Tumour promoting HER2 splice variant 16HER2 : regulation and implication in breast cancer

PhD ThesisResults from high-throughput studies of the last decade have shown an important connection between regulated pre-mRNA splicing and tumorigenesis as well as therapy resistance. To date only relatively few such interactions have been studied in depth. The human epidermal growth factor receptor 2 (HER2) is an important biomarker in cancer, especially in 20-30% of breast cancer cases. This subset of breast cancer patients are treated with HER2-targeting therapies in addition to chemotherapy. Although these therapies are very successful, in a subset of patients with primary tumours as well as in advanced metastatic breast cancer resistance occurs. The underlying mechanisms are not well understood; one potential cause is the alternative splicing of HER2 pre-mRNA yielding protein variants which can evade or prevent action of these therapeutics. This PhD thesis details progress in understanding the mechanisms regulating the production of the HER2 variant Δ16HER2, which has been shown to have high oncogenic potential. Four key splicing factors that regulate this splicing event were identified as well as a specific RNA:protein binding site in the Δ16HER2 splicing region. In addition, a potential novel HER2 mRNA transcript was identified involving retention of intron 25 (termed I25HER2). I25HER2 was shown to be expressed in both normal human tissues and breast cancer tissue samples. Interestingly, the expression level of I25HER2 is not proportional to wild type HER2 in all tissues. Although, I25HER2 would be expected to undergo nonsense-mediated decay, this study indicated that I25HER2 mRNA has similar stability to Δ16HER2 mRNA. An ongoing challenge in both clinical and non-clinical studies is the inability to distinguish different HER2 protein variants. Initial results presented in this thesis, from a comparative study between immunohistochemistry (using three different HER2 antibodies, including the clinically used HercepTestTM) and HER2 gene amplification (using HER2 CISH pharmDxTM) in a cohort of primary human ductal carcinomas showed a subset of discordant cases. In a few of these tumours elevated levels of HER2 splice variants could be detected. The iii possible implication for successful treatment of these patients’ highlights the importance of linking the basic processing of HER2 transcripts and protein variants with pathological information from patients

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Modelling Distributions of Rove Beetles in Mountainous Areas Using Remote Sensing Data

Mountain ecosystems are biodiversity hotspots that are increasingly threatened by climate and land use/land cover changes. Long-term biodiversity monitoring programs provide unique insights into resulting adverse impacts on plant and animal species distribution. Species distribution models (SDMs) in combination with satellite remote sensing (SRS) data offer the opportunity to analyze shifts of species distributions in response to these changes in a spatially explicit way. Here, we predicted the presence probability of three different rove beetles in a mountainous protected area (Gran Paradiso National Park, GPNP) using environmental variables derived from Landsat and Aster Global Digital Elevation Model data and an ensemble modelling approach based on five different model algorithms (maximum entropy, random forest, generalized boosting models, generalized additive models, and generalized linear models). The objectives of the study were (1) to evaluate the potential of SRS data for predicting the presence of species dependent on local-scale environmental parameters at two different time periods, (2) to analyze shifts in species distributions between the years, and (3) to identify the most important species-specific SRS predictor variables. All ensemble models showed area under curve (AUC) of the receiver operating characteristics values above 0.7 and true skills statistics (TSS) values above 0.4, highlighting the great potential of SRS data. While only a small proportion of the total area was predicted as highly suitable for each species, our results suggest an increase of suitable habitat over time for the species Platydracus stercorarius and Ocypus ophthalmicus, and an opposite trend for Dinothenarus fossor. Vegetation cover was the most important predictor variable in the majority of the SDMs across all three study species. To better account for intra- and inter-annual variability of population dynamics as well as environmental conditions, a continuation of the monitoring program in GPNP as well as the employment of SRS with higher spatial and temporal resolution is recommended

A Possible Mobile Health Solution in Orthopedics and Trauma Surgery: Development Protocol and User Evaluation of the Ankle Joint App

Background: Ankle sprains are one of the most frequent sports injuries. With respect to the high prevalence of ankle ligament injuries and patients’ young age, optimizing treatment and rehabilitation is mandatory to prevent future complications such as chronic ankle instability or osteoarthritis. Objective: In modern times, an increasing amount of smartphone usage in patient care is evident. Studies investigating mobile health (mHealth)–based rehabilitation programs after ankle sprains are rare. The aim of this study was to expose any issues present in the development process of a medical app as well as associated risks and chances. Methods: The development process of the Ankle Joint App was defined in chronological order using a protocol. The app’s quality was evaluated using the (user) German Mobile App Rating Scale (MARS-G) by voluntary foot and ankle surgeons (n=20) and voluntary athletes (n=20). Results: A multidisciplinary development team built a hybrid app with a corresponding backend structure. The app’s content provides actual medical literature, training videos, and a log function. Excellent interrater reliability (interrater reliability=0.92; 95% CI 0.86-0.96) was obtained. The mean overall score for the Ankle Joint App was 4.4 (SD 0.5). The mean subjective quality scores were 3.6 (surgeons: SD 0.7) and 3.8 (athletes: SD 0.5). Behavioral change had mean scores of 4.1 (surgeons: SD 0.7) and 4.3 (athletes: SD 0.7). The medical gain value, rated by the surgeons only, was 3.9 (SD 0.6). Conclusions: The data obtained demonstrate that mHealth-based rehabilitation programs might be a useful tool for patient education and collection of personal data. The achieved (user) MARS-G scores support a high quality of the tested app. Medical app development with an a priori defined target group and a precisely intended purpose, in a multidisciplinary team, is highly promising. Follow-up studies are required to obtain funded evidence for the ankle joints app’s effects on economical and medical aspects in comparison with established nondigital therapy paths

Smartphone and App Usage in Orthopedics and Trauma Surgery: Survey Study of Physicians Regarding Acceptance, Risks, and Future Prospects in Germany

Background: In the course of digitization, smartphones are affecting an increasing number of areas of users’ lives, giving them almost ubiquitous access to the internet and other web applications. Mobile health (mHealth) has become an integral part of some areas of patient care. In contrast to other disciplines, routine integration of mobile devices in orthopedics and trauma surgery in Germany is still in its infancy. Objective: This study aimed to investigate physicians’ current state of opinion regarding acceptance, future prospects, and risks of medical apps in the field of orthopedics and trauma surgery in Germany. Methods: A web-based survey among orthopedics and trauma surgeons in German university hospitals on the use of medical apps in everyday clinical practice was conducted between September 2018 and February 2019. The survey consisted of 13 open- and closed-ended or multiple-choice questions. A logistic regression analysis was performed to ascertain the effects of interindividual characteristics on the likelihood of participants’ app and smartphone usage behavior. Results: A total of 206 physicians participated in the survey. All of the participants (206/206, 100%) owned a smartphone, and 79.1% (159/201) used the device, while 64.7% (130/201) used apps regularly in everyday clinical practice. Medical apps were perceived as beneficial, given their substantial future promise, by 90.1% (181/201) of the participants. However, 62.5% (120/192) of the participants were not satisfied with the current supply of medical apps in app stores. Desired specifications for future apps were “intuitive usability” (167/201, 83.1%), “no advertising” (145/201, 72.1%), and “free apps” (92/201, 45.8%). The attributes “transparent app development and app sponsoring” (75/201, 37.3%) and the existence of an “easy-to-understand privacy statement” (50/201, 24.9%) were of minor relevance. The majority of the participants (162/194, 83.5%) considered that future apps in the field of “medical research” would provide the greatest benefit. The greatest predicted risks were “data misuse” (147/189, 77.8%), “usage of untrustworthy apps” (135/189, 71.4%), and “alienation from patients” (51/189, 27.0%). Increasing age was significantly associated with a reduction in the likelihood of regular smartphone (odds ratio [OR] 0.91, 95% CI 0.86-0.97; P=.002) and app (OR 0.90, 95% CI 0.85-0.96; P=.001) usage, while the medical profession grade had no significant impact on the usage behavior. Conclusions: The study demonstrates that young German doctors in orthopedics and trauma surgery already use smartphones and apps in everyday clinical practice. Medical apps are considered to play an important role in the future. However, a significant discrepancy exists between the supply and demand of mHealth applications, which creates a legal and ethical vacuum with regard to data protection

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a ‘function follows form’ model of differentiation

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes

Association of Chronic Kidney Disease With Plasma NfL and Other Biomarkers of Neurodegeneration: The H70 Birth Cohort Study in Gothenburg

BACKGROUND AND OBJECTIVES: Studies associate chronic kidney disease (CKD) with neurodegeneration. This study investigated the relation between kidney function, blood, cerebrospinal fluid (CSF), and structural brain MRI markers of neurodegeneration, in a sample including individuals with and without CKD. METHODS: Participants from the Gothenburg H70 Birth Cohort Study, with data on plasma-neurofilament light (P-NfL), estimated glomerular filtration rate (eGFR) and structural brain MRI were included. Participants were invited to also have CSF collected. The primary endpoint of the present study was to determine any association between CKD and P-NfL. Secondary endpoints included cross-sectional associations between CKD, eGFR and cerebrospinal fluid (CSF)- and MRI-derived markers of neurodegeneration and Alzheimer's disease (AD) pathology (MRI: cortical thickness, hippocampal volume, lateral ventricle volume, white matter lesion volume; CSF: β-amyloid (Aβ) 42, Aβ42/40, Aβ42/p-tau, t-tau, p-tau, NfL). Participants with P-NfL and eGFR at baseline were re-examined on eGFR, 5.5 (5.3; 6.1) years (median; IQR) after the first visit, and the predictive value of P-NfL levels on incident CKD was estimated longitudinally, using a Cox proportional hazards model. RESULTS: We included 744 participants, 668 without CKD (Age 71 (70; 71) years, 50% males) and 76 with CKD (age 71 (70;71) years, 39% males). Biomarkers from cerebrospinal fluid (CSF) were analysed in 313 participants. 558 individuals returned for a re-examination of eGFR (75% response rate, age 76 (76; 77), 48% males, 76 new cases of CKD). Participants with CKD had higher P-NfL levels than those with normal kidney function (median; 18.8 versus 14.0 pg/mL, p<0.001), while MRI and CSF markers were similar between the groups. P-NfL was independently associated with CKD after adjustment for confounding variables, including hypertension and diabetes (OR; 3.231, p<0.001), in a logistic regression model. eGFR, and CSF Aβ 42/40: R=0.23, p=0.004 correlated in participants with Aβ42 pathology. P-NfL levels in the highest quartile were associated with incident CKD at follow-up (HR; 2.08 (1.14: 4.50)). DISCUSSION: In a community-based cohort of 70-year olds, P-NfL was associated with both prevalent and incident CKD, while CSF and/or imaging measures did not differ by CKD status. Participants with CKD and dementia presented similar levels of P-NfL

Plasma and CSF NfL are differentially associated with biomarker evidence of neurodegeneration in a community-based sample of 70-year-olds.

Neurofilament light protein (NfL) in cerebrospinal fluid (CSF) and plasma (P) are suggested to be interchangeable markers of neurodegeneration. However, evidence is scarce from community-based samples. NfL was examined in a small-scale sample of 287 individuals from the Gothenburg H70 Birth cohort 1944 study, using linear models in relation to CSF and magnetic resonance imaging (MRI) biomarker evidence of neurodegeneration. CSF-NfL and P-NfL present distinct associations with biomarker evidence of Alzheimer's disease (AD) pathology and neurodegeneration. P-NfL was associated with several markers that are characteristic of AD, including smaller hippocampal volumes, amyloid beta (Aβ)42, Aβ42/40, and Aβ42/t-tau (total tau). CSF-NfL demonstrated associations with measures of synaptic and neurodegeneration, including t-tau, phosphorylated tau (p-tau), and neurogranin. Our findings suggest that P-NfL and CSF-NfL may exert different effects on markers of neurodegeneration in a small-scale community-based sample of 70-year-olds