Repeated birth injuries lead to long-term pelvic floor muscle dysfunction in the preclinical rat model

BackgroundVaginal childbirth is a key risk factor for pelvic floor muscle injury and dysfunction, and subsequent pelvic floor disorders. Multiparity further exacerbates these risks. Using the rat model, validated for the studies of the human pelvic floor muscles, we have previously identified that a single simulated birth injury results in pelvic floor muscle atrophy and fibrosis.ObjectiveTo test the hypothesis that multiple birth injuries would further overwhelm the muscle regenerative capacity, leading to functionally relevant pathological alterations long-term.Study designSprague-Dawley rats underwent simulated birth injury and were allowed to recover for 8 weeks before undergoing additional birth injury. Animals were sacrificed at acute (3 and 7 days postinjury), subacute (21, 28, and 35 days postinjury), and long-term (8 and 12 weeks postinjury) time points post second injury (N=3-8/time point), and the pubocaudalis portion of the rat levator ani complex was harvested to assess the impact of repeated birth injuries on muscle mechanical and histomorphological properties. The accompanying transcriptional changes were assessed by a customized NanoString panel. Uninjured animals were used as controls. Data with a parametric distribution were analyzed by a 2-way analysis of variance followed by post hoc pairwise comparisons using Tukey's or Sidak's tests; nonparametrically distributed data were compared with Kruskal-Wallis test followed by pairwise comparisons with Dunn's test. Data, analyzed using GraphPad Prism v8.0, San Diego, CA, are presented as mean ± standard error of the mean or median (range).ResultsFollowing the first simulated birth injury, active muscle force decreased acutely relative to uninjured controls (12.9±0.9 vs 25.98±2.1 g/mm2, P<.01). At 4 weeks, muscle active force production recovered to baseline and remained unchanged at 8 weeks after birth injury (P>.99). Similarly, precipitous decrease in active force was observed immediately after repeated birth injury (18.07±1.2 vs 25.98±2.1 g/mm2, P<.05). In contrast to the functional recovery after a single birth injury, a long-term decrease in muscle contractile function was observed up to 12 weeks after repeated birth injuries (18.3±1.6 vs 25.98±2.1 g/mm2, P<.05). Fiber size was smaller at the long-term time points after second injury compared to the uninjured group (12 weeks vs uninjured control: 1485 (60.7-5000) vs 1989 (65.6-4702) μm2, P<.0001). The proportion of fibers with centralized nuclei, indicating active myofiber regeneration, returned to baseline at 8 weeks post-first birth injury, (P=.95), but remained elevated as far as 12 weeks post-second injury (12 weeks vs uninjured control: 7.1±1.5 vs 0.84±0.13%, P<0.0001). In contrast to the plateauing intramuscular collagen content after 4 weeks post-first injury, fibrotic degeneration increased progressively over 12 weeks after repeated injury (12 weeks vs uninjured control: 6. 7±1.1 vs 2.03±0.2%, P<.001). Prolonged expression of proinflammatory genes accompanied by a greater immune infiltrate was observed after repeated compared to a single birth injury.ConclusionOverall, repeated birth injuries lead to a greater magnitude of pathological alterations compared to a single injury, resulting in more pronounced pelvic floor muscle degeneration and muscle dysfunction in the rat model. The above provides a putative mechanistic link between multiparity and the increased risk of pelvic floor dysfunction in women

Myricetin Inhibits Islet Amyloid Polypeptide (IAPP) Aggregation and Rescues Living Mammalian Cells from IAPP Toxicity

The aggregation of the amyloidogenic polypeptide IAPP (Islet Amyloid Polypeptide, amylin) is believed to play a direct role in the death of pancreatic β-islet cells in type II diabetes. Preventing the initial aggregation event of IAPP is one strategy for slowing, and possibly preventing, the progression of this disease. Here, we investigate myricetin’s potential as an inhibitor of IAPP aggregation. We show that myricetin prevented thioflavin T binding in a concentration dependent manner. Atomic force microscopy revealed that myricetin prevented fiber formation under rigorous conditions conducive to forming IAPP aggregates. Using an IAPP-EGFP (Enhanced Green Fluorescent Protein) protein construct, we find that high concentrations of myricetin slowed the in vivo aggregation of IAPP-EGFP. Myricetin was also found to rescue living mammalian cells from the toxic effects of IAPP. These results indicate that myricetin is a strong inhibitor of IAPP amyloid aggregation and a potential lead molecule for the development of an amyloid inhibiting therapeutic

Ready, Set, Fuse! The Coronavirus Spike Protein and Acquisition of Fusion Competence

Coronavirus-cell entry programs involve virus-cell membrane fusions mediated by viral spike (S) proteins. Coronavirus S proteins acquire membrane fusion competence by receptor interactions, proteolysis, and acidification in endosomes. This review describes our current understanding of the S proteins, their interactions with and their responses to these entry triggers. We focus on receptors and proteases in prompting entry and highlight the type II transmembrane serine proteases (TTSPs) known to activate several virus fusion proteins. These and other proteases are essential cofactors permitting coronavirus infection, conceivably being in proximity to cell-surface receptors and thus poised to split entering spike proteins into the fragments that refold to mediate membrane fusion. The review concludes by noting how understanding of coronavirus entry informs antiviral therapies

Cleavage of the SARS Coronavirus Spike Glycoprotein by Airway Proteases Enhances Virus Entry into Human Bronchial Epithelial Cells In Vitro

Background: Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases. Methodology/Principal Findings: Purified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment. Conclusions/Significance: These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV. © 2009 Kam et al.published_or_final_versio

Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly

Characterization of the avian trojan gene family reveals contrasting evolutionary constraints

"Trojan" is a leukocyte-specific, cell surface protein originally identified in the chicken. Its molecular function has been hypothesized to be related to anti-apoptosis and the proliferation of immune cells. The Trojan gene has been localized onto the Z sex chromosome. The adjacent two genes also show significant homology to Trojan, suggesting the existence of a novel gene/protein family. Here, we characterize this Trojan family, identify homologues in other species and predict evolutionary constraints on these genes. The two Trojan-related proteins in chicken were predicted as a receptor-type tyrosine phosphatase and a transmembrane protein, bearing a cytoplasmic immuno-receptor tyrosine-based activation motif. We identified the Trojan gene family in ten other bird species and found related genes in three reptiles and a fish species. The phylogenetic analysis of the homologues revealed a gradual diversification among the family members. Evolutionary analyzes of the avian genes predicted that the extracellular regions of the proteins have been subjected to positive selection. Such selection was possibly a response to evolving interacting partners or to pathogen challenges.We also observed an almost complete lack of intracellular positively selected sites, suggesting a conserved signaling mechanism of the molecules. Therefore, the contrasting patterns of selection likely correlate with the interaction and signaling potential of the molecules

An in vivo platform for identifying inhibitors of protein aggregation

Protein aggregation underlies an array of human diseases, yet only one small molecule therapeutic has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of IAPP aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation