Fatigue in multiple sclerosis: cross-sectional correlation with brain MRI findings in 71 patients.

Fatigue is an unexplained but common and disabling symptom in MS. We assessed fatigue in 71 patients with MS and identified MS-fatigue (MSF) and MS-nonfatigue (MSNF) groups. Fatigue severity did not correlate with regional or global MRI plaque load or atrophy assessed by conventional sequences. No significant differences were noted in any MRI measures between MSF and MSNF groups. We suggest that brain MRI disease topography or severity does not explain fatigue in MS and that fatigue is likely due to mechanisms poorly characterized by conventional MRI.     

Chemokine pattern in relation to disease state in human immunodeficiency virus-infected children.

Although beta chemokines can block human immunodeficiency virus (HIV) entry into target cells, their role in HIV disease progression is controversial. To determine the association of RANTES with HIV disease state, we examined constitutive mRNA expression by reverse-transcribed polymerase chain reaction (RT-PCR) in peripheral blood mononuclear cells (PBMCs) and induction of RANTES secretion by enzyme-linked immunosorbent assay (ELISA) in anti-CD3 monoclonal antibody (MAb)-stimulated cultures of PBMCs, and in CD4+ and CD8+ T cell subsets of 17 HIV-infected children. In comparison with uninfected subjects, PBMCs of HIV-infected children were deficient in both constitutive RANTES mRNA expression as well as in stimulus-induced RANTES production. Children in clinical category C were found to be more deficient than children in clinical category A. Expression of RANTES mRNA in PMBCs was inversely correlated with plasma virus load and correlated directly with CD4+ T cell counts. In T cell subsets, RANTES production was equivalent between CD4+ and CD8+ T cells in patients and controls but CD8+ T cells of children in clinical category A produced higher RANTES levels than those of children in clinical category C. The beta-chemokine RANTES may play an important role in slowing clinical disease progression in HIV-infected children.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK’s multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.

Clathrin-mediated endocytosis (CME) regulates the uptake of nutrients, growth factors, adhesion molecules, transmembrane ion channels, transporters, signaling receptors, and other ligand–receptor complexes. Thus, CME plays a crucial role in cell homeostasis by constantly remodeling and controlling the composition of the plasma membrane (PM) in response to various extracellular and intracellular stimuli. Consequently, dysregulated CME has been extensively linked to disease (1, 2). CME is a multistep process involving 1) “priming,” i.e., the regulated and localized activation of clathrin assembly proteins, predominantly adaptor protein 2 (AP2) complexes at the PM; 2) initiation of clathrin assemblies; 3) stabilization of clathrin-coated pits (CCPs) in the form of a macromolecular complex; 4) productive CCP growth and maturation, which culminates in 5) fission and the release of newly formed clathrin-coated vesicles (CCVs) into the cytosol (3, 4). Many nascent CCPs fail to complete this multistep process and instead rapidly disassemble as early or late abortive pits (5–7). In addition to the major coat proteins, clathrin and AP2, successful completion of CME requires the activities of a myriad of endocytic accessory proteins (EAPs). These EAPs, many of which are multidomain proteins, encode multiple biochemical activities, including curvature generation and sensing, cargo recruitment, scaffolding, and lipid modification (1, 4, 8).The activities of EAPs, or of their individual functional domains, were largely identified through in vitro biochemical assays. The in vivo functions of EAPs in CME are frequently measured by cargo uptake, which scores the net accumulation of cargo inside cells, but lacks the temporal resolution and sensitivity to capture early steps or the regulation of CCP growth. Indeed, several publications showed that measurements of cargo uptake alone are unable to reveal alterations in the early kinetics of CCP maturation caused by the absence of one or several EAPs (9–11). An alternative approach to assess stage-specific EAP functions has been to measure the temporal hierarchy of their recruitment to CCPs. Using a pH-sensitive fluorescent cargo to mark scission events, Merrifield and colleagues (7) measured the recruitment profiles of 34 EAPs to CCPs with high temporal resolution, providing insight into their sequential roles in CME. The study also highlighted the nonuniform molecular composition of individual CCPs. However, its major limitation was that, prior to the advent of genome-editing technologies, fluorescently labeled EAPs were transiently overexpressed, which is especially problematic given the likely competition arising from widely shared protein interaction domains and binding motifs. Moreover, as individually tracked CCPs, referred to hereafter as intensity traces, were aligned to the terminal fission event, early molecular signatures of these EAPs at CCPs were missed, especially given the heterogeneity of CCP lifetimes (7, 12). Nonetheless, these and similar pioneering studies in yeast (13, 14) have led to the concept that the CME machinery is organized into functional modules that act sequentially, and in a stereotypic manner, during CCP maturation (1, 4).It has become increasingly evident that CME and cell surface receptor signaling are reciprocally regulated by feedback loops (15–20). This has led to the understanding that CME is not a passive process, but that it can respond and adapt to multiple inputs. Moreover, consistent with the essential role of CME in cellular physiology, the process is robust and exhibits plasticity, in that compensatory mechanisms can restore CME even when individual stages of CCP maturation are significantly perturbed (9, 19, 21). This robustness and plasticity likely derive from the overlapping functions, and thus redundancies, of EAPs regulating clathrin assembly and CCP maturation.Although the individual activities of many EAPs have been extensively studied, many controversies exist as to how, and at which stage(s), these activities contribute to the overall process of CME (22–29). In part, these controversies may reflect the wide range of cell types, experimental systems, and assays used to study EAP function. A more comprehensive analysis under identical experimental conditions has never been undertaken and could lead to a better understanding of the functional hierarchy and the role played by each EAP during CME. Such a systematic analysis requires a readout that directly measures discrete early stages of CCP nucleation, initiation, and maturation. Confocal and total internal reflection fluorescence microscopy (TIR-FM)-based live-cell imaging (5, 6, 30, 31) paired with unbiased and high-content image analyses (6, 9, 11, 32) are essential for the detection of these alterations in early regulatory stages of CCV formation.Equipped with live cell TIR-FM and computer vision tools to quantify several stage-specific parameters of CME, including rates of CCP initiation, stabilization, and maturation (9, 12, 33), we have quantified the knockdown (kd) effect (i.e., phenotype) of most known or suspected EAPs on CCP dynamics within a uniform and rigorous experimental framework. Based on previous studies and their biochemically defined activities, the 67 proteins studied can be assigned to functionally distinct modules (1, 4, 8). Here, we have clustered EAPs based on their phenotypic signatures. Interestingly, these phenotypically defined clusters do not overlap with the biochemically defined modules. Our results highlight the functional complexity of protein–protein interactions and EAP activity during CCV formation. The overlapping activities and hence functional redundancies of multidomain EAPs provide a mechanistic basis for the robustness of CME and the ability of cells to counter defects in CCP maturation by activation of compensatory mechanisms.     

Evaluation of bone marrow reticulin in patients with chronic immune thrombocytopenia treated with eltrombopag: Data from the EXTEND study

Thrombopoietin receptor agonists, which raise platelet counts in patients with chronic immune thrombocytopenia, may be associated with increases in bone marrow (BM) reticulin. Patients with chronic immune thrombocytopenia participating in the Eltrombopag Extended Dosing (EXTEND) study underwent BM biopsies to identify clinically relevant BM fibrosis‐related increases. Specimens were centrally reviewed by 2 hematopathologists. Two hundred thirty‐two biopsy specimens were collected from 117 patients treated for ≤5.5 years. Moderate to marked reticulin fibrosis was found in 2 patients. After withdrawing from the study, the biopsy of 1 patient reverted to normal. There were no other pathologic changes identified among on‐treatment specimens, and no pattern of abnormal reticulin deposition associated with eltrombopag treatment was evident. Am. J. Hematol. 90:598–601, 2015. © 2015 Wiley Periodicals, Inc.     

Health Promotion Board–Ministry of Health Clinical Practice Guidelines: Falls Prevention among Older Adults Living in the Community

The Health Promotion Board (HPB) has developed the Clinical Practice Guidelines (CPG) on Falls Prevention among Older Adults Living in the Community to provide health professionals in Singapore with recommendations for evidence-based assessments and interventions for falls prevention. This article reproduces the introduction and executive summary of the key recommendations from the HPB-MOH CPG on Falls Prevention among Older Adults Living in the Community for the information of SMJ readers. The chapters and page numbers mentioned in the reproduced extract refer to the full text of the guidelines, which are available from the Health Promotion Board website: http://www.hpb.gov. sg/cpg-falls-prevention. The recommendations should be used with reference to the full text of the guidelines. Following this article are multiple choice questions based on the full text of the guidelines.     

Achieving an esthetic smile with fixed and removal prosthesis using extracoronal castable precision attachments

Satisfactory restoration in a patient with a partially edentulous situation can be challenging especially when unilateral or bilateral posterior segment of teeth is missing. Successful restoration can be done with various conventional and contemporary treatment options. One such treatment modality is attachment-retained cast partial dentures. A key to success for an attachment retained cast partial denture is the strategic selection of teeth for retention. This clinical report discusses rehabilitation of a patient with the help of a combined prosthesis in the upper arch and stud retained overdenture in the lower arch.Key Words: Overdenture, precision attachment, preci-vertix attachment