The RNA-binding protein La-related protein 1 (LARP1) plays a central role in ribosome biosynthesis. Its C-terminal DM15 region binds the 7-methylguanosine (m7G) cap and 5' terminal oligopyrimidine (TOP) motif characteristic of transcripts encoding ribosomal proteins and translation factors. Under the control of mammalian target of rapamycin complex 1 (mTORC1), LARP1 regulates translation of these transcripts. Characterizing the dynamics of DM15-TOP recognition is essential to understanding this fundamental biological process. We use molecular dynamics simulations, biophysical assays, and X-ray crystallography to reveal the mechanism of DM15 binding to TOP transcripts. Residues C-terminal to the m7G-binding site play important roles in cap recognition. Furthermore, we show that the unusually static pocket that recognizes the +1 cytosine characteristic of TOP transcripts drives binding specificity. Finally, we demonstrate that the DM15 pockets involved in TOP-specific m7GpppC-motif recognition are likely druggable. Collectively, these studies suggest unique opportunities for further pharmacological development.

The mechanism by which monosodium urate (MSU) crystals induce inflammation is not completely understood. Few studies have shown that MSU is capable of stimulating the release of IL-1β in the absence of LPS treatment. The purinergic P2X7 receptor is involved in the release of IL-1β in inflammatory settings caused by crystals, as is the case in silicosis.

M20 aminopeptidases, such as Peptidase T (PepT), are implicated in the hydrolysis of oligopeptides during the terminal stages of protein degradation pathway to maintain turnover. Therefore, specific inhibition of PepT bores well for the development of novel next-generation antileishmanials. This work describes the metal dependence, substrate preferences and inhibition of PepT, and demonstrates in detail the role of its two conserved substrate binding residues.

Cardiac dysfunction is a common adverse effect of subarachnoid hemorrhage (SAH). Autopsy of SAH patients shows immunocyte infiltration into the heart. In this study, a SAH model of endovascular perforation was performed in adult male mice in order to test whether SAH causes cardiac dysfunction in non-primary cardiac disease young adult male mice and whether immune response mediates SAH induced cardiac and neurological deficit. Splenectomy was performed on a subpopulation of mice one week prior to induction of the SAH. Neurological functional tests, transthoracic Doppler echocardiography, immunofluorescent staining, and flow cytometry were performed to investigate neurological and cardiac function and immune/inflammatory effects of SAH in mice with or without splenectomy. We found that SAH significantly induces ventricular fibrillation and cardiac dysfunction identified by significantly reduced left ventricular ejection fraction, left ventricular fractional shortening, decreased heart rate, as well as increased macrophage and neutrophil infiltration into heart and inflammatory factor expression in the heart compared to sham control mice. SAH also induces neurological deficit, increases astrocyte and microglial activity, and inflammatory cell infiltration into brain as well as up-regulates inflammatory factor expression in the brain tissue. Splenectomy not only significantly improves neurological function, but also reduces cardiac dysfunction compared to SAH alone mice. Splenectomy in SAH mice significantly reduces inflammatory cell infiltration, and decreases NADPH oxidase-2 and macrophage chemokine protein-1 expression in heart and brain when compared to non-splenectomy SAH mice. Our data suggest that, SAH induces acute cardiac dysfunction in non-primary cardiac disease mice. Secondary immune response may play an important role in mediating brain-heart damage after SAH.

Sensorineural hearing loss due to aging, noise exposure, trauma or drug ototoxicity is irreversible because cochlear hair cells and neurons cannot regenerate. Recently, therapeutic strategies involving nanoparticles have been developed as innovative drug delivery systems. Thermodynamically stable liquid crystalline nanoparticles based on the polar lipid glycerol monooleate (GMO NP, cubosomes), nontoxic and able to encapsulate both hydrophilic and hydrophobic compounds, were produced and tested for biocompatibility in an immortalized Organ of Corti derived cell line (OC-k3), through cell viability and cytomorphological assays, and Western blot expression profiles of apoptotic markers. Overall, the GMO NP were biocompatible in OC-k3 at the doses and time tested, supporting previous data obtained in a neuronal cell line (PC12). The results encourage further tests on GMO NP-mediated drug release with improved target specificity and could be useful to develop innovative therapies against sensorineural hearing loss.

Biologics are potentially immunogenic and can elicit immune response. Complex biologics, such as bispecific antibodies or multi-domain molecules can induce anti-drug antibodies (ADA) with specificity to different domains. Domain specific ADAs may differently affect drug efficacy and safety, and thus, characterization of ADA domain specificity has become a regulatory expectation for multi-domain biologics. Unlike well-established methods for screening, confirmation, titer and neutralizing ADA detection, characterization of ADA domain specificity is an emerging field. The conventional approach for determination of ADA domain specificity is a competitive inhibition with domain-containing molecules. When developing a conventional domain specificity assay for moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin, comprised of two functional domains (CD22-binding fragment and truncated Pseudomonas exotoxin A (PE38), we encountered a bioanalytical challenge. The method was able to detect immunodominant anti-PE38 (ADA-PE) but generated false negative results for low abundant CD22-binding domain ADA (ADA-BD) in a polyclonal sample. Troubleshooting experiments using control samples with varying levels of each ADA subtype demonstrated that a major factor for successful ADA identification was the ratio of the ADA signals contributed by each ADA subtype. To overcome this unique bioanalytical challenge, we developed a novel approach, which ensures detection of a domain-specific ADA subtype regardless of its relative level in a polyclonal ADA sample by evaluating signal inhibition by a respective domain-containing molecule at the condition when signals from all other ADAs are fully blocked. The method has been used for characterization of ADA domain specificity in moxetumomab pasudotox clinical trials, including study 1053, the pivotal Phase III study in hairy cell leukemia patients. It allowed for successful detection of ADA-BD in the presence of immunodominant ADA-PE, enabling accurate determination of domain specificity for moxetumomab pasudotox. The results demonstrated that the method was superior than the conventional approach. The method could be applied broadly to other biologics with two or more domains when there is a need to detect a minor ADA subtype in polyclonal samples.

Zika virus (ZIKV) is an emergent flavivirus associated with severe neurological disorders. ZIKV NS3 protein is a viral protease that cleaves the ZIKV polyprotein precursor into individual viral proteins. In this study, we found that ZIKV NS3 by itself exhibited mitochondrial localization, which was quite different from its endoplasmic reticulum (ER) localization in ZIKV-infected cells. We screened viral proteins and identified NS2B as the bona fide recruiter of NS3 to the ER. The NS2B C-terminal tail interacted with NS3 protease domain to retain NS3 on the ER. β-Sheet motifs that formed between NS2B and the NS3 protease domain played important roles in their interaction, while mutation in the β-strand of NS2B attenuated NS2B-NS3 interaction and impaired the ability of NS3 protease to cleave the polyprotein precursor into multiple viral proteins. Consequently, NS2B mutations led to severe inhibition of ZIKV replication and production due to insufficient NS3 protease activity. In summary, our study reveals the critical role of NS2B in NS3 recruitment and protease function and provides mechanistic insight into ZIKV replication.

Acetaminophen (APAP) is one of the most commonly used drugs worldwide, and APAP-induced liver injury is the most frequent cause of acute liver failure in developed countries. However, the mechanisms of APAP-induced hepatotoxicity are not well understood, and treatment options for the disorder are very limited. Here, we show that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a major mediator of APAP-induced liver injury in mice, and its blockade markedly ameliorates the liver failure. In APAP-treated mice, TRAIL was expressed in the liver, spleen, and peripheral blood primarily by CD11b+Gr1+ neutrophils. The concentration of soluble TRAIL in the blood, and the frequencies of TRAIL+ leukocytes in the spleen and liver positively correlated with the severity of liver injury. APAP sensitized hepatocytes to TRAIL-induced apoptosis by upregulating the expression of the TRAIL receptor DR5 (death receptor 5), presumably through its transcription factor CHOP (C/EBP homologous protein). Importantly, blocking TRAIL with a soluble DR5-Fc fusion protein (sDR5-Fc) significantly attenuated APAP-induced liver injury, the hepatic infiltration of leukocytes, the levels of inflammatory cytokines, and the mortality of mice. When administered alongside N-acetylcysteine, sDR5-Fc further protected against APAP-induced acute liver injury. Thus, the TRAIL-DR5 signaling pathway plays a key role in APAP-induced liver inflammation and failure, and its blockade represents an effective new strategy to treat the liver disease.

The reduction in muscle mass and strength with age, sarcopenia, is a prevalent condition among the elderly, linked to skeletal muscle dysfunction and cell apoptosis. We demonstrated that testosterone protects against H2O2-induced apoptosis in C2C12 muscle cells. Here, we analyzed the effect of testosterone on mitochondrial gene expression in C2C12 skeletal muscle cells. We found that testosterone increases mRNA expression of genes encoded by mitochondrial DNA, such as NADPH dehydrogenase subunit 1 (ND1), subunit 4 (ND4), cytochrome b (CytB), cytochrome c oxidase subunit 1 (Cox1) and subunit 2 (Cox2) in C2C12. Additionally, the hormone induced the expression of the nuclear respiratory factors 1 and 2 (Nrf-1 and Nrf-2), the mitochondrial transcription factors A (Tfam) and B2 (TFB2M), and the optic atrophy 1 (OPA1). The simultaneous treatment with testosterone and the androgen receptor antagonist, Flutamide, reduced these effects. H2O2-oxidative stress induced treatment, significantly decreased mitochondrial gene expression. Computational analysis revealed that mitochondrial DNA contains specific sequences, which the androgen receptor could recognize and bind, probably taking place a direct regulation of mitochondrial transcription by the receptor. These findings indicate that androgen plays an important role in the regulation of mitochondrial transcription and biogenesis in skeletal muscle.

There is a lack of systematic descriptions and characterization of strokes and their effects in both the cerebral hippocampus and cortex. Shuxuetong (SXT) injection was reported to have good therapeutic effects in the clinic; therefore, it was selected as a drug intervention method for cerebral ischemia repair in rat models. The aim of this study was to understand the features of molecules and pathways and to reveal key processes of SXT repair.

No abstract available

Circadian clocks coordinate physiological and behavioral rhythms that allow the organism to anticipate and adapt to daily changes in environment. The clock-driven cellular oscillations are highly tissue specific to efficiently fine-tune local signaling, manage energy use and segregate incompatible processes. In most peripheral tissues, food acts as the main cue that entrains the oscillations to external time. Food intake and energy balance are under control of endocannabinoid (EC) signaling. Despite this obvious link between the circadian and EC systems, evidence for their interaction started to emerge only recently. We used targeted lipidomics to analyze circadian variations in EC tone in rat plasma, liver and adrenal tissue. The results provide the evidence that ECs, monoacylglycerols, N-acylethanolamines and their precursors oscillate with a tissue-specific circadian phase in plasma and liver. We then identified a set of rhythmically expressed genes likely responsible for the variations in EC tissue tone. In contrast to the liver, EC levels did not oscillate in the adrenal glands. Instead, we revealed that local EC receptor genes are under circadian regulation. To explore the impact of metabolic signals on expression of these genes, we used daytime-restricted feeding schedule. We subsequently showed that daytime feeding strongly suppressed liver-expressed fatty acid binding protein 5 (Fabp5) and adrenal-expressed non-canonical endocannabinoid receptors Gpr55 and Trpv1, whereas it upregulated liver-expressed Trpv1 and glycerophosphodiester phosphodiesterase 1 (Gde1). Our results reveal tissue-specific mechanisms involved in interaction between endocannabinoid signaling, circadian system and metabolism.

Highly elevated plasma levels of interleukin-10 (IL-10) are causally associated with "Disappearing HDL Syndrome" and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages.

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A-CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A-binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A-CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.

Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear size and shape are important factors in regulating the mechanical properties of cells during their migration through such tight spaces. At the onset of migratory behavior, cells often initiate the expression of vimentin, an intermediate filament protein that polymerizes into networks extending from a juxtanuclear cage to the cell periphery. However, the role of vimentin intermediate filaments (VIFs) in regulating nuclear shape and mechanics remains unknown. Here, we use wild-type and vimentin-null mouse embryonic fibroblasts to show that VIFs regulate nuclear shape and perinuclear stiffness, cell motility in 3D, and the ability of cells to resist large deformations. These changes increase nuclear rupture and activation of DNA damage repair mechanisms, which are rescued by exogenous reexpression of vimentin. Our findings show that VIFs provide mechanical support to protect the nucleus and genome during migration.

People with recurrent or drug-resistant TB require long courses of intramuscular injections. We evaluate a novel system in which patient-nominated lay carers were trained to deliver intramuscular injections to patients in their own homes.

Lowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn't clear.

Autophagy is an intracellular catabolic process prominent in starvation, aging and disease. Neuronal autophagy is particularly important, as it affects the development and function of the nervous system, and is heavily implicated in neurodegenerative disease. Nonetheless, how autophagy is regulated in neurons remains poorly understood. Using an unbiased proteomics approach, we demonstrate that the primary initiator of autophagy, the UNC-51/ULK kinase, is negatively regulated by the ubiquitin ligase RPM-1. RPM-1 ubiquitin ligase activity restricts UNC-51 and autophagosome formation within specific axonal compartments, and exerts effects broadly across the nervous system. By restraining UNC-51 activity, RPM-1 inhibits autophagosome formation to affect axon termination, synapse maintenance and behavioral habituation. These results demonstrate how UNC-51 and autophagy are regulated subcellularly in axons, and unveils a mechanism for restricting initiation of autophagy across the nervous system. Our findings have important implications beyond nervous system development, given growing links between altered autophagy regulation and neurodegenerative diseases.

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.

Deep-sea sponge grounds are vulnerable marine ecosystems, which through their benthic-pelagic coupling of nutrients, are of functional relevance to the deep-sea realm. The impact of fishing bycatch is here evaluated for the first time at a bathyal, sponge-dominated ecosystem in the high seas managed by the Northwest Atlantic Fisheries Organization. Sponge biomass surfaces created from research survey data using both random forest modeling and a gridded surface revealed 231,140 t of sponges in the area. About 65% of that biomass was protected by current fisheries closures. However, projections of trawling tracks estimated that the sponge biomass within them would be wiped out in just 1 year by the current level of fishing activity if directed on the sponges. Because these sponges filter 56,143 ± 15,047 million litres of seawater daily, consume 63.11 ± 11.83 t of organic carbon through respiration, and affect the turnover of several nitrogen nutrients, their removal would likely affect the delicate ecological equilibrium of the deep-sea benthic ecosystem. We estimated that, on Flemish Cap, the economic value associated with seawater filtration by the sponges is nearly double the market value of the fish catch. Hence, fishery closures are essential to reach sponge conservation goals as economic drivers cannot be relied upon.