A Versatile Virus-Mimetic Engineering Approach for Concurrent Protein Nanocage Surface-Functionalization and Cargo Encapsulation.

Naturally occurring protein nanocages like ferritin are self-assembled from multiple subunits. Because of their unique cage-like structure and biocompatibility, there is a growing interest in their biomedical use. A multipurpose and straightforward engineering approach does not exist for using nanocages to make drug-delivery systems by encapsulating hydrophilic or hydrophobic drugs and developing vaccines by surface functionalization with a protein like an antigen. Here, a versatile engineering approach is described by mimicking the HIV-1 Gap polyprotein precursor. Various PREcursors of nanoCages (PREC) are designed and created by linking two ferritin subunits via a flexible linker peptide containing a protease cleavage site. These precursors can have additional proteins at their N-terminus, and their protease cleavage generates ferritin-like nanocages named protease-induced nanocages (PINCs). It is demonstrated that PINC formation allows concurrent surface decoration with a protein and hydrophilic or hydrophobic drug encapsulation up to fourfold more than the amount achieved using other methods. The PINCs/Drug complex is stable and efficiently kills cancer cells. This work provides insight into the precursors' design rules and the mechanism of PINCs formation. The engineering approach and mechanistic insight described here will facilitate nanocages' applications in drug delivery or as a platform for making multifunctional therapeutics like mosaic vaccines.

Perineural Injection Therapy for Chronic Exertional Compartment Syndrome Refractory to Initial Compartment Release: A Case Report.

This is a case of a 26-year-old active duty male with a 1-year history of distal anterolateral leg pain and numbness which would persist following activity cessation. He was referred to physical therapy and eventually orthopedic surgery for bilateral anterior exertional compartment syndrome and underwent bilateral anterolateral fasciotomies. One year after surgery, he continued to have pain along the posterior aspect of his lower legs with residual numbness over his left dorsomedial foot. He was referred to sports medicine for further evaluation and Botox injections without significant symptomatic changes. He subsequently underwent diagnostic ultrasound of his lower legs which showed multiple entrapment points of the left superficial peroneal nerve along the fasciotomy scar. An additional electrodiagnostic study showed left superficial peroneal sensory mononeuropathy. Eighteen months following surgery, he received his first perineural injection therapy (PIT) treatment. A mixture of lidocaine and D5W was prepared to achieve 1 mg/cc which was then injected along his tibial, saphenous, and sural nerves. Following four PIT sessions, the patient's overall lower extremity pain, weakness, and functionality had improved. This case demonstrates potential benefit with PIT in patients with refractory symptoms following surgery for chronic exertional compartment syndrome. These symptoms may be due to chronic irritation of cutaneous nerves and they may benefit from treatment with PIT. Our case may represent a possible paradigm shift in the conservative treatment of chronic exertional compartment syndrome, especially when refractory to surgical compartment release.

Sequential Organ Failure Assessment (SOFA) Score for predicting mortality in patients with sepsis in Vietnamese intensive care units: a multicentre, cross-sectional study.

OBJECTIVES: To compare the accuracy of the Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation II (APACHE II) Scores in predicting mortality among intensive care unit (ICU) patients with sepsis in a low-income and middle-income country. DESIGN: A multicentre, cross-sectional study. SETTING: A total of 15 adult ICUs throughout Vietnam. PARTICIPANTS: We included all patients aged ≥18 years who were admitted to ICUs for sepsis and who were still in ICUs from 00:00 to 23:59 of the specified study days (ie, 9 January, 3 April, 3 July and 9 October of the year 2019). PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was hospital all-cause mortality (hospital mortality). We also defined the secondary outcome as all-cause deaths in the ICU (ICU mortality). RESULTS: Of 252 patients, 40.1% died in hospitals, and 33.3% died in ICUs. SOFA Score (areas under the receiver operating characteristic curve (AUROC): 0.688 (95% CI 0.618 to 0.758); cut-off value≥7.5; PAUROC<0.001) and APACHE II Score (AUROC: 0.689 (95% CI 0.622 to 0.756); cut-off value ≥20.5; PAUROC<0.001) both had a poor discriminatory ability for predicting hospital mortality. However, the discriminatory ability for predicting ICU mortality of SOFA (AUROC: 0.713 (95% CI 0.643 to 0.783); cut-off value≥9.5; PAUROC<0.001) was fair and was better than that of APACHE II Score (AUROC: 0.672 (95% CI 0.603 to 0.742); cut-off value≥18.5; PAUROC<0.001). A SOFA Score≥8 (adjusted OR (AOR): 2.717; 95% CI 1.371 to 5.382) and an APACHE II Score≥21 (AOR: 2.668; 95% CI 1.338 to 5.321) were independently associated with an increased risk of hospital mortality. Additionally, a SOFA Score≥10 (AOR: 2.194; 95% CI 1.017 to 4.735) was an independent predictor of ICU mortality, in contrast to an APACHE II Score≥19, for which this role did not. CONCLUSIONS: In this study, SOFA and APACHE II Scores were worthwhile in predicting mortality among ICU patients with sepsis. However, due to better discrimination for predicting ICU mortality, the SOFA Score was preferable to the APACHE II Score in predicting mortality.Clinical trials registry - India: CTRI/2019/01/016898.

Synergy between Winter Flounder antimicrobial peptides.

Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.

Predictive validity of the quick Sequential Organ Failure Assessment (qSOFA) score for the mortality in patients with sepsis in Vietnamese intensive care units.

BACKGROUND: The simple scoring systems for predicting the outcome of sepsis in intensive care units (ICUs) are few, especially for limited-resource settings. Therefore, this study aimed to evaluate the accuracy of the quick Sequential (Sepsis-Related) Organ Failure Assessment (qSOFA) score in predicting the mortality of ICU patients with sepsis in Vietnam. METHODS: We did a multicenter cross-sectional study of patients with sepsis (≥18 years old) presenting to 15 adult ICUs throughout Vietnam on the specified days (i.e., 9th January, 3rd April, 3rd July, and 9th October) representing the different seasons of 2019. The primary and secondary outcomes were the hospital and ICU all-cause mortalities, respectively. The area under the receiver operating characteristic curve (AUROC) was calculated to determine the discriminatory ability of the qSOFA score for deaths in the hospital and ICU. The cut-off value of the qSOFA scores was determined by the receiver operating characteristic curve analysis. Upon ICU admission, factors associated with the hospital and ICU mortalities were assessed in univariable and multivariable logistic models. RESULTS: Of 252 patients, 40.1% died in the hospital, and 33.3% died in the ICU. The qSOFA score had a poor discriminatory ability for both the hospital (AUROC: 0.610 [95% CI: 0.538 to 0.681]; cut-off value: ≥2.5; sensitivity: 34.7%; specificity: 84.1%; PAUROC = 0.003) and ICU (AUROC: 0.619 [95% CI: 0.544 to 0.694]; cutoff value: ≥2.5; sensitivity: 36.9%; specificity: 83.3%; PAUROC = 0.002) mortalities. However, multivariable logistic regression analyses show that the qSOFA score of 3 was independently associated with the increased risk of deaths in both the hospital (adjusted odds ratio, AOR: 3.358; 95% confidence interval, CI: 1.756 to 6.422) and the ICU (AOR: 3.060; 95% CI: 1.651 to 5.671). CONCLUSION: In our study, despite having a poor discriminatory value, the qSOFA score seems worthwhile in predicting mortality in ICU patients with sepsis in limited-resource settings. CLINICAL TRIAL REGISTRATION: Clinical trials registry-India: CTRI/2019/01/016898.

Biomarkers for Cyclin-Dependent Kinase 4/6 Inhibitors in the Treatment of Hormone Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Advanced/Metastatic Breast Cancer: Translation to Clinical Practice.

PURPOSE: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have emerged as effective treatments for patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) advanced/metastatic breast cancer (mBC). Dedicated research efforts have been undertaken to find predictive biomarkers of response or resistance to these therapies although no molecular biomarkers for mBC have reached the clinic so far. This review aims to summarize and evaluate the performance of biomarkers in predicting progression-free survival in phase II and III clinical trials of CDK4/6 inhibitors in HR+/HER2- mBC. METHODS: For this narrative review, a structured literature search of PubMed, Embase, and the Cochrane library (CENTRAL) was performed. Phase II or III clinical trials of a CDK4/6 inhibitor in patients with HR+/HER2- mBC reporting on at least one molecular biomarker analysis of progression-free survival were included. Publications and selected conference abstracts were included up until November 2021. RESULTS: Twenty-two articles reporting biomarker results of 12 clinical trials were included. Retinoblastoma protein status and cyclin E1 mRNA expression were promising baseline biomarkers, whereas PIK3CA circulating tumor DNA ratio on treatment relative to baseline, change in plasma thymidine kinase activity, and circulating tumor cell count were potential dynamic biomarkers of response. A number of biomarkers were unsuccessful, despite a strong mechanistic rationale, and others are still being explored. CONCLUSION: Our review of clinical trials showed that there are a number of promising biomarkers at baseline and several dynamic biomarkers that might predict response to CDK4/6 inhibitors. Validation of these findings and assessment of clinical utility are crucial to make the final translation to clinical practice. Better understanding of disease heterogeneity and further elucidation of resistance mechanisms could inform future studies of rationally selected biomarkers.

Continuing Medical Education in Vietnam: A Weighted Analysis from Healthcare Professionals' Perception and Evaluation.

PURPOSE: Continuing medical education (CME) is a compulsory requirement for every health professional. However, to date, little is known about the effectiveness of CME in Vietnam. This study assessed CME programs based on attendees' perception and evaluation. METHODS: A cross-sectional study was conducted during a five-month period in all 62 CME courses at a university hospital. A self-report, anonymous questionnaire was distributed to the participants during the course and was collected at the end of the course. The questionnaire included questions about demographic characteristics, experiences during the course and participants' perception and evaluation as measured by the 19-item Program Evaluation Instrument (PEI). A higher score on the PEI indicates a higher level of positive reaction toward CME programs. RESULTS: Among 1312 participants in the analysis, the majority were females (58.1%) with a mean age of 34.5 (SD = 10.6) years. Almost all participants had good, positive perceptions toward CME. However, about 5% of participants reported CME a waste of time. Participants reported a high score on the PEI (95.0±8.9) and all four dimensions including program objectives (20.7±2.2), learner's objectives (18.8±2.3), teacher's behavior (25.7±2.7) and program satisfaction (29.7±3.4). While there was no association between demographic characteristics and PEI score, attendance rate during the courses and perceptions toward CME were positively associated with PEI score. CONCLUSION: CME programs receive positive reaction and evaluation from healthcare professionals and are helpful in providing and updating knowledge, attitude and practice in Vietnam. However, further studies are needed in other settings and specialties to fully understand the effectiveness of CME in Vietnam.

Healthcare Workers' Emotions, Stressor Experiences and Coping Strategies During the COVID-19 Pandemic in Vietnam.

PURPOSE: Healthcare workers (HCWs) are a crucial resource in the battle against the COVID-19 pandemic but are vulnerable to both SARS-CoV-2 infection and negative psychological consequences. This study evaluated HCWs' emotions, stressor experiences and coping strategies during the pandemic. METHODS: A cross-sectional study was conducted among HCWs at the University Medical Center in Ho Chi Minh City. The questionnaire was adapted from the MERS-CoV Staff Questionnaire to measure HCWs' emotions, stressor experiences and coping strategies during the COVID-19 pandemic. RESULTS: Among the 1423 participants eligible in the data analysis, the majority were female (71.1%) with a mean age of 34.2 (standard deviation 7.8) years. While most participants reported that they did their job because of their professionalism and duty as HCWs (87.4%), a high number reported feeling nervous and scared (86.0%). Most participants reported worry about transmitting SARS-CoV-2 to their families or friends (76.6%) and concern that a small mistake or lapse in concentration could infect themselves and others (76.7%). The most common coping strategies were following strict personal protective measures (95.3%), avoiding going out (92.5%) and reading about SARS-CoV-2 (92.3%). Females who had a higher educational level and less than 5-years work experience and those who worked at clinical departments and subclinical departments were more vulnerable. CONCLUSION: This study indicates an urgent need for psychological support for HCWs, especially for those at high risk of having stress. Interventions and support should utilize psychological resources and approaches effectively to adapt to the new situation during the COVID-19 pandemic.

Factors relating to mortality in septic patients in Vietnamese intensive care units from a subgroup analysis of MOSAICS II study.

Sepsis is the most common cause of in-hospital deaths, especially from low-income and lower-middle-income countries (LMICs). This study aimed to investigate the mortality rate and associated factors from sepsis in intensive care units (ICUs) in an LMIC. We did a multicenter cross-sectional study of septic patients presenting to 15 adult ICUs throughout Vietnam on the 4 days representing the different seasons of 2019. Of 252 patients, 40.1% died in hospital and 33.3% died in ICU. ICUs with accredited training programs (odds ratio, OR: 0.309; 95% confidence interval, CI 0.122-0.783) and completion of the 3-h sepsis bundle (OR: 0.294; 95% CI 0.083-1.048) were associated with decreased hospital mortality. ICUs with intensivist-to-patient ratio of 1:6 to 8 (OR: 4.533; 95% CI 1.621-12.677), mechanical ventilation (OR: 3.890; 95% CI 1.445-10.474) and renal replacement therapy (OR: 2.816; 95% CI 1.318-6.016) were associated with increased ICU mortality, in contrast to non-surgical source control (OR: 0.292; 95% CI 0.126-0.678) which was associated with decreased ICU mortality. Improvements are needed in the management of sepsis in Vietnam such as increasing resources in critical care settings, making accredited training programs more available, improving compliance with sepsis bundles of care, and treating underlying illness and shock optimally in septic patients.

Lipid domain formation and non-lamellar structures associated with varied lysylphosphatidylglycerol analogue content in a model Staphylococcal plasma membrane.

Dipalmitoyl-3-aza-dehydroxy-lysylphosphatidylglycerol (DP3adLPG), is a chemically stable synthetic analogue of the bacterial lipid lysylphosphatidylglycerol (LPG), designed as a substitute for the notoriously labile native lipid in biophysical investigations. In Staphylococcus aureus, LPG is known to play a role in resistance to antibiotics by altering membrane charge properties in response to environmental stress, but little is known about how LPG influences other bilayer physicochemical properties or lateral organisation, through the formation of complexes with lipids such as phosphatidylglycerol (PG). In this study we have investigated the different phases formed by biomimetic mixtures of 3adLPG and PG in different thermotropic states, using neutron diffraction and electron microscopy. In a DPPG/DP3adLPG 70:30 mol% mixture, two distinct lamellar phases were observed below the lipid melting transition: Lß' 1 and Lß' 2 with respective periodicities of 82 and 62 Å. Increasing the proportion of DP3adLPG to mimic the effects of environmental stress led to the disappearance of the Lß' 1 phase and the formation of an inverse hexagonal phase. The compositions of these different phases were identified by investigating the thermotropic properties of the two mixtures, and probing their interaction with the antimicrobial peptide magainin 2 F5W. We propose that the observed polymorphism results from the preferential formation of either triplet PG-3adLPG-PG, or paired PG-3adLPG complexes, dependent upon the mixing proportions of the two lipids. The relevance of these findings to the role native LPG in S. aureus, are discussed with respect to their influence on antibiotic resistance and lateral membrane organisation.

Binding Interactions of Peptide Aptamers.

Peptide aptamers are short amino acid chains that are capable of binding specifically to ligands in the same way as their much larger counterparts, antibodies. Ligands of therapeutic interest that can be targeted are other peptide chains or loops located on the surface of protein receptors (e.g., GCPR), which take part in cell-to-cell communications either directly or via the intermediary of hormones or signalling molecules. To confer on aptamers the same sort of conformational rigidity that characterises an antibody binding site, aptamers are often constructed in the form of cyclic peptides, on the assumption that this will encourage stronger binding interactions than would occur if the aptamers were simply linear chains. However, no formal studies have been conducted to confirm the hypothesis that linear peptides will engage in stronger binding interactions with cyclic peptides than with other linear peptides. In this study, the interaction of a model cyclic decamer with a series of linear peptide constructs was compared with that of a linear peptide with the same sequence, showing that the cyclic configuration does confer benefits by increasing the strength of binding.

Characterization of Posttranslationally Modified Multidrug Efflux Pumps Reveals an Unexpected Link between Glycosylation and Antimicrobial Resistance.

The substantial rise in multidrug-resistant bacterial infections is a current global imperative. Cumulative efforts to characterize antimicrobial resistance in bacteria has demonstrated the spread of six families of multidrug efflux pumps, of which resistance-nodulation-cell division (RND) is the major mechanism of multidrug resistance in Gram-negative bacteria. RND is composed of a tripartite protein assembly and confers resistance to a range of unrelated compounds. In the major enteric pathogen Campylobacter jejuni, the three protein components of RND are posttranslationally modified with N-linked glycans. The direct role of N-linked glycans in C. jejuni and other bacteria has long been elusive. Here, we present the first detailed account of the role of N-linked glycans and the link between N-glycosylation and antimicrobial resistance in C. jejuni We demonstrate the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilizing protein complexes and the promotion of protein-protein interaction, thus mediating antimicrobial resistance via enhancing multidrug efflux pump activity. This affirms that glycosylation is critical for multidrug efflux pump assembly. We present a generalized strategy that could be used to investigate general glycosylation system in Campylobacter genus and a potential target to develop antimicrobials against multidrug-resistant pathogens.IMPORTANCE Nearly all bacterial species have at least a single glycosylation system, but the direct effects of these posttranslational protein modifications are unresolved. Glycoproteome-wide analysis of several bacterial pathogens has revealed general glycan modifications of virulence factors and protein assemblies. Using Campylobacter jejuni as a model organism, we have studied the role of general N-linked glycans in the multidrug efflux pump commonly found in Gram-negative bacteria. We show, for the first time, the direct link between N-linked glycans and multidrug efflux pump activity. At the protein level, we demonstrate that N-linked glycans play a role in enhancing protein thermostability and mediating the assembly of the multidrug efflux pump to promote antimicrobial resistance, highlighting the importance of this posttranslational modification in bacterial physiology. Similar roles for glycans are expected to be found in other Gram-negative pathogens that possess general protein glycosylation systems.

A pleurocidin analogue with greater conformational flexibility, enhanced antimicrobial potency and in vivo therapeutic efficacy.

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.

Synthetic Antimicrobial Peptide Tuning Permits Membrane Disruption and Interpeptide Synergy.

The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.

Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase.

Isothermal titration calorimetry (ITC) is conventionally used to acquire thermodynamic data for biological interactions. In recent years, ITC has emerged as a powerful tool to characterize enzyme kinetics. In this study, we have adapted a single-injection method (SIM) to study the kinetics of human soluble epoxide hydrolase (hsEH), an enzyme involved in cardiovascular homeostasis, hypertension, nociception, and insulin sensitivity through the metabolism of epoxy-fatty acids (EpFAs). In the SIM method, the rate of reaction is determined by monitoring the thermal power, while the substrate is being depleted, overcoming the need for synthetic substrates and reducing postreaction processing. Our results show that ITC enables the detailed, rapid, and reproducible characterization of the hsEH-mediated hydrolysis of several natural EpFA substrates. Furthermore, we have applied a variant of the single-injection ITC method for the detailed description of enzyme inhibition, proving the power of this approach in the rapid screening and discovery of new hsEH inhibitors using the enzyme's physiological substrates. The methods described herein will enable further studies on EpFAs' metabolism and biology, as well as drug discovery investigations to identify and characterize hsEH inhibitors. This also promises to provide a general approach for the characterization of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques.

Temporin L and aurein 2.5 have identical conformations but subtly distinct membrane and antibacterial activities.

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.

15-deoxy-Δ12,14-Prostaglandin J2 inhibits human soluble epoxide hydrolase by a dual orthosteric and allosteric mechanism.

Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.

LARP4A recognizes polyA RNA via a novel binding mechanism mediated by disordered regions and involving the PAM2w motif, revealing interplay between PABP, LARP4A and mRNA.

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A-oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein-protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.

Minor sequence modifications in temporin B cause drastic changes in antibacterial potency and selectivity by fundamentally altering membrane activity.

Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required to understand their native function and for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMPs activity. Relatively modest modifications to AMPs primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Gram-positive bacteria while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.

Real-World Effectiveness of Palbociclib Versus Clinical Trial Results in Patients With Advanced/Metastatic Breast Cancer That Progressed on Previous Endocrine Therapy.

OBJECTIVE: The aim of this study was to assess the real-world effectiveness and tolerability of palbociclib combined with endocrine therapy for the treatment of hormone receptor positive (HR-positive), human epidermal growth factor receptor 2 negative (HER2-negative), advanced/metastatic breast cancer that progressed on previous endocrine therapy, and to compare these results with the outcomes of the PALOMA-3 clinical trial. METHODS: This study was a retrospective observational cohort study including all patients who started with palbociclib in the St. Antonius Hospital between September 1, 2016 and April 1, 2018 for the treatment of HR-positive, HER2-negative advanced/metastatic breast cancer that progressed on previous endocrine therapy. Individual patient data were collected from electronic medical records. Primary study outcomes were progression-free survival (PFS) and the number of permanent treatment discontinuations before disease progression due to adverse events (AEs). Secondary outcomes were the frequency of all (serious) AEs and the frequency of and reasons for dose reductions, -interruptions and cycle delays. RESULTS: A total of 46 patients were studied with a median follow-up of 13.0 months. Overall, the median PFS in real-world clinical practice was 10.0 months (95% confidence interval (CI) 4.9-15.1), compared with 9.5 months in PALOMA-3 (95% CI 9.2-11.0). Two patients discontinued treatment because of AEs. Neutropenia was the most frequent grade 3-4 AE, but with no febrile neutropenia events. Most AEs were managed with palbociclib dose modifications. Regarding these modifications, more cycle delays, less dose reductions, and less dose interruptions occurred in clinical practice compared with PALOMA-3 (59 vs 36%, 22 vs 34%, and 9 vs 54%, respectively). Patients who did not meet the PALOMA-3 study eligibility criteria (n = 16) showed a lower median PFS of 5.5 months (95% CI 4.7-6.4). CONCLUSIONS: The effectiveness and tolerability of palbociclib in real-world clinical practice corresponded well with the results obtained in the PALOMA-3 clinical trial. Despite the differences in dose modifications, this study suggests that there is no efficacy-effectiveness gap in this patient population.