Outcome of long-term biliary stenting for stones in the 2010s: beware the cholecystectomised!

Objective: Endoscopic retrograde cholangiopancreatography (ERCP) is the mainstay of management for most patients with common bile duct stones (CBDS). Duct clearance at initial ERCP may not be achieved in a third of patients, many of whom may be elderly with multiple comorbidities rendering them at potentially high risk for further procedures. We aimed to quantify the rate of biliary sequelae and mortality among a large cohort undergoing a single ERCP with sphincterotomy and stent insertion without having undergone complete ductal clearance (permanent stent insertion, PSI), and to examine factors that may predispose to adverse outcomes. Design/method: Outcomes of all ERCPs undertaken on the intact papilla between February 2010 and January 2020 were distilled to identify a cohort who had undergone PSI for initially irretrievable CBDS. These were subjected to retrospective follow-up until the development of biliary sequelae, death or survival into 2023. Results: There were 2175 index ERCPs for CBDS, of whom 114 met the PSI criteria. Eleven did not survive their index hospitalisation, leaving 103 for follow-up. Of these, 25 (24%) developed late biliary sequelae, 19 (18%) required at least one further ERCP and 8 (8%) died from biliary sequelae. Adverse outcomes were found to be more common among those who had undergone cholecystectomy prior to ERCP, and those with periampullary diverticula. Conclusions: Long-term biliary stenting following sphincterotomy remains a valid option for selected patients with initially irretrievable bile duct stones who could be at high risk from repeat procedures.

Biophysical and Biochemical Characterization of Structurally Diverse Small Molecule Hits for KRAS Inhibition.

We describe six compounds as early hits for the development of direct inhibitors of KRAS, an important anticancer drug target. We show that these compounds bind to KRAS with affinities in the low micromolar range and exert different effects on its interactions with binding partners. Some of the compounds exhibit selective binding to the activated form of KRAS and inhibit signal transduction through both the MAPK or the phosphatidylinositide 3-kinase PI3K-protein kinase B (AKT) pathway in cells expressing mutant KRAS. Most inhibit intrinsic and/or SOS-mediated KRAS activation while others inhibit RAS-effector interaction. We propose these compounds as starting points for the development of non-covalent allosteric KRAS inhibitors.

Review of inhalable nanoparticles for the pulmonary delivery of anti-tuberculosis drugs.

Tuberculosis is an airborne disease caused by the pathogen, Mycobacterium tuberculosis, which predominantly affects the lungs. World Health Organization (WHO) has reported that about 85% of TB patients are cured with the existing 6-month antibiotic regimen. However, the lengthy oral administration of high-dose anti-TB drugs is associated with significant side effects and leads to drug resistance cases. Alternatively, reformulating existing anti-tubercular drugs into inhalable nanoparticulate systems is a promising strategy to overcome the challenges associated with oral treatment as they could enhance drug retention in the pulmonary region to achieve an optimal drug concentration in the infected lungs. Hence, this review provides an overview of the literature on inhalable nano-formulations for the delivery of anti-TB drugs, including their formulation techniques and preclinical evaluations between the years 2000 and 2020, gathered from electronic journals via online search engines such as Google Scholar and PubMed. Previous in vitro and in vivo studies highlighted that the nano-size, low toxicity, and high efficacy were among the factors influencing the fate of nanoparticulate system upon deposition in the lungs. Although many preclinical studies have shown that inhalable nanoparticles increased therapeutic efficacy and minimised adverse drug reactions when delivered through the pulmonary route, none of them has progressed into clinical trials to date. This could be attributed to the high cost of inhaled regimes due to the expensive production and characterisation of the nanoparticles as well as the need for an inhalation device as compared to the oral treatment. Another barrier could be the lack of medical acceptance due to insufficient number of trained staff to educate the patients on the correct usage of the inhalation device. Hence, these barriers should be addressed satisfactorily to make the inhaled nanoparticles regimen a reality for the treatment of TB.

Advances in Polyhydroxyalkanoate Nanocarriers for Effective Drug Delivery: An Overview and Challenges.

Polyhydroxyalkanoates (PHAs) are natural polymers produced under specific conditions by certain organisms, primarily bacteria, as a source of energy. These up-and-coming bioplastics are an undeniable asset in enhancing the effectiveness of drug delivery systems, which demand characteristics like non-immunogenicity, a sustained and controlled drug release, targeted delivery, as well as a high drug loading capacity. Given their biocompatibility, biodegradability, modifiability, and compatibility with hydrophobic drugs, PHAs often provide a superior alternative to free drug therapy or treatments using other polymeric nanocarriers. The many formulation methods of existing PHA nanocarriers, such as emulsion solvent evaporation, nanoprecipitation, dialysis, and in situ polymerization, are explained in this review. Due to their flexibility that allows for a vessel tailormade to its intended application, PHA nanocarriers have found their place in diverse therapy options like anticancer and anti-infective treatments, which are among the applications of PHA nanocarriers discussed in this article. Despite their many positive attributes, the advancement of PHA nanocarriers to clinical trials of drug delivery applications has been stunted due to the polymers' natural hydrophobicity, controversial production materials, and high production costs, among others. These challenges are explored in this review, alongside their existing solutions and alternatives.

Determinants of Membrane Orientation Dynamics in Lipid-Modified Small GTPases.

The transient membrane engagement and reorientation of the soluble catalytic domain of Ras proteins has emerged as an important modulator of their functions. However, there has been limited information on whether this phenomenon is applicable to other members of the Ras superfamily. To address this issue, we conducted long-time-scale atomistic molecular dynamics simulations (55 µs aggregate simulation time) on representatives of the Ras, Rho, and Arf family proteins that differ in sequence, lipid modification, and the rigidity of the linker between the lipid anchor and the catalytic G-domain. The results show that the concept of membrane reorientation is generalizable to most but not all members of the Ras superfamily. Specifically, C-terminally prenylated small GTPases that are anchored to membranes via a single flexible linker adopt multiple orientations, whereas those that are N-terminally myristoylated and harbor a rigid linker experience limited orientational dynamics. Combined with published reports on Ras proteins, these observations provide insights into the common principles and determinants of the orientational dynamics of lipidated small GTPases on membrane surfaces and offer new ways of thinking about the regulation and druggability of the Ras superfamily proteins.

Management of marginal tissue recession using platelet-rich fibrin: A case report.

BACKGROUND: Marginal tissue recession leads to exposure of root surfaces of teeth resulting in root sensitivity, caries, or an unsightly appearance. Its management is carried out both by eliminating contributing factors as well as using surgical techniques. Platelet-rich fibrin (PRF) is a seond-generation platelet concentrate first described by Choukroun et al. in 2001. This case report is of a Millers Class II recession in 41 regions managed using PRF with a double pedicle flap. METHOD: A 25-year-old patient presented with gingival recession height of 5 mm, measured from the cemento-enamel junction (CEJ) to gingival margin. The recession width was 3 mm at the CEJ and PD of 1 mm with respect to 41 region. There was no keratinized tissue apical to recession. However, adequate keratinized tissue was clinically present on either side of the recession defect wrt 41 region. A partial thickness double pedicle flap was raised wrt 41 region. PRF was prepared by centrifuging whole blood at 2,700 RPM for 12 min. The membrane was folded to create bulk and secured over the recession defect. RESULTS: The patient was reviewed at regular intervals of 01, 06, months and 01 year postoperatively. Significant coverage of denuded root was achieved with good color match and relief from sensitivity. Results were stable one year postoperatively. CONCLUSION: Cases that present with a deep and wide recession are challenging to address owing to the extensive loss of keratinized tissue. In such cases, double pedicle flap can be carried out to augment gingiva by utilizing keratinized tissue adjacent to the defect site. Platelet concentrates like PRF contain platelet-derived growth factors that exhibit chemotactic and mitogenic properties that promote and modulate cellular functions involved in tissue healing, regeneration, and cell proliferation. PRF is an autologous biomaterial which may be used in root coverage procedures. Long-term studies with a larger sample size are required to establish PRF as a predictable method of gingival recession coverage.

RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention.

RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, they activate intracellular signal transduction cascades. Membrane-bound RAS molecules segregate into multimers, known as nanoclusters. These nanoclusters, held together through weak protein-protein and protein-lipid associations, are highly dynamic and respond to cellular input signals and fluctuations in the local lipid environment. Disruption of RAS nanoclusters results in downregulation of RAS-mediated mitogenic signaling. In this review, we discuss the propensity of RAS proteins to display clustering behavior and the interfaces that are associated with these assemblies. Strategies to therapeutically disrupt nanocluster formation or the stabilization of signaling incompetent RAS complexes are discussed.

The KRAS and other prenylated polybasic domain membrane anchors recognize phosphatidylserine acyl chain structure.

KRAS interacts with the inner leaflet of the plasma membrane (PM) using a hybrid anchor that comprises a lysine-rich polybasic domain (PBD) and a C-terminal farnesyl chain. Electrostatic interactions have been envisaged as the primary determinant of interactions between KRAS and membranes. Here, we integrated molecular dynamics (MD) simulations and superresolution spatial analysis in mammalian cells and systematically compared four equally charged KRAS anchors: the wild-type farnesyl hexa-lysine and engineered mutants comprising farnesyl hexa-arginine, geranylgeranyl hexa-lysine, and geranylgeranyl hexa-arginine. MD simulations show that these equally charged KRAS mutant anchors exhibit distinct interactions and packing patterns with different phosphatidylserine (PtdSer) species, indicating that prenylated PBD-bilayer interactions extend beyond electrostatics. Similar observations were apparent in intact cells, where each anchor exhibited binding specificities for PtdSer species with distinct acyl chain compositions. Acyl chain composition determined responsiveness of the spatial organization of different PtdSer species to diverse PM perturbations, including transmembrane potential, cholesterol depletion, and PM curvature. In consequence, the spatial organization and PM binding of each KRAS anchor precisely reflected the behavior of its preferred PtdSer ligand to these same PM perturbations. Taken together these results show that small GTPase PBD-prenyl anchors, such as that of KRAS, have the capacity to encode binding specificity for specific acyl chains as well as lipid headgroups, which allow differential responses to biophysical perturbations that may have biological and signaling consequences for the anchored GTPase.

A regulatory role of membrane by direct modulation of the catalytic kinase domain.

Cell membrane modulates the function and activity of specific proteins and acts more than just a non-specific scaffolding machinery. In this review, I focus on studies that highlight a direct membrane-mediated modulation of the catalytic kinase domain of a variety of kinases thereby regulating the kinase activity. It emerges that membrane provides a second level of regulation once kinase domain is relieved of its inactive auto-inhibitory state. For the first time a generalized regulatory role of membrane is proposed that governs the kinase activity by modulating the catalytic kinase domain. Striking similarities among a variety of multi-domain kinases as well as single-domain lipidated enzymes such as RAS proteins are presented.

Long-Range Coupled Motions Underlie Ligand Recognition by a Chemokine Receptor.

Chemokines are unusual class-A G protein-coupled receptor agonists because of their large size (∼10 kDa) and binding at two distinct receptor sites: N-terminal domain (Site-I, unique to chemokines) and a groove defined by extracellular loop/transmembrane helices (Site-II, shared with all small molecule class-A ligands). Structures and sequence analysis reveal that the receptor N-terminal domains (N-domains) are flexible and contain intrinsic disorder. Using a hybrid NMR-MD approach, we characterized the role of Site-I interactions for the CXCL8-CXCR1 pair. NMR data indicate that the CXCR1 N-domain becomes structured on binding and that the binding interface is extensive with 30% CXCL8 residues participating in this initial interaction. MD simulations indicate that CXCL8 bound at Site-I undergoes extensive reorganization on engaging Site-II with several residues initially engaged at Site-I also engaging at Site-II. We conclude that structural plasticity of Site-I interactions plays an active role in driving ligand recognition by a chemokine receptor.

Probing the Conformational and Energy Landscapes of KRAS Membrane Orientation.

Membrane reorientation of oncogenic RAS proteins is emerging as an important modulator of their functions. Previous studies have shown that the most common orientations include those with either the three C-terminal α-helices (OS1) or N-terminal ß-strands (OS2) of the catalytic domain facing the membrane. OS1 and OS2 differ by the degree to which the effector-interacting surface is occluded by the membrane. However, the relative stability of these states and the rates of transition between them remained undetermined. How mutations might modulate preferences for specific orientation states is also far from clear. The current work attempted to address these questions through a comprehensive analysis of two 20 µs-long atomistic molecular dynamics simulations. The simulations were conducted on the oncogenic G12D and Q61H KRAS mutants bound to an anionic lipid bilayer. G12D and Q61H are among the most prevalent cancer-causing mutations at the P-loop and switch 2 regions of KRAS, respectively. We found that both mutants fluctuate in a similar manner between OS1 and OS2 via an intermediate orientation OS0, and both favor the signaling competent OS1 and OS0 over the occluded OS2. However, they differ in the details, such as in the extent to which they sample OS1. Analysis of the orientation free-energy landscapes estimated from the simulations indicate that OS1 and OS2 are the most stable states. However, the overall free energy surface is rugged, indicating a large diversity of conformations including at least two substates in each orientation state that differ in stability only by about 0.5-1.0 kcal/mol. Reversible transitions between OS1 and OS2 occur via two well-defined pathways that traverse OS0. In the minimum energy path, helix 4 remains close to the membrane as the angle of the catalytic domain from the membrane plane changes, resulting in a barrier of ∼1 kcal/mol for OS1/OS2 interconversions. Estimation of the rates of the various transitions based on survival probabilities yielded two rate constants in the order of 107 and 106 s-1, which we attribute to intrinsic protein conformational dynamics and transient protein-lipid interactions, respectively. The faster process dominates every transition, confirming a previous suggestion that RAS membrane reorientation is driven by conformational fluctuations rather than protein-lipid interactions.

Clinical efficacy of periosteal pedicle graft with subepithelial connective tissue graft in gingival recession coverage.

INTRODUCTION: Correction of gingival recession (GR) involves eliminating the cause of recession and it often requires surgical correction. Subepithelial connective tissue graft (SCTG) technique by Langer and Langer provides excellent esthetics and is considered most predictable in obtaining marginal tissue recession coverage. However, the requirement of a second surgical procedure for harvesting CTG remote from planned site of recession coverage increases the chances of postoperative infection. There is also a limitation in the amount of graft that can be harvested and an increased operative time contributing to patient discomfort. MATERIALS AND METHODS: Overcoming these difficulties has become possible with the introduction of "periosteal pedicle graft" (PPG). The study is a split-mouth design where patients with bilateral, isolated GR defects were treated using two different surgical techniques, i.e., PPG versus the SCTG in obtaining GR coverage. The objective was to analyze the comparative amount of root coverage and the gain in width of attached gingiva achieved by both the techniques. RESULTS: All the clinical parameters evaluated were comparable between SCTG and PPG when recorded at 6 and 9 months posttreatment. CONCLUSION: Both techniques produced satisfactory and predictable results. PPG eliminated the need for the 2nd operative site, lesser intraoperative time, and hence, patient comfort was enhanced.

Multi-target, ensemble-based virtual screening yields novel allosteric KRAS inhibitors at high success rate.

RAS mutations account for >15% of all human tumors, and of these ~85% are due to mutations in a particular RAS gene: KRAS. Recent studies revealed that KRAS harbors four druggable allosteric sites. Here, we have (a) used molecular simulations to generate ensembles of wild type and four major oncogenic KRAS mutants (G12V, G12D, G13D, and Q61H); (b) characterized the druggability of each allosteric pocket in each protein; (c) conducted extensive ensemble-based virtual screening using pocket-tailored ligand libraries; (d) prioritized hits through hierarchical postdocking analysis; and (e) validated predicted hits with NMR. Of the 785 diverse potential hits identified by our in silico analysis, we tested 90 for their ability to bind KRAS using NMR and found that nine cause backbone amide chemical shift perturbations of residues near the functionally responsive switch loops, suggesting potential binding. We conducted detailed biophysical analyses on a novel indole-based compound to demonstrate the potential of our workflow to yield lead compounds. We believe the detailed information documented in this work regarding the druggability profile of each allosteric site and the chemical fingerprints of compounds that target them will serve as vital resources for future structure-based drug design efforts against KRAS, a high-value target for cancer therapy.

Discovery of High-Affinity Noncovalent Allosteric KRAS Inhibitors That Disrupt Effector Binding.

Approximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and notorious oncogene. Mutations that render KRAS constitutively active will lead to uncontrolled cell growth and cancer. However, despite aggressive efforts in recent years, there are no drugs on the market that directly target KRAS and inhibit its aberrant functions. In the current work, we combined structure-based design with a battery of cell and biophysical assays to discover a novel pyrazolopyrimidine-based allosteric KRAS inhibitor that binds to activated KRAS with sub-micromolar affinity and disrupts effector binding, thereby inhibiting KRAS signaling and cancer cell growth. These results show that pyrazolopyrimidine-based compounds may represent a first-in-class allosteric noncovalent inhibitors of KRAS. Moreover, by studying two of its analogues, we identified key chemical features of the compound that interact with a set of specific residues at the switch regions of KRAS and play critical roles for its high-affinity binding and unique mode of action, thus providing a blueprint for future optimization efforts.

Three distinct regions of cRaf kinase domain interact with membrane.

Raf kinases are downstream effectors of small GTPase Ras. Mutations in Ras and Raf are associated with a variety of cancers and genetic disorders. Of the three Raf isoforms, cRaf is most frequently involved in tumor initiation by Ras. Cytosolic Raf is auto-inhibited and becomes active upon recruitment to the plasma membrane. Since the catalytic domain of Raf is its kinase domain, we ask the following: does the kinase domain of Raf has potential to interact with membrane and if yes, what role does the membrane interaction play? We present a model of cRaf kinase domain in complex with a heterogeneous membrane bilayer using atomistic molecular dynamics simulation. We show that the kinase domain of cRaf has three distinct membrane-interacting regions: a polybasic motif (R.RKTR) from the regulatory αC-helix, an aromatic/hydrophobic cluster from the N-terminal acidic region (NtA) and positively charged/aromatic cluster from the activation segment (AS). We show that residues from these regions form an extended membrane-interacting surface that resembles the membrane-interacting residues from known membrane-binding domains. Activating phosphorylatable regions (NtA and AS), make direct contact with the membrane whereas R.RKTR forms specific multivalent salt bridges with PA. PA lipids dwell for longer times around the R.RKTR motif. Our results suggest that membrane interaction of monomeric cRaf kinase domain likely orchestrates the Raf activation process and modulates its function. We show that R.RKTR is a hotspot that interacts with membrane when cRaf is monomeric and becomes part of the interface upon Raf dimerization. We propose that in terms of utilizing a specific hotspot to form membrane interaction and dimer formation, both Raf and its upstream binding partner KRas, are similar.

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs.

Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal ß-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 µs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.

Quality of Life Among Children Who Had Undergone Ventriculoperitoneal Shunt Surgery.

BACKGROUND: Ventriculoperitoneal (VP) shunting is the most common neurosurgical treatment for hydrocephalus. In spite of significant developments in the technology and design of shunt systems, shunt surgery is still associated with morbidity. AIM: To identify the problems faced by children on VP shunt and assess their quality of life (QOL). SETTING AND DESIGN: A cross-sectional exploratory study. MATERIALS AND METHODS: A total of 31 children on VP shunt were selected through consecutive sampling technique, and hydrocephalus outcome questionnaire was used to collect the data, with the converted metric score ranging from 0 to 1. Hydrocephalus due to stroke, hemorrhage, and malignant tumors was excluded. RESULTS: The mean age of patients was 11.51 ± 4.26 years. Headache and generalized pain were the common problems experienced (42%). The mean score of QOL was 0.67 ± 0.21. Among the three domains, cognitive domain was the most affected. Among the clinicoradiological factors, multiple surgeries (P = 0.02) had the most significant impact on QOL. CONCLUSION: Children who underwent VP shunt face various health-related problems in different domains and low QOL. Although cognitive domain was the most affected, multiple surgeries had the most significant impact on QOL. Appropriately focused interventions and holistic management are essential to improve the QOL of children undergoing VP shunt.

A "Tug of War" Maintains a Dynamic Protein-Membrane Complex: Molecular Dynamics Simulations of C-Raf RBD-CRD Bound to K-Ras4B at an Anionic Membrane.

Association of Raf kinase with activated Ras triggers downstream signaling cascades toward regulating transcription in the cells' nucleus. Dysregulation of Ras-Raf signaling stimulates cancers. We investigate the C-Raf RBD and CRD regions when bound to oncogenic K-Ras4B at the membrane. All-atom molecular dynamics simulations suggest that the membrane plays an integral role in regulating the configurational ensemble of the complex. Remarkably, the complex samples a few states dynamically, reflecting a competition between C-Raf CRD- and K-Ras4B- membrane interactions. This competition arises because the interaction between the RBD and K-Ras is strong while the linker between the RBD and CRD is short. Such a mechanism maintains a modest binding for the overall complex at the membrane and is expected to facilitate fast signaling processes. Competition of protein-membrane contacts is likely a common mechanism for other multiprotein complexes, if not multidomain proteins at membranes.