Mechanisms of actin filament severing and elongation by formins.

Humans express fifteen formins, playing crucial roles in actin-based processes, such as cytokinesis, cell motility, and mechanotransduction 1,2. However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. While formins are known for their ability to nucleate and elongate F-actin 3-7, some formins can additionally depolymerize, sever, or bundle F-actin. Two mammalian formins, inverted formin-2 (INF2) and diaphanous-1 (Dia1), exemplify this diversity. INF2 displays potent severing activity but elongates weakly 8-11, whereas Dia1 has potent elongation activity but does not sever 4,8. Using cryo-electron microscopy (cryo-EM), we reveal five structural states of INF2 and two of Dia1 bound to the middle and barbed end of F-actin. INF2 and Dia1 bind differently to these sites, consistent with their distinct activities. The FH2 and WH2 domains of INF2 are positioned to sever F-actin, whereas Dia1 appears unsuited for severing. Structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.

Molecular mechanisms linking missense ACTG2 mutations to visceral myopathy.

Visceral myopathy is a life-threatening disease characterized by muscle weakness in the bowel, bladder, and uterus. Mutations in smooth muscle γ-actin (ACTG2) are the most common cause of the disease, but the mechanisms by which the mutations alter muscle function are unknown. Here, we examined four prevalent ACTG2 mutations (R40C, R148C, R178C, and R257C) that cause different disease severity and are spread throughout the actin fold. R178C displayed premature degradation, R148C disrupted interactions with actin-binding proteins, R40C inhibited polymerization, and R257C destabilized filaments. Because these mutations are heterozygous, we also analyzed 50/50 mixtures with wild-type (WT) ACTG2. The WT/R40C mixture impaired filament nucleation by leiomodin 1, and WT/R257C produced filaments that were easily fragmented by smooth muscle myosin. Smooth muscle tropomyosin isoform Tpm1.4 partially rescued the defects of R40C and R257C. Cryo-electron microscopy structures of filaments formed by R40C and R257C revealed disrupted intersubunit contacts. The biochemical and structural properties of the mutants correlate with their genotype-specific disease severity.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

Ensembles of human myosin-19 bound to calmodulin and regulatory light chain RLC12B drive multimicron transport.

Myosin-19 (Myo19) controls the size, morphology, and distribution of mitochondria, but the underlying role of Myo19 motor activity is unknown. Complicating mechanistic in vitro studies, the identity of the light chains (LCs) of Myo19 remains unsettled. Here, we show by coimmunoprecipitation, reconstitution, and proteomics that the three IQ motifs of human Myo19 expressed in Expi293 human cells bind regulatory light chain (RLC12B) and calmodulin (CaM). We demonstrate that overexpression of Myo19 in HeLa cells enhances the recruitment of both Myo19 and RLC12B to mitochondria, suggesting cellular association of RLC12B with the motor. Further experiments revealed that RLC12B binds IQ2 and is flanked by two CaM molecules. In vitro, we observed that the maximal speed (∼350 nm/s) occurs when Myo19 is supplemented with CaM, but not RLC12B, suggesting maximal motility requires binding of CaM to IQ-1 and IQ-3. The addition of calcium slowed actin gliding (∼200 nm/s) without an apparent effect on CaM affinity. Furthermore, we show that small ensembles of Myo19 motors attached to quantum dots can undergo processive runs over several microns, and that calcium reduces the attachment frequency and run length of Myo19. Together, our data are consistent with a model where a few single-headed Myo19 molecules attached to a mitochondrion can sustain prolonged motile associations with actin in a CaM- and calcium-dependent manner. Based on these properties, we propose that Myo19 can function in mitochondria transport along actin filaments, tension generation on multiple randomly oriented filaments, and/or pushing against branched actin networks assembled near the membrane surface.

Network Models and Simulation Analytics for Multi-scale Dynamics of Biological Invasions.

Globalization and climate change facilitate the spread and establishment of invasive species throughout the world via multiple pathways. These spread mechanisms can be effectively represented as diffusion processes on multi-scale, spatial networks. Such network-based modeling and simulation approaches are being increasingly applied in this domain. However, these works tend to be largely domain-specific, lacking any graph theoretic formalisms, and do not take advantage of more recent developments in network science. This work is aimed toward filling some of these gaps. We develop a generic multi-scale spatial network framework that is applicable to a wide range of models developed in the literature on biological invasions. A key question we address is the following: how do individual pathways and their combinations influence the rate and pattern of spread? The analytical complexity arises more from the multi-scale nature and complex functional components of the networks rather than from the sizes of the networks. We present theoretical bounds on the spectral radius and the diameter of multi-scale networks. These two structural graph parameters have established connections to diffusion processes. Specifically, we study how network properties, such as spectral radius and diameter are influenced by model parameters. Further, we analyze a multi-pathway diffusion model from the literature by conducting simulations on synthetic and real-world networks and then use regression tree analysis to identify the important network and diffusion model parameters that influence the dynamics.

Patient preferences in retinal drug delivery.

Retinal vascular diseases (RVDs) are often treated with intravitreally (IVT) injected drugs, with relatively low patient compliance and potential risks. Ongoing research explores alternative RVD treatments, including eye drops and oral tablets. This study surveyed RVD patients treated with IVT injections to establish factors influencing low compliance rates while gauging treatment delivery method preferences. Demographics, perspectives, and treatment preferences were collected via IRB-approved, self-administered survey sent to Glick Eye Institute patients treated via IVT injections. Demographics, diagnoses, and treatments were ascertained from respondents' medical records. Gender, age, and number of IVT injections received were used as stratifications. Five-level Likert-style scales and t-tests evaluated responses and stratification comparisons. The most common diagnoses in the respondent population (n = 54; response rate = 5%) were age-related macular degeneration, macular edema, and diabetic retinopathy. Respondents had varying levels of education, income, and age. Most (83%) admitted feeling anxious prior to their first IVT injection, but 80% reported willingness to receive IVT injections indefinitely, with a preference for ophthalmologist visits every 1-3 months. Eye drops would be preferred over IVT injections by 76% of respondents, while 65% preferred oral tablets, due to several perceived negative factors of IVT injections and positive factors for eye drops. Stratified groups did not differ in responses to survey questions. RVD patients will accept IVT injections for vision preservation, but alternative delivery methods like eye drops or oral tablets would be preferred. Thus, development of eye drop and oral therapeutics for RVD treatment is further emphasized by these findings.

Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

Patient perspectives of target weight management and ultrafiltration in haemodialysis: a multi-center survey.

BACKGROUND: Decisions around planned ultrafiltration volumes are the only part of the haemodialysis prescription decided upon at every session. Removing too much fluid or too little is associated with both acute symptoms and long-term outcomes. The degree to which patients engage with or influence decision-making is not clear. We explored patient perspectives of prescribing ultrafiltration volumes, their understanding of the process and engagement with it. METHODS: A questionnaire developed for this study was administered to 1077 patients across 10 UK Renal Units. Factor analysis reduced the dataset into factors representing common themes. Relationships between survey results and factors were investigated using regression models. ANCOVA was used to explore differences between Renal Units. RESULTS: Patients generally felt in control of their fluid management and that they were given the final say on planned ultrafiltration volumes. Around half of the respondents reported they take an active role in their treatment. However, respondents were largely unable to relate signs and symptoms to fluid management practice and a third said they would not report common signs and symptoms to clinicians. A fifth of patients reported not to know how ultrafiltration volumes were calculated. Patients responded positively to questions relating to healthcare staff, though with significant variation between units, highlighting differences in perception of care. CONCLUSIONS: Despite a lack of formal acknowledgement in fluid management protocols, patients have significant involvement in decisions regarding fluid removal during dialysis. Furthermore, substantial gaps remain in patient knowledge and engagement. Formalizing the role of patients in these decisions, including patient education, may improve prescription and achievement of target weights.

Molecular Dynamics for Antimicrobial Peptide Discovery.

Although antimicrobial resistance is an increasingly significant public health concern, there have only been two new classes of antibiotics approved for human use since the 1960s. Understanding the mechanisms of action of antibiotics is critical for novel antibiotic discovery, but novel approaches are needed that do not exclusively rely on experiments. Molecular dynamics simulation is a computational tool that uses simple models of the atoms in a system to discover nanoscale insights into the dynamic relationship between mechanism and biological function. Such insights can lay the framework for elucidating the mechanism of action and optimizing antibiotic templates. Antimicrobial peptides represent a promising solution to escalating antimicrobial resistance, given their lesser tendency to induce resistance than that of small-molecule antibiotics. Simulations of these agents have already revealed how they interact with bacterial membranes and the underlying physiochemical features directing their structure and function. In this minireview, we discuss how traditional molecular dynamics simulation works and its role and potential for the development of new antibiotic candidates with an emphasis on antimicrobial peptides.

Non-Equilibrium Mass Exchange in AOT Reverse Micelles.

Reverse micelles (RMs) composed of water and sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane have a remarkably narrow size distribution around a mean value determined by the water loading ratio of the system. It has been proposed that RMs establish this equilibrium size distribution either by the diffusion of individual components through the isooctane phase or by cycles of fusion and fission. To examine these mechanisms, a 24 µs all-atom molecular dynamics simulation of a system containing one small RM and one large RM was performed. Results show that the net movement of water from the small RM to the large RM occurred in a direction that made the small RM smaller and the large RM larger-according to water loading ratios that would have been appropriate for their size. Changes in AOT number that would bring the water loading ratio of each RM closer to that of the overall system only occurred via cycles of RM fusion and fission. These behaviors are most likely driven by the electrostatics of sodium AOT and the dielectric effects of water.

DPP1 Inhibitors: Exploring the Role of Water in the S2 Pocket of DPP1 with Substituted Pyrrolidines.

A series of pyrrolidine amino nitrile DPP1 inhibitors have been developed and characterized. The S2 pocket structure-activity relationship for these compounds shows significant gains in potency for DPP1 from interacting further with target residues and a network of water molecules in the binding pocket. Herein we describe the X-ray crystal structures of several of these compounds alongside an analysis of factors influencing the inhibitory potency toward DPP1 of which stabilization of the water network, demonstrated using Grand Canonical Monte Carlo simulations and free energy calculations, is attributed as a main factor.

Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish.

Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.

Renal association clinical practice guideline in post-operative care in the kidney transplant recipient.

These guidelines cover the care of patients from the period following kidney transplantation until the transplant is no longer working or the patient dies. During the early phase prevention of acute rejection and infection are the priority. After around 3-6 months, the priorities change to preservation of transplant function and avoiding the long-term complications of immunosuppressive medication (the medication used to suppress the immune system to prevent rejection). The topics discussed include organization of outpatient follow up, immunosuppressive medication, treatment of acute and chronic rejection, and prevention of complications. The potential complications discussed include heart disease, infection, cancer, bone disease and blood disorders. There is also a section on contraception and reproductive issues.Immediately after the introduction there is a statement of all the recommendations. These recommendations are written in a language that we think should be understandable by many patients, relatives, carers and other interested people. Consequently we have not reworded or restated them in this lay summary. They are graded 1 or 2 depending on the strength of the recommendation by the authors, and AD depending on the quality of the evidence that the recommendation is based on.

Evading the fate of Pheidippides: acute coronary thrombosis in a young marathon runner with minimal atherosclerosis but sickle cell trait.

Marathon running transiently increases the risk of sudden cardiac death. Some previous studies have suggested that this is due to relatively advanced but asymptomatic atherosclerosis. Other theories suggest that potentiation of inflammation and the coagulation cascade, by extremes of exertion, is more important. We present a clinical case of a young, previously fit athlete who felt chest discomfort eight miles into a marathon but finished the race. Shortly after completion he felt very unwell and had chest pain. Ambulance electrocardiograms showed evidence of an evolving anterior myocardial infarction. Invasive assessment with coronary angiography and intravascular ultrasound was able to show the mechanism of thrombosis. Fissuring of a small rim of atherosclerosis potentiated a large pro-thrombotic response, the patient was also found to have sickle cell trait. Medical treatment with blood thinning drugs was able to restore normality to the vessel over a period of two weeks, without the need for angioplasty or stent implantation.

Retrograde Wiring of Collateral Channels of the Heart in Chronic Total Occlusions: A Systematic Review and Meta-Analysis of Safety, Feasibility, and Incremental Value in Achieving Revascularization.

AIM: To conduct a systematic review and meta-analysis on retrograde wiring in chronic total occlusions (CTOs) with focus on its safety and feasibility. METHODS AND RESULTS: We searched publications from 1990 to December 2013 in PubMed, Ovid, EMBASE, and the Cochrane database inserting a number of terms relating to the collateral circulation of the heart in CTOs. A total of 18 case series (n range17-462) with a total of 2280 CTO revascularization attempts fulfilled criteria for a study of retrograde wiring of collateral channels in CTOs. There were no randomized studies comparing a primary antegrade with a primary retrograde approach. Procedural CTO revascularization rates ranged from 67% to 90.6% with a large proportion having previously failed an "antegrade" approach. The septal perforator collaterals and epicardial channels were used in 73.2% (n = 1670) and 21.7% (n = 495) of cases. Although collateral/coronary perforation was not infrequent (n = 90, 5%), serious acute complications were uncommon; in the combined population 18 cases of cardiac tamponade (0.8%) and 3 deaths (0.1%). Septal perforating wiring (79.3%) was significantly more likely to be successful compared to epicardial coronary artery wiring (72.5%) when chosen by the operator as a route of retrograde access to the CTO body (relative risk 1.11 [95% confidence interval: 1.02-1.20; P = .013]). CONCLUSION: Successful retrograde wiring of collateral channels in selected patients undertaken by "CTO dedicated" operators can significantly enhance the chances of revascularization of complex CTOs with a low risk of acute serious complications. Septal perforator channels are significantly more likely to be successfully retrogradely wired compared to epicardial vessels when either is selected, by reference to their anatomical suitability by the operator, as a route of access.

Site-specific intravascular ultrasound analysis of remodelling index and calcified necrosis patterns reveals novel blueprints for coronary plaque instability.

AIMS: Post-mortem pathological studies have shown that a "vulnerable" plaque is the dominant patho-physiological mechanism responsible for acute coronary syndromes (ACS). One way to improve our understanding of these plaques in vivo is by using histological "surrogates" created by intravascular ultrasound derived virtual histology (IVUS-VH). Our aim in this analysis was to determine the relationship between site-specific differences in individual plaque areas between ACS plaques and stable plaques (SP), with a focus on remodelling index and the pattern of calcifying necrosis. METHODS AND RESULTS: IVUS-VH was performed before percutaneous intervention in both ACS culprit plaques (CP) n=70 and stable disease (SP) n=35. A total of 210 plaque sites were examined in 105 lesions at the minimum lumen area (MLA) and the maximum necrotic core site (MAX NC). Each plaque site had multiple measurements made including some novel calculations to ascertain the plaque calcification equipoise (PCE) and the calcified interface area (CIA). CP has greater amounts of positive remodelling at the MLA (RI@MLA): 1.1 (±0.17) vs. 0.95 (±0.14) (P<0.001); lower values for PCE 30% vs. 54% (P<0.001) but a higher CIA 5.38 (±2.72) vs. 3.58 (±2.26) (P=0.001). These features can provide discriminatory ability between plaque types with area under the curve (AUC) measurements between 0.65-0.86. The cut-off values with greatest sensitivity and specificity to discriminate CP morphologies were: RI @ MLA >1.12; RI @ MAX NC >1.22; PCE @ MLA <47.1%; PCE @MAX NC <47.3%; CIA @ MLA >2.6; CIA @ MAX NC >3.1. CONCLUSIONS: Determining the stage of calcifying necrosis, along with the remodelling index can discriminate between stable and ACS related plaques. These findings could be applied in the future to help detect plaques that have a vulnerable phenotype.