.834 Å and 0.82 Å respectively, which is in good agreement with the reported experimental2 heterobilayer structures35. A 4 × 4 supercell of MoS2 with a 5 × 5 supercell of graphene was considered due to the 23% difference in the lattice constants between graphene (2.42 Å) and MoS2 (3.12 Å) and the graphene layer was further stretched by ~3% to meet the commensurability criterion. Also, Chen et al.14 investigated the electronic and optical property of graphene/stanene heterobilayers where the lattice constants of graphene and stanene differ by about 46%. Hence, they14 proposed a supercell composed of 4 × 4 graphene and 2 × 2 stanene and further stretched the stanene layer by 4.7% to form commensurable structures.The basic unit cell consists of two Sn atoms, four B atoms and four N atoms. Our Sn/h-BN heterobilayer configuration can be compared to the reported literature on the graphene/MoS36, graphene on copper substrate36, germanene/BeO37 and graphene/stanene14.Three stacking patterns of stanene-hexagonal boron nitride (Sn/h-BN) have been considered in this study as shown in Fig. et al.32 for graphene/h-BN heterobilayer. Our obtained equilibrium distances are also comparable to the reported38 interlayer distances (3.57 Å to 3.75 Å) for bilayer h-BN using GGA in the scheme of PBE. It is also comparable to the experimentally obtained interlayer distance of the Sn bilayers (3.5 Å ± 0.2 Å) during the epitaxial growth of stanene8 as well as with the calculated interlayer distance of the Sn bilayers (3.6 Å) by Evazzade et al.39 using GGA and PBE exchange correlation in the Quantum Espresso package where DFT-D modeling has been employed. On the other hand, vdW-DF2 has been reported to yield an equilibrium interlayer distance higher than the experimentally determined interlayer distance for some heterobilayers (3.77 Å for graphene-SiC32). In fact, the equilibrium interlayer distance is found to be sensitive with the choice of the exchange-correlation functionals40.Figure 15.Moreover, as suggested by the binding energies of the three configurations shown in Fig. 41 than the binding energies of the weak vdW interactions42. This indicates that the stanene monolayer is bound to the h-BN substrate via interactions somewhat stronger than the weak vdW interactions43. The heterobilayers such as graphene/h-BN36, stanene/MoS244 and graphene/SiO242 are reported to have weak vdW interactions between the heterobilayers with a binding energy of less than 100 meV per unit cell. On the other hand, in the graphene/HfO2 heterobilayer structures, the graphene is strongly bound to the HfO2 with a binding energy of 110 meV43. The similar phenomenon has been reported in the study of the graphene/stanene heterobilayers14 with a binding energy of more than 180 meV per unit cell at optimum spacing. As an aside, the optimized interlayer distances for the three stacking configurations of the Sn/h-BN heterobilayers considered in this study are 3.7 Å to 3.78 Å, which are larger than the typical bond length of the Sn-B bonds (2.24 Å)13 and the bond length of the Sn-N bonds (2.14 Å)13. This indicates that, the Sn-B and the Sn-N covalent bonds are absent in the Sn/h-BN heterostructures and further conforms to the similar phenomena reported regarding the graphene/stanene14 and the graphene/HfO243 heterobilayers.Furthermore, the calculated binding energies in our study for the Sn/h-BN heterobilayers per unit cell at equilibrium are more than 250 meV which is higheret al.45 (70 meV direct band gap in the presence of SOC) as well as by Xiong et al.46 (73 meV direct band gap with SOC), thereby confirming the validity of our simulations. Furthermore, in the presence of SOC a Fermi-level band crossing phenomenon of bands is observed at gamma (Γ) point. For the band structure of stanene as well as stanene nanoribbons in the presence of SOC, at gamma point similar phenomenon of either bands touching or crossing the Fermi level has been reported by Broek et al.5, Modarresi et al.45, Lu et al.47, as well as by Nagarajan et al.48. Despite this phenomenon, in view of the opening of the band gap at K point Broek et al.5 concluded that, pristine stanene offers perspectives of an all-Sn field effect transistor (FET). However, as discussed by Xiong et al.46 and Nagarajan et al.48, the effect of SOC on the band structure of stanene and the Fermi level touching or crossing phenomenon indicates its possibility of applications in spinelectronics and quantum spin Hall fields. Hence, the overall effect of SOC in the band structure of stanene and stanene based nanostructures requires consideration in terms of their promising application in nanoelectronics, spinelectronics, quantum spin Hall insulators as well as field effect transistor.For further benchmarking, we have computed the band structure of the 2D monolayer stanene without SOC as well as in the presence of SOC as shown in Fig. Figure 49. However, at gamma point (Γ) a Fermi-level band crossing phenomenon of bands can be observed which is similar to pristine stanene with the effect of SOC indicating the possibility of applying Sn/h-BN heterobilayers in the application of spinelectronics and quantum spin Hall insulators48.With the inclusion of SOC, the observed band gap for the structure I, II and III are 74 meV, 109 meV and 90 meV, respectively as shown in Fig. mo respectively, which are small enough to provide high carrier mobility. The computed electron effective masses for the other two configurations are 0.054  × 106 ms−1, 0.526 × 106 ms−1 and 0.538 × 106 ms−1 for structure I, II and III, respectively which are in the similar order of magnitude to that of stanene5 (0.97 × 106 ms−1) and graphene50 (106 ms−1).The linear Dirac disperison near K point suggests a high charge carrier mobility. To further analyze this fact, we have calculated the effective mass of electrons et al.51 for the stanene/MoS2 heterostructure where the strong interaction breaks the sublattice symmetry of stanene and opens a band gap. Similarly, the strong interaction with the BeO monolayer substrate introduces a finite band gap in the germanene/BeO heterostructure by breaking the symmetry of the germanene37. Also, due to the symmetry breaking of the two carbon atoms in the hexagonal graphene induced by the interaction with the h-BN monolayer, a finite band gap of ~50 meV opens as reported in the literature36.Due to the interaction between the stanene and the h-BN layer, the sublattice symmetry of the zero band gap hexagonal stanene structure is broken, therefore opening a direct band gap. Similar phenomenon has been reported by Xiong 5. Furthermore, as shown in Fig. 52, h-BN orbitals do not hybridize with stanene orbitals near the Fermi level ensuring non-intensive interactions of the two layers. This is further supported by the real space charge density distribution of the conduction and valence bands for structure III as shown in Fig. et al.18, the band structure for the graphene/BN bilayer is dominated by the bands associated with the carbon atoms near the Fermi level, with a band gap of ~100 meV at the Dirac point. Similarly, silicene is reported53 to dominate the electronic transport in the silicene/h-BN heterobilayers and the Dirac cone of the silicene layer is preserved after contacting the BN layer, which is in accordance with our study for the stanene/h-BN heterobilayers. In fact, the heterostructures consisting of single layer silicene and graphene sandwiched between h-BN bilayers also show a band gap opening and high carrier mobility while the silicene and graphene layer remain nearly unaffected by h-BN55. In addition, the periodic heterostructures with a graphene sheet sandwiched between four h-BN layers also show similar phenomenon56.Next, to analyze the electronic properties and gain deeper insight on the interlayer interactions, we have plotted the total as well as the projected density of states (PDOS) for structure III of the stanene/h-BN heterobilayer as shown in Fig. In this section, we focus our study to tune up the band gap of the Sn/h-BN heterostructure for its possible use in the high performance nanoelectronic and spintronic devices.58 therefore reducing the band gap. This is in line with the study of graphene/h-BN heterobilayers15 of various stacking patterns where the band gaps increase with the decrease in interlayer spacing.Firstly, we have calculated the energy band gaps with the variation of the interlayer spacing of the stanene and h-BN layers for all three considered configurations as presented in Fig. 5. On the other hand, the reverse phenomenon i.e. the upward shift of Fermi level is expected for the compressive strain.Figure Finally, we have investigated one non-high symmetry configuration for the Sn/h-BN heterostructure as shown in Fig. et al.36.The electronic band structure for the non-high symmetry configuration is shown in Fig. In summary, we have investigated the structural and electronic properties of the Sn/h-BN heterobilayers from DFT studies considering van der Waals interaction between two layers. We have considered three different stacking patterns and all of these are found to be electrically stable in terms of the high binding energy. The band structures of the Sn/h-BN heterobilayer shows a direct band gap of about 30 meV at the Fermi energy level while linear Dirac dispersion relation is closely maintained. The real space charge distribution and density of states of the heterostructure suggest the localization of stanene π orbital in the conduction as well as in the valence bands, further confirming the superior role of stanene in shaping the electronic properties of the stanene/h-BN heterostructure. Moreover, our calculated small effective mass and the emergence of Dirac cone suggest high charge carrier mobility for the Sn/h-BN heterostructure. Furthermore, our analysis concludes that, the band gap can be effectively tuned by varying the interlayer spacing between the Sn and h-BN monolayer while preserving the stability. A small increase in the band gap is observed with the increasing percentage of tensile biaxial strain. At a higher percentage of strain, the Fermi level shifts downward and self hole doping characteristics with hole carriers appear. Such unique and tunable electronic properties of the Sn/h-BN heterobilayer would further encourage the Sn-based nanoelectronic and spintronic device applications.


Graphene is an excellent modifier for the surface modification of electrochemical electrodes due to its exceptional physical properties and, for the development of graphene-based chemical and biosensors, is usually coated on glassy carbon electrodes (GCEs) via drop casting. However, the ease of aggregation and high defect content of reduced graphene oxides degrade the electrical properties. Here, we fabricated low-defect graphene electrodes by catalytically thermal treatment of HPHT diamond substrate, followed by the electrodeposition of Au nanoparticles (AuNPs) with an average size of ≈60 nm on the electrode surface using cyclic voltammetry. The Au nanoparticle-decorated graphene electrodes show a wide linear response range to vanillin from 0.2 to 40 µM with a low limit of detection of 10 nM. This work demonstrates the potential applications of graphene-based hybrid electrodes for highly sensitive chemical detection. Vanillin  is the flavor component of vanilla bean, which is widely used in foods, beverages, and medicines ,2, becau2-bonded carbon atoms. It has extensive influence in the field of electrochemistry due to its exceptional electrical properties, like high carrier mobility ) and high specific surface area (2600 m2/g) [Graphene is a two-dimensional material composed of hexagonally constructed sp00 m2/g) ,17,18,1900 m2/g) ,21,22,2300 m2/g) . Moreove00 m2/g) .Compared to rGO, chemical-vapour-deposited (CVD) graphene exhibits lower electrical resistance and fewer structural defects. However, the lack of functional groups on the surface of CVD graphene limits its electrochemical activity. Therefore, it is efficient to improve the device sensitivity based on CVD graphene via further surface modification, such as molecular doping, the grafting of functional groups, and the decoration of noble metal nanoparticles ,27,28. R3-to-sp2 conversion process. Then, AuNPs were electrodeposited on the graphene surface using a simple cyclic voltammetry (CV) method. The fabricated electrode exhibits a linear electrochemical detection range of 0.2–4 µM with a low detection limit of 10 nM, demonstrating potential applications of graphene-based hybrid electrodes for highly sensitive chemical detection.In this work, we fabricated graphene-diamond hybrid electrodes by a catalytic annealing process using a thin Ni layer as catalyst. High-quality graphene films were formed directly on the diamond surface through an in-situ sp2O2, H2SO4, CuSO4, KCl, HCl, Na2HPO4, NaH2PO4, and vanillin were purchased from Sinopharm Chemical Reagent Co. Ltd. Shanghai, China.High-pressure and high-temperature (HPHT) diamonds with dimensions of 3.5 × 3.5 × 1 mm were purchased from Shenzhen Tiantian Xiangshang Diamond Co. Ltd., Shenzhen, China. Nickel (99.995%) was brought from New Material Technology Co. Ltd., Beijing, China. H2SO4:H2O2 = 7 mL:3 mL) solution at 50 °C for 4 h to remove surface contaminants, followed by ultrasonic cleaning in deionised water and ethanol for 10 min, respectively. A 20-nm-thick Ni film was deposited on the diamond surface via an e-beam system  at a base pressure and deposition rate of 1.4 × 10−5 Torr and 0.5 Å/s, respectively. The Ni/diamond substrate was then thermally treated at 1020 °C for 15 min in a tube furnace system , with H2 flow of 8 sccm. Finally, the graphene–diamond hybrid electrodes were obtained after Ni removal in an etching solution (10 g CuSO4 and 50 mL HCl in 50 mL deionised water). The graphene–diamond hybrid sample was immobilised on a plastic substrate and connected by silver paints to silver wire. Except for the graphene surface, other exposed areas were protected by silicone resin. Au nanoparticles were then electrodeposited on the graphene surface via CV at a scan rate of 100 mV/s with 0.2 mL of 1% AuCl4 solution diluted in 10 mL PBS (PH = 7.0) solution with deposition times varying from 2 to 16 min.The HPHT diamond substrate used in this study was comprised of (1 0 0) singe-crystal diamond. The diamond substrate was first immersed in a mixture of sulfuric acid and hydrogen peroxide  and X-ray photoelectron spectroscopy . The surface morphology was observed using a field emission scanning electron microscope . The electrochemical experiments were carried out with an Autolab workstation . The mercurous chloride reference and Pt counter electrode were obtained from Aida Hengsheng Co. Ltd., Tianjin, China.3-to-sp2 conversion process, in which the HPHT diamond was used as a carbon source. The sp3-bonded carbon atoms on the diamond surface were dissolved into a thin nickel film (≈20 nm) at high temperature (1020 °C) and then precipitated from the nickel film to form sp2-bonded carbons during a rapid cooling process. The success of sp3-to-sp2 conversion can be demonstrated by XPS analysis, which was used to determine the bonding information of the diamond surface before and after catalytic thermal treatment. As shown in 3 bonds (C–C), while the ratio of sp2–sp3 bonds of the graphene–diamond surface increased to 1:1 after annealing. The quality of the obtained graphene was investigated with the Raman spectrometer, as shown in −1, 1586 cm−1, and 2698 cm−1 can be assigned to the D-band, G-band, and 2D-band of graphene, respectively. The I2D/IG ratio (≈0.62) and full width at half maximum (FWHM) of the 2D-band (≈64.5 cm−1) suggest that the graphene is composed of few layers [D/IG ratio (<0.1) reflects that the graphene converted from diamond has low defect content [−1, which can be assigned to the single-crystal diamond [2D/IG ratio of graphene film mostly varied between 1.0 and 1.5, showing that the diamond surface was covered by few layers of graphene [As schematically illustrated in w layers . In addi content . We noti diamond , confirmgraphene .−2 and ≈8 μm−2 for the samples after electrodeposition for 4 and 16 min, respectively, as exhibited in In order to enhance the sensitivity of graphene hybrid electrodes for detecting vanillin, the as-prepared electrode was further modified with AuNPs via electrodeposition. The mechanism of AuNP-decorated graphene electrodes for vanillin detection was investigated by changing the scan rate of CV measurement. As shown in ed works ,21. Our 3-to-sp2 conversion process, the graphene–diamond hybrid electrodes were fabricated via catalytic thermal treatment. The low-defect graphene electrodes were further modified by electrodeposition of AuNPs using the CV method, and the deposition time was optimised to 4 min for achieving a good trade-off between the size (≈60 nm) and surface density of AuNPs, resulting in the highest electrochemical sensitivity for vanillin detection. As a result, a wide linear dynamic range of the hybrid electrodes can be achieved from 0.2 to 40 µM with a very low limit of detection of 10 nM. The AuNP/graphene sensing platform is highly efficient for vanillin determination in food, cosmetic, and pharmaceutical analysis.Based on an sp


This performance seems not to be connected to the strong orbital hybridization that Ni and graphene interface present, indicating electron transfer might not be playing a main role in the robust response of this nanostructured system. The observed protection from biological environment can be understood in terms of graphene impermeability to transfer Ni2+ ions, which is enhanced for few layers of graphene grown on Ni. We expect our work will provide a new route for application of graphene as a protection coating for metals in biological environments, where current strategies have shown short-term efficiency and have raised health concerns.In this work we present a study on the performance of CVD  graphene coatings grown and transferred on Ni as protection barriers under two scenarios that lead to unwanted metal ion release, microbial corrosion and allergy test conditions. These phenomena have a strong impact in different fields considering nickel  is one of the most widely used metals in industrial and consumer products. Microbial corrosion costs represent fractions of national gross product in different developed countries, whereas Ni allergy is one of the most prevalent allergic conditions in the western world, affecting around 10% of the population. We found that grown graphene coatings act as a protective membrane in biological environments that decreases microbial corrosion of Ni and reduces release of Ni When metals are in contact with biofilms, which naturally form under the exposure of materials to ambient moisture, miscellaneous metabolic activities lead to the production of organic and inorganic acids, volatile compounds and other chemical reactions with the metals. These processes induce a highly accelerated deterioration of the substrate known as microbiologically influenced corrosion or microbial corrosion . This phCurrent strategies to prevent microbial corrosion in metallic structures aim to attack biofilms that cause microbial corrosion. They mainly include physical methods  and chemical methods  ,4. The u2+ ions from Ni metallic objects that come in direct contact with human skin has been related to a phenomenon known as the allergic contact hypersensitivity response to Ni [On the other hand, there is a phenomenon that, similar to microbial corrosion, includes metal ion release when a metallic material is in contact with other living systems, such as the one provided by human skin. Among metallic materials, Ni and its alloys are one of the most widely used in diverse domestic objects that come in contact with human skin. Ni has been chosen for most of these applications due to its mechanical properties, corrosion resistance, durability, appearance and availability. However, remarkable as these properties are, they do not seem to withstand direct interaction with human skin conditions. Release of Nise to Ni ,7. This se to Ni ,12,13,142+ is not only released from coins [2+-induced contact dermatitis [2+ ions have been reported to trigger an innate immune response in resident skin cells [Niom coins  but alsormatitis ,17,18. Ain cells  they arein cells . When niin cells . This acin cells ,22. Reduin cells ,24, whicin cells ,26. Evenin cells .2+, by capturing nickel ions [Different nanoscale approaches have been used to produce certain degrees of control over contact allergy and microbial corrosion issues. For instance, the application of calcium phosphate and calcium carbonate nanoparticles to the skin seems to be effective in preventing the allergy towards Nikel ions , which akel ions ,30. Simikel ions ,32. HoweRecently our group and others have reported Cu microbial corrosion passivation using single-layer CVD graphene (SLG) grown on that metal ,33,34. AMotivated by the need of studies exploring the protection that graphene coatings offer to other relevant metals (other than copper), we present a study on the efficiency of graphene grown and transferred on Ni as an ionic barrier under two biological conditions, microbial corrosion and sweat immersion. Chemical vapor deposition (CVD) graphene, with surface areas in the centimeter square range, has been chosen as coating due to its versatility and lack of intrinsic cytotoxicity . This coIn this work we explored the efficiency of as-grown few layer graphene (FLG) and transferred graphene onto Ni foils. We mainly focus on the way in which these nanostructured coatings modify interaction between: (1) Ni and bacteria  and (2) Ni and sweat .−5 hPa whereas during graphene growth was kept at 1.2 × 10−2 hPa. A slight modification of this methodology is introduced by supplying this mixed flux in five steps of 20 min each. Between each step, the sample was held only under the hydrogen flux for 10 min to ensure a high coverage. The final methane step was followed by rapid cooling under a hydrogen-argon flux of 10 sccm and 20 sccm respectively. Similarly, few-layer graphene grown on nickel was obtained through methodology reported in ref. [Graphene grown on Ni and graphene transferred onto Ni surfaces were included in this study to establish a comparison between performances of these graphene coatings obtained through different methodologies. The performance of graphene transferred on Ni will allow a comparison between the same type of nanometric membrane but with different electronic interaction (hybridization) with metallic substrate; weak coupling for graphene transferred on Ni and strong for grown samples ,36. Grap in ref. , using aSingle-layer graphene was transferred onto Ni substrate using the PMMA-assisted method . For thi2 surface area were used for all measurements.Corresponding Ni control substrates were prepared treating fresh Ni (NiF) foils  under the same temperature and hydrogen pressure conditions used for graphene growth but without the carbon precursor gas. Thermally treated Ni samples (control samples) will be called NiT. Samples of 1 cm−10 torr), samples were annealed at 100 °C for 20 min to remove all gases adsorbed on sample surface. Graphitic quality of as-grown FLG was investigated by MicroRaman measurements  at ambient conditions at low laser power (<1 mW).A combination of scanning electron microscopy (SEM) , scanning tunneling microscopy  and X-ray photoelectron spectrometry  was used to characterize samples morphology and chemical environment. Prior to STM and XPS analysis, where ultra-high vacuum conditions are required  by filtration of the suspension using negative pressure. The filters with a bacteria film were dried at room temperature during 90 min in order to obtain a stable water contact angle measured by sessile drop method using 1 μL of distilled. The contact angle was measured based on image analysis [Surface hydrophobicity of coated and uncoated Ni samples was determined by contact angle measurements. For this characterization a drop of milliQ water (2 μL) was placed on the surface of coated and uncoated samples and images were immediately captured using a high-resolution camera. Bacterial hydrophobicity was measured following standard methods  with somanalysis  using thanalysis .Escherichia coli MG1655 cultures, which have been reported to cause metal microbial corrosion [−1) and yeast extract (5 g·L−1). The bacterial cultures were concentrated by centrifugation , washed three times with Milli-Q water, and finally resuspended up to a turbidity of 3.0 at 600 nm. The turbidity of this stock dispersion is equivalent to a bacterial concentration of ~1 × 109 CFU/mL−1 (Colony forming units per milliliter). This high bacterial concentration was chosen to ensure response of graphene coatings is robust enough, even for large number of bacteria. Milli-Q water was used as dispersant to avoid bacterial duplication and the forthcoming accumulation of mineral crystals that may interfere with the collection of microscopy images or cause unwanted chemical reactions with the samples. Nickel release from metallic samples exposed to bacteria was determined by Inductively Coupled Plasma (ICP) . Control samples (NiT) were exposed to Milli-Q water without bacteria. After 24 h, bacteria were recovered, poured into 2.5 mL of 15 μM EDTA (ethylenediaminetetraacetic acid)  solution (pH 10) and centrifuged at 5000× g during 10 min. The supernatant was recovered, and the total Ni concentration was also quantified by ICP.To explore response of graphene coatings (FLG and 4MLG) to biological environments and their ability to prevent microbial corrosion of Ni, we used orrosion ,52. BactE. coli MG1655 stock dispersion was placed on each sample surface in order to obtain a final bacterial density of 60 μL/cm2. Sample + bacteria systems were incubated at 37 °C during 24 h in a humidity chamber to avoid evaporation. Once this incubation period completed, bacteria were recovered with 3 volumes of Milli-Q water using a standard micropipette. Cell viability at 0 and 24 h was determined using the microdot methodology [v/v) glutaraldehyde and dehydrated by washing with a graded ethanol series (from 10% to 100%), followed by critical-point drying and gold coating.Cell viability (inverse to cell death) was monitored to evaluate the antibacterial activity of coated and uncoated Ni samples. Such antibacterial performance is a direct consequence of Ni ions release and, in our case, will be an indirect indicator of Ni microbial corrosion (in addition to previously mentioned ICP measurements). One volume (100 μL) of hodology . Each exTo study the response of graphene-coated Ni samples to sweat exposure immersion tests were performed for FLG, 4MLG and Ni samples. The artificial sweat solution was prepared similar to EN1811 , using 0Morphological characterization of samples prior to bacterial and sweat contact was obtained by SEM a–d. FresScanning tunneling microscopy in ultrahigh vacuum conditions was used to visualize FLG grown on Ni with nanoscale and atomic resolution e. Charac−1) and 2D (~2688 cm−1) bands, with a small G/2D (~0.23), consistent with literature [−1). Raman spectra displayed typically sharp G (1584 cm−1) and broad 2D (~2701 cm−1) bands for FLG  and FLG coatings, we performed microRaman measurements on these samples. Multiple areas of these samples were analyzed, and representative spectra are shown in terature . Upshift for FLG h, consissharp G 14 cm−1 an3/2 spectrum, which contains information on metallic Ni , NiO (peak in green), and Ni(OH)2 (peak in blue) surface contents that are present when nickel is exposed to ambient conditions [2 signals (in blue) are present for NiF and NiT samples (and to lesser extent for FLG), they are stronger for NiF. This is in agreement with previous reports where an initial Ni oxide growth in metallic Ni saturates at few layers after which oxygen in air leads to formation of Ni(OH)2 on the surface, that continue growing for longer exposure to atmospheric gas pressure [2 at the air-oxide interface and NiO between the metal and hydroxide layer. Nickel metallic signal is stronger for graphene-coated Ni than for bare Ni samples, regardless heat treatment. However, it is clear that NiT samples, due to oxide removal treatment, are similar to graphene-coated Ni in terms of metallic Ni composition, indicating that NiT is in fact the control sample for viability, microbial corrosion and sweat exposure measurements.Surface composition information of Ni samples was obtained by means of XPS analysis a. In parnditions ,60. Stropressure . The resE. coli [Contact angle measurements were performed to characterize surface hydrophobicity of coated and uncoated Ni samples b–d. HydrE. coli , tends tE. coli . In the E. coli . HydrophE. coli . AccordiE. coli after 24 h of incubation on NiT, NiF and graphene-coated Ni samples, together with morphology of bacteria after incubation  exhibit a wide range of significant abnormalities (E. coli on FLG grown on Ni (2)  and the consequent release of Ni ions.SEM images of malities d,e, suchmalities f, in agrwn on Ni b, similan Ni (2) a. This in Ni (2) f. For 4M2 (control) samples indicates that a connection between charge transfer from Ni to bacteria through graphene and their bactericide activity is less likely.Electron transfer from microbial membranes to graphene has been reported to produce a strong antibacterial effect in graphene grown on copper system , where tE. coli cells was chosen as control sample. Solutions with and without bacteria (control) do not contain salts than might affect electrochemical interaction with metal. Dissolved Ni from graphene-coated Ni samples (FLG) in contact with Milli-Q water with bacteria is close to the concentration obtained in control sample. Ni ions release is reduced up to 98.7% when using FLG coating (compare to NiT—control sample of FLG) and up to 46% when using transferred graphene (compare to NiF—control sample of 4MLG). These results demonstrate grown graphitic membranes efficiently protect underlying Ni from microbial corrosion due to bacterial interaction. In particular, Ni dissolution is found to be inversely related to cell viability  while for graphene-coated samples Ni ion release reaches a similar value (~0.6 µg/cm2/week).To study the performance of graphitic coatings under Ni allergy conditions we performed ICP measurements on Ni dissolution for graphene-coated and uncoated Ni samples under sweat immersion conditions  . Nickel ith skin ,22. Our Different coatings have been reported to be used to reduce Ni release from biomaterials in contact with artificial sweat ,70. In v2+ ion release for NiF, when compare with NiT.SEM micrographs of uncoated Ni samples after sweat exposure (insets in 2+ (70 pm) [Our results suggest that the observed effectiveness of graphene as a barrier against interaction between Ni and bacteria or sweat can be understood in terms of graphene permeability. Graphene’s p orbitals form a dense and delocalized cloud that blocks the gap within its aromatic rings , creatin (70 pm) , which a2+ ions release from Cu foils in contact with bacteria [bacteria . The pro2+ ions, which is enhanced for a few layers of graphene.In conclusion, we studied the performance of graphitic coatings as a protection barrier under microbial corrosion and Ni allergy conditions. From the microbial corrosion point of view, cell viability and Ni dissolution results indicate these nanostructured coatings effectively block interaction between bacteria and underlying metal. The absence of bactericide effect for this highly electronically hybridized system suggests that most of the protection from the observed protection from biological environment can be understood in terms of graphene impermeability to transfer NiThis protective effect of impermeable graphene coatings is extended to the case of Ni allergy conditions. Although Ni dissolution does not remain below the value established by international Directive 94/27/EC in graphene-coated Ni samples, its proximity to this value confirms the great potential of graphitic coatings to prevent the significant amounts of nickel ions usually released from metallic devices, which may interact with the human body causing adverse health effects. The most effective way of preventing nickel allergy is the avoidance of exposure. The ideal case would be the complete ban of nickel use, which is irrelevant due the wide distribution of the metal worldwide. In this scenario the observed performance of graphitic coatings to prevent Ni ion release suggests a promising new venue to prevent allergy reactions. Further in vivo experiments are needed to evaluate biocompatibility of graphene-coated materials on humans and animals.