A high sedentary time is associated with increased mortality risk. Previous studies indicate that replacement of sedentary time with light- and moderate-to-vigorous physical activity attenuates the ...risk for adverse outcomes and improves cardiovascular risk factors. Patients with cardiovascular disease are more sedentary compared to the general population, while daily time spent sedentary remains high following contemporary cardiac rehabilitation programmes. This clinical trial investigated the effectiveness of a sedentary behaviour intervention as a personalised secondary prevention strategy (SIT LESS) on changes in sedentary time among patients with coronary artery disease participating in cardiac rehabilitation.
Patients were randomised to usual care (n = 104) or SIT LESS (n = 108). Both groups received a comprehensive 12-week centre-based cardiac rehabilitation programme with face-to-face consultations and supervised exercise sessions, whereas SIT LESS participants additionally received a 12-week, nurse-delivered, hybrid behaviour change intervention in combination with a pocket-worn activity tracker connected to a smartphone application to continuously monitor sedentary time. Primary outcome was the change in device-based sedentary time between pre- to post-rehabilitation. Changes in sedentary time characteristics (prevalence of prolonged sedentary bouts and proportion of patients with sedentary time ≥ 9.5 h/day); time spent in light-intensity and moderate-to-vigorous physical activity; step count; quality of life; competencies for self-management; and cardiovascular risk score were assessed as secondary outcomes.
Patients (77% male) were 63 ± 10 years and primarily diagnosed with myocardial infarction (78%). Sedentary time decreased in SIT LESS (- 1.6 - 2.1 to - 1.1 hours/day) and controls (- 1.2 ─1.7 to - 0.8), but between group differences did not reach statistical significance (─0.4 ─1.0 to 0.3) hours/day). The post-rehabilitation proportion of patients with a sedentary time above the upper limit of normal (≥ 9.5 h/day) was significantly lower in SIT LESS versus controls (48% versus 72%, baseline-adjusted odds-ratio 0.4 (0.2-0.8)). No differences were observed in the other predefined secondary outcomes.
Among patients with coronary artery disease participating in cardiac rehabilitation, SIT LESS did not induce significantly greater reductions in sedentary time compared to controls, but delivery was feasible and a reduced odds of a sedentary time ≥ 9.5 h/day was observed.
Netherlands Trial Register: NL9263. Outcomes of the SIT LESS trial: changes in device-based sedentary time from pre-to post-cardiac rehabilitation (control group) and cardiac rehabilitation + SIT LESS (intervention group). SIT LESS reduced the odds of patients having a sedentary time >9.5 hours/day (upper limit of normal), although the absolute decrease in sedentary time did not significantly differ from controls. SIT LESS appears to be feasible, acceptable and potentially beneficial, but a larger cluster randomised trial is warranted to provide a more accurate estimate of its effects on sedentary time and clinical outcomes. CR: cardiac rehabilitation.
The nephron naturally provides a concave conformation for epithelial cells, yet biomedical applications such as bioartificial kidney can employ convex seeding. Frequently glass or ...polydimethylsiloxane are utilized as base materials to study renal epithelial cell response to curvatures. Insights on relevant materials for biomedical applications remain limited. Here it is investigated how human immortalized renal proximal tubule epithelial cells (RPTEC) respond to a range of concave and convex curvatures made from a bis‐urea modified polycaprolactone material. Solvent cast chips containing a 50–500 µm diameter range of both concave and convex semicylindrical structures are successfully produced. Concave structures are completely covered by cells under all conditions. Yet cell layers present gaps on the summits of convex structures; the relative gap size enlarges as the diameter decreased. Increased proliferation time and cell seeding density allow for cells to overcome summit avoidance and nearly engulf convex structures. Interestingly, sample inversion also results in gap closure on convex structures during live imaging. Polarization of RPTECs is more prominent on concave compared to convex structures. These findings suggest that biomedical applications such as the bioartificial kidney should focus on concave seeding of cells to acquire more functional cell monolayers.
Curvature on biomaterial interfaces affects cell behavior, however insights remain limited on renal epithelial cell response to curvature on relevant biomaterials. Cell behavior is investigated on a polycaprolactone‐based chips containing a 50–500 µm diameter range of both concave and convex semicylindrical structures. Concave structures induce increased cell coverage and polarization compared to convex structures.
Nuclear factor III (NFIII) is a protein from HeLa cells that stimulates the initiation of adenovirus type 2 (Ad2) DNA replication by binding to a specific nucleotide sequence in the origin, adjacent ...to the nuclear factor I recognition site. DNA sequences sharing a high degree of homology to the NFIII binding site in Ad2 were found in a number of transcription regulatory elements, all containing the octanucleotide sequence ATGCAAAT. We have analysed the interaction between NFIII and the octamer‐containing sequences in a histone H2B promoter, immunoglobulin light and heavy chain promoters, an immunoglobulin heavy chain enhancer, a U2 snRNA enhancer and the SV40 enhancer as well as Ad4. All sequences were recognized by NFIII as indicated by gel retardation assays, DNase I footprinting and methylation protection experiments. A comparison of the relative binding affinities using competition assays indicated that mutations in the octanucleotide sequence reduced the binding affinity considerably. Small but significant differences in affinity were also observed depending on the sequences bordering the conserved octanucleotide. The methylation protection patterns indicate that both major and minor groove contacts are involved in NFIII binding. The data suggest that NFIII could function both in adenovirus DNA replication and in the transcriptional control of several groups of genes sharing the octanucleotide sequence.
Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding ...proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system.
The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine S-HPMPA) is a potent and selective inhibitor of adenovirus (Ad) replication in cell culture. We studied the mechanism ...of inhibition using a reconstituted in vitro DNA replication system. The diphosphoryl derivative (S)-HPMPApp, but not (S)-HPMPA, inhibited the DNA replication of origin containing fragments strongly. The inhibitory effect was exerted at the level of elongation, while initiation was resistant to the drug. Remarkably, the elongation of short strands was only slightly impaired, while inhibition was maximal upon synthesis of long DNA fragments. (S)-HPMPApp appeared to be competitive with dATP, suggesting that the Ad DNA polymerase is the prime target for the drug. We purified the Ad DNA polymerase in complex to the precursor terminal protein to homogeneity from cells infected with overproducing recombinant vaccinia viruses. Employing gapped DNA or poly(dT).oligo(dA) templates, only a weak inhibition was observed. However, inhibition was strongly enhanced in the presence of the adenovirus DNA binding protein (DBP). We interpret this to mean that the increased processivity of the polymerization reaction in the presence of DBP leads to increased drug sensitivity.
Polymorphisms among HLA class II molecules expressed by cells with different HLA-DR4 haplotypes were analysed biochemically (isoelectrofocussing and 2D gels), cellularly (HLA-Dw) and serologically ...(monoclonal antibodies). The results confirm the correlation which exists between HLA-D specificity and DR beta chain isoelectric point polymorphism. Furthermore, a biochemical polymorphism was observed among DQw3 molecules. No correlation was found with HLA-Dw types. On the other hand a correlation was found between DQ-polymorphism and TA10 and 2B3 specificities defined by monoclonal antibodies. The comparison of different methods defining polymorphisms of HLA class II molecules will be discussed.
Enhanced epidermal growth factor receptor (EGFR) activity has been strongly linked to breast cancer progression and mediators of EGFR endocytosis may well be involved. We developed a semi-automated ...high-content fluorescence microscopy-based EGFR endocytosis screen to identify proteins that mediate EGFR endocytosis in human HBL100 breast cancer cells. Knockdown of 172 individual endocytosis and actin-regulatory genes with small interfering RNAs led to the identification of 14 genes of which the contribution to EGFR endocytosis in breast cancer is until now poorly defined, including DNAJC6, GDI2, FGD6, HAX1, NECAP2 and AnxA2. We show that depletion of the actin and endocytosis regulatory protein annexin A2 (AnxA2) in a panel of four triple negative breast cancer (TNBC) cell lines affected EGFR endocytosis. Depletion of AnxA2 in the aggressive and highly metastatic MDA-MB-231 TNBC cell line resulted in the inhibition of EGFR transport beyond the early endosomes. This inhibition coincided with enhanced epidermal growth factor (EGF)-induced cell migration and downstream signaling via c-Jun N-terminal kinase (JNK) and Akt. Moreover, AnxA2 knockdown increased lung metastasis formation in mice. The effect of AnxA2 knockdown on EGFR endocytosis in MDA-MB-231 was related to dephosphorylation/activation of the actin-severing protein cofilin, as re-expression of an inactive S3E-cofilin mutant, but not an active S3A-cofilin mutant, re-established EGFR endocytosis to control levels. Together, our data provide evidence for AnxA2 as a mediator of EGFR endocytosis and signaling in breast cancer via regulation of cofilin activation.
BRCA1-mutated breast cancer is primarily driven by DNA copy-number alterations (CNAs) containing large numbers of candidate driver genes. Validation of these candidates requires novel approaches for ...high-throughput in vivo perturbation of gene function. Here we develop genetically engineered mouse models (GEMMs) of BRCA1-deficient breast cancer that permit rapid introduction of putative drivers by either retargeting of GEMM-derived embryonic stem cells, lentivirus-mediated somatic overexpression or in situ CRISPR/Cas9-mediated gene disruption. We use these approaches to validate Myc, Met, Pten and Rb1 as bona fide drivers in BRCA1-associated mammary tumorigenesis. Iterative mouse modeling and comparative oncogenomics analysis show that MYC-overexpression strongly reshapes the CNA landscape of BRCA1-deficient mammary tumors and identify MCL1 as a collaborating driver in these tumors. Moreover, MCL1 inhibition potentiates the in vivo efficacy of PARP inhibition (PARPi), underscoring the therapeutic potential of this combination for treatment of BRCA1-mutated cancer patients with poor response to PARPi monotherapy.
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