No abstract

Circular dichroism (CD) spectroscopy is widely used to characterize the secondary structure composition of proteins. To derive accurate and detailed structural information from the CD spectra, we have developed the Beta Structure Selection (BeStSel) method (PNAS, 112, E3095), which can handle the spectral diversity of β-structured proteins. The BeStSel webserver provides this method with useful accessories to the community with the main goal to analyze single or multiple protein CD spectra. Uniquely, BeStSel provides information on eight secondary structure components including parallel β-structure and antiparallel β-sheets with three different groups of twist. It overperforms any available method in accuracy and information content, moreover, it is capable of predicting the protein fold down to the topology/homology level of the CATH classification. A new module of the webserver helps to distinguish intrinsically disordered proteins by their CD spectrum. Secondary structure calculation for uploaded PDB files will help the experimental verification of protein MD and in silico modelling using CD spectroscopy. The server also calculates extinction coefficients from the primary sequence for CD users to determine the accurate protein concentrations which is a prerequisite for reliable secondary structure determination. The BeStSel server can be freely accessed at https://bestsel.elte.hu.

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with K d values of 9 and 40 M, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy-and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: K d values of 37 and 3.5 nM for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent K d value (3 M). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network.

Summary Background Ustekinumab is effective in Crohn's disease. However, a substantial proportion of patients will not respond or lose response to ustekinumab. The current evidence to support the effectiveness of dose‐optimisation for ustekinumab nonresponse is limited. Aim To assess the effectiveness of dose escalation of ustekinumab. Methods This was a multicentre retrospective cohort study. We included active Crohn's disease patients who received a standard‐dose intravenous induction and at least one subcutaneous ustekinumab 90 mg dose. All enrolled patients received dose escalation by either shortening the interval between the doses to every 4 or 6 weeks, intravenous reinduction or a combination of strategies. The primary outcome of the study was clinical response at week 16 after dose escalation. Results A total of 142 patients (22 centres/14 countries) were included. The patients were dose‐escalated after a median treatment duration of 30 weeks. At week 16 from escalation, 73/142 (51.4%) responded to treatment, including 55/142 (38.7%) in clinical remission. Corticosteroid‐free remission was achieved in 6/34 (17.6%) patients on corticosteroids at the time of escalation; 118/142 (83%) continued treatment beyond week 16. Follow‐up data beyond week 16 were available for 74/118 (62.7%) patients. On the last follow‐up, 51/98 (52%) patients with available data responded to treatment, including 41/98 (42%) in clinical remission. Conclusions Intensification of ustekinumab maintenance dosage was effective in over 50% of the patients. This strategy should be considered in patients who are nonresponsive to every 8 weeks ustekinumab maintenance dosing.

The results of the present study show that following thoracotomy in the early postoperative period continuous EDA is a better pain relieving method than continuous IC block, as indicated by the VAS scores.

The majority of lung cancers (LC) belong to the non-small cell lung carcinoma (NSCLC) type. The two main NSCLC sub-types, namely adenocarcinoma (AC) and squamous cell carcinoma (SCC), respond differently to therapy. Whereas the link between cigarette smoke and lung cancer risk is well established, the relevance of non-canonical Wnt pathway up-regulation detected in SCC remains poorly understood. The present study was undertaken to investigate further the molecular events in canonical and non-canonical Wnt signalling during SCC development. A total of 20 SCC and AC samples with matched non-cancerous controls were obtained after surgery. TaqMan array analysis confirmed up-regulation of non-canonical Wnt5a and Wnt11 and identified down-regulation of canonical Wnt signalling in SCC samples. The molecular changes were tested in primary small airway epithelial cells (SAEC) and various lung cancer cell lines (e.g. A549, H157, etc). Our studies identified Wnt11 and Wnt5a as regulators of cadherin expression and potentiated relocation of β-catenin to the nucleus as an important step in decreased cellular adhesion. The presented data identifies additional details in the regulation of SCC that can aid identification of therapeutic drug targets in the future.

PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation) is the presented nuclear magnetic resonance (NMR) approach to identify at once the location of proteins’ exposed surface, hindered accessibility, and exchange processes occurring on a μs–ms time scale. In addition to mapping the protein surface accessibility, the application of this method under specific conditions makes it possible to distinguish conformational mobility and chemical exchange processes, thereby providing an alternative to characterization by more demanding techniques (transverse relaxation dispersion, saturation transfer, and high-pressure NMR). Moreover, its high sensitivity enables studying samples at low, physiologically more relevant concentrations. Association, dynamics, and oligomerization are addressed by PENELOP for a component of SARS-CoV-2 replication transcription complex and an amyloidogenic protein.

There are four matters of uncertainty considered in this working group report, which are distilled into four clinical questions: (1) What is the evidence for the need for palpation of the lung in modern era of imaging? (2) Is there evidence of a difference in outcome for an open versus a closed approach? (3) Is there evidence of a difference in outcome for an initial policy of bilateral versus unilateral exploration? (4) In patients with known bilateral disease, is there a difference in outcome with a simultaneous versus a staged approach?We searched the literature formally and supplemented this with knowledge from all other sources. We provide evidence tables on the first two questions by relying on a group consensus and frame recommendations for the other two.There are no randomized trials to guide us but there are comparative studies addressing the need for palpation and the need to and open operation in all cases. The evidence is equivocal, and opinions are divided in the literature.Palpation of the lung is still seen as necessary in a therapeutic metastasectomy as opposed to a diagnostic procedure when videothoracoscopy is adequate. However, the importance of palpation becomes less clear with advances in imaging. Routine bilateral exploration for unilateral disease was not favored. For bilateral disease, an initial median sternotomy has a place for some cases but sequenced thoracotomy was preferred.