Capturing RAS oligomerization on a membrane.

RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.

ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance.

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

BRD4354 Is a Potent Covalent Inhibitor against the SARS-CoV-2 Main Protease.

Numerous organic molecules are known to inhibit the main protease (MPro) of SARS-CoV-2, the pathogen of Coronavirus Disease 2019 (COVID-19). Guided by previous research on zinc-ligand inhibitors of MPro and zinc-dependent histone deacetylases (HDACs), we identified BRD4354 as a potent inhibitor of MPro. The in vitro protease activity assays show that BRD4354 displays time-dependent inhibition against MPro with an IC50 (concentration that inhibits activity by 50%) of 0.72 ± 0.04 µM after 60 min of incubation. Inactivation follows a two-step process with an initial rapid binding step with a KI of 1.9 ± 0.5 µM followed by a second slow inactivation step, kinact,max of 0.040 ± 0.002 min-1. Native mass spectrometry studies indicate that a covalent intermediate is formed where the ortho-quinone methide fragment of BRD4354 forms a covalent bond with the catalytic cysteine C145 of MPro. Based on these data, a Michael-addition reaction mechanism between MPro C145 and BRD4354 was proposed. These results suggest that both preclinical testing of BRD4354 and structure-activity relationship studies based on BRD4354 are warranted to develop more effective anti-COVID therapeutics.

Nonlinear Frequency Modulation for Fourier Transform Ion Mobility Mass Spectrometry Improves Experimental Efficiency.

Through optimization of terminal frequencies and effective sampling rates, we have developed nonlinear sawtooth-shaped frequency sweeps for efficient Fourier transform ion mobility mass spectrometry (FT-IM-MS) experiments. This is in contrast to conventional FT-IM-MS experiments where ion gates are modulated according to a linear frequency sweep. Linear frequency sweeps are effective but can be hindered by the amount of useful signal obtained using a single sweep over a large frequency range imposed by ion gating inefficiencies, particularly small ion packets, and gate depletion. These negative factors are direct consequences of the inherently low gate pulse widths of high-frequency ion gating events, placing an upper bound on FT-IM-MS performance. Here, we report alternative ion modulation strategies. Sawtooth frequency sweeps may be constructed for the purpose of either extending high-SNR transients or conducting efficient signal-averaging experiments for low-SNR transients. The data obtained using this approach show high-SNR signals for a set of low-mass tetraalkylammonium salts (<1000 m/z) where resolving powers in excess of 500 are achieved. Data for low-SNR obtained for multimeric protein complexes streptavidin (53 kDa) and GroEL (800 kDa) also reveal large increases in the signal-to-noise ratio for reconstructed arrival time distributions.

A reliable external calibration method for reaction monitoring with benchtop NMR.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique with the ability to acquire both quantitative and structurally insightful data for multiple components in a test sample. This makes NMR spectroscopy a desirable tool to understand, monitor, and optimize chemical transformations. While quantitative NMR (qNMR) approaches relying on internal standards are well-established, using an absolute external calibration scheme is beneficial for reaction monitoring as resonance overlap complications from an added reference material to the sample can be avoided. Particularly, this type of qNMR technique is of interest with benchtop NMR spectrometers as the likelihood of resonance overlap is only enhanced with the lower magnetic field strengths of the used permanent magnets. The included study describes a simple yet robust methodology to determine concentration conversion factors for NMR systems using single- and multi-analyte linear regression models. This approach is leveraged to investigate a pharmaceutically relevant amide coupling batch reaction. An on-line stopped-flow (i.e., interrupted-flow or paused-flow) benchtop NMR system was used to monitor both the 1,1'-carbonyldiimidazole (CDI) promoted acid activation and the amide coupling. The results highlight how quantitative measurements in benchtop NMR systems can provide valuable information and enable analysts to make decisions in real time.

2024 Australia-New Zealand Expert Consensus Statement on Cardiac Amyloidosis.

Over the past 5 years, early diagnosis of and new treatments for cardiac amyloidosis (CA) have emerged that hold promise for early intervention. These include non-invasive diagnostic tests and disease modifying therapies. Recently, CA has been one of the first types of cardiomyopathy to be treated with gene editing techniques. Although these therapies are not yet widely available to patients in Australia and New Zealand, this may change in the near future. Given the rapid pace with which this field is evolving, it is important to view these advances within the Australian and New Zealand context. This Consensus Statement aims to update the Australian and New Zealand general physician and cardiologist with regards to the diagnosis, investigations, and management of CA.

Clinical perspectives on sampling and processing approaches for the management of infection in diabetic foot ulceration: A qualitative study.

Diabetic foot ulcers (DFUs) often become infected and are treated with antimicrobials, with samples collected to inform care. Swab samples are easier than tissue sampling but report fewer organisms. Compared with culture and sensitivity (C&S) methods, molecular microbiology identifies more organisms. Clinician perspectives on sampling and processing are unknown. We explored clinician perspectives on DFU sampling-tissue samples/wound swabs-and on processing techniques, culture and sensitivity or molecular techniques. The latter provides information on organisms which have not survived transport to the laboratory for culture. We solicited feedback on molecular microbiology reports. Qualitative study using semi-structured interview, with analysis using a Framework approach. CODIFI2 clinicians from UK DFU clinics. Seven consultants agreed to take part. They reported, overall, a preference for tissue samples over swabbing. Clinicians were not confident replacing C&S with molecular microbiology as the approach to reporting was unfamiliar. The study was small and did not recruit any podiatrists or nurses, who may have discipline-specific attitudes or perspectives on DFU care. Both sampling approaches appear to be used by clinicians. Molecular microbiology reports would not be, at present, suitable for replacement of traditional culture and sensitivity.

Increasing Isolation Efficiency Using a Segmented Quadrupole Mass Filter Operated with Rectangular Waveforms.

The performance of a segmented quadrupole mass filter operated with rectangular waveforms and capacitively coupled rectangular waveforms applied to the prefilters was examined on a home-built quadrupole-Orbitrap platform. For peak widths of 50 m/z, 100% isolation efficiency was achieved, which fell to approximately 20% for 5 m/z peak width for a rectangular waveform of 150 V0-p. Due to a small exit aperture following the mass filter, peak structure was observed in both experimental peak shapes and those simulated using SIMION. A larger radius quadrupole was examined and achieved similar performance. While the segmented quadrupole does remove the defocusing effects of the fringing fields, the ion beam is only slightly refocused due to the low RF voltage which limits achievable gains in isolation efficiency.

CD38+ Alveolar macrophages mediate early control of M. tuberculosis proliferation in the lung.

Tuberculosis, caused by M.tuberculosis (Mtb), remains an enduring global health challenge, especially given the limited efficacy of current therapeutic interventions. Much of existing research has focused on immune failure as a driver of tuberculosis. However, the crucial role of host macrophage biology in controlling the disease remains underappreciated. While we have gained deeper insights into how alveolar macrophages (AMs) interact with Mtb, the precise AM subsets that mediate protection and potentially prevent tuberculosis progression have yet to be identified. In this study, we employed multi-modal scRNA-seq analyses to evaluate the functional roles of diverse macrophage subpopulations across different infection timepoints, allowing us to delineate the dynamic landscape of controller and permissive AM populations during the course of infection. Our analyses at specific time-intervals post-Mtb challenge revealed macrophage populations transitioning between distinct anti- and pro-inflammatory states. Notably, early in Mtb infection, CD38- AMs showed a muted response. As infection progressed, we observed a phenotypic shift in AMs, with CD38+ monocyte-derived AMs (moAMs) and a subset of tissue-resident AMs (TR-AMs) emerging as significant controllers of bacterial growth. Furthermore, scATAC-seq analysis of naïve lungs demonstrated that CD38+ TR-AMs possessed a distinct chromatin signature prior to infection, indicative of epigenetic priming and predisposition to a pro-inflammatory response. BCG intranasal immunization increased the numbers of CD38+ macrophages, substantially enhancing their capability to restrict Mtb growth. Collectively, our findings emphasize the pivotal, dynamic roles of different macrophage subsets in TB infection and reveal rational pathways for the development of improved vaccines and immunotherapeutic strategies.

Host stress drives tolerance and persistence: The bane of anti-microbial therapeutics.

Antibiotic resistance, typically associated with genetic changes within a bacterial population, is a frequent contributor to antibiotic treatment failures. Antibiotic persistence and tolerance, which we collectively term recalcitrance, represent transient phenotypic changes in the bacterial population that prolong survival in the presence of typically lethal concentrations of antibiotics. Antibiotic recalcitrance is challenging to detect and investigate-traditionally studied under in vitro conditions, our understanding during infection and its contribution to antibiotic failure is limited. Recently, significant progress has been made in the study of antibiotic-recalcitrant populations in pathogenic species, including Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enterica, and Yersiniae, in the context of the host environment. Despite the diversity of these pathogens and infection models, shared signals and responses promote recalcitrance, and common features and vulnerabilities of persisters and tolerant bacteria have emerged. These will be discussed here, along with progress toward developing therapeutic interventions to better treat recalcitrant pathogens.

Variation in Systemic Antibiotic Treatment for Diabetic Foot Osteomyelitis in England and Wales: A Multi-Centre Case Review.

Background: Diabetic foot osteomyelitis (DFO) is a major complication and can lead to significant morbidity and mortality. Systemic antibiotic therapy is often initiated first line to achieve quiescence of infection. To perform a multi-centre case review of systemic antibiotic intervention to treat adults with DFO in England and Wales and compare with national guidelines 'Diabetic foot problems: prevention and management'. Methods: Eight centres from England and Wales retrospectively collated data from a minimum of five adults (aged ≥ 18 years) from electronic case records. All patients were treated with systemic antibiotics following a new diagnosis of DFO (1 June 2021-31 December 2021). Results: 40 patients (35 males and 5 females) were included; the mean age was 62.3 years (standard deviation (SD) 13.0). Patients commenced systemic oral 14 (35%) or intravenous 26 (65%) antibiotic therapy following a new diagnosis of DFO. Twenty-seven (67.5%) patients were medically or surgically managed in the 12-week period with clinical quiescence of infection. Twenty-one patients (52.5%) had no recurrence of DFO infection within 12 weeks; seventeen (42.5%) of these patients had clinical quiescence of infection with systemic antibiotics alone without surgical intervention and nine (22.5%) of these cases had no recurrence of DFO. There were no cases of major amputation or death. All centres showed significant in-centre variability in systemic antibiotic management; variability was reported in the clinical and quantity indicators specifically to antibiotic selection, single versus dual therapy, mode of delivery and duration of treatment. Conclusions: This case review identifies there is existing variation when treating adults with systemic antibiotics for DFO. Further national guidance is required to standardise service delivery and care to improve patient outcomes.

Impaired fatty acid import or catabolism in macrophages restricts intracellular growth of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) infection of macrophages reprograms cellular metabolism to promote lipid retention. While it is clearly known that intracellular Mtb utilize host derived fatty acids and cholesterol to fuel the majority of its metabolic demands, the role of macrophage lipid catabolism on the bacteria's ability to access the intracellular lipid pool remains undefined. We utilized a CRISPR genetic knockdown approach to assess the impact of sequential steps in fatty acid metabolism on the growth of intracellular Mtb. Our analyzes demonstrate that knockdown of lipid import, sequestration and metabolism genes collectively impair the intracellular growth of Mtb in macrophages. We further demonstrate that modulating fatty acid homeostasis in macrophages impairs Mtb replication through diverse pathways like enhancing production of pro- inflammatory cytokines, autophagy, restricting the bacteria access to nutrients and increasing oxidative stress. We also show that impaired macrophage lipid droplet biogenesis is restrictive to intracellular Mtb replication, but increased induction of the same by blockade of downstream fatty acid oxidation fails to rescue Mtb growth. Our work expands our understanding of how host fatty acid homeostasis impacts Mtb growth in the macrophage.

Comparing Unmet Service Needs Between Rural and Urban Family Caregivers of People Living With Alzheimer's Disease and Related Dementias: A Multisite Study.

BACKGROUND AND OBJECTIVES: The scarcity of resources and available caregiving services in rural areas in the United States has been well documented. However, less research has compared unmet service needs between caregivers of people with Alzheimer's disease and related dementias (ADRD) in rural versus urban areas. RESEARCH DESIGN AND METHODS: Using semistructured interviews guided by theories of health service use and dependent care, we interviewed 20 family caregivers residing in rural areas of Western North Carolina and 18 caregivers within the urban setting of Houston, Texas, and compared their unmet service needs and contextual factors that facilitate their service use. RESULTS: Thematic analyses revealed similar unmet service needs among rural and urban caregivers; however, the ways they approached and solved their challenges differed. Caregivers in rural areas wished for more information and caregiver support whereas urban caregivers looked for information they needed until they found the answers. Rural caregivers expressed guilt about using services because they felt they were limited and zero-sum whereas urban caregivers shared available resources so that other caregivers could use them as well. Unmet service needs for urban caregivers included more racially and ethnically specific services for people with ADRD in their ethnic-specific languages and foods while rural caregivers' cultural needs were not racially and ethnically specific but for more place-specific services. DISCUSSION AND IMPLICATIONS: Recommendations for rural caregivers included utilizing online and virtual opportunities and expanding their reach across the United States. For urban caregivers, increasing culturally tailored service options would likely increase access and use.

Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions.

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5'-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as -100 kJ/mol at 298 K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.

A cross sectional pilot study utilising STrain Analysis and Mapping of the Plantar Surface (STAMPS) to measure plantar load characteristics within a healthy population.

BACKGROUND: No in-shoe systems, measuring both components of plantar load (plantar pressure and shear stress) are available for use in patients with diabetes. The STAMPS (STrain Analysis and Mapping of the Plantar Surface) system utilises digital image correlation (DIC) to determine the strain sustained by a deformable insole, providing a more complete understanding of plantar shear load at the foot-surface interface. RESEARCH QUESTIONS: What is the normal range and pattern of strain at the foot-surface interface within a healthy population as measured by the STAMPS system? Is STAMPS a valid tool to measure the effects of plantar load? METHODS: A cross-sectional study of healthy participants was undertaken. Healthy adults without foot pathology or diabetes were included. Participants walked 20 steps with the STAMPS insole in a standardised shoe. Participants also walked 10 m with the Novel Pedar® plantar pressure measurement insole within the standardised shoe. Both measurements were repeated three times. Outcomes of interest were global and regional values for peak resultant strain (SMAG) and peak plantar pressure (PPP). RESULTS: In 18 participants, median peak SMAG and PPP were 35.01 % and 410.6kPa respectively. The regions of the hallux and heel sustained the highest SMAG (29.31 % (IQR 24.56-31.39) and 20.50 % (IQR 15.59-24.12) respectively) and PPP (344.8kPa (IQR 268.3 - 452.5) and 279.3kPa (IQR 231.3-302.1) respectively). SMAG was moderately correlated with PPP (r= 0.65, p < 0.001). Peak SMAG was located at the hallux in 55.6 % of participants, at the 1st metatarsal head (MTH) in 16.7 %, the heel in 16.7 %, toes 3-5 in 11.1 % and the MTH2 in 5.6 %. SIGNIFICANCE: The results demonstrate the STAMPS system is a valid tool to measure plantar strain. Further studies are required to investigate the effects of elevated strain and the relationship with diabetic foot ulcer formation.

Native mass spectrometry of membrane protein-lipid interactions in different detergent environments.

Native mass spectrometry (MS) is revealing the role of specific lipids in modulating membrane protein structure and function. Membrane proteins solubilized in detergents are often introduced into the mass spectrometer; however, commonly used detergents for structural studies, such as dodecylmaltoside, tend to generate highly charged ions, leading to protein unfolding, thereby diminishing their utility for characterizing protein-lipid interactions. Thus, there is a critical need to develop approaches to investigate protein-lipid interactions in different detergents. Here, we demonstrate how charge-reducing molecules, such as spermine and trimethylamine-N-oxide, enable characterization of lipid binding to the bacterial water channel (AqpZ) and ammonia channel (AmtB) in complex with regulatory protein GlnK in different detergent environments. We find protein-lipid interactions are not only protein-dependent but can also be influenced by the detergent and type of charge-reducing molecule. AqpZ-lipid interactions are enhanced in LDAO (n-dodecyl-N,N-dimethylamine-N-oxide), whereas the interaction of AmtB-GlnK with lipids is comparable among different detergents. A fluorescent lipid binding assay also shows detergent dependence for AqpZ-lipid interactions, consistent with results from native MS. Taken together, native MS will play a pivotal role in establishing optimal experimental parameters that will be invaluable for various applications, such as drug discovery, as well as biochemical and structural investigations.

Expanding Native Mass Spectrometry to the Masses.

At the 33rd ASMS Sanibel Meeting, on Membrane Proteins and Their Complexes, a morning roundtable discussion was held discussing the current challenges facing the field of native mass spectrometry and approaches to expanding the field to nonexperts. This Commentary summarizes the discussion and current initiatives to address these challenges.

Water Plays Key Roles in Stabilities of Wild Type and Mutant Transthyretin Complexes.

Transthyretin (TTR), a 56 kDa homotetramer that is involved in the transport of thyroxine and retinol, has been linked to amyloidosis through disassembly of tetramers to form monomers, dimers, and trimers that then reassemble into higher order oligomers and/or fibrils. Hybrid TTR (hTTR) tetramers are found in heterozygous individuals that express both wild type TTR (wt-TTR) and mutant TTR (mTTR) forms of the protein, and these states display increased rates of amyloidosis. Here we monitor subunit exchange (SUE) reactions involving homomeric and mixed tetramers using high resolution native mass spectrometry (nMS). Our results show evidence that differences in TTR primary structure alter tetramer stabilities, and hTTR products can form spontaneously by SUE reactions. In addition, we find that solution temperature has strong effects on TTR tetramer stabilities and formation of SUE products. Lower temperatures promote formation of hTTR tetramers containing L55P and V30M subunits, whereas small effects on the formation of hTTR tetramers containing F87A and T119M subunits are observed. We hypothesize that the observed temperature dependent stabilities and subsequent SUE behavior are a result of perturbations to the network of "two kinds of water": hydrating and structure stabilizing water molecules (Spyrakis et al. J. Med. Chem. 2017, 60 (16), 6781-6827; Xu et al. Soft Matter 2012, 8, 324-336) that stabilize wt-TTR and mTTR tetramers. The results presented in this work illustrate the utility of high resolution nMS for studies of the structures, stabilities, and dynamics of protein complexes that directly influence SUE reactions.

Home Care Worker Continuity in Home-Based Long-Term Care: Associated Factors and Relationships With Client Health and Well-Being.

Background and Objectives: Despite the importance of provider continuity across healthcare settings, continuity among home care workers who provide hands-on long-term care is understudied. This project describes home care worker continuity, identifies factors associated with increased continuity, and examines associations between continuity and client outcomes. Research Design and Methods: We conducted a retrospective cohort study of clients receiving Medicaid-funded home-based long-term care (n = 3,864) using insurance plan and home care agency data from a large nonprofit organization. We estimated home care worker continuity for clients between 6-month clinical assessments using Bice-Boxerman scores. We then used generalized estimating equations to model associations between home care worker continuity and (1) client characteristics (e.g., cognitive impairment), and (2) client functional, health, and psychosocial outcomes. Results: While home care worker continuity was lowest for clients receiving the most weekly care hours, a range of continuity existed across all levels of care need. Those who were male, older, Asian/Pacific Islander/Native American, cognitively impaired, and functionally impaired had lower continuity. Higher home care worker continuity was significantly associated (p < .05) with fewer falls, a higher likelihood of functional improvement/stabilization, and fewer depressive symptoms. Discussion and Implications: The finding that home care worker continuity is associated with the health and well-being of home-based long-term care clients underscores the importance of building high-quality relationships in long-term care. Continued efforts are necessary to understand and advance home care worker continuity and to identify other aspects of the home care experience that benefit those receiving long-term care at home.