Molecular characterization of the breast cancer associated antigen NY-BR-1

Breast cancer is one of the most common malignancies with increasing incidence every year and a leading cause of death among women. Although early stage breast cancer can be effectively treated, there are limited numbers of treatment options available for patients with advanced and metastatic disease. The breast cancer associated antigen NY-BR-1 was identified by a serological screening strategy (SEREX). NY-BR-1 is expressed in the majority of breast tumours (>70 %) as well as in metastases, in normal breast tissue, in testis, and occasionally in prostate tissue. Due to its restricted expression pattern, its immunogenicity and its subcellular localization to the cytoplasm and plasma-membrane, NY-BR-1 represents a promising target for immunotherapeutic approaches in breast cancer patients. The aim of this thesis was the molecular and functional characterization of NY-BR-1 to reveal its biological function in normal and tumorigenic breast tissues. To examine the effect of NY-BR-1 over-expression on cellular processes, such as proliferation, apoptosis and the cell cycle, HEK293, HEK293T, MCF-10A, and MCF-7 were transiently transfected with NY-BR-1. The results showed that NY-BR-1+ cells do not proliferate, do not have an increased apoptosis rate and accumulate in the G1 phase compared to the control cells. This finding is supported by IHC stainings of normal breast tissue sections for NY-BR-1 and Ki-67 (proliferation marker) displaying NY-BR-1+/Ki-67- cells. Another approach to clarify the biological function of NY-BR-1 was the identification of specific protein interaction partners by co-immunoprecipitations with lysates of NY-BR-1 transfected cells (HEK293, MCF-10A, MCF-7) followed by mass spectrometry. The protein identified in all three analysed cell lines is the tubulin beta-4B chain, which amongst other is involved in mitosis, underlining a crucial role of NY-BR-1 during the cell cycle. To investigate the transcriptional regulation of the NY-BR-1 gene, an in silico prediction for potential transcription factor binding sites in the NY-BR-1 promoter region was performed with the Alibaba 2.0 algorithm. Tissue pieces as well as isolated epithelial cells from healthy breast tissue and tumour cells from pleural effusions were treated with different hormones, a demethylation agent and histone deacetylase inhibitors. The NY-BR-1 expression was analysed via qPCR. Several estrogen receptor binding sites were mapped in the NY-BR-1 promoter region and within the gene (up to Intron 21) by ChIP-Seq. The NY-BR-1 protein and the estrogen receptor (ER) are mainly co-expressed in normal breast tissues as assessed by IHC stainings and in silico analyses of RNA-seq data showed that NY-BR-1 is predominantly expressed in ER positive luminal A and B breast cancers. However, the stimulation of normal breast cells as well as tumour cells from pleural effusions with estrogen, tamoxifen or progesterone did not systematically show an up- or down-regulatory effect. On the other hand, short term stimulations of tumour cells from pleural effusions with progesterone, estrogen, vitamin D3 and retinoic acid lead to an up-regulation of NY-BR-1 expression (up to 8 fold) compared to the untreated controls. VPA treatment also induced NY-BR-1 expression. Of note, the combination of VPA with the above mentioned hormones only showed a weak up-regulation of NY-BR-1 expression (up to 3 fold) compared to the controls. These results suggest that NY-BR-1 expression is further regulated on histone level and/or non-histone proteins such as transcription factors modified by acetylation. Treatment of the cells with the demethylation agent 5´Aza-deoxycytidine did not show a distinct induction or up-regulation of NY-BR-1 expression. Moreover, the methylation status of selected promoter regions containing the predicted CpG islands was assessed in a selection of treated samples, but no association between methylation patterns and NY-BR-1 expression was observed. This finding could be confirmed by an in silico analysis of the TCGA database. Strikingly, a decreased NY-BR-1 expression in cultured normal breast tissue pieces and isolated epithelial cells versus snap-frozen cells was seen, which was not observed in breast tumour cells from pleural effusions cultured in conditioned medium. An in depth analysis of the conditioned medium containing the cell free effusion supernatant as well as cell culture experiments with normal breast epithelial cells in conditioned medium will reveal, which factors, e.g. cytokines, chemokines, hormones or growth factors, lead to a sustained NY-BR-1 expression. Considering the complex regulation of NY-BR-1 gene expression, the mosaic-like protein expression in normal breast tissue and the inhibitory effect of NY-BR-1 over-expression on proliferation and the G1 phase cell cycle arrest in normal cells it was hypothesized, that NY-BR-1 may be expressed by a specific breast progenitor cell population. Thus, mammospheres from isolated normal breast epithelial cells were generated and analysed by immunofluorescent staining and qPCR for NY-BR-1 expression and the presence of progenitor cell markers. In four analysed patients, NY-BR-1 was expressed in primary spheres and co-expression with integrin-α 6, HER2, GATA-3 and FOXA1 could be observed whereas no ER or progesterone receptor (PR) mRNA was detected. IHC staining with ER and NY-BR-1 in normal breast tissue showed that as well ER+/NY-BR-1+ as ER-/NY-BR-1+ cells in the mammary gland are existing. These results suggest that NY-BR-1 is expressed in the ER+/ER- luminal progenitor cells of the mammary gland. The NY-BR-1 gene was investigated regarding genetical variations to receive a better understanding of splice variants. Thereby, 69 damaging nsSNPs within the coding region of NY-BR-1 gene and 39 potential splicing SNPs could be identified by using in silico anlaysis. Taken together, NY-BR-1 is expressed mainly in well differentiated hormone sensitive estrogen receptor positive breast cancer subtypes and it is likely that NY-BR-1 expression is influenced by ERα and/or PR expression, but the association of NY- BR-1 expression and ER signaling still needs to be elucidated. Furthermore, transiently NY-BR-1 expressing cells show an inhibited proliferation rate and accumulate in the G1 phase. In vivo, endogenous NY-BR-1 is expressed in non proliferating (Ki-67 negative) cells in normal breast tissue. The protein potentially interacts with the tubulin beta-4B chain suggesting a crucial role during mitosis. Moreover, NY-BR-1 was shown to be expressed in progenitor cells of the mammary gland. The phenotypic characterization of these progenitor cells and the question whether the protein is also expressed in cancer stem cells is part of ongoing studies. In summary, the presented work could show for the first time that NY-BR-1 is a progenitor cell marker in the mammary gland and is influences the mitotic process

In silico SNP analysis of the breast cancer antigen NY-BR-1

Background: Breast cancer is one of the most common malignancies with increasing incidences every year and a leading cause of death among women. Although early stage breast cancer can be effectively treated, there are limited numbers of treatment options available for patients with advanced and metastatic disease. The novel breast cancer associated antigen NY-BR-1 was identified by SEREX analysis and is expressed in the majority (>70%) of breast tumors as well as metastases, in normal breast tissue, in testis and occasionally in prostate tissue. The biological function and regulation of NY-BR-1 is up to date unknown. Methods: We performed an in silico analysis on the genetic variations of the NY-BR-1 gene using data available in public SNP databases and the tools SIFT, Polyphen and Provean to find possible functional SNPs. Additionally, we considered the allele frequency of the found damaging SNPs and also analyzed data from an in-house sequencing project of 55 breast cancer samples for recurring SNPs, recorded in dbSNP. Results: Over 2800 SNPs are recorded in the dbSNP and NHLBI ESP databases for the NY-BR-1 gene. Of these, 65 (2.07%) are synonymous SNPs, 191 (6.09%) are non-synoymous SNPs, and 2430 (77.48%) are noncoding intronic SNPs. As a result, 69 non-synoymous SNPs were predicted to be damaging by at least two, and 16 SNPs were predicted as damaging by all three of the used tools. The SNPs rs200639888, rs367841401 and rs377750885 were categorized as highly damaging by all three tools. Eight damaging SNPs are located in the ankyrin repeat domain (ANK), a domain known for its frequent involvement in protein-protein interactions. No distinctive features could be observed in the allele frequency of the analyzed SNPs. Conclusion: Considering these results we expect to gain more insights into the variations of the NY-BR-1 gene and their possible impact on giving rise to splice variants and therefore influence the function of NY-BR-1 in healthy tissue as well as in breast cancer

How do parents access, appraise, and apply health information on early childhood allergy prevention? A focus group and interview study

BackgroundWhen parents want to make health-related decisions for their child, they need to be able to handle health information from a potentially endless range of sources. Early childhood allergy prevention (ECAP) is a good example: recommendations have shifted from allergen avoidance to early introduction of allergenic foods. We investigated how parents of children under 3 years old access, appraise and apply health information about ECAP, and their respective needs and preferences.MethodsWe conducted 23 focus groups and 24 interviews with 114 parents of children with varied risk for allergies. The recruitment strategy and a topic guide were co-designed with the target group and professionals from public health, education, and medicine. Data were mostly collected via video calls, recorded and then transcribed verbatim. Content analysis according to Kuckartz was performed using MAXQDA and findings are presented as a descriptive overview.ResultsParents most frequently referred to family members, friends, and other parents as sources of ECAP information, as well as healthcare professionals (HCPs), particularly pediatricians. Parents said that they exchanged experiences and practices with their peers, while relying on HCPs for guidance on decision-making. When searching for information online, they infrequently recalled the sources used and were rarely aware of providers of “good” health information. While parents often reported trying to identify the authors of information to appraise its reliability, they said they did not undertake more comprehensive information quality checks. The choice and presentation of ECAP information was frequently criticized by all parent groups; in particular, parents of at-risk children or with a manifested allergy were often dissatisfied with HCP consultations, and hence did not straightforwardly apply advice. Though many trusted their HCPs, parents often reported taking preventive measures based on their own intuition.ConclusionOne suggestion to react upon the many criticisms expressed by parents regarding who and how provides ECAP information is to integrate central ECAP recommendations into regular child care counseling by HCPs—provided that feasible ways for doing so are identified. This would assist disease prevention, as parents without specific concerns are often unaware of the ECAP dimension of issues such as nutrition

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Synthesis of Cu Single Atoms Supported on Mesoporous Graphitic Carbon Nitride and Their Application in Liquid-Phase Aerobic Oxidation of Cyclohexene

Different loadings of Cu single atoms were anchored on a graphitic carbon nitride (g-C3N4) matrix using a two-step thermal synthesis method and applied in liquid-phase cyclohexene oxidation under mild conditions using molecular O2 as the oxidizing agent. The oxidation state of Cu was determined to be Cu+, which is in linear coordination with two neighboring nitrogen atoms at a distance of 1.9 Å. The catalyst with 0.9 wt % Cu pyrolyzed at 380 °C was found to exhibit the best catalytic performance with the highest conversion up to 82% with an allylic selectivity of 55%. It also showed high reusability over four catalytic runs without any detectable Cu leaching. Cyclohexene oxidation followed first-order kinetics with an apparent activation energy of 66.2 kJ mol–1. The addition of hydroquinone as a radical scavenger confirmed that cyclohexene oxidation proceeds via a radical mechanism. Time-resolved in situ attenuated total reflection infrared (ATR-IR) spectroscopy was carried out to qualitatively monitor the cyclohexene oxidation pathways. The comparison with the homogeneous analogue Cu(I) iodide indirectly verified the linearly N-coordinated single Cu(I) species to be the active sites for cyclohexene oxidation

Health Literacy in Health Professionals Two Years into the COVID-19 Pandemic: Results From a Scoping Review

BackgroundHealth literacy (HL) is an important public health goal but also crucial in individuals providing medical care. During the pandemic, COVID-19–related HL of health professionals (HPs) has gained momentum; it helps to minimize the risk of self-infection, on the one hand, and to protect patients and relatives from infection, on the other. However, comprehensive information about the levels of individual pandemic-related HL in HPs is scarce. ObjectiveIn this paper, we aimed at describing the extent of existing research on HL (concept) conducted in HPs (population) in the COVID-19 pandemic (context). The review intends to map the literature on HL in HPs, thereby highlighting research gaps. MethodsThis scoping review was conducted using the methodology of Khalil et al (2016). This involved an electronic search of PubMed (MEDLINE) and PsycInfo and a hand search. The included studies were iteratively examined to find items representing the four HL dimensions of access, understand, critically appraise, and apply COVID-19–related health information. ResultsThe search yielded a total of 3875 references. Only 7 (1.4%) of the 489 included studies explicitly stated to have addressed HL; 2 (0.4%) studies attempted to develop an instrument measuring COVID-19–related HL in HPs; 6 (1.2%) studies included an HL measure in an observational survey design. Of the remainder, the vast majority used a cross-sectional design. The dimensions access and understand were frequently examined, but few studies looked at the dimensions critical appraisal or apply. Very few studies reported an intervention aiming to improve a COVID-19–related HL outcome. ConclusionsHigh levels of COVID-19–related HL among HPs are necessary to ensure not only safe practice with necessary protection of HPs, their patients, and relatives, but also successful care delivery and subsequently improved health outcomes in the long term. To advance our understanding of how high COVID-19–related HL manifests itself in HPs, how it relates to health outcomes, and how it can be improved, more research is necessary. Trial RegistrationOpen Science Framework dbfa5; https://osf.io/dbfa5

Table_3_How do parents access, appraise, and apply health information on early childhood allergy prevention? A focus group and interview study.DOCX

BackgroundWhen parents want to make health-related decisions for their child, they need to be able to handle health information from a potentially endless range of sources. Early childhood allergy prevention (ECAP) is a good example: recommendations have shifted from allergen avoidance to early introduction of allergenic foods. We investigated how parents of children under 3 years old access, appraise and apply health information about ECAP, and their respective needs and preferences.MethodsWe conducted 23 focus groups and 24 interviews with 114 parents of children with varied risk for allergies. The recruitment strategy and a topic guide were co-designed with the target group and professionals from public health, education, and medicine. Data were mostly collected via video calls, recorded and then transcribed verbatim. Content analysis according to Kuckartz was performed using MAXQDA and findings are presented as a descriptive overview.ResultsParents most frequently referred to family members, friends, and other parents as sources of ECAP information, as well as healthcare professionals (HCPs), particularly pediatricians. Parents said that they exchanged experiences and practices with their peers, while relying on HCPs for guidance on decision-making. When searching for information online, they infrequently recalled the sources used and were rarely aware of providers of “good” health information. While parents often reported trying to identify the authors of information to appraise its reliability, they said they did not undertake more comprehensive information quality checks. The choice and presentation of ECAP information was frequently criticized by all parent groups; in particular, parents of at-risk children or with a manifested allergy were often dissatisfied with HCP consultations, and hence did not straightforwardly apply advice. Though many trusted their HCPs, parents often reported taking preventive measures based on their own intuition.ConclusionOne suggestion to react upon the many criticisms expressed by parents regarding who and how provides ECAP information is to integrate central ECAP recommendations into regular child care counseling by HCPs—provided that feasible ways for doing so are identified. This would assist disease prevention, as parents without specific concerns are often unaware of the ECAP dimension of issues such as nutrition.</p

Table_2_How do parents access, appraise, and apply health information on early childhood allergy prevention? A focus group and interview study.DOCX

BackgroundWhen parents want to make health-related decisions for their child, they need to be able to handle health information from a potentially endless range of sources. Early childhood allergy prevention (ECAP) is a good example: recommendations have shifted from allergen avoidance to early introduction of allergenic foods. We investigated how parents of children under 3 years old access, appraise and apply health information about ECAP, and their respective needs and preferences.MethodsWe conducted 23 focus groups and 24 interviews with 114 parents of children with varied risk for allergies. The recruitment strategy and a topic guide were co-designed with the target group and professionals from public health, education, and medicine. Data were mostly collected via video calls, recorded and then transcribed verbatim. Content analysis according to Kuckartz was performed using MAXQDA and findings are presented as a descriptive overview.ResultsParents most frequently referred to family members, friends, and other parents as sources of ECAP information, as well as healthcare professionals (HCPs), particularly pediatricians. Parents said that they exchanged experiences and practices with their peers, while relying on HCPs for guidance on decision-making. When searching for information online, they infrequently recalled the sources used and were rarely aware of providers of “good” health information. While parents often reported trying to identify the authors of information to appraise its reliability, they said they did not undertake more comprehensive information quality checks. The choice and presentation of ECAP information was frequently criticized by all parent groups; in particular, parents of at-risk children or with a manifested allergy were often dissatisfied with HCP consultations, and hence did not straightforwardly apply advice. Though many trusted their HCPs, parents often reported taking preventive measures based on their own intuition.ConclusionOne suggestion to react upon the many criticisms expressed by parents regarding who and how provides ECAP information is to integrate central ECAP recommendations into regular child care counseling by HCPs—provided that feasible ways for doing so are identified. This would assist disease prevention, as parents without specific concerns are often unaware of the ECAP dimension of issues such as nutrition.</p