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Structure-function relationships of biological macromolecules, in particular proteins, provide crucial insights for fundamental biochemistry, medical research and early drug discovery. However, production of recombinant proteins, either for structure determination, functional studies, or to be used as biopharmaceutical products, is often hampered by their instability and propensity to aggregate in solution in vitro. Protein samples of poor quality are often associated with reduced reproducibility as well as high research and production expenses. Several biophysical methods are available for measuring protein aggregation and stability. Yet, discovering and developing means to improve protein behaviour and structure-function integrity remains a demanding task. Here, we discuss workflows that are made possible by adapting established biophysical methods to high-throughput screening approaches. Rapid identification and optimisation of conditions that promote protein stability and reduce aggregation will support researchers and industry to maximise sample quality, stability and reproducibility, thereby reducing research and development time and costs.

Chlamydia pneumoniae is a Gram-negative bacterium responsible for a number of human respiratory diseases and linked to some chronic inflammatory diseases. The major outer membrane protein (MOMP) of Chlamydia is a conserved immunologically dominant protein located in the outer membrane, which, together with its surface exposure and abundance, has led to MOMP being the main focus for vaccine and antimicrobial studies in recent decades. MOMP has a major role in the chlamydial outer membrane complex through the formation of intermolecular disulphide bonds, although the exact interactions formed are currently unknown. Here, it is proposed that due to the large number of cysteines available for disulphide bonding, interactions occur between cysteine-rich pockets as opposed to individual residues. Such pockets were identified using a MOMP homology model with a supporting low-resolution (~4 Å) crystal structure. The localisation of MOMP in the E. coli membrane was assessed using direct stochastic optical reconstruction microscopy (dSTORM), which showed a decrease in membrane clustering with cysteine-rich regions containing two mutations. These results indicate that disulphide bond formation was not disrupted by single mutants located in the cysteine-dense regions and was instead compensated by neighbouring cysteines within the pocket in support of this cysteine-rich pocket hypothesis.

Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA+ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA+ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA+ domain of a bEBP bound to RNAP-σ54-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA+ proteins utilises ATPase activities to allow σ54-dependent transcription initiation.

Transcatheter aortic valve implantation (TAVI) has revolutionised the management of aortic valve disease, offering a less invasive alternative to traditional surgical valve replacement for severe aortic stenosis (AS). TAVI for pure aortic regurgitation (AR) is less well established, and, in fact, it was previously labelled as a relative contraindication. However, TAVI has been utilised for selected cases of pure or predominant AR. The primary limitations regarding the use of TAVI in AR are related to the absence of anatomical factors seen in patients with AS that have contributed to the safe and stable functioning of current-generation prostheses. These include aortic root dilatation, mobile valve leaflets and labile blood pressure within the aortic root, which may further increase the risk of valve migration and periprosthetic leak after deployment. Furthermore, patients with AR have more heterogeneous aortic root anatomies when compared to the population of patients with calcific or degenerative AS. This review article describes the current evidence for the off-label use of TAVI in pure AR and the various clinical syndromes associated with AR where there may be specific challenges in the application of TAVI.

Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Exercise training reduces inflammation in breast cancer survivors; however, the mechanism is not fully understood., Objectives: The effects of acute and chronic exercise on monocyte toll-like receptor (TLR2 and 4) expression and intracellular cytokine production were examined in sedentary breast cancer survivors., Methods: Eleven women with stage I, II, or III breast cancer within one year of treatment completion performed an acute, intermittent aerobic exercise trial. Blood samples were obtained before, immediately, and 1 h after a 45-min acute exercise trial that was performed before and after 16 weeks of combined aerobic and resistance. LPS-stimulated intracellular IL-1ß, TNF, and IL-6 production, and TLR2 and TLR4 expression were evaluated in CD14 + CD16 - and CD14 + CD16 + monocytes using flow cytometry., Results: Exercise training decreased IL-1ß + CD14 + CD16 - proportion (24.6%, p=0.016), IL-1ß + CD14 + CD16 - mean fluorescence intensity (MFI) (-9989, p=0.014), IL-1ß + CD14 + CD16 + MFI (-11101, p=0.02), and IL-6 + CD14 + CD16 - proportion (16.9%, P=0.04). TLR2 and TLR4 expression did not change following exercise training but decreased 1 h after acute exercise in CD14 + CD16 - (-63, p=0.002) and CD14 + CD16 + (-18, p=0.006) monocytes, respectively. Immediately after the acute exercise, both monocyte subgroup cell concentration increased, with CD14 + CD16 + concentrations being decreased at 1 h post without changes in intracellular cytokine production., Conclusions: Exercise training reduced monocyte intracellular pro-inflammatory cytokine production, especially IL-1ß, although these markers did not change acutely. While acute exercise downregulated the expression of TLR2 and TLR4 on monocytes, this was not sustained over the course of training. These results suggest that the anti-inflammatory effect of combined aerobic and resistance exercise training in breast cancer survivors may be, in part, due to reducing resting monocyte pro-inflammatory cytokine production., Competing Interests: None., (© 2021 Published by Elsevier Inc.)

Mucosal-associated invariant T (MAIT) cells have properties of both the innate and adaptive immune systems but are an understudied population within exercise immunology. These lymphocytes aggregate at the mucous membranes, but it is unknown if submaximal exercise alters their circulating numbers or function., Purpose: To determine the MAIT cell response to submaximal exercise on activation and homing marker expression and stimulated cytokine production., Methods: Twenty healthy, young, recreationally active males cycled for 40 min at 86% of VT after an overnight fast. Peripheral blood mononuclear cells were isolated and labeled to identify specific MAIT cell populations using flow cytometry. Cytokine production after stimulation was also determined., Results: Mucosal-associated invariant T cells were 2.9% of T cells and increased to 3.9% after exercise and with recovery whereas cell numbers significantly increased by 91.5% after exercise before returning to resting levels. Chemokine and activation marker absolute cell number significantly increased while expression levels remained constant but the high levels of CCR5 may help direct MAIT cells to sites of inflammation. After stimulation, TNFα expression significantly increased after exercise before returning to baseline with a similar trend for IFNγ., Conclusions: The MAIT cell numbers undergo a partial biphasic response after submaximal exercise and appear to be preferentially mobilized within T cells; however, the magnitude of the submaximal response was attenuated relative to maximal exercise. Stimulated MAIT cells increase TNFα expression, indicating greater responsiveness to pathogens after acute exercise.

Purpose: Mucosal associated invariant T (MAIT) cells have properties of the innate and acquired immune systems. While the response to vigorous exercise has been established for most leukocytes, MAIT cells have not been investigated. Therefore, the purpose was to determine if MAIT cell lymphocytosis occurs with acute maximal aerobic exercise and if this response is influenced by exercise duration, cardiovascular fitness, or body composition., Methods: Twenty healthy young males with moderate fitness levels performed an extended graded exercise test until volitional fatigue. Peripheral blood mononuclear cells were isolated from venous blood obtained prior and immediately after exercise and were labeled to identify specific T cell populations using flow cytometry., Results: The percentage of MAIT cells relative to total T cells significantly increased from 3.0 to 3.8% and absolute MAIT cell counts increased by 2.2-fold following maximal exercise. MAIT cell subpopulation proportions were unchanged with exercise. Within cytotoxic T lymphocytes (CTL), MAIT cells consisted of 8% of these cells and this remained constant after exercise. MAIT cell counts and changes with exercise were not affected by body composition, VO 2peak , or exercise duration., Conclusions: Maximal exercise doubled MAIT cell numbers and showed preferential mobilization within total T cells but the response was not influenced by fitness levels, exercise duration, or body composition. These results suggest that acute exercise could be used to offset MAIT cell deficiencies observed with certain pathologies. MAIT cells also make up a substantial proportion of CTLs, which may have implications for cytotoxicity assays using these cells.

Cardiac tamponade is a rare complication of coronary intervention to chronic total occlusions (CTO PCI). We report a case of persistent bleeding from a venous source following successful anterograde dissection-reentry (ADR) CTO PCI. Pericardiocentesis was performed 1 h post-procedure for tamponade. Persistent bleeding was investigated with contrast transesophageal echocardiography, pericardial manometry and blood analysis. Coronary venography revealed subtle extravasation from a cardiac vein adjacent to the site of luminal re-entry. Coronary venous perforation using ADR CTO PCI has not previously been described; however, the volume of blood loss may be significant and surgical exploration may be appropriate.

Coronary artery disease (CAD) is common in patients with severe aortic stenosis undergoing transcatheter aortic valve implantation (TAVI), but its clinical relevance is controversial. At present, the optimal means of defining CAD in patients undergoing TAVI with respect to its prognostic implications and the assessment of myocardial ischaemia is not known. For this reason, the best treatment options are a matter for debate, and current guidelines do not recommend revascularization. As the indications for TAVI expand, the lack of any rigorous means of guiding coronary revascularization might negatively affect the clinical outcomes of future patients. In this Review, we summarize the methods of assessing CAD in TAVI populations, and the data on the safety and efficacy of percutaneous coronary intervention in patients undergoing TAVI. We discuss the putative effects of aortic stenosis on the functional assessment of CAD using pressure or flow wires or by noninvasive stress testing. We propose that a new, well-validated method of assessing CAD as a cause of myocardial ischaemia--which distinguishes it from myocardial infarction, previous revascularization, or non-flow-limiting disease--in patients with severe aortic stenosis is needed to guide revascularization in the current era of TAVI.