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Background: It is common for women to explore and plan strategies to cope during labour. These strategies are usually focused on pain control and described as either pharmacological or non-pharmacological. As labour is an individual experience, each woman should be enabled to choose strategies that best suit them, and that reflect what they feel influences their sense of capacity to cope., Aim: By exploring women's intentions and choices of strategies, this study aimed to understand how coping strategies can better reflect women's individual needs and expectations., Methods: Fifty-six primiparous women were recruited from one tertiary hospital in Melbourne, Australia between February and May 2021. Data were collected via a survey in late pregnancy using open-ended questions. Content and thematic analyses were used to analyse responses., Results: Themes related to how women frame the intensity of labour, how they strive for a relationally safe environment and a need to be prepared and knowledgeable. Strategies chosen by women could be grouped into two categories: intrinsic and extrinsic. Intrinsic strategies could be self-generated by women (such as breathing techniques and movement), while extrinsic strategies required either equipment (such as a bath) or others to administer (such as epidural analgesia)., Conclusions: Women value having a range of intrinsic and extrinsic strategies that enable autonomy or require external support. This moves beyond the 'pharmacological and non-pharmacological' categorisation of strategies, and we propose that reframing strategies as intrinsic and extrinsic could have a number of benefits on women's sense of autonomy and utilisation of strategies. The findings provide a foundation for more targeted research into how women can be supported to individualise and implement these coping strategies in labour., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Matrix metalloproteinase-9 (MMP-9) is an endopeptidase that remodels the extracellular matrix. MMP-9 has been implicated in several diseases including neurodegeneration, arthritis, cardiovascular diseases, fibrosis and several types of cancer, resulting in a high demand for MMP-9 inhibitors for therapeutic purposes. For such drug design efforts, large amounts of MMP-9 are required. Yet, the catalytic domain of MMP-9 (MMP-9Cat) is an intrinsically unstable enzyme that tends to auto-cleave within minutes, making it difficult to use in drug design experiments and other biophysical studies. We set our goal to design MMP-9Cat variant that is active but stable to auto-cleavage. For this purpose, we first identified potential auto-cleavage sites on MMP-9Cat using mass spectroscopy and then eliminated the auto-cleavage site by predicting mutations that minimize auto-cleavage potential without reducing enzyme stability. Four computationally designed MMP-9Cat variants were experimentally constructed and evaluated for auto-cleavage and enzyme activity. Our best variant, Des2, with 2 mutations, was as active as the wild-type enzyme but did not exhibit auto-cleavage after 7 days of incubation at 37°C. This MMP-9Cat variant, with an identical with MMP-9Cat WT active site, is an ideal candidate for drug design experiments targeting MMP-9 and enzyme crystallization experiments. The developed strategy for MMP-9CAT stabilization could be applied to redesign other proteases to improve their stability for various biotechnological applications., (© 2023 The Author(s).)

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4 S207A,L185D , a first-of-a-kind 'decoy' PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4 S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This 'substrate-capturing' KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 'proteinase substrate capture' approach to regulate proteinase-mediated PAR1 signaling., (© 2021. The Author(s).)

Introduction: Liberia in West Africa has one of the highest maternal mortality ratios in the world (990/100,000 live births). Many women in Liberia live in rural, remote villages with little access to safe maternity services. The World Health Organization has identified maternity waiting homes (MWHs) as one strategy to minimize the barrier of distance in accessing a skilled birth attendant. However, limited data exist on satisfaction with MWHs or maternal health care in Liberia., Methods: This mixed-methods case study examines women's satisfaction with their stay at a MWH and compares utilization rates before and during the Ebola outbreak. From 2012 to 2014, 650 women who stayed at one of 6 MWHs in rural Liberia during the perinatal or postnatal period were surveyed. Additionally, 60 semi-structured interviews were conducted with traditional providers, skilled birth attendants, and women utilizing the MWHs. Quantitative analyses assessed satisfaction rates before and during the Ebola outbreak. Content analysis of semi-structured interviews supplemented the quantitative data and provided a lens into the elements of satisfaction with the MWHs., Results: The majority of women who utilized the MWHs stated they would suggest the MWH to a friend or relative who was pregnant (99.5%), and nearly all would utilize the home again (98.8%). Although satisfaction with the MWHs significantly decreased during the Ebola outbreak (P < .001), participants were satisfied overall with the MWHs. Content analysis identified areas of satisfaction that encompassed the themes of restful and supportive environment as well as areas for improvement such as lacking necessary resources and loneliness., Discussion: This case study demonstrated that women using MWHs in Bong County, Liberia are generally satisfied with their experience and plan to use an MWH again during future pregnancies to access a skilled birth attendant for birth. Women are also willing to encourage family and friends to use MWHs., (© 2017 by the American College of Nurse-Midwives.)

Background: To compare advanced musculoskeletal physiotherapists with other health professionals by measuring outcomes for patients presenting to the emergency department with lower limb soft tissue injuries or acute low back pain., Methods: A prospective study was conducted (Lower limb soft tissue injury cohort, n=88), (Acute low back pain cohort, n=29) at the emergency departments of two urban hospitals. A univariate analysis was completed for a number of outcome measures: Lower Extremity Functional Scale, Roland Morris Disability Questionnaire, imaging requirements, Patient Satisfaction Questionnaire, Numerical Pain Rating Scale and medication use. Data was obtained at discharge, two weeks and six weeks post-discharge., Results: Advanced musculoskeletal physiotherapists ordered less imaging, had less opioids (lower limb soft tissue injury) administered to patients, and patients' described equal or more satisfaction when compared to another health professional (p<0.05). No significant differences were found between groups for functional or pain related outcomes in both cohorts (p>0.05)., Conclusion: Advanced musculoskeletal physiotherapists are less likely to order imaging, obtain similar outcomes regarding pain medications and display equal or more patient satisfaction when compared to other health professionals for patients presenting to the emergency department with lower limb soft tissue injuries or acute low back pain., (Copyright © 2016 College of Emergency Nursing Australasia. All rights reserved.)

Our understanding of protein evolution would greatly benefit from mapping of binding landscapes, i.e., changes in protein-protein binding affinity due to all single mutations. However, experimental generation of such landscapes is a tedious task due to a large number of possible mutations. Here, we use a simple computational protocol to map the binding landscape for two homologous high-affinity complexes, involving a snake toxin fasciculin and acetylcholinesterase from two different species. To verify our computational predictions, we experimentally measure binding between 25 Fas mutants and the 2 enzymes. Both computational and experimental results demonstrate that the Fas sequence is close to the optimum when interacting with its targets, yet a few mutations could further improve Kd, kon, and koff. Our computational predictions agree well with experimental results and generate distributions similar to those observed in other high-affinity PPIs, demonstrating the potential of simple computational protocols in capturing realistic binding landscapes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Multispecific proteins play a major role in controlling various functions such as signaling, regulation of transcription/translation, and immune response. Hence, a thorough understanding of the atomic-level principles governing multispecific interactions is important not only for the advancement of basic science but also for applied research such as drug design. Here, we study evolution of an exemplary multispecific protein, a Tissue Inhibitor of Matrix Metalloproteinases 2 (TIMP2) that binds with comparable affinities to more than twenty-six members of the Matrix Metalloproteinase (MMP) and the related ADAMs families. We postulate that due to its multispecific nature, TIMP2 is not optimized to bind to any individual MMP type, but rather embodies a compromise required for interactions with all MMPs. To explore this hypothesis, we perform computational saturation mutagenesis of the TIMP2 binding interface and predict changes in free energy of binding to eight MMP targets. Computational results reveal the non-optimality of the TIMP2 binding interface for all studied proteins, identifying many affinity-enhancing mutations at multiple positions. Several TIMP2 point mutants predicted to enhance binding affinity and/or binding specificity towards MMP14 were selected for experimental verification. Experimental results show high abundance of affinity-enhancing mutations in TIMP2, with some point mutations producing more than ten-fold improvement in affinity to MMP14. Our computational and experimental results collaboratively demonstrate that the TIMP2 sequence lies far from the fitness maximum when interacting with its target enzymes. This non-optimality of the binding interface and high potential for improvement might characterize all proteins evolved for binding to multiple targets.

The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein scaffold H10A24 [¿CH(3)()CONH-CGGGELWKL.HEELLKK.FEELLKL.AEERLKK. L-CONH(2)()¿(2)](2). Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the E(m8.5) by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV [Fe(III)PPIX](+) - [Fe(III)PPIX](+) interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in the E(m) values of synthetic cytochromes.

We have taken a computational approach to design mutations that stabilize a large protein domain of approximately 200 residues in two alternative conformations. Mutations in the hydrophobic core of the alphaMbeta2 integrin I domain were designed to stabilize the crystallographically defined open or closed conformers. When expressed on the cell surface as part of the intact heterodimeric receptor, binding of the designed open and closed I domains to the ligand iC3b, a form of the complement component C3, was either increased or decreased, respectively, compared to wild type. Moreover, when expressed in isolation from other integrin domains using an artificial transmembrane domain, designed open I domains were active in ligand binding, whereas designed closed and wild type I domains were inactive. Comparison to a human expert designed open mutant showed that the computationally designed mutants are far more active. Thus, computational design can be used to stabilize a molecule in a desired conformation, and conformational change in the I domain is physiologically relevant to regulation of ligand binding.

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Proton exchange with aqueous media coupled to heme oxidation/reduction is commonly seen but not understood in natural cytochromes. Our synthetic tetrahelix bundle heme protein maquettes successfully reproduce natural proton coupling to heme oxidation/reduction. Potentiometry reveals major pK shifts from 4.2 to 7.0 and from 9.4 to 10.3 in the maquette-associated acid/base group(s) upon heme reduction. Consequently, a 210 mV decrease in the heme redox potential is observed between the two extremes of pH. Potentiometry with resonance Raman and FTIR spectroscopy performed over a wide pH range strongly implicates glutamate side chains as the source of proton coupling below pH 8.0, whereas lysine side chains are suggested above pH 8.0. Remarkably, the pK values of several glutamates in the maquette are elevated from their solution value (4.4) to values as high as 7.0. It is suggested that these glutamates are recruited into the interior of the bundle as part of a structural rearrangement that occurs upon heme binding. Glutamate to glutamine variants of the prototype protein demonstrate that removal of the glutamate closest to the heme diminishes but does not abolish proton exchange. It is necessary to remove additional glutamates before pH-independent heme oxidation/reduction profiles are achieved. The mechanism of redox-linked proton coupling appears to be rooted in distributed partial charge compensation, the magnitude of which is governed by the dielectric distance between the ferric heme and acid/base side chains. A similar mechanism is likely to exist in native redox proteins which undergo charge change upon cofactor oxidation/reduction.