As a fundamental component of the host cellular cytoskeleton, actin is routinely engaged by infecting viruses. Furthermore, viruses from diverse groups, and infecting diverse hosts, have convergently evolved an array of mechanisms for manipulating the actin cytoskeleton for efifcacious infection. An ongoing chorus of research now indicates that the actin cytoskeleton is critical for viral replication at many stages of the viral life cycle, including binding, entry, nuclear localization, genomic transcription and reverse transcription, assembly, and egress/dissemination. Speciifcally, viruses subvert the force-generating and macromolecular scaffolding properties of the actin cytoskeleton to propel viral surifng, internalization, and migration within the cell. Additionally, viruses utilize the actin cytoskeleton to support and organize assembly sites, and eject budding virions for cell-to-cell transmission. It is the purpose of this review to provide an overview of current research, focusing on the various mechanisms and themes of virus-mediated actin modulation described therein.

Cardiac arrhythmias occur in a wide variety of conditions where increased intracellular Ca2+ is known to occur, including myocardial ischemia, cardiac glycoside toxicity, congestive heart failure, and excessive prolongation of action potential duration. The multifunctional Ca2+/calmodulin dependent protein kinaseⅡ(CaM kinase) is an important physiologic target for intracellular Ca2+ and regulates key control points for intracellular Ca2+ homeostasis, including L-type Ca2+ current, release and uptake of Ca2+ from the sarcoplasmic reticulum. Since CaM kinase is uniquely positioned to affect Ca2+ sensitive ionic currents and resultant arrhythmias, CaM kinase may also be an effective antiarrhythmic drug target. The transient inward current (Iti) triggers delayed afterdepolarizations, which are a likely cause of Ca2+ overload arrhythmias. The identity of Iti is controversial, but appears to result from different ionic currents depending on the experimental conditions. These currents are likely activated by intracellular Ca2+ because Iti always follows excessive intracellular Ca2+ concentrations (［Ca2+］i). The studies from our laboratory indicate that three different ［Ca2+］i sensitive currents, which could contribute to Iti, are present in rabbit ventricular myocytes. These currents are the Na+/Ca2+ exchanger, the Ca2+ activated Cl- current, and the Ca2+ activated non-selective cation current. We also found that Iti was suppressed by CaM kinase inhibitory peptides in the presence of physiologic solutions. These data support the hypothesis: CaM kinase can augment Iti during clinically relevant conditions, which are associated with increased ［Ca2+］i, and thus act as a proarrhythmic signaling molecule.

Objective:To investigate the expression of endoplasmic reticulum stress proteins in photoreceptor apoptosis in light-induced retinal degeneration. Methods: Exposure to excessive levels of light induced photoreceptor apoptosis and had been previously used as a model for the study of retinal degeneration. Photoreceptor apoptosis was detected by terminal dUTP transferase nick end labeling (TUNEL). The protein expression levels of ER stress sensors including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2? (eIF2?) and phospho-double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) were examined by immuno-fluorescence and Western blot analysis. Results: Following light exposure, the protein expression levels of GRP78/BiP, caspase-12, phospho-eIF2? and phospho-PERK were up-regulated in a time dependent manner. The up-regulation of these proteins coincided with or preceded the photoreceptor apoptosis. At the peak of their expression, they were mainly located in the photoreceptor inner segments and/or outer nuclear layers (ONL). Conclusion: Activation of endoplasmic reticulum stress proteins appears to play an important role in light-induced retinal degeneration. Therefore endoplasmic reticulum stress modulators could become a strong candidate for a therapeutic agent in treatment of these diseases.

The adapter protein Lamellipodin (Lpd) plays an important role in cell migration. In particular, Lpd mediates lamellipodia formation by regulating actin dynamics via interacting with Ena/VASP proteins. Its RA-PH tandem domain configuration suggests that like its paralog RIAM, Lpd may also mediate particular Ras GTPase signaling. We determined the crystal structures of the Lpd RA-PH domains alone and with an N-terminal coiled-coil region (cc-RA-PH). These structures reveal that apart from the anticipated coiled-coil interaction, Lpd may also oligomerize through a second intermolecular contact site. We then validated both oligomerization interfaces in solution by mutagenesis. A fluorescence-polarization study demonstrated that Lpd binds phosphoinositol with low affinity. Based on our crystallographic and biochemical data, we propose that Lpd and RIAM serve diverse functions: Lpd plays a predominant role in regulating actin polymerization, and its function in mediating Ras GTPase signaling is largely suppressed compared to RIAM.
Actins ; metabolism ; Amino Acid Sequence ; Binding Sites ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Humans ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Mutagenesis ; Phosphatidylinositols ; metabolism ; Polymerization ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Signal Transduction ; ras Proteins ; metabolism

Purpose: Outpatient classified total hip arthroplasty (THA) is a safe option for a select group of patients. An analysis of a national database was conducted to understand the risk factors for unplanned discharge to a skilled nursing facility (SNF) or acute rehabilitation (rehab) after outpatient classified THA. Materials and Methods: A query of the National Surgical Quality Improvement Program (NSQIP) database for THA (Current Procedural Terminology [CPT] 27130) performed from 2015 to 2018 was conducted. Patient demographics, American Society of Anesthesiologists (ASA) classification, functional status, NSQIP morbidity probability, operative time, length of stay (LOS), 30-day reoperation rate, readmission rate, and associated complications were collected. Results: A total of 2,896 patients underwent outpatient classified THA. The mean age of patients was 61.2 years. The mean body mass index (BMI) was 29.6 kg/m 2 with median ASA 2. The results of univariate comparison of SNF/rehab versus home discharge showed that a significantly higher percentage of females (58.7% vs. 46.8%), age >70 years (49.3% vs. 20.9%), ASA ≥3 (58.0% vs. 25.8%), BMI >35 kg/m 2 (23.3% vs. 16.2%), and hypoalbuminemia (8.0% vs. 1.5%) (P<0.0001) were discharged to SNF/rehab. The results of multivariable logistic regression showed that female sex (odds ratio [OR] 1.47; P=0.03), age >70 years (OR 3.08; P=0.001), ASA≥3 (OR 2.56; P=0.001), and preoperative hypoalbuminemia (<3.5 g/dL) (OR 3.76; P=0.001) were independent risk factors for SNF/rehab discharge. Conclusion Risk factors associated with discharge to a SNF/rehab after outpatient classified THA were identified. Surgeons will be able to perform better risk stratification for patients who may require additional postoperative intervention.

Purpose: The number of obese patients seeking total hip arthroplasty (THA) continues to expand despite body mass index (BMI) cutoffs. We sought to determine the outcomes of THA in the morbidly obese patient, and hypothesized they would have comparable outcomes to two cohorts of obese, and normal weight patients. Materials and Methods: THA performed on morbidly obese patients (BMI >40 kg/m2 ) at a single academic center from 2010 until 2020 were retrospectively reviewed. Eighty morbidly obese patients were identified, and matched in a 1:3:3 ratio to control cohorts with BMI 30-40 kg/m2 and BMI <30 kg/m2 . Acute postoperative outcomes and BMI change after surgery were evaluated for clinical significance with univariate and regression analyses. Cox proportional hazard ratio was calculated to evaluate prosthetic joint infection (PJI) and revision surgery through follow-up. Mean follow-up was 3.9 years. Results: In the acute postoperative period, morbidly obese patients trended towards increased hospital length of stay, facility discharge and 90-day hospital returns. At final follow-up, a higher percentage of morbidly obese patients had clinically significant (>5%) BMI loss; however, this was not significant. Cox hazard ratio with BMI <30 kg/m 2 as a reference demonstrated no significant difference in survival to PJI and all-cause revision in the morbidly obese cohort. Conclusion Morbidly obese patients (BMI >40 kg/m2 ) require increased resource expenditure in the acute postoperative period. However, they are not inferior to the control cohorts (BMI <30 kg/m2 , BMI 30-40 kg/m2 ) in terms of PJI or all-cause revisions at mid-term follow-up.

Asthma ; complications ; therapy ; Humans ; Invasive Pulmonary Aspergillosis ; complications ; drug therapy ; Male ; Middle Aged ; Pulmonary Disease, Chronic Obstructive ; complications ; therapy

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45γ [corrected], revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45γ [corrected] identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45γ [corrected] is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.
Amino Acid Motifs ; Animals ; Apoptosis ; radiation effects ; CDC2 Protein Kinase ; Cell Cycle ; Cell Survival ; Crystallography, X-Ray ; Cyclin B ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinases ; HeLa Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Mice ; Mutagenesis, Site-Directed ; Mutation, Missense ; Proliferating Cell Nuclear Antigen ; metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Protein Structure, Quaternary ; Ultraviolet Rays

Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupled-enzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PP(i)), which is generated during nascent strand synthesis. The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid high-throughput drug screening targeting anti-HIV therapies due to its high speed and convenience. Moreover, this assay can be used to measure primase activity in an easy and sensitive manner, which suggests that this novel approach could be wildly used to analyze the activity of PP(i)-generated and ATP-free enzyme reactions.
Anti-HIV Agents ; pharmacology ; Colorimetry ; Diphosphates ; analysis ; metabolism ; Drug Evaluation, Preclinical ; HIV ; drug effects ; enzymology ; HIV Reverse Transcriptase ; analysis ; antagonists & inhibitors ; metabolism ; Humans ; In Vitro Techniques ; Nevirapine ; pharmacology ; Reverse Transcriptase Inhibitors ; pharmacology ; Sequence Analysis, DNA ; Thymine Nucleotides ; metabolism

Enterovirus 71 (EV71), one of the major causative agents for hand-foot-and-mouth disease (HFMD), has caused more than 100 deaths among Chinese children since March 2008. The EV71 genome encodes an RNAdependent RNA polymerase (RdRp), denoted 3D(pol), which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics. Here we report the crystal structures of EV71 RdRp (3D(pol)) and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4 Å resolution. The structure of EV71 RdRp (3D(pol)) has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp. And the EV71 RdRp (3D(pol)) complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding. The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus (FMDV) 3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase. These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.
Amino Acid Sequence ; Child ; China ; epidemiology ; Crystallography, X-Ray ; Drug Discovery ; Enterovirus A, Human ; chemistry ; enzymology ; Hand, Foot and Mouth Disease ; drug therapy ; epidemiology ; virology ; Humans ; Models, Molecular ; Molecular Sequence Data ; Molecular Targeted Therapy ; Protein Conformation ; Protein Folding ; RNA Replicase ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Substrate Specificity