Association of known SARS-CoV-2 serostatus and adherence to personal protection measures and the impact of personal protective measures on seropositivity in a population-based cross-sectional study (MuSPAD) in Germany.

BACKGROUND: In 2020/2021 in Germany, several non-pharmacological interventions were introduced to lower the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated to what extent knowledge of prior infection with SARS-CoV-2 or vaccination status influenced the use of personal protection measures (PPM). Further, we were interested in the effect of compliance with PPM on SARS-CoV-2 serostatus. METHODS: Data was based on a sequential, multilocal seroprevalence study (MuSPAD), carried out in eight locations from July 2020 to August 2021. We estimated the association between a known SARS-CoV-2 serostatus (reported positive PCR test or vaccination) and self-reported PPM behavior (hand hygiene, physical distancing, wearing face mask), just as the association of PPM compliance with seropositivity against nucleocapsid (NC), receptor-binding domain (RBD), and spike protein (S) antigens. We identified relevant variables and deduced adjustment sets with directed acyclic graphs (DAG), and applied mixed logistic regression. RESULTS: Out of the 22,297 participants (median age: 54 years, 43% male), 781 were classified as SARS-CoV-2-infected and 3,877 had a vaccinated immune response. Vaccinated individuals were less likely to keep 1.5 m distance [OR = 0.74 (95% CI: 0.57-0.97)] and only partly physically distanced [OR = 0.71 (95% CI: 0.58-0.87)]. Participants with self-reported positive PCR test had a lower chance of adhering partly to physical distancing [OR = 0.70 (95% CI: 0.50-0.99)] in comparison to the reference group. Higher odds of additionally wearing a face mask was observed in vaccinated [OR = 1.28 (95% CI: 1.08-1.51)] even if it was not obligatory. Overall, among unvaccinated participants, we found little evidence of lower odds of seropositivity given mask wearing [OR: 0.91 (95% CI: 0.71-1.16)], physical distancing [OR: 0.84 (95% CI: 0.59-1.20)] and no evidence for completely adhering to hand cleaning [OR: 0.97 (95% CI: 0.29-3.22)]. CONCLUSIONS: A known confirmed prior infection and vaccination may have the potential to influence adherence to PPM.

Erythritol as sweetener-wherefrom and whereto?

Erythritol is a naturally abundant sweetener gaining more and more importance especially within the food industry. It is widely used as sweetener in calorie-reduced food, candies, or bakery products. In research focusing on sugar alternatives, erythritol is a key issue due to its, compared to other polyols, challenging production. It cannot be chemically synthesized in a commercially worthwhile way resulting in a switch to biotechnological production. In this area, research efforts have been made to improve concentration, productivity, and yield. This mini review will give an overview on the attempts to improve erythritol production as well as their development over time.

Concurrent validity of single and groups of walking assessments following acute spinal cord injury.

STUDY DESIGN: Retrospective analysis of prospectively collected longitudinal data. Variables of interest are timed and untimed walking assessments (10MWT, 6MWT, TUG, WISCI, SCIM3a, SCIM3b) and lower extremities motor scores (LEMS) from both sides' lower limb motor segments, measured five times within the first year after acute spinal cord injury (SCI). OBJECTIVES: Assessing concurrent validity of single and groups of walking assessments in comparison with LEMS in SCI patients. SETTING: European Multicenter study about Spinal Cord Injury, a collaboration of 22 centers. METHODS: Canonical correlation analysis (CCA) was applied to single and groups of assessments at each time point, separately for patients able to perform timed walking assessments (less impaired; patient subgroup I) and for all patients (no selection; patient subgroup II). RESULTS: In patient subgroup I, SCIM3b, WISCI, 10MWT and 6MWT all had high and similar concurrent validity one year after injury. Among all groups of three walking assessments, SCIM3a, WISCI and 10MWT had highest concurrent validity, similar to all six walking assessments together. Timed walking assessments generally had higher concurrent validity than untimed ones. In patient subgroup II, WISCI distinctly had highest concurrent validity one year after injury, similar to all three untimed walking assessments together. CONCLUSIONS: CCA can assess concurrent validity of single and groups of assessments. Minimal sets of walking assessments with comparable concurrent validity as all assessments together were proposed. As these sets differ by patient group, walking assessments should be specified according to expected walking ability to allow for targeted, cost-effective application of assessments.

Nanoparticle-mediated delivery of small RNA molecules in tumor therapy.

In principle, RNA interference (RNAi) allows for the inhibition of any oncogene of choice, thus leading to novel concepts in tumor therapy. For their delivery, the RNAi-inducing small RNA molecules (small interfering RNAs, siRNAs) can be formulated in various nanoparticle systems, prior to testing them in preclinical animal models. The same is true for miRNAs that have more recently been explored in therapeutic miRNA replacement strategies. This puts high demands on the properties of the nanoparticles. This review article discusses various nanoparticulate systems for RNA delivery in vivo and gives an overview of preclinical studies on siRNA- or miRNA-based tumor therapy.

A secreted FGF-binding protein can serve as the angiogenic switch in human cancer.

The growth and metastatic spread of cancer is directly related to tumor angiogenesis, and the driving factors need to be understood to exploit this process therapeutically. However, tumor cells and their normal stroma express a multitude of candidate angiogenic factors, and very few specific inhibitors have been generated to assess which of these gene products are only innocent bystanders and which contribute significantly to tumor angiogenesis and metastasis. Here we investigated whether the expression in tumors of a secreted fibroblast growth factor (FGF)-binding protein (FGF-BP) that mobilizes and activates locally stored FGFs (ref. 11) can serve as an angiogenic switch molecule. Developmental expression of the retinoid-regulated FGF-BP gene is prominent in the skin and intestine during the perinatal phase and is down-modulated in the adult. The gene is, however, upregulated in carcinogen-induced skin tumors, in squamous cell carcinoma (SCC) and in some colon cancer cell lines and tumor samples. To assess the significance of FGF-BP expression in tumors, we depleted human SCC (ME-180) and colon carcinoma (LS174T) cell lines of their endogenous FGF-BP by targeting with specific ribozymes. We found that the reduction of FGF-BP reduced the release of biologically active basic FGF (bFGF) from cells in culture. Furthermore, the growth and angiogenesis of xenograft tumors in mice was decreased in parallel with the reduction of FGF-BP. This suggests that human tumors can utilize FGF-BP as an angiogenic switch molecule.

CUX1: target of Akt signalling and mediator of resistance to apoptosis in pancreatic cancer.

BACKGROUND AND AIMS: The transcription factor CUX1 is known as a regulator of cell differentiation and cell cycle progression. Previously, CUX1 was identified as a modulator of invasiveness in various cancers. Based on expression profiles suggesting a role for CUX1 in mediating chemoresistance, the aim of this study was to characterise the effect of CUX1 on apoptosis as well as its regulation by signalling pathways modulating drug resistance in pancreatic cancer. METHODS: The effect of CUX1 on TRAIL- (tumour necrosis factor-related apoptosis-inducing ligand) and drug-induced apoptosis was analysed using overexpression and knock-down strategies. Regulation of CUX1 by phosphatidylinositol-3-kinase (PI3K)/Akt signalling was examined at the mRNA and protein level. The effect of CUX1 knock-down by nanoparticle-complexed small interfering RNA (siRNA) in vivo was analysed in a murine xenograft model. Furthermore, CUX1 RNA and protein expression was evaluated in human pancreatic cancer and adjacent normal tissues. RESULTS: Knock-down of CUX1 resulted in significantly enhanced TRAIL- and drug-induced apoptosis, associated with increased PARP (poly ADP-ribose polymerase) cleavage and caspase activity. Vice versa, overexpression of CUX1 inhibited apoptosis. CUX1 expression was induced by activation of Akt/protein kinase B signalling, and decreased by PI3K inhibitors. The antiapoptotic effect of CUX1 was associated with upregulation of BCL2 and downregulation of tumour necrosis factor alpha. CUX1 was significantly overexpressed in pancreatic cancers, as analysed by in situ hybridisation and immunohistochemistry. In vivo, silencing of CUX1 by intratumourally administered polyethylenimine-complexed siRNA led to reduced tumour growth and increased apoptosis in pancreatic cancer xenografts. CONCLUSION: CUX1 was identified as an important mediator of tumour cell survival in pancreatic cancer in vitro and in vivo.

The phenomenon of degeneration of industrial Trichoderma reesei strains.

BACKGROUND: Even if the loss of production capacity of a microorganism is said to be a serious problem in various biotechnology industries, reports in literature are rather rare. Strains of the genera Trichoderma reesei are used for large-scale production of cellulases, which are needed in food and feed, textile, paper industries and biofuel production. RESULTS: Here, we describe the phenomenon of spontaneous degeneration of T. reesei strains during large-scale cultivation. The phenotype of the degenerated population is characterized most importantly by a loss of any cellulase formation. Interestingly, promoter regions of relevant genes had a more compact chromatin in the (cel -) strains compared to productive strains. For a systematic investigation of the phenomenon a protocol for artificially induced and lab-scaled strain degeneration was developed. This workflow allows to determine the degeneration rate and thus, to compare the occurrence of a degenerated population in differently productive strains on the one hand, and to monitor the success of any strategies to prevent or decrease the degeneration on the other hand. While highly productive strains have higher degeneration rates compared to moderate producers, the degeneration can hardly be triggered in moderate producers. The observed (cel -) phenotype is not caused by a mutation in the gene encoding the essential transactivator Xyr1. The development of a non-producing population is also not triggered by any compounds released by either producing or non-producing cells. CONCLUSIONS: The extent of the occurrence of a degenerated strain population relates to the production capacity of the strain and goes along with chromatin condensation in relevant promoter regions.

Emergency Conversion to General Anesthesia Is a Tolerable Risk in Patients Undergoing Mechanical Thrombectomy.

BACKGROUND AND PURPOSE: Mechanical thrombectomy for acute ischemic stroke is performed with the patient under local anesthesia, conscious sedation, or general anesthesia. According to recent trials, up to 16% of patients require emergency conversion to general anesthesia during mechanical thrombectomy. This study investigated the procedural and clinical outcomes after emergency conversion in comparison with local anesthesia, conscious sedation, and general anesthesia. MATERIALS AND METHODS: This retrospective study included 254 patients undergoing mechanical thrombectomy for acute large-vessel occlusion. The procedure was started with the patient either under local anesthesia, conscious sedation, or general anesthesia. Emergency conversion was defined as induction of general anesthesia during mechanical thrombectomy. The primary outcomes were successful reperfusion (TICI 2b/3) and functional independence (mRS at 90 days, ≤2). RESULTS: Twenty-five patients (9.8%) required emergency conversion to general anesthesia. The time from admission to flow restoration was increased under general anesthesia (median, 137 minutes) and emergency conversion (median, 138 minutes) compared with local anesthesia (median 110 minutes). After adjustment for confounders, emergency conversion to general anesthesia and primary general anesthesia had comparable chances of successful reperfusion (OR = 1.28; 95% CI, 0.31-5.25). Patients with emergency conversion had a tendency toward higher chances of functional independence (OR = 4.48; 95% CI, 0.49-40.86) compared with primary general anesthesia, but not compared with local anesthesia (OR = 0.86; 95% CI, 0.14-5.11) and conscious sedation (OR = 1.07; 95% CI, 0.17-6.53). CONCLUSIONS: Patients with emergency conversion did not have lower chances of successful reperfusion or functional independence compared those with primary general anesthesia, and time to flow restoration was also similar. We found no evidence supporting the primary induction of general anesthesia in patients at risk for emergency conversion.

Inhibition of PIM1 blocks the autophagic flux to sensitize glioblastoma cells to ABT-737-induced apoptosis.

Overcoming apoptosis resistance is one major issue in glioblastoma (GB) therapies. Accumulating evidence indicates that resistance to apoptosis in GB is mediated via upregulation of pro-survival BCL2-family members. The synthetic BH3-mimetic ABT-737 effectively targets BCL2, BCL2 like 1 and BCL2 like 2 but still barely affects cell survival which is presumably due to its inability to inhibit myeloid cell leukemia 1 (MCL1). The constitutively active serine/threonine kinase proviral integration site for moloney murine leukemia virus 1 (PIM1) was recently found to be overexpressed in GB patient samples and to maintain cell survival in these tumors. For different GB cell lines, Western Blot, mitochondrial fractionation, fluorescence microscopy, effector caspase assays, flow cytometry, and an adult organotypic brain slice transplantation model were used to investigate the putative PIM1/MCL1 signaling axis regarding potential synergistic effects with ABT-737. We demonstrate that combination of the PIM1 inhibitor SGI-1776 or the pan-PIM kinase inhibitor AZD1208 with ABT-737 strongly sensitizes GB cells to apoptosis. Unexpectedly, this effect was found to be MCL1-independent, but could be partially blocked by caspase 8 (CASP8) inhibition. Remarkably, the analysis of autophagy markers in combination with the observation of massive accumulation and hampered degradation of autophagosomes suggests a completely novel function of PIM1 as a late stage autophagy regulator, maintaining the autophagic flux at the level of autophagosome/lysosome fusion. Our data indicate that PIM1 inhibition and ABT-737 synergistically induce apoptosis in an MCL1-independent but CASP8-dependent manner in GB. They also identify PIM1 as a suitable target for overcoming apoptosis resistance in GB.

Diffusion Tensor Imaging of Dystrophic Skeletal Muscle : Comparison of Two Segmentation Methods Adapted to Chemical-shift-encoded Water-fat MRI.

PURPOSE: To compare the influence of two different regions of interest (ROIs) on diffusion tensor metrics in dystrophic thigh muscles using a custom-made (whole muscle) ROI including and a selective ROI excluding areas of fatty replacement. METHODS: Diffusion tensor imaging (DTI) and chemical-shift-encoded water-fat magnetic resonance imaging (MRI) of the thigh was conducted on a 3-Tesla system in 15 cases with muscular dystrophy and controls. The ROIs were chosen according to patterns of fatty replacement on co-registered axial DTI and gradient echo sequence (GRE) images. Fractional anisotropy (FA), apparent diffusion coefficient (ADC), fiber track length (FTL), and muscle fat fractions (MFF) were compared between both ROI segmentations. These comparisons, muscle-specific correlation coefficients, and the influence of ROI localization on tensor metrics were derived based on linear mixed effects regression models. RESULTS: In the cases a high correlation was observed for ADC and FA with MFF using a custom ROI. The correlation was weaker but still significant with a selective ROI method. Using the custom ROI, FTL correlated significantly with MFF in 3 out of 4 muscles (r ≤ -0.51). A correlation was not found for the selective ROI method. Interaction analysis revealed that the association of ADC and FA with MFF was not significantly influenced by the ROI localization. For FTL the ROI localization significantly reduced the negative association with MFF. CONCLUSION: The DTI metrics and FTL of custom ROI segmentation are significantly influenced by MFF. Contrary to ADC and FA, the effect of MFF on FTL is significantly reduced when applying selective ROI segmentation, which could therefore be a better option for MR tractography.

Sustained delivery of siRNA poly- and lipopolyplexes from porous macromer-crosslinked gelatin gels.

RNA interference (RNAi) is a promising technique to treat severe diseases on a pre-protein level. We and others postulate that the release of nanoparticle-complexed small interfering RNA (siRNA) from implanted biomaterials could provide structural support for tissue repair, combined with local siRNA transfection of invading and regenerating cells. In this study, we systematically investigated cross-linked gelatin based hydrogel formulations (cGEL) as degradable controlled release matrices for siRNA. Aiming at the definition of correlations between cGEL composition, siRNA nanoparticle formulation, release kinetics of complexed siRNA and transfection efficiency, we combined five different cGEL formulations and three transfection systems, i.e. polyplexes with polyethyleneimine (PEI), PEI in combination with liposomes (lipopolyplexes) and polyplexes based on tyrosin-modified PEI (P10Y). It was found that the distribution of these poly-/lipopolyplexes, when applied onto the negatively charged hydrogels, was strongly dependent on their zeta potential. Furthermore, siRNA release from the hydrogel was a multifactorial process, as diffusion, hydrogel degradation and nanoparticle decomplexation overlapped over time. This resulted in a prolonged release of siRNA for up to 21days. In the case of PEI complexes and lipopolyplexes, release kinetics depended on the cGEL formulation. In contrast, when employing P10Y polyplexes, an initial burst release was observed with no further release thereafter. Silencing activity was determined using constitutively luciferase-expressing SKOV-3-Luc reporter cells. Surface and bulk porosity in hydrogels was introduced by addition of soluble polyethylene glycol during fabrication, leading to improved knockdown. The rapid onset of knockdown efficacy will also provide the basis for the determination of long-term effects.

The novel MKL target gene myoferlin modulates expansion and senescence of hepatocellular carcinoma.

Megakaryoblastic Leukemia 1 and 2 (MKL1/2) are transcriptional coactivators of Serum Response Factor (SRF) with an essential role for hepatocellular carcinoma (HCC) growth and oncogene-induced senescence. In this report, we identified myoferlin as a novel MKL/SRF target gene by gene expression profiling and verification in vivo in HCC xenografts. Myoferlin was overexpressed in human and murine HCCs triggered by conditional expression of constitutively active SRF-VP16 protein in hepatocytes. Furthermore, myoferlin was required for HCC cell invasion, proliferation and anchorage-independent cell growth. We provide evidence that myoferlin is a crucial gene target of MKL1/2 mediating its effect on oncogene-induced senescence by modulating the activation state of the EGFR and downstream MAPK and p16-/Rb pathways. Depletion of myoferlin in tumour cells from SRF-VP16-derived murine HCCs induced a senescence phenotype. These findings identify MKL1/2 and myoferlin as novel therapeutic targets to treat human HCC by a senescence-inducing strategy.

The human papillomavirus (HPV) 16 E6 oncoprotein leads to an increase in gene expression of the angiogenic switch molecule FGF-BP in non-immortalized human keratinocytes.

Fibroblast growth factor binding protein (FGF-BP) is a secreted protein that binds FGF-1 and FGF-2 and is involved in mobilization and activation of FGFs from the extracellular matrix. FGF-BP overexpression as well as ribozyme-mediated reduction of endogenous FGF-BP revealed that FGF-BP can be rate-limiting for tumor growth and angiogenesis. Recent studies showed that FGF-BP expression is up-regulated during early phases of tumorigenesis, indicating that the role of FGF-BP in angiogenesis is a critical early step in the development and progression of tumors. Human papillomavirus type 16 (HPV 16) is highly associated with the development of anogenital cancers. Here we demonstrate that the stable expression of the E6 oncogene of HPV 16 leads to an activation of the FGF-BP promoter in primary human foreskin keratinocytes (one of the natural host cells of these viruses). This is associated with an increase in the steady state levels of FGF-BP mRNA and FGF-BP protein in cells stably expressing E6. Transient E6 expression revealed that the observed activation of the FGF-BP promoter by the viral oncogene is an early process which is independent from immortalization/transformation events in the cells.

Expression of a truncated 100 kDa HER2 splice variant acts as an endogenous inhibitor of tumour cell proliferation.

Overexpression of the HER2 (neu/c-erbB-2) oncogene frequently coincides with an aggressive clinical course of certain human adenocarcinomas. Expression and secretion of aberrant HER2 splice variants has been reported in various cell lines and tissues and can interfere with the oncogenic HER2 activity. Here we demonstrate, using two different approaches, that expression of a truncated 100 kDa HER2 variant which encodes the extracellular domain of HER2 (HER-ECD) inhibits growth factor-mediated tumour cell proliferation. A HER2-ECD cDNA encoding the truncated variant was overexpressed in MCF7 breast cancer cells. HER2-ECD overexpression decreased spontaneous proliferation of MCF7 cells as well as heregulin-mediated soft agar colony formation. Concomitantly, heregulin-induced phosphorylation of HER4 as well as downstream activation of p44/p42 MAP-kinases was decreased. To confirm these data, ribozymes were targeted to the 3'-untranslated region of the 2.3 kb HER2-ECD mRNA which is spontaneously expressed in MKN7 gastric cancer cells. HER2-ECD-targeted ribozymes downregulated HER2-ECD expression and enhanced EGF-mediated soft agar colony formation of MKN7 cells. In parallel, EGF-induced activation of p44/p42 MAP-kinases and activation of c-Fos expression were increased in ribozyme-transfected MKN7 cells. Finally, in RT-PCR we found a trend towards a progressive loss of 2.3 kb HER2-ECD mRNA expression in more advanced gastric tumours. These data show that the HER2-ECD variant inhibits growth factor-mediated tumour cell proliferation suggesting an important role during the progression of human cancer.

Ribozyme targeting of HER-2 inhibits pancreatic cancer cell growth in vivo.

We have analysed HER-2 expression and function in pancreatic cancer cells to determine whether HER-2 has a rate-limiting role for pancreatic cancer cell growth in vitro and in vivo. To specifically assess HER-2 function, we used HER-2-targeted ribozymes expressed under the control of the tet-off promoter system. Six out of 11 human pancreatic cancer cell lines expressed all four epidermal growth factor (EGF)-receptor family members (HER-1 (EGF-R), HER-2, HER-3, and HER-4), including Panc89 cells. Expression of the ribozymes quenched endogenous HER-2 mRNA levels in Panc89 cells by approximately 40-60% which was reflected by a 40-50% reduction of the HER-2 surface glycoprotein. HER-2 depletion inhibited the in vitro proliferation rate by approximately 40% and decreased in vivo tumour growth by approximately 60% (P<0.05). Our study demonstrates for the first time a rate-limiting role for HER-2 in pancreatic cancer cell proliferation and suggests HER-2 targeting as a potential approach in pancreatic cancer therapy.

Reversal of HER-2 over-expression renders human ovarian cancer cells highly resistant to taxol.

Currently, the treatment options for advanced ovarian cancer are limited. Thus, the majority of the patients are treated with drugs with considerable side effects but in many cases without clinical benefit. The relationship between activation of an oncogene like the HER-2 receptor and drug sensitivity, is of considerable interest as this molecular marker may allow to better predict response to chemotherapy. The aim of this study was to evaluate whether over-expression of the HER-2 receptor would modulate drug responsiveness to doxorubicin, cisplatin and taxol in ovarian cancer cells. An anti-HER-2-targeted ribozyme approach was used to abrogate HER-2 expression in human SK-OV-3 ovarian cancer cells. SK-OV-3 cells expressing very low residual levels of HER-2 protein, were then assessed for their sensitivity to doxorubicin, cisplatin and taxol and compared to control cells. HER-2 expression had no effect on the cytotoxicity of doxorubicin (IC50=10 nM) or cisplatin (IC50=5 microM) in proliferation assays. In contrast, the sensitivity to taxol was increased approximately 70-fold in SK-OV-3 ovarian cancer cells expressing high levels of HER-2 (IC50=10(-5) nM) compared to HER-2 depleted cells (IC50=7x10(-4) nM). If these findings can be confirmed in patients, it could be possible that HER-2 expression may serve as a marker for response to taxol treatment in ovarian cancer patients.

[Aneurysm of Valsalva's sinus (author's transl)]. / Aneurysma des Sinus Valsalvae.

An aneurysm of Valsalva's sinus is one of the rarest malformations of the aortic root. The case of a non-ruptured aneurysm is reported and the possible pathogenic mechanisms of the congenital and the acquired forms of this aneurysm are discussed, as well as the clinical symptoms and the diagnostic procedures. Finally, the necessity of surgical treatment of the aneurysm is pointed out.

[Diagnostic and clinical features of atrial myxoma (author's transl)]. / Zur diagnostik und Klinik des Vorhofmyxoms.

The most common benign cardiac tumours are artial myxomas. Characteristically they may interfere with the normal functioning of atrioventricular valves or manifest themselves by embolism to cerebral or peripheral arteries or they may mimic systemic diseases. Hence, early diagnosis and immediate surgical treatment are important. A case of left atrial myxoma is reported and the various non-invasive and invasive diagnositc methods and their significance are outlined. If the angiocardiographic findings are questionable, the echocardiographic features are believed to be of greater reliability. Finally, the histopathology of atrial myxoma, the rate of tumour growth, as well as the possibility of postoperative reappearance of the myxoma are discussed.

[Myocardial performance during therapeutic starvation in obese subjects]. / Kardiale Leistungsfähigkeit von Fettsüchtigen während Nullkalorien-Diät

The effect was studied of therapeutic total starvation of two weeks' duration on myocardial performance in 12 obeses patients. Half of these subjects were trained for 10 minutes daily on a bicycle ergometer, whilst the other half had no training. After two weeks of total fasting no significant changes were observed either inter- or intracollectively with regard to systolic time intervals, ejection fraction and the index of myocardial contractility, nor in respect to serum electrolytes. Physical training had no influence on the systolic time intervals and the other noninvasive parameters of left ventricular performance. The inclusion of previous data obtained in a similar investigation enabled the establishment of a correlation between the serum potassium level and the ejection franction.