Characterization of the promoter of the human farnesyltransferase beta subunit and the impact of the transcription factor OCT-1 on its expression.

Farnesyltransferase (FTase) enables about 100 proteins to interact with cellular membranes by catalyzing the posttranslational addition of a farnesyl group. Farnesylated proteins provide important functions and inhibitors against the ß-subunit of the heterodimer of FTase are intensively studied in clinical and preclinical trials. However, very little is known about the transcriptional regulation of the ß-subunit. The examined promoter region of the human FTase ß-subunit gene (FNTB) showed significant basal promoter activity in HEK-293 and in HeLa cells. We were able to locate the core promoter at -165 to -74. Ten potential binding sites of the transcription factor OCT-1 were detected. Three could be confirmed using EMSA super shift experiments. OCT-1 overexpression and knockdown confirmed it as an important regulator of FNTB expression. Our results provide a basis for further research on FNTB/OCT-1 regulation, its inhibitors and diseases influenced by both such as colon carcinoma or diabetes mellitus.

Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B.

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

The small molecule Bcl-2/Mcl-1 inhibitor TW-37 shows single-agent cytotoxicity in neuroblastoma cell lines.

BACKGROUND: High-risk neuroblastoma with N-Myc amplification remains a therapeutic challenge in paediatric oncology. Antagonism of pro-death Bcl-2 homology (BH) proteins to pro-survival BH members such as Mcl-1 and Bcl-2 has become a treatment approach, but previous studies suggest that a combined inhibition of Bcl-2 and Mcl-1 is necessary. TW-37 inhibits Mcl-1 and Bcl-2 with almost the same affinity. However, single-agent cytotoxicity of TW-37 in neuroblastoma cell lines has not been investigated. METHODS: Cell viability, apoptosis, proliferation and changes in growth properties were determined in SKNAS, IMR-5, SY5Y and Kelly cells after treatment with TW-37. After transfection with Mcl-1 or Bcl-2 siRNA, apoptosis and proliferation were investigated in Kelly cells. Mice with Kelly cell line xenografts were treated with TW-37 and tumor growth, survival and apoptosis were determined. RESULTS: Cell lines with N-Myc amplification were more sensitive to TW-37 treatment, IC50 values for IMR-5 and Kelly cells being 0.28 µM and 0.22 µM, compared to SY5Y cells and SKNAS cells (IC50 0.96 µM and 0.83 µM). Treatment with TW-37 resulted in increased apoptosis and reduced proliferation rates, especially in IMR5 and Kelly cells. Bcl-2 as well as Mcl-1 knockdown induced apoptosis in Kelly cells. TW-37 led to a decrease in tumor growth and a favorable survival (p = 0.0379) in a Kelly neuroblastoma xenografts mouse model. CONCLUSION: TW-37 has strong single-agent cytotoxicity in vitro and in vivo. Therefore, combined inhibition of Bcl-2/Mcl-1 by TW-37 in N-Myc amplified neuroblastoma may represent an interesting therapeutic strategy.

Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers.

The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H-Ras, N-Ras, and K-Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best-known member and has gained significant scientific as well as public attention as an endocrine disrupting chemical, a fact that eventually led to its replacement. However, compounds used to replace BPA still contain the molecular scaffold of bisphenols. BPA, BPAF, BPB, BPE, BPF, and an amine-substituted BPAF-derivate all interact with all GDP-bound Ras-Isoforms through binding to a common site on these proteins. NMR-, SOScat-, and GDI- assay-based data revealed a new bisphenol-induced, allosterically activated GDP-bound Ras conformation that define these plasticisers as Ras allosteric agonists.

Hypoxia Inducible Factor-2 Alpha and Prolinhydroxylase 2 Polymorphisms in Patients with Acute Respiratory Distress Syndrome (ARDS).

Hypoxia-inducible-factor-2α (HIF-2α) and HIF-2 degrading prolyl-hydroxylases (PHD) are key regulators of adaptive hypoxic responses i.e., in acute respiratory distress syndrome (ARDS). Specifically, functionally active genetic variants of HIF-2α (single nucleotide polymorphism (SNP) [ch2:46441523(hg18)]) and PHD2 (C/T; SNP rs516651 and T/C; SNP rs480902) are associated with improved adaptation to hypoxia i.e., in high-altitude residents. However, little is known about these SNPs' prevalence in Caucasians and impact on ARDS-outcome. Thus, we tested the hypotheses that in Caucasian ARDS patients SNPs in HIF-2α or PHD2 genes are (1) common, and (2) independent risk factors for 30-day mortality. After ethics-committee approval, 272 ARDS patients were prospectively included, genotyped for PHD2 (Taqman SNP Genotyping Assay) and HIF-2α-polymorphism (restriction digest + agarose-gel visualization), and genotype dependent 30-day mortality was analyzed using Kaplan-Meier-plots and multivariate Cox-regression analyses. Frequencies were 99.62% for homozygous HIF-2α CC-carriers (CG: 0.38%; GG: 0%), 2.3% for homozygous PHD2 SNP rs516651 TT-carriers (CT: 18.9%; CC: 78.8%), and 3.7% for homozygous PHD2 SNP rs480902 TT-carriers (CT: 43.9%; CC: 52.4%). PHD2 rs516651 TT-genotype in ARDS was independently associated with a 3.34 times greater mortality risk (OR 3.34, CI 1.09-10.22; p = 0.034) within 30-days, whereas the other SNPs had no significant impact (p = ns). The homozygous HIF-2α GG-genotype was not present in our Caucasian ARDS cohort; however PHD2 SNPs exist in Caucasians, and PHD2 rs516651 TT-genotype was associated with an increased 30-day mortality suggesting a relevance for adaptive responses in ARDS.

Hypoxia inducible factor-1 alpha and prolinhydroxlase 2 polymorphisms in patients with severe sepsis: a prospective observational trial.

BACKGROUND: Hypoxia-inducible-factor-1α (HIF-1α) and HIF-1 degrading prolyl-hydroxylases (PHD) are key regulators of the hypoxic-inflammatory response. Functionally active genetic variants in the HIF-1α (C/T; Single Nucleotide Polymorphism (SNP) rs11549465) and the PHD2 gene (EGLN1; C/T; SNP rs516651 and T/C; SNP rs480902) are associated with altered HIF-1α mRNA nuclear translocation and an altered adaptation to hypoxia. Furthermore, the HIF system is important in surviving inflammatory disorders and sepsis. Thus, we tested the hypotheses, that SNPs in the HIF-1α or PHD2 genes are (1) common in Caucasians, with 2) the HIF-1α genetic variant being associated with an altered HIF-1α mRNA expression; and 3) independent risk factors for 30-day mortality in severe sepsis. METHODS: After ethics approval, 128 septic patients (Caucasian descent) were included prospectively within 24 h after first diagnosing sepsis. Patients characteristics and severity of illness (simplified acute physiology score II), genotypes (Taqman assay), and their influence on leukocyte HIF-1α-mRNA-expression (Real-Time PCR) and 30-day mortality were determined. RESULTS: Frequencies were 0.8 % for homozygous HIF-1α TT-carriers (CT 17.6 %; CC 81.6 %), 2.5 % for homozygous PHD2 SNP rs516651 TT-allele carriers (CT 17.5 % and CC 80 %), and 9.4 % for homozygous PHD2 SNP rs480902 TT-allele carriers (CT 34.4 % and CC 56.3 %). While HIF-1α T-allele carriers had a borderline decrease in HIF-1α-mRNA-expression (p = 0.06) neither HIF-1α nor PHD2 SNPs were (independent) risk factors for 30-day mortality. CONCLUSIONS: Genetic variants in HIF-1α and PHD2 genes exist in Caucasians but do not appear to alter 30-day mortality in sepsis.

A 3'UTR polymorphism modulates mRNA stability of the oncogene and drug target Polo-like Kinase 1.

BACKGROUND: The Polo-like Kinase 1 (PLK1) protein regulates cell cycle progression and is overexpressed in many malignant tissues. Overexpression is associated with poor prognosis in several cancer entities, whereby expression of PLK1 shows high inter-individual variability. Although PLK1 is extensively studied, not much is known about the genetic variability of the PLK1 gene. The function of PLK1 and the expression of the corresponding gene could be influenced by genomic variations. Hence, we investigated the gene for functional polymorphisms. Such polymorphisms could be useful to investigate whether PLK1 alters the risk for and the course of cancer and they could have an impact on the response to PLK1 inhibitors. METHODS: The coding region, the 5' and 3'UTRs and the regulatory regions of PLK1 were systematically sequenced. We determined the allele frequencies and genotype distributions of putatively functional SNPs in 120 Caucasians and analyzed the linkage and haplotype structure using Haploview. The functional analysis included electrophoretic mobility shift assay (EMSA) for detected variants of the silencer and promoter regions and reporter assays for a 3'UTR polymorphism. RESULTS: Four putatively functional polymorphisms were detected and further analyzed, one in the silencer region (rs57973275), one in the core promoter region (rs16972787), one in intron 3 (rs40076) and one polymorphism in the 3'untranslated region (3'UTR) of PLK1 (rs27770). Alleles of rs27770 display different secondary mRNA structures and showed a distinct allele-dependent difference in mRNA stability with a significantly higher reporter activity of the A allele (p < 0.01). CONCLUSION: The present study provides evidence that at least one genomic variant of PLK1 has functional properties and influences expression of PLK1. This suggests polymorphisms of the PLK1 gene as an interesting target for further studies that might affect cancer risk, tumor progression as well as the response to PLK1 inhibitors.

The BCL2-938 C > A promoter polymorphism is associated with risk group classification in children with acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. While current treatment regimens achieve almost 80% overall survival, long-term side effects of chemotherapeutic agents can be severe. The functional BCL2-938C > A promoter polymorphism is known to influence the balance between survival and apoptosis of malignant hematolymphoid cells. We investigated its usefulness as a marker for treatment stratification for children with ALL. METHODS: We analyzed DNA from 182 children suffering from ALL in this study to determine genotypes of the -938 C > A polymorphism by "slow-down" PCR. RESULTS: ALL patients with the BCL2-938CC genotype had an approximately 3-fold higher risk of belonging to a high-risk group. Within the high-risk group, 50% of BCL2-938CC patients were classified as high-risk due to poor prednisone response whereas only 33% of patients with AC and AA genotypes were classified as high-risk for the same reason. CONCLUSIONS: Our results suggest that BCL2-938C > A genotyping may be beneficial for therapy response prediction in ALL patients, and warrant examination in a larger cohort to validate its usefulness for treatment stratification of pediatric ALL patients.

Expression of aquaporin 5 and the AQP5 polymorphism A(-1364)C in association with peritumoral brain edema in meningioma patients.

Aquaporins (AQP) are a growing family of water-channel proteins, numbering 13 to date. Recent studies have reported AQP1 and AQP4 to be involved in the development and resorption of brain edemas of different origin. Other AQPs have also been detected in brain tissue, but their impact on brain edema remains to be shown. To evaluate a possible role of AQP5 in brain edema, we investigated the association of AQP5 expression and the functional AQP5 promoter polymorphism A(-1364)C with occurrence and intensity of peritumoral edema in meningioma patients. Peritumoral edema was classified in three degrees based on preoperative imaging in 89 meningioma patients treated at the University Hospital Essen between 2003 and 2006. AQP5 expression was assessed immunohistochemically in tumor tissue obtained during neurosurgical tumor resection. Genotypes of the A(-1364)C polymorphism were determined using the "slowdown" polymerase chain reaction. Higher levels of AQP5 expression were significantly correlated with the AQP5-1364 AA genotype (P = 0.02). AQP5 expression was positively correlated with edema (P = 0.04). AQP5 genotypes were not significantly associated with the occurrence, but with the intensity of peritumoral brain edema (P = 0.04). In our cohort, 40 % of patients with grade I, 66.7 % with grade II, and 76.5 % with grade III edema possessed at least one A allele. Development and intensity of peritumoral edema in meningiomas are associated with AQP5 expression. The intensity of edema correlates with the AQP5 A(-1364)C genotype. This suggests AQP5 as an interesting new candidate involved in peritumoral brain edema in meningioma patients.

Regulation of protein prenylation.

Prenyltransferases (PTases) are known to play a role in embryonic development, normal tissue homeostasis and cancer by posttranslationally modifying proteins involved in these processes. They are being discussed as potential drug targets in an increasing number of diseases, ranging from Alzheimer's disease to malaria. Protein prenylation and the development of specific PTase inhibitors (PTIs) have been subject to intense research in recent decades. Recently, the FDA approved lonafarnib, a specific farnesyltransferase inhibitor that acts directly on protein prenylation; and bempedoic acid, an ATP citrate lyase inhibitor that might alter intracellular isoprenoid composition, the relative concentrations of which can exert a decisive influence on protein prenylation. Both drugs represent the first approved agent in their respective substance class. Furthermore, an overwhelming number of processes and proteins that regulate protein prenylation have been identified over the years, many of which have been proposed as molecular targets for pharmacotherapy in their own right. However, certain aspects of protein prenylation, such as the regulation of PTase gene expression or the modulation of PTase activity by phosphorylation, have attracted less attention, despite their reported influence on tumor cell proliferation. Here, we want to summarize the advances regarding our understanding of the regulation of protein prenylation and the potential implications for drug development. Additionally, we want to suggest new lines of investigation that encompass the search for regulatory elements for PTases, especially at the genetic and epigenetic levels.

Prenyltransferase gene expression reveals an essential role of prenylation for the inflammatory response in human gingival fibroblasts.

BACKGROUND: Prenyltrasferases (PTases) are a class of enzymes known to be responsible for promoting posttranslational modification at the carboxyl terminus of proteins containing a so-called CaaX-motif. The process is responsible for proper membrane localization and the appropriate function of several intracellular signaling proteins. Current research demonstrating the pathomechanistic importance of prenylation in inflammatory illnesses emphasizes the requirement to ascertain the differential expression of PT genes under inflammatory settings, particularly in periodontal disease. METHODS: Telomerase-immortalized human gingival fibroblasts (HGF-hTert) were cultured and treated with either inhibitors of prenylation (PTI) lonafarnib, tipifarnib, zoledronic acid, or atorvastatin at concentrations of 10 µM in combination with or without 10 µg Porphyromonas gingivalis lipopolysaccharide (LPS) for 24 h. Prenyltransferase genes FNTB, FNTA, PGGT1B, RABGGTA, RABGGTB, and PTAR1 as well as inflammatory marker genes MMP1 and IL1B were detected using quantitative real-time polymerase chain reaction (RT-qPCR). Immunoblot and protein immunoassay were used to confirm the results on the protein level. RESULTS: RT-qPCR experiments revealed significant upregulation of IL1B, MMP1, FNTA, and PGGT1B upon LPS treatment. PTase inhibitors caused significant downregulation of the inflammatory cytokine expression. Interestingly, FNTB expression was significantly upregulated in response to any PTase inhibitor in combination with LPS, but not upon LPS treatment only, indicating a vital role of protein farnesyltransferase in the proinflammatory signaling cascade. CONCLUSIONS: In this study, distinct PTase gene expression patterns in pro-inflammatory signaling were discovered. Moreover, PTase inhibiting drugs ameliorated inflammatory mediator expression by a significant margin, indicating that prenylation is a major pre-requisite for innate immunity in periodontal cells.

Farnesyltransferase-inhibitors exert in vitro immunosuppressive capacity by inhibiting human B-cells.

Objectives: Farnesyltransferase inhibitors (FTI), which inhibit the prenylation of Ras GTPases, were developed as anti-cancer drugs. As additional target proteins for prenylation were identified in the past, it is likely that FTI have potential value for therapeutic purposes beyond cancer. The effect of FTI on B-cells remains unclear. To address this issue, we investigated the effects of in vitro FTI treatment on effector and regulatory B-cells in healthy controls and renal transplant patients. Methods: For this purpose, B-cells were isolated from the peripheral blood of healthy controls and renal transplant patients. Purified B-cells were stimulated via Toll-like-receptor 9 (TLR-9) in the presence or absence of FTI. Regulatory functions, such as IL-10 and Granzyme B (GrB) secretion, were assessed by flow cytometry. In addition, effector B-cell functions, such as plasma cell formation and IgG secretion, were studied. Results: The two FTI Lonafarnib and tipifarnib both suppressed TLR-9-induced B-cell proliferation. Maturation of IL-10 producing B-cells was suppressed by FTI at high concentrations as well as induction of GrB-secreting B-cells. Plasma blast formation and IgG secretion were potently suppressed by FTI. Moreover, purified B-cells from immunosuppressed renal transplant patients were also susceptible to FTI-induced suppression of effector functions, evidenced by diminished IgG secretion. Conclusion: FTI suppress in vitro B-cell proliferation and plasma cell formation while partially preserving IL-10 as well as GrB production of B-cells. Thus, FTI may have immunosuppressive capacity encouraging further studies to investigate the potential immunomodulatory value of this agent.

Evidence-based selection of reference genes for RT-qPCR assays in periodontal research.

OBJECTIVE: To underline the necessity of adequate reference genes for real-time quantitative polymerase chain reaction (RT-qPCR) and evaluate a novel tool for condition-specific reference gene selection. BACKGROUND: RT-qPCR is a commonly used experimental technique that allows for highly sensitive analysis of gene transcription. Moreover, the use of internal reference genes as a means for relative quantification has rendered RT-qPCR a straightforward method for a variety of sciences, including dentistry. However, the expressional stability of internal reference genes must be evaluated for every assay in order to account for possible quantification bias. MATERIALS AND METHODS: Herein, we used the software tool RefGenes to identify putatively stable reference genes with the help of microarray datasets and evaluated them. Additionally, we propose an evidence-based workflow for adequate normalization of thusly identified genes. Human gingival fibroblasts (HGF-hTert), human acute leukemia-derived monocytes (THP-1), and telomerase immortalized gingival keratinocytes (TIGKs) were subjected to set-ups simulating various glycemic conditions and lipopolysaccharide challenges. Five common housekeeping genes (HKGs) and five genes from RefGenes were selected as targets and RT-qPCR was performed subsequently. Then, normalization algorithms Bestkeeper, Normfinder, and geNorm were used for further analysis of the putative reference gene stability. RESULTS: RefGenes-derived targets exhibited the highest stability values in THP-1 and TIGK cell lines. Moreover, unacceptable standard variations were observed for some common HKG like ß-actin. However, common HKG exhibited good stability values in HGF-hTert cells. CONCLUSION: The results indicate that microarray-based preselection of putative reference genes is a valuable refinement for RT-qPCR studies. Accordingly, the present study proposes a straightforward workflow for evidence-based preselection and validation of internal reference genes.

Diuretics: a contemporary pharmacological classification?

Diuretics are drugs that increase the flow of urine. They are commonly used to treat edema, hypertension, and heart failure. Typically, the pharmacological group consists of five classes: thiazide diuretics, loop diuretics, potassium-sparing diuretics, osmotic diuretics, and carbonic anhydrase inhibitors. This traditional classification and the nomenclature of diuretics have not changed over the last decades, which means that it was not adapted to current pharmacological research. Modern approaches in the field of pharmacological nomenclature suggest the introduction of mechanism-based drug class designations, which is not yet reflected in the group of diuretics. Moreover, included drug classes have lost their relevance as diuretic agents. Carbonic anhydrase inhibitors, for example, are mainly used in the treatment of glaucoma. Newer agents such as vasopressin-2 receptor antagonists or SGLT2 inhibitors possess diuretic properties but are not included in the pharmacological group. This review discusses the currentness of the pharmacological classification of diuretics. We elaborate changes in the field of nomenclature, the contemporary medical use of classical diuretics, and new diuretic agents.

Regulatory BCL2 promoter polymorphism (-938C>A) is associated with adverse outcome in patients with prostate carcinoma.

Molecular markers predictive of prostate cancer prognosis are urgently needed. Overexpression of the antiapoptotic protein, Bcl-2, has repeatedly been shown to be associated with adverse outcome in this malignancy. We hypothesized that a regulatory BCL2 -938C>A promoter polymorphism, which significantly affects promoter activity and Bcl-2 expression in different malignancies, may influence survival. Reporter assays and electrophoretic mobility shift assays reveled that the -938C>A BCL2 promoter polymorphism significantly affects promoter activity and transcription factor binding in prostate cancer cells. Significantly higher BCL2 mRNA expression was observed in primary prostate carcinomas derived from patients with the AA, compared to CC, genotype. Survival analysis showed that the -938AA genotype was an independent, unfavorable prognostic factor for relapse-free survival in a primary cohort of 142 patients and in an independent replication cohort of 148 patients, with hazard ratios (HR) of 4.4 (95% CI, 1.3-15.1; p = 0.018) and 4.6 (95% CI, 1.5-14.2; p = 0.009). Furthermore, the -938AA genotype was independently associated with worse overall survival in the replication series, with a HR of 10.9 (95% CI, 1.2-99.3; p = 0.034). We conclude that the BCL2 -938C>A polymorphism is an independent predictor of relapse-free and overall survival in patients with prostate cancer. The BCL2 -938C>A polymorphism should be evaluated prospectively and may also have promise in assisting optimal patient choice for treatment with BCL2-targeted drugs already in evaluation for prostate cancer treatment.

Toll-like receptor 4 single-nucleotide polymorphisms Asp299Gly and Thr399Ile in head and neck squamous cell carcinomas.

BACKGROUND: Chronic inflammation plays an important role in head and neck squamous cell carcinomas (HNSCC). This study addresses the impact of two single nucleotide polymorphisms (SNP) Asp299Gly and Thr399Ile of the toll-like receptor (TLR) 4 gene on the clinical outcome while accounting for the influence of adjuvant systemic therapy in a large cohort of HNSCC patients. METHODS: Genotype analysis was done using DNA from tissue samples from 188 patients with HNSCC; TLR4 protein expression was assessed immunohistochemically in tissue microarrays. Classical survival models were used for statistical analyses. RESULTS: Ten percent of patients with HNSCC presented with the TLR4 299Gly and 17% with the TLR4 399Ile allele. Patients with the heterozygous genotype TLR4 Asp299Gly had a significantly reduced disease-free and overall survival. Also, patients with the heterozygous genotype TLR4 Thr399Ile had a reduced disease-free survival. Notably, these associations seem to be attributable to relatively poor therapy response as e.g. reflected in a significantly shorter DFS among HNSCC patients carrying the Asp299Gly variant and receiving adjuvant systemic therapy. CONCLUSION: According to this study, TLR4 299Gly und 399Ile alleles may serve as markers for prognosis of head and neck cancer in patients with adjuvant systemic therapy, particularly chemotherapy, and might indicate therapy resistance.

Calcitonin substitution in calcitonin deficiency reduces particle-induced osteolysis.

BACKGROUND: Periprosthetic osteolysis is a major cause of aseptic loosening in joint arthroplasty. This study investigates the impact of CT (calcitonin) deficiency and CT substitution under in-vivo circumstances on particle-induced osteolysis in Calca -/- mice. METHODS: We used the murine calvarial osteolysis model based on ultra-high molecular weight polyethylene (UHMWPE) particles in 10 C57BL/6J wild-type (WT) mice and twenty Calca -/- mice. The mice were divided into six groups: WT without UHMWPE particles (Group 1), WT with UHMWPE particles (Group 2), Calca -/- mice without UHMWPE particles (Group 3), Calca -/- mice with UHMWPE particles (Group 4), Calca -/- mice without UHMWPE particles and calcitonin substitution (Group 5), and Calca -/- mice with UHMWPE particle implantation and calcitonin substitution (Group 6). Analytes were extracted from serum and urine. Bone resorption was measured by bone histomorphometry. The number of osteoclasts was determined by counting the tartrate-resistant acid phosphatase (TRACP) + cells. RESULTS: Bone resorption was significantly increased in Calca -/- mice compared with their corresponding WT. The eroded surface in Calca -/- mice with particle implantation was reduced by 20.6% after CT substitution. Osteoclast numbers were significantly increased in Calca -/- mice after particle implantation. Serum OPG (osteoprotegerin) increased significantly after CT substitution. CONCLUSIONS: As anticipated, Calca -/- mice show extensive osteolysis compared with wild-type mice, and CT substitution reduces particle-induced osteolysis.

Motivations and Experiences of Volunteering Medical Students in the COVID-19 Pandemic-Results of a Survey in Germany.

Introduction: During the first lockdown of the COVID-19 pandemic, several medical students volunteered as assistants in hospitals, public health departments, and other healthcare services to support and substitute permanent staff. The underlying motivations to help are unclear. Therefore, we aimed to assess medical students' motivations and influencing variables such as perceived stress and burden, compassion, and indicators of spirituality. Materials and Methods: Cross-sectional survey (convenience sample) from May to June 2020, directly after the first lockdown, among medical students with standardized instruments. One of them is the 12-item Motivations to Help Scale (MtHS) which was designed to fit to the population of medical students. Results: Among the 731 completers, 52% were working as volunteers during the pandemic in different medical areas, most in hospitals and only a few in other areas (9% in public health departments, 6% in outpatient services), 37% would have liked to work but did not get an appropriate employment, and 21% did not intend to voluntarily support the hospital staff. Their mental burden during work was rather low, while they were somewhat affected by the personal fate of the patients. With respect to their motivations to volunteer as measured with the MtHS, Altruistic Intentions/Helping (Cronbach's alpha = 0.898) scored highest, followed by Practical Application/Learning (Cronbach's alpha = 0.808), while Role Testing/Recognition (Cronbach's alpha = 0.702) scored lowest. Those who volunteered had significantly higher scores for Altruistic Intentions/Helping and Practical Application/Learning, while the different phases of medical study (preclinical phase, clinical phase, and higher semester) had no influence on the extent of the students' motivation. The motivations to help were not at all or only marginally (inversely) related to indicators of stress and burden, while Altruistic Intentions/Helping was weakly related to affections by patients' fate. Conclusions: Medical students' intention to support healthcare professionals as supplementary assistants were both prosocial and proself motivated. With this opportunity to practically apply their current knowledge and to improve their skills and competences, volunteering students might be more motivated for their further studies and their future career as compassionate medical doctors.

Pharmacogenetic association of diabetes-associated genetic risk score with rapid progression of coronary artery calcification following treatment with HMG-CoA-reductase inhibitors -results of the Heinz Nixdorf Recall Study.

HMG-CoA-Reductase inhibitors (HMGRIs) are currently the most widely used group of drugs in patients with coronary artery disease (CAD) and are given preemptively to patients with high levels of cholesterol, including those with diabetes mellitus (DM). However, intake of HMGRIs also increases the progression of coronary artery calcification (CAC) and the risk of developing DM. This study aimed to investigate whether HMGRI intake interacts with the diabetes-associated genetic risk score (GRS) to affect CAC progression using data from the population-based Heinz Nixdorf Recall (HNR) study. CAC was measured in 3157 participants using electron-beam computed tomography twice, at baseline (CACb) and 5 years later (CAC5y). CAC progression was classified as slow, expected, or rapid based on predicted values. Weighted DM GRS was constructed using 100 diabetes mellitus-associated single nucleotide polymorphisms (SNPs). We used log-linear regression to evaluate the interaction of HMGRI intake with diabetes-associated GRS and individual SNPs on CAC progression (rapid vs. expected/slow), adjusting for age, sex, and log(CACb + 1). The prevalence of rapid CAC progression in the HNR study was 19.6%. We did not observe any association of the weighted diabetes mellitus GRS with the rapid progression of CAC (relative risk (RR) [95% confidence interval (95% CI)]: 1.01 [0.94; 1.10]). Furthermore, no indication of an interaction between GRS and HMGRI intake was observed (1.08 [0.83; 1.41]). Our analyses showed no indication that the impact of HMGRIs on CAC progression is significantly more severe in patients with a high genetic risk of developing DM than in those with a low GRS.