Optimized PAR-2 RING dimerization mediates cooperative and selective membrane binding for robust cell polarity.

Cell polarity networks are defined by quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while also ensuring that networks remain responsive to dynamically changing cellular states and/or spatial cues during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by the dimerization of the polarity protein PAR-2 via its N-terminal RING domain. Combining theory and experiment, we show that dimer affinity is optimized to achieve dynamic, selective, and cooperative binding of PAR-2 to the plasma membrane during polarization. Reducing dimerization compromises positive feedback and robustness of polarization. Conversely, enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to the anterior polarity kinase, aPKC/PKC-3. Thus, our data reveal a key role for a dynamically oligomeric RING domain in optimizing interaction affinities to support a robust and responsive cell polarity network, and highlight how optimization of oligomerization kinetics can serve as a strategy for dynamic and cooperative intracellular targeting.

14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to inhibit G proteins.

Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.

Control of atypical PKCι membrane dissociation by tyrosine phosphorylation within a PB1-C1 interdomain interface.

Atypical PKCs are cell polarity kinases that operate at the plasma membrane where they function within multiple molecular complexes to contribute to the establishment and maintenance of polarity. In contrast to the classical and novel PKCs, atypical PKCs do not respond to diacylglycerol cues to bind the membrane compartment. Until recently, it was not clear how aPKCs are recruited; whether aPKCs can directly interact with membranes or whether they are dependent on other protein interactors to do so. Two recent studies identified the pseudosubstrate region and the C1 domain as direct membrane interaction modules; however, their relative importance and coupling are unknown. We combined molecular modeling and functional assays to show that the regulatory module of aPKCι, comprising the PB1 pseudosubstrate and C1 domains, forms a cooperative and spatially continuous invariant membrane interaction platform. Furthermore, we show the coordinated orientation of membrane-binding elements within the regulatory module requires a key PB1-C1 interfacial ß-strand (beta-strand linker). We show this element contains a highly conserved Tyr residue that can be phosphorylated and that negatively regulates the integrity of the regulatory module, leading to membrane release. We thus expose a hitherto unknown regulatory mechanism of aPKCι membrane binding and release during cell polarization.

Into the fold: advances in understanding aPKC membrane dynamics.

Atypical protein kinase Cs (aPKCs) are part of the PKC family of protein kinases and are atypical because they don't respond to the canonical PKC activators diacylglycerol (DAG) and Ca2+. They are central to the organization of polarized cells and are deregulated in several cancers. aPKC recruitment to the plasma membrane compartment is crucial to their encounter with substrates associated with polarizing functions. However, in contrast with other PKCs, the mechanism by which atypical PKCs are recruited there has remained elusive until recently. Here, we bring aPKC into the fold, summarizing recent reports on the direct recruitment of aPKC to membranes, providing insight into seemingly discrepant findings and integrating them with existing literature.

The effect of power stretchers on occupational injury rates in an urban emergency medical services system.

BACKGROUND: To examine occupational injury rates in a dual-response emergency medical services (EMS) system before and after implementation of a power-lift stretcher system. METHODS: The seasonally-adjusted occupational injury rate was estimated relative to medical call volume (per 1000 calls) and workers (per 100 FTEs) from 2009 to 2019, and stratified by severity (lost-time, healthcare only), role (EMS, FIRE) and type (patient-handling). Power-lift stretchers were adopted between 2013 and 2015. Preinjury versus postinjury rates were compared using binomial tests. Interrupted time series (ITS) analysis was used to estimate the trend and change in injuries related to patient-handling, with occupational illnesses serving as control. RESULTS: Binomial tests revealed varied results, with reductions in the injury rate per 1000 calls (-14.0%) and increases in the rate per 100 FTEs (+14.1%); rates also differed by EMS role and injury severity. ITS analysis demonstrated substantial reductions in patient-handling injuries following implementation of power-lift stretchers, both in the injury rate per 1000 calls (-50.4%) and per 100 FTEs (-46.6%), specifically among individuals deployed on the ambulance. Injury rates were slightly elevated during the winter months (+0.8 per 100 FTEs) and lower during spring (-0.5 per 100 FTEs). CONCLUSIONS: These results support the implementation of power-lift stretchers for injury prevention in EMS systems and demonstrate advantages of ITS analysis when data span long preintervention and postintervention periods.

Oncogenic RET kinase domain mutations perturb the autophosphorylation trajectory by enhancing substrate presentation in trans.

To decipher the molecular basis for RET kinase activation and oncogenic deregulation, we defined the temporal sequence of RET autophosphorylation by label-free quantitative mass spectrometry. Early autophosphorylation sites map to regions flanking the kinase domain core, while sites within the activation loop only form at later time points. Comparison with oncogenic RET kinase revealed that late autophosphorylation sites become phosphorylated much earlier than wild-type RET, which is due to a combination of an enhanced enzymatic activity, increased ATP affinity, and surprisingly, by providing a better intermolecular substrate. Structural analysis of oncogenic M918T and wild-type RET kinase domains reveal a cis-inhibitory mechanism involving tethering contacts between the glycine-rich loop, activation loop, and αC-helix. Tether mutations only affected substrate presentation but perturbed the autophosphorylation trajectory similar to oncogenic mutations. This study reveals an unappreciated role for oncogenic RET kinase mutations in promoting intermolecular autophosphorylation by enhancing substrate presentation.

Co-ordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention.

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

Paramedic attitudes towards prehospital spinal care: a cross-sectional survey.

BACKGROUND: The optimal application of spinal motion restriction (SMR) in the prehospital setting continues to be debated. Few studies have examined how changing guidelines have been received and interpreted by emergency medical services (EMS) personnel. This study surveys paramedics' attitudes, observations, and self-reported practices around the treatment of potential spine injuries in the prehospital setting. METHODS: This was a cross-sectional survey of a North American EMS agency. After development and piloting, the final version of the survey contained four sections covering attitudes towards 1) general practice, 2) specific techniques, 3) assessment protocols, and 4) mechanisms of injury (MOI). Questions used Likert-scale, multiple-choice, yes/no, and free-text responses. Exploratory factor analysis (EFA) was used to identify latent constructs within responses, and factor scores were analyzed by ordinal logistic regression for associations with demographic characteristics (including qualification level, gender, and years of experience). MOI evaluations were assessed for inter-rater reliability (Fleiss' kappa). Inductive qualitative content analysis, following Elo & Kyngäs (2008), was used to examine free-text responses. RESULTS: Two hundred twenty responses were received (36% of staff). Raw results indicated that respondents felt that SMR was seen as less important than in the past, that they were treating fewer patients than previously, and that they follow protocol in most situations. The EFA identified two factors: one (Judging MOIs) captured paramedics' estimation that the presented MOI could potentially cause a spine injury, and another (Treatment Value) reflected respondents' composite view of the effectiveness, importance, and applicability of SMR. Respondents with advanced life support (ALS) qualification were more likely to be skeptical of the value of SMR compared to those at the basic life support (BLS) level (OR: 2.40, 95%CI: 1.21-4.76, p = 0.01). Overall, respondents showed fair agreement in the evaluation of MOIs (k = 0.31, 95%CI: 0.09-0.49). Content analysis identified tension expressed by respondents between SMR-as-directed and SMR-as-applied. CONCLUSION: Results of this survey show that EMS personnel are skeptical of many elements of SMR but use various strategies to balance protocol adherence with optimizing patient care. While identifying several areas for future research, these findings argue for incorporating provider feedback and judgement into future guideline revision.

Oligonucleotide-Recognizing Topoisomerase Inhibitors (OTIs): Precision Gene Editors for Neurodegenerative Diseases?

Topoisomerases are essential enzymes that recognize and modify the topology of DNA to allow DNA replication and transcription to take place. Topoisomerases are divided into type I topoisomerases, that cleave one DNA strand to modify DNA topology, and type II, that cleave both DNA strands. Topoisomerases normally rapidly religate cleaved-DNA once the topology has been modified. Topoisomerases do not recognize specific DNA sequences, but actively cleave positively supercoiled DNA ahead of transcription bubbles or replication forks, and negative supercoils (or precatenanes) behind, thus allowing the unwinding of the DNA-helix to proceed (during both transcription and replication). Drugs that stabilize DNA-cleavage complexes with topoisomerases produce cytotoxic DNA damage and kill fast-dividing cells; they are widely used in cancer chemotherapy. Oligonucleotide-recognizing topoisomerase inhibitors (OTIs) have given drugs that stabilize DNA-cleavage complexes specificity by linking them to either: (i) DNA duplex recognizing triplex forming oligonucleotide (TFO-OTIs) or DNA duplex recognizing pyrrole-imidazole-polyamides (PIP-OTIs) (ii) or by conventional Watson-Crick base pairing (WC-OTIs). This converts compounds from indiscriminate DNA-damaging drugs to highly specific targeted DNA-cleaving OTIs. Herein we propose simple strategies to enable DNA-duplex strand invasion of WC-OTIs giving strand-invading SI-OTIs. This will make SI-OTIs similar to the guide RNAs of CRISPR/Cas9 nuclease bacterial immune systems. However, an important difference between OTIs and CRISPR/Cas9, is that OTIs do not require the introduction of foreign proteins into cells. Recent successful oligonucleotide therapeutics for neurodegenerative diseases suggest that OTIs can be developed to be highly specific gene editing agents for DNA lesions that cause neurodegenerative diseases.

The Rho family GEF FARP2 is activated by aPKCι to control tight junction formation and polarity.

The elaboration of polarity is central to organismal development and to the maintenance of functional epithelia. Among the controls determining polarity are the PAR proteins, PAR6, aPKCι and PAR3, regulating both known and unknown effectors. Here, we identify FARP2 as a 'RIPR' motif-dependent partner and substrate of aPKCι that is required for efficient polarisation and junction formation. Binding is conferred by a FERM/FA domain-kinase domain interaction and detachment promoted by aPKCι-dependent phosphorylation. FARP2 is shown to promote GTP loading of Cdc42, which is consistent with it being involved in upstream regulation of the polarising PAR6-aPKCι complex. However, we show that aPKCι acts to promote the localised activity of FARP2 through phosphorylation. We conclude that this aPKCι-FARP2 complex formation acts as a positive feedback control to drive polarisation through aPKCι and other Cdc42 effectors.This article has an associated First Person interview with the first author of the paper.

Emergency care with lay responders in underserved populations: a systematic review.

OBJECTIVE: To assess the individual and community health effects of task shifting for emergency care in low-resource settings and underserved populations worldwide. METHODS: We systematically searched 13 databases and additional grey literature for studies published between 1984 and 2019. Eligible studies involved emergency care training for laypeople in underserved or low-resource populations, and any quantitative assessment of effects on the health of individuals or communities. We conducted duplicate assessments of study eligibility, data abstraction and quality. We synthesized findings in narrative and tabular format. FINDINGS: Of 19 308 papers retrieved, 34 studies met the inclusion criteria from low- and middle-income countries (21 studies) and underserved populations in high-income countries (13 studies). Targeted emergency conditions included trauma, burns, cardiac arrest, opioid poisoning, malaria, paediatric communicable diseases and malnutrition. Trainees included the general public, non-health-care professionals, volunteers and close contacts of at-risk populations, all trained through in-class, peer and multimodal education and public awareness campaigns. Important clinical and policy outcomes included improvements in community capacity to manage emergencies (14 studies), patient outcomes (13 studies) and community health (seven studies). While substantial effects were observed for programmes to address paediatric malaria, trauma and opioid poisoning, most studies reported modest effect sizes and two reported null results. Most studies were of weak (24 studies) or moderate quality (nine studies). CONCLUSION: First aid education and task shifting to laypeople for emergency care may reduce patient morbidity and mortality and build community capacity to manage health emergencies for a variety of emergency conditions in underserved and low-resource settings.

Head-Neck Motion in Prehospital Trauma Patients under Spinal Motion Restriction: A Pilot Study.

BACKGROUND: Spinal precautions are intended to limit motion of potentially unstable spinal segments. The efficacy of various treatment approaches for motion restriction in the cervical spine has been rigorously investigated using healthy volunteers and, to a lesser extent, cadaver samples. No previous studies have objectively measured this motion in trauma patients with potential spine injuries during prehospital care. Objective: The purpose of this study was to characterize head-neck (H-N) kinematics in a sample of trauma patients receiving spinal precautions in the field. Methods: This was a prospective observational study of trauma patients in the prehospital setting. Trauma patients meeting criteria for spinal precautions were eligible for inclusion. Participants received usual care, consisting of either a long backboard, cervical collar, and head blocks (BC) or a cervical collar only (CO), and behavior was categorized as compliant (C) or non-compliant (N). Three inertial measurement units (IMUs), placed on each participant's forehead, sternum, and stretcher, yielded data on H-N motion. Outcomes were described in terms of H-N displacement and acceleration, including single- and multi-planar values, root mean square (RMS), and bouts of continuous motion above pre-determined thresholds. Data were analyzed to compare H-N motion by phase of prehospital care, as well as treatment type and patient behavior. RESULTS: Substantial single- and multi-plane H-N motion was observed among all participants. Maximum single-plane displacements were between 11.3 ± 3.0 degrees (rotation) and 19.0 ± 16.6 degrees (flexion-extension). Maximum multi-plane displacements averaged 31.2 ± 7.2 degrees (range: 7.2 to 82.1 degrees). Maximum multi-plane acceleration averaged 5.8 ± 1.4 m/s2 (range: 1.2 to 19.9 m/s2). There were no significant differences among participants between prehospital phase and treatment type. Non-compliant participants showed significantly more motion than compliant participants. Conclusion: Among actual patients, movement appears to be greater than previously recorded in simulation studies, and to be associated with patient behavior. Miniature IMUs are a feasible approach to field-based measurement of H-N kinematics in trauma patients. Future research should evaluate the effects of patient compliance, treatment, and phase of care using larger samples. Key words: spinal immobilization; cervical spine; cervical collar; long backboard.

Effects of Spinal Immobilization and Spinal Motion Restriction on Head-Neck Kinematics during Ambulance Transport.

Objective: To determine the influence of ambulance motion on head-neck (H-N) kinematics and to compare the effectiveness of two spinal precaution (SP) protocols: spinal immobilization (SI) and spinal motion reduction (SMR). Methods: Eighteen healthy volunteers (7 females) underwent a series of standardized ambulance transport tasks, across various speeds, under the two SP protocols in a balanced order (n = 12 drivers, n = 7 ambulances). Inertial measurement units were placed on participants' heads and sternums, with another affixed to the stretcher mattress frame. Outcome measures included H-N displacement and acceleration. Results: Ambulance accelerations varied across driving tasks (2.5-9.5 m/s2) and speeds (3.0-6.2 m/s2) and resulted in a wide range of H-N displacements (7.2-22.6 deg) and H-N accelerations (1.4-10.9 m/s2). Relative to SMR, SI resulted in reduced H-N motion during turning, accelerating, and speed bumps (1.9-10.7 deg; 0.4-2.6 m/s2), but increased H-N accelerations during abrupt starts/stops and some higher speed tasks (0.4-2.5 m/s2). Ambulance acceleration was moderately correlated to H-N acceleration (r = 0.68) and displacement (r = 0.42). Conclusion: H-N motion was somewhat coupled to ambulance acceleration and varied across a wide range, regardless of SP approach. In general, SI resulted in a modest reduction in H-N displacement and acceleration, with some exceptions. The results inform clinical decisions on SP practice during prehospital transport and demonstrate a novel approach to quantifying H-N motion in prehospital care.

Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction.

Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET(Δ) (E3) and RET(Δ) (E345), respectively. Here we report the presence of Ret(Δ) (E3) and Ret(Δ) (E345) in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RET(ΔE3) and RET(ΔE345) are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RET(ΔE3) is phosphorylated in a similar manner to full-length RET. RET(ΔE345), in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr(905), and also on one of the most important signaling residues, Tyr(1062) These data provide the first evidence for a physiologic role of these isoforms in RET pathway function.

Outcomes and characteristics of non-immobilised, spine-injured trauma patients: a systematic review of prehospital selective immobilisation protocols.

OBJECTIVES: This review assesses prehospital selective immobilisation protocols across a range of outcomes, including neurological deterioration and characteristics of injured, non-immobilised patients. METHODS: Six electronic reference databases and eight grey literature sources were systematically searched. We included studies that enrolled acute trauma patients in the prehospital setting who were assessed for spine injury according to predefined clinical criteria and either immobilised or not. Data items included instances of neurological deterioration among patients with spine injuries, as well as available characteristics of those who were injured and not immobilised. Available data and study heterogeneity prevented meta-analyses. Bias was assessed for both individual studies and across studies by outcome. RESULTS: 604 unique articles were retrieved, of which 7 met inclusion criteria. There was moderate or high risk of bias across studies in all outcomes. Of 76 patients with spine injuries who were not immobilised, 72 had no neurological deficit that appeared after emergency medical services contact, and the remaining four were not followed. Within this group, there appears to be a trend towards elderly patients who suffered a thoracic or lumbar injury from a low-risk mechanism of injury. Among studies that report both the results of the protocol assessment and immobilisation status, there is variable correspondence between the two. CONCLUSIONS: Data limitations and study biases suggest caution when interpreting and applying the results of this review. Its findings are consistent with the conclusions of individual studies. The characteristics of injured, non-immobilised patients point to areas of future research to investigate apparent trends.

Structures of the human Pals1 PDZ domain with and without ligand suggest gated access of Crb to the PDZ peptide-binding groove.

Many components of epithelial polarity protein complexes possess PDZ domains that are required for protein interaction and recruitment to the apical plasma membrane. Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs). This study describes the molecular interaction between the Crb carboxy-terminal motif (ERLI), which is required for Drosophila cell polarity, and the Pals1 PDZ domain using crystallography and fluorescence polarization. Only the last four Crb residues contribute to Pals1 PDZ-domain binding affinity, with specificity contributed by conserved charged interactions. Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove. Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein-protein interaction.

Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.

BACKGROUND: Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. METHODS: To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. RESULTS: Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. CONCLUSIONS: Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).

Pharmacological inhibition of DDAH1 improves survival, haemodynamics and organ function in experimental septic shock.

The aim of the present study was to investigate the therapeutic effects of pharmacological inhibition of DDAH1 (dimethylarginine dimethylaminohydrolase 1), an enzyme that metabolizes endogenously produced nitric oxide synthase inhibitors, principally ADMA (asymmetric dimethylarginine). The present study employs a series of rodent models to evaluate the effectiveness a DDAH1-selective inhibitor (L-257). Short-term models involved the development of endotoxaemia using lipopolysaccharide and long-term models involved the intraperitoneal administration of faecal slurry. In order to generate the most relevant model possible, following induction of severe sepsis, animals received appropriate fluid resuscitation and in some models vasopressor therapy. The effects of L-257 on survival, haemodynamics and organ function were subsequently assessed. Survival was significantly longer in all L-257 treatment groups (P<0.01) and no adverse effects on haemodynamics and organ function were observed following L-257 administration to either animals with sepsis or naïve animals. Haemodynamic performance was preserved and the noradrenaline dose required to maintain target blood pressure was reduced in the treated animals (P<0.01). Animals receiving L-257 had significantly increased plasma ADMA concentrations. Plasma nitrite/nitrate was reduced as was severity of sepsis-associated renal dysfunction. The degree of tachycardia was improved as were indices of tissue and microvascular perfusion. The results of the present study show that the selective DDAH-1 inhibitor L-257 improved haemodynamics, provided catecholamine sparing and prolonged survival in experimental sepsis. Further studies will determine its potential utility in human septic shock.

A winged helix domain in human MUS81 binds DNA and modulates the endonuclease activity of MUS81 complexes.

The MUS81-EME1 endonuclease maintains metazoan genomic integrity by cleaving branched DNA structures that arise during the resolution of recombination intermediates. In humans, MUS81 also forms a poorly characterized complex with EME2. Here, we identify and determine the structure of a winged helix (WH) domain from human MUS81, which binds DNA. WH domain mutations greatly reduce binding of the isolated domain to DNA and impact on incision activity of MUS81-EME1/EME2 complexes. Deletion of the WH domain reduces the endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes, and incisions made by MUS81-EME2 are made closer to the junction on substrates containing a downstream duplex, such as fork structures and nicked Holliday junctions. WH domain mutation or deletion in Schizosaccharomyces pombe phenocopies the DNA-damage sensitivity of strains deleted for mus81. Our results indicate an important role for the WH domain in both yeast and human MUS81 complexes.

G-actin regulates the shuttling and PP1 binding of the RPEL protein Phactr1 to control actomyosin assembly.

The Phactr family of PP1-binding proteins is implicated in human diseases including Parkinson's, cancer and myocardial infarction. Each Phactr protein contains four G-actin binding RPEL motifs, including an N-terminal motif, abutting a basic element, and a C-terminal triple RPEL repeat, which overlaps a conserved C-terminus required for interaction with PP1. RPEL motifs are also found in the regulatory domains of the MRTF transcriptional coactivators, where they control MRTF subcellular localisation and activity by sensing signal-induced changes in G-actin concentration. However, whether G-actin binding controls Phactr protein function - and its relation to signalling - has not been investigated. Here, we show that Rho-actin signalling induced by serum stimulation promotes the nuclear accumulation of Phactr1, but not other Phactr family members. Actin binding by the three Phactr1 C-terminal RPEL motifs is required for Phactr1 cytoplasmic localisation in resting cells. Phactr1 nuclear accumulation is importin α-ß dependent. G-actin and importin α-ß bind competitively to nuclear import signals associated with the N- and C-terminal RPEL motifs. All four motifs are required for the inhibition of serum-induced Phactr1 nuclear accumulation when G-actin is elevated. G-actin and PP1 bind competitively to the Phactr1 C-terminal region, and Phactr1 C-terminal RPEL mutants that cannot bind G-actin induce aberrant actomyosin structures dependent on their nuclear accumulation and on PP1 binding. In CHL-1 melanoma cells, Phactr1 exhibits actin-regulated subcellular localisation and is required for stress fibre assembly, motility and invasiveness. These data support a role for Phactr1 in actomyosin assembly and suggest that Phactr1 G-actin sensing allows its coordination with F-actin availability.