Kinesin-1 captures RNA cargo in its adaptable coils.

The prototypic and ubiquitous microtubule motor, kinesin-1, uses a variety of adaptor proteins to facilitate the selective transport of diverse cargo within the cell. These cargo adaptors bind to the motor complex through interactions with the kinesin light or heavy chains (KLCs or KHCs). In this issue of Genes & Development, Dimitrova-Paternoga et al. (pp. 976-991) present the first structural characterization of a KHC-cargo adaptor interface. They describe an antiparallel heterotrimeric coiled-coil complex between the carboxy tail of KHC and Tm1-I/C (aTm1), the atypical tropomyosin that is important for oskar mRNA transport in Drosophila oocytes. This interaction enhances direct binding between KHC and RNA. Their findings demonstrate the structural plasticity of the KHC tail as a platform for protein-protein interactions and reveal how a cargo adaptor protein can modify a motor-RNA interface to promote transport.

A de novo designed coiled coil-based switch regulates the microtubule motor kinesin-1.

Many enzymes are allosterically regulated via conformational change; however, our ability to manipulate these structural changes and control function is limited. Here we install a conformational switch for allosteric activation into the kinesin-1 microtubule motor in vitro and in cells. Kinesin-1 is a heterotetramer that accesses open active and closed autoinhibited states. The equilibrium between these states centers on a flexible elbow within a complex coiled-coil architecture. We target the elbow to engineer a closed state that can be opened with a de novo designed peptide. The alternative states are modeled computationally and confirmed by biophysical measurements and electron microscopy. In cells, peptide-driven activation increases kinesin transport, demonstrating a primary role for conformational switching in regulating motor activity. The designs are enabled by our understanding of ubiquitous coiled-coil structures, opening possibilities for controlling other protein activities.

De novo designed peptides for cellular delivery and subcellular localisation.

Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes.

Supporting choice: an innovative model of integrated palliative care funded by a private health insurer.

BACKGROUND: The number of Australians dying each year is predicted to double in the next 25 years and there is an urgent need to establish sustainable models for providing high quality end-of-life care. An innovative community care model (Bupa Palliative Care Choices Program or BPCCP) was developed and piloted with the purpose of supporting patients in achieving their choices surrounding end-of-life care. AIMS: This study evaluates whether BPCCP patients were more likely to die in their place of choice compared with patients receiving standard care. Additional aims were evaluating patient and carer satisfaction and insurer cost. METHODS: This prospective, comparative cohort study comprises a clinical chart audit and survey of patient and carer experience. RESULTS: More BPCCP participants preferred to die at home (53% vs 31%). A lower proportion of BPCCP patients died in acute hospitals (10% vs 19%) and more of this cohort died at home (46% vs 26%). In both cohorts, nearly 90% of patients were able to die in their preferred location. Patient and carer satisfaction with the programme was very high in the small cohort who responded to the survey. There was a decrease in average claims spend per patient enrolled in the programme during the first 12-month period of implementation compared with historical claims spend for inpatients only. CONCLUSIONS: This evaluation of an innovative community palliative care intervention indicates that the extra services available to patients support the choice of dying at home and the ability to do so while generating claims cost efficiencies.

The role of point-of-care tests in antibiotic stewardship for urinary tract infections in a resource-limited setting on the Thailand-Myanmar border.

OBJECTIVE: Published literature from resource-limited settings is infrequent, although urinary tract infections (UTI) are a common cause of outpatient presentation and antibiotic use. Point-of-care test (POCT) interpretation relates to antibiotic use and antibiotic resistance. We aimed to assess the diagnostic accuracy of POCT and their role in UTI antibiotic stewardship. METHODS: One-year retrospective analysis in three clinics on the Thailand-Myanmar border of non-pregnant adults presenting with urinary symptoms. POCT (urine dipstick and microscopy) were compared to culture with significant growth classified as pure growth of a single organism >10(5)  CFU/ml. RESULTS: In 247 patients, 82.6% female, the most common symptoms were dysuria (81.2%), suprapubic pain (67.8%) and urinary frequency (53.7%). After excluding contaminated samples, UTI was diagnosed in 52.4% (97/185); 71.1% (69/97) had a significant growth on culture, and >80% of these were Escherichia coli (20.9% produced extended-spectrum ß-lactamase (ESBL)). Positive urine dipstick (leucocyte esterase ≥1 and/or nitrate positive) compared against positive microscopy (white blood cell >10/HPF, bacteria ≥1/HPF, epithelial cells <5/HPF) had a higher sensitivity (99% vs. 57%) but a lower specificity (47% vs. 89%), respectively. Combined POCT resulted in the best sensitivity (98%) and specificity (81%). Nearly one in ten patients received an antimicrobial to which the organism was not fully sensitive. CONCLUSION: One rapid, cost-effective POCT was too inaccurate to be used alone by healthcare workers, impeding antibiotic stewardship in a high ESBL setting. Appropriate prescribing is improved with concurrent use and concordant results of urine dipstick and microscopy.

Corrigendum: A highly potent anti-VISTA antibody KVA12123 - a new immune checkpoint inhibitor and a promising therapy against poorly immunogenic tumors.

[This corrects the article DOI: 10.3389/fimmu.2023.1311658.].

A highly potent anti-VISTA antibody KVA12123 - a new immune checkpoint inhibitor and a promising therapy against poorly immunogenic tumors.

Background: Immune checkpoint therapies have led to significant breakthroughs in cancer patient treatment in recent years. However, their efficiency is variable, and resistance to immunotherapies is common. VISTA is an immune-suppressive checkpoint inhibitor of T cell response belonging to the B7 family and a promising novel therapeutic target. VISTA is expressed in the immuno-suppressive tumor microenvironment, primarily by myeloid lineage cells, and its genetic knockout or antibody blockade restores an efficient antitumor immune response. Methods: Fully human monoclonal antibodies directed against VISTA were produced after immunizing humanized Trianni mice and single B cell sequencing. Anti-VISTA antibodies were evaluated for specificity, cross-reactivity, monocyte and T cell activation, Fc-effector functions, and antitumor efficacy using in vitro and in vivo models to select the KVA12123 antibody lead candidate. The pharmacokinetics and safety profiles of KVA12123 were evaluated in cynomolgus monkeys. Results: Here, we report the development of a clinical candidate anti-VISTA monoclonal antibody, KVA12123. KVA12123 showed high affinity binding to VISTA through a unique epitope distinct from other clinical-stage anti-VISTA monoclonal antibodies. This clinical candidate demonstrated high specificity against VISTA with no cross-reactivity detected against other members of the B7 family. KVA12123 blocked VISTA binding to its binding partners. KVA12123 induced T cell activation and demonstrated NK-mediated monocyte activation. KVA12123 treatment mediated strong single-agent antitumor activity in several syngeneic tumor models and showed enhanced efficacy in combination with anti-PD-1 treatment. This clinical candidate was engineered to improve its pharmacokinetic characteristics and reduce Fc-effector functions. It was well-tolerated in preclinical toxicology studies in cynomolgus monkeys, where hematology, clinical chemistry evaluations, and clinical observations revealed no indicators of toxicity. No cytokines associated with cytokine release syndrome were elevated. Conclusion: These results establish that KVA12123 is a promising drug candidate with a distinct but complementary mechanism of action of the first generation of immune checkpoint inhibitors. This antibody is currently evaluated alone and in combination with pembrolizumab in a Phase 1/2 open-label clinical trial in patients with advanced solid tumors.

Differential sensing with arrays of de novo designed peptide assemblies.

Differential sensing attempts to mimic the mammalian senses of smell and taste to identify analytes and complex mixtures. In place of hundreds of complex, membrane-bound G-protein coupled receptors, differential sensors employ arrays of small molecules. Here we show that arrays of computationally designed de novo peptides provide alternative synthetic receptors for differential sensing. We use self-assembling α-helical barrels (αHBs) with central channels that can be altered predictably to vary their sizes, shapes and chemistries. The channels accommodate environment-sensitive dyes that fluoresce upon binding. Challenging arrays of dye-loaded barrels with analytes causes differential fluorophore displacement. The resulting fluorimetric fingerprints are used to train machine-learning models that relate the patterns to the analytes. We show that this system discriminates between a range of biomolecules, drink, and diagnostically relevant biological samples. As αHBs are robust and chemically diverse, the system has potential to sense many analytes in various settings.

Molecular architecture of the autoinhibited kinesin-1 lambda particle.

Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the α-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative-stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1 in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drives kinesin-1 activation.

Battlefield orthopaedic injuries cause the majority of long-term disabilities.

Extremity injuries make up 54% of combat wounds sustained in Operation Iraqi Freedom and Operation Enduring Freedom. In a cohort of war-wounded service members, we identified the conditions secondary to battle injury that result in disqualification from continued service. The Army Physical Evaluation Board records of 464 wounded service members who were injured between October 2001 and January 2005 were reviewed to determine the codes indicating unfitting conditions. Sixty-nine percent of these conditions were orthopaedic. Fifty-seven percent of the injured had unfitting conditions that were orthopaedic only. Of those evacuated from theater with a primary diagnosis of injury to the head, thorax, or abdomen and who suffered an orthopaedic injury as well, 76% had an orthopaedic diagnosis as the primary unfitting condition. Orthopaedic-related disability has a significant impact on the affected patient, the health care system, and, in the case of wounded service members, on military strength and readiness.

Mortality in female war veterans of operations enduring freedom and Iraqi freedom.

BACKGROUND: Combat-wounded service members are surviving battle injuries more than ever. Given different combat roles held by men and women, female service members should survive wounds at an unprecedented rate. QUESTIONS/PURPOSES: We determined whether the casualty rates for females differ from their male counterparts and characterized wounds sustained by female casualties. METHODS: We calculated the percentage of the 5141 deaths among the 40,531 casualties by gender for those serving in Operations Enduring Freedom (OEF) and Iraqi Freedom (OIF) from Defense Manpower Statistics between 2001 and 2009. We searched the Joint Theatre Trauma Registry for female casualties and described their injury characteristics. No matched cohort of male casualties was searched. RESULTS: Female veterans comprised 1.9% of all casualties and 2.4% of all deaths. In OIF, the percent death for women was 14.5% (103 deaths) versus 12.0% (4226 deaths) for men. In OEF, the percent death for women was 35.9% (19 deaths) versus 17.0% (793 deaths) for men. Battle-injured females had a greater proportion of facial and external injuries and more severe extremity injuries compared with those nonbattle-injured. CONCLUSIONS: The casualty death rate appears higher for women than men although the mechanisms of fatal injuries are not known and may not be comparable. Although facial, external, and extremity injuries were common among battle-injured females, no conclusion can be made as to whether male casualties sustain similar wounding patterns. LEVEL OF EVIDENCE: Level II, prognostic study. See Guidelines for Authors for a complete description of levels of evidence.

Data-driven disaster management requires data: implementation of a military orthopaedic trauma registry.

The Military Orthopaedic Trauma Registry (MOTR) is a comprehensive joint service registry of military orthopaedic injuries. Conceived in 2006, MOTR is now operational for retrospective data entry and prospective data collection of extremity injuries sustained by U.S. service members serving in current Overseas Contingency Operations. Running in tandem with data from the United States Army Institute of Surgical Research's Joint Theater Trauma Registry (JTTR), MOTR augments the casualty data included in JTTR with additional orthopaedic specific data (i.e., the injury patterns, characteristics, treatment, and complications associated with extremity war injuries). Extremity war injuries are the major clinical burden of the current conflicts. However, the scope of the injuries in detail useful to the orthopaedic researcher has never been prospectively collected. MOTR is designed to fill that gap in extremity trauma research. As such, MOTR represents an evolutionary step in the refinement of data-driven disaster management.

Fragment-linking peptide design yields a high-affinity ligand for microtubule-based transport.

Synthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides that make improved interactions with the target. Here, we devise a fragment-linking strategy-"mash-up" design-to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolar-affinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.

Molecular mechanism for kinesin-1 direct membrane recognition.

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1-dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1-mediated organelle transport.

Organ-specific attenuation of murine hepatitis virus strain A59 by replacement of catalytic residues in the putative viral cyclic phosphodiesterase ns2.

The Murine hepatitis virus (MHV) strain A59 ns2 protein is a 30-kDa nonstructural protein that is expressed from a subgenomic mRNA in the cytoplasm of virus-infected cells. Its homologs are also encoded in other closely related group 2a coronaviruses and more distantly related toroviruses. Together, these proteins comprise a subset of a large superfamily of 2H phosphoesterase proteins that are distinguished by a pair of conserved His-x-Thr/Ser motifs encompassing catalytically important residues. We have used a vaccinia virus-based reverse genetic system to produce recombinant viruses encoding ns2 proteins with single-amino-acid substitutions in, or adjacent to, these conserved motifs, namely, inf-ns2 H46A, inf-ns2 S48A, inf-ns2-S120A, and inf-ns2-H126R. All of the mutant viruses replicate in mouse 17 clone 1 fibroblast cells and mouse embryonic cells to the same extent as the parental wild-type recombinant virus, inf-MHV-A59. However, compared to inf-MHV-A59, the inf-ns2 H46A and inf-ns2-H126R mutants are highly attenuated for replication in mouse liver following intrahepatic inoculation. Interestingly, none of the mutant viruses were attenuated for replication in mouse brain following intracranial inoculation. These results show that the ns2 protein of MHV-A59 has an important role in virus pathogenicity and that a substitution of the histidine residues of the MHV-A59 ns2 His-x-Thr/Ser motifs is critical for virus virulence in the liver but not in the brain. This novel phenotype suggests a strategy to investigate the function of the MHV-A59 ns2 protein involving the search for organ-specific proteins or RNAs that react differentially to wild-type and mutant ns2 proteins.

Prevalence of QTc Prolongation in Patients With Advanced Cancer Receiving Palliative Care-A Cause for Concern?

CONTEXT: Medications commonly used for symptom control along with other known risk factors have the potential to prolong ventricular repolarization as measured by the QT interval (the time from the start of the Q wave to the end of the T wave) on a standard electrocardiogram (ECG). OBJECTIVES: To document the prevalence of a prolonged QT interval corrected for heart rate (QTc) interval in the palliative/oncology setting, compare automatic ECG QTc measurements with manual readings and identify any correlation between QTc prolongation and the use of drugs or other risk factors. METHODS: A convenience sample of consecutive patients with cancer, admitted under or known to the palliative/supportive care teams in two metropolitan hospitals, and willing to provide an ECG recording and basic demographic information including QTc risk factors were included. Both automated and manually calculated QTc intervals were recorded. Multivariable analysis was used to determine risk factors independently associated with prolonged QTc intervals. RESULTS: Of the 389 participants, there was a significant difference in mean QTc between sites using automated but not manual calculations. Manual readings were therefore used with predetermined cutoffs of 0.44 seconds (males) and 0.46 seconds (females). Seventy-two (18.5%) of the participants had a prolonged QTc with six (1.5%) having a prolongation of >0.50 seconds. At-risk drugs were being taken by 218 participants (56.0% of total cohort). Factors shown to be associated with QTc prolongation included age, gender, performance status, and hypocalcemia. No specific medication was associated with increased risk. CONCLUSION: Although almost 20% of patients receiving palliative care had prolongation of QTc, the possibility of serious consequences appeared to be low despite the frequent occurrence of risk factors.

Controls on surface water carbonate chemistry along North American ocean margins.

Syntheses of carbonate chemistry spatial patterns are important for predicting ocean acidification impacts, but are lacking in coastal oceans. Here, we show that along the North American Atlantic and Gulf coasts the meridional distributions of dissolved inorganic carbon (DIC) and carbonate mineral saturation state (Ω) are controlled by partial equilibrium with the atmosphere resulting in relatively low DIC and high Ω in warm southern waters and the opposite in cold northern waters. However, pH and the partial pressure of CO2 (pCO2) do not exhibit a simple spatial pattern and are controlled by local physical and net biological processes which impede equilibrium with the atmosphere. Along the Pacific coast, upwelling brings subsurface waters with low Ω and pH to the surface where net biological production works to raise their values. Different temperature sensitivities of carbonate properties and different timescales of influencing processes lead to contrasting property distributions within and among margins.

Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia.

The coronavirus mouse hepatitis virus (MHV) induces a minimal type I interferon (IFN) response in several cell types in vitro despite the fact that the type I IFN response is important in protecting the mouse from infection in vivo. When infected with MHV, mice deficient in IFN-associated receptor expression (IFNAR(-/-)) became moribund by 48 h postinfection. MHV also replicated to higher titers and exhibited a more broad tissue tropism in these mice, which lack a type I IFN response. Interestingly, MHV induced IFN-beta in the brains and livers, two main targets of MHV replication, of infected wild-type mice. MHV infection of primary cell cultures indicates that hepatocytes are not responsible for the IFN-beta production in the liver during MHV infection. Furthermore, macrophages and microglia, but not neurons or astrocytes, are responsible for IFN-beta production in the brain. To determine the pathway by which MHV is recognized in macrophages, IFN-beta mRNA expression was quantified following MHV infection of a panel of primary bone marrow-derived macrophages generated from mice lacking different pattern recognition receptors (PRRs). Interestingly, MDA5, a PRR thought to recognize primarily picornaviruses, was required for recognition of MHV. Thus, MHV induces type I IFN in macrophages and microglia in the brains of infected animals and is recognized by an MDA5-dependent pathway in macrophages. These findings suggest that secretion of IFN-beta by macrophages and microglia plays a role in protecting the host from MHV infection of the central nervous system.

Motor-cargo adaptors at the organelle-cytoskeleton interface.

Cytoskeletal motors of the dynein, kinesin and myosin superfamilies maintain and adapt subcellular organelle organization to meet functional demands and support the vesicular transport of material between organelles. These motors require the capacity to specifically recognize the vesicle/organelle to be transported and are capable of selective recognition of multiple cargo. Recent studies have begun to uncover the molecular basis for motor recruitment and have highlighted the role of organelle-associated 'cargo-adaptor' proteins in cellular transport. These adaptors possess sequences and/or structural features that enable both motor recruitment and activation from regulated, inactive, states to enable motility on the cytoskeleton. Motor-cargo adaptor interactions define a key organelle-cytoskeleton interface, acting as crucial regulatory hubs to enable the cell to finely control membrane trafficking and organelle dynamics. Understanding the molecular basis of these interactions may offer new opportunities to control and manipulate cytoskeletal and organelle dynamics for the development of new research tools and potentially therapeutics.

Impact of TennCare reform on care of patients with rheumatic diseases.

BACKGROUND: As a consequence of cost-cutting changes, termed "TennCare reform," to Tennessee's state-funded health care plan, a number of beneficiaries have either had their benefits substantially reduced or have been disenrolled entirely. The purpose of this study was to determine the impact of these reforms on the health of patients with chronic rheumatic diseases. METHODS: We determined differences with adherence to scheduled appointments in an urban academic rheumatology clinic in the 3 months before and in the 3 months after TennCare reform. A telephone survey was conducted to determine plans for future rheumatic care among those patients scheduled to be seen in this clinic in the 3 months after TennCare reform. RESULTS: Overall, 402 of the 601 patients scheduled for a rheumatology clinic appointment before TennCare reform (67%) adhered to their scheduled rheumatology appointment, compared with 362 of 595 patients (61%) scheduled for an appointment after TennCare reform, a difference that was statistically significant (P = 0.034). After TennCare reform, patients who did not adhere with clinic follow-up were more likely to have been disenrolled from TennCare (P < 0.001). By telephone survey, among those who were disenrolled from TennCare, almost half indicated that they did not know where they were going to receive future rheumatic disease care. Among those who retained TennCare, less than half indicated that they could afford to purchase all of their medications. CONCLUSIONS: TennCare reform has significantly limited care for patients with rheumatic diseases. The long-term consequences of this merit future study.