by
Kimberly M. Ramonell;
Wenxiao Zhang;
Annette Hadley;
Ching-wen Chen;
Katherine T. Fay;
John D. Lyons;
Nathan J. Klingensmith;
Kevin McConnell;
Craig Coopersmith;
Mandy L Ford
Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4 + and CD8 + T cells and CD4 + central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4 + and CD8 + T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4 + T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients.
Pulmonary hypertension (PH) is a serious disorder that causes significant morbidity and mortality. The pathogenesis of PH involves complex derangements in multiple pathways including reductions in peroxisome proliferator-activated receptor gamma (PPARγ). Hypoxia, a common PH stimulus, reduces PPARγ in experimental models. In contrast, activating PPARγ attenuates hypoxia-induced PH and endothelin 1 (ET-1) expression. To further explore mechanisms of hypoxia-induced PH and reductions in PPARγ, we examined the effects of hypoxia on selected microRNA (miRNA or miR) levels that might reduce PPARγ expression leading to increased ET-1 expression and PH. Our results demonstrate that exposure to hypoxia (10% O 2 ) for 3-weeks increased levels of miR-27a and ET-1 in the lungs of C57BL/6 mice and reduced PPARγ levels. Hypoxia-induced increases in miR-27a were attenuated in mice treated with the PPARγ ligand, rosiglitazone (RSG, 10 mg/kg/d) by gavage for the final 10 d of exposure. In parallel studies, human pulmonary artery endothelial cells (HPAECs) were exposed to control (21% O 2 ) or hypoxic (1% O 2 ) conditions for 72 h. Hypoxia increased HPAEC proliferation, miR-27a and ET-1 expression, and reduced PPARγ expression. These alterations were attenuated by treatment with RSG (10 μM) during the last 24 h of hypoxia exposure. Overexpression of miR-27a or PPARγ knockdown increased HPAEC proliferation and ET-1 expression and decreased PPARγ levels, whereas these effects were reversed by miR-27a inhibition. Further, compared to lungs from littermate control mice, miR-27a levels were upregulated in lungs from endothelial-targeted PPARγ knockout (ePPARγ KO) mice. Knockdown of either SP1 or E GR1 was sufficient to significantly attenuate miR-27a expression in HPAECs. Collectively, these studies provide novel evidence that miR-27a and PPARγ mediate mutually repressive actions in hypoxic pulmonary vasculature and that targeting PPARγ may represent a novel therapeutic approach in PH to attenuate proliferative mediators that stimulate proliferation of pulmonary vascular cells.
Y-box binding protein 1 (YBX1 or YB1) is a therapeutically relevant oncoprotein capable of RNA and DNA binding and mediating protein–protein interactions that drive proliferation, stemness, and resistance to platinum-based therapies. Given our previously published findings, the potential for YB1-driven cisplatin resistance in medulloblastoma (MB), and the limited studies exploring YB1-DNA repair protein interactions, we chose to investigate the role of YB1 in mediating radiation resistance in MB. MB, the most common pediatric malignant brain tumor, is treated with surgical resection, cranio-spinal radiation, and platinum-based chemotherapy, and could potentially benefit from YB1 inhibition. The role of YB1 in the response of MB to ionizing radiation (IR) has not yet been studied but remains relevant for determining potential anti-tumor synergy of YB1 inhibition with standard radiation therapy. We have previously shown that YB1 drives proliferation of cerebellar granular neural precursor cells (CGNPs) and murine Sonic Hedgehog (SHH) group MB cells. While others have demonstrated a link between YB1 and homologous recombination protein binding, functional and therapeutic implications remain unclear, particularly following IR-induced damage. Here we show that depleting YB1 in both SHH and Group 3 MB results not only in reduced proliferation but also synergizes with radiation due to differential response dynamics. YB1 silencing through shRNA followed by IR drives a predominantly NHEJ-dependent repair mechanism, leading to faster γH2AX resolution, premature cell cycle re-entry, checkpoint bypass, reduced proliferation, and increased senescence. These findings show that depleting YB1 in combination with radiation sensitizes SHH and Group 3 MB cells to radiation.
Glioblastoma (GBM) is a hypervascular and aggressive primary malignant tumor of the central nervous system. Recent investigations showed that traditional therapies along with antiangiogenic therapies failed due to the development of post-therapy resistance and recurrence. Previous investigations showed that there were changes in the cellular and metabolic compositions in the tumor microenvironment (TME). It can be said that tumor cell-directed therapies are ineffective and rethinking is needed how to treat GBM. It is hypothesized that the composition of TME-associated cells will be different based on the therapy and therapeutic agents, and TME-targeting therapy will be better to decrease recurrence and improve survival. Therefore, the purpose of this study is to determine the changes in the TME in respect of T-cell population, M1 and M2 macrophage polarization status, and MDSC population following different treatments in a syngeneic model of GBM. In addition to these parameters, tumor growth and survival were also studied following different treatments. The results showed that changes in the TME-associated cells were dependent on the therapeutic agents, and the TME-targeting therapy improved the survival of the GBM bearing animals. The current GBM therapies should be revisited to add agents to prevent the accumulation of bone marrow-derived cells in the TME or to prevent the effect of immune-suppressive myeloid cells in causing alternative neovascularization, the revival of glioma stem cells, and recurrence. Instead of concurrent therapy, a sequential strategy would be better to target TME-associated cells.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Recent evidence suggests that grammatical aspect can bias how individuals perceive criminal intentionality during discourse comprehension. Given that criminal intentionality is a common criterion for legal definitions (e.g., first-degree murder), the present study explored whether grammatical aspect may also impact legal judgments. In a series of four experiments participants were provided with a legal definition and a description of a crime in which the grammatical aspect of provocation and murder events were manipulated. Participants were asked to make a decision (first- vs. second-degree murder) and then indicate factors that impacted their decision. Findings suggest that legal judgments can be affected by grammatical aspect but the most robust effects were limited to temporal dynamics (i.e., imperfective aspect results in more murder actions than perfective aspect), which may in turn influence other representational systems (i.e., number of murder actions positively predicts perceived intentionality). In addition, findings demonstrate that the influence of grammatical aspect on situation model construction and evaluation is dependent upon the larger linguistic and semantic context. Together, the results suggest grammatical aspect has indirect influences on legal judgments to the extent that variability in aspect changes the features of the situation model that align with criteria for making legal judgments.
by
Gregory Melikian;
Stephanie M Bester;
Guochao Wei;
Haiyan Zhao;
Daniel Adu-Ampratwum;
Naseer Iqbal;
Valentine V Courouble;
Ashwanth Francis;
Arun S Annamalai;
Parmat K Singh;
Nikoloz Shkriabai;
Peter Van Blerkom;
James Morrison;
Eric M Poeschla;
Alan N Engelman;
Gregory B Melikyan;
Patrick R Griffin;
James R Fuchs;
Francisco J Asturias;
Mamuka Kvaratskhelia
The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.
by
Samantha T Reyes;
Robert MJ Deacon;
Scarlett G Guo;
Franscisco J Altimiras;
Jessa B Castillo;
Berend van der Wildt;
Aimara P Morales;
Jun Hyung Park;
Daniel Klamer;
Jarrett Rosenberg;
Lindsay M Oberman;
Nell Rebowe;
Jeffrey Sprouse;
Christopher U Missling;
Christopher R McCurdy;
Patricia Cogram;
Walter Kaufmann;
Frederick T Chin
Fragile X syndrome (FXS), a disorder of synaptic development and function, is the most prevalent genetic form of intellectual disability and autism spectrum disorder. FXS mouse models display clinically-relevant phenotypes, such as increased anxiety and hyperactivity. Despite their availability, so far advances in drug development have not yielded new treatments. Therefore, testing novel drugs that can ameliorate FXS’ cognitive and behavioral impairments is imperative. ANAVEX2-73 (blarcamesine) is a sigma-1 receptor (S1R) agonist with a strong safety record and preliminary efficacy evidence in patients with Alzheimer’s disease and Rett syndrome, other synaptic neurodegenerative and neurodevelopmental disorders. S1R’s role in calcium homeostasis and mitochondrial function, cellular functions related to synaptic function, makes blarcamesine a potential drug candidate for FXS. Administration of blarcamesine in 2-month-old FXS and wild type mice for 2 weeks led to normalization in two key neurobehavioral phenotypes: open field test (hyperactivity) and contextual fear conditioning (associative learning). Furthermore, there was improvement in marble-burying (anxiety, perseverative behavior). It also restored levels of BDNF, a converging point of many synaptic regulators, in the hippocampus. Positron emission tomography (PET) and ex vivo autoradiographic studies, using the highly selective S1R PET ligand [18F]FTC-146, demonstrated the drug’s dose-dependent receptor occupancy. Subsequent analyses also showed a wide but variable brain regional distribution of S1Rs, which was preserved in FXS mice. Altogether, these neurobehavioral, biochemical, and imaging data demonstrates doses that yield measurable receptor occupancy are effective for improving the synaptic and behavioral phenotype in FXS mice. The present findings support the viability of S1R as a therapeutic target in FXS, and the clinical potential of blarcamesine in FXS and other neurodevelopmental disorders.
Traditional restriction endonuclease-based cloning has been routinely used to generate replication-competent simian-human immunodeficiency viruses (SHIV) and simian tropic HIV (stHIV). This approach requires the existence of suitable restriction sites or the introduction of nucleotide changes to create them. Here, using an In-Fusion cloning technique that involves homologous recombination, we generated SHIVs and stHIVs based on epidemio-logically linked clade C transmitted/founder HIV molecular clones from Zambia. Replacing vif from these HIV molecular clones with vif of SIVmac239 resulted in chimeric genomes used to generate infectious stHIV viruses. Likewise, exchanging HIV env genes and introducing N375 mutations to enhance macaque CD4 binding site and cloned into a SHIVAD8-EObackbone. The generated SHIVs and stHIV were infectious in TZMbl and ZB5 cells, as well as macaque PBMCs. Therefore, this method can replace traditional methods and be a valuable tool for the rapid generation and testing of molecular clones of stHIV and SHIV based on primary clinical isolates will be valuable to generate rapid novel challenge viruses for HIV vaccine/cure studies.
Fragile X syndrome is a common cause of intellectual disability and autism spectrum disorder. The gene underlying the disorder, fragile X mental retardation 1 (FMR1), is silenced in most cases by a CGGrepeat expansion mutation in the 5? untranslated region (UTR). Recently, we identified a variant located in the 3?UTR of FMR1 enriched among developmentally delayed males with normal repeat lengths. A patient-derived cell line revealed reduced levels of endogenous fragile X mental retardation protein (FMRP), and a reporter containing a patient 3?UTR caused a decrease in expression. A control reporter expressed in cultured mouse cortical neurons showed an expected increase following synaptic stimulation that was absent when expressing the patient reporter, suggesting an impaired response to neuronal activity. Mobility-shift assays using a control RNA detected an RNA-protein interaction that is lost with the patient RNA, and HuR was subsequently identified as an associated protein. Cross-linking immunoprecipitation experiments identified the locus as an in vivo target of HuR, supporting our in vitro findings. These data suggest that the disrupted interaction of HuR impairs activity-dependent translation of FMRP, which may hinder synaptic plasticity in a clinically significant fashion.