Distinct Modulation of Spontaneous and GABA-Evoked Gating by Flurazepam Shapes Cross-Talk Between Agonist-Free and Liganded GABAA Receptor Activity

GABAA receptors (GABAARs) play a crucial inhibitory role in the CNS. Benzodiazepines (BDZs) are positive modulators of specific subtypes of GABAARs, but the underlying mechanism remains obscure. Early studies demonstrated the major impact of BDZs on binding and more recent investigations indicated gating, but it is unclear which transitions are affected. Moreover, the upregulation of GABAAR spontaneous activity by BDZs indicates their impact on receptor gating but the underlying mechanisms remain unknown. Herein, we investigated the effect of a BDZ (flurazepam) on the spontaneous and GABA-induced activity for wild-type (WT, α1β2γ2) and mutated (at the orthosteric binding site α1F64) GABAARs. Surprisingly, in spite of the localization at the binding site, these mutations increased the spontaneous activity. Flurazepam (FLU) upregulated this activity for mutants and WT receptors to a similar extent by affecting opening/closing transitions. Spontaneous activity affected GABA-evoked currents and is manifested as an overshoot after agonist removal that depended on the modulation by BDZs. We explain the mechanism of this phenomenon as a cross-desensitization of ligand-activated and spontaneously active receptors. Moreover, due to spontaneous activity, FLU-pretreatment and co-application (agonist + FLU) protocols yielded distinct results. We provide also the first evidence that GABAAR may enter the desensitized state in the absence of GABA in a FLU-dependent manner. Based on our data and model simulations, we propose that FLU affects agonist-induced gating by modifying primarily preactivation and desensitization. We conclude that the mechanisms of modulation of spontaneous and ligand-activated GABAAR activity concerns gating but distinct transitions are affected in spontaneous and agonist-evoked activity

Oligomerization of ZFYVE27 (Protrudin) Is Necessary to Promote Neurite Extension

ZFYVE27 (Protrudin) was originally identified as an interacting partner of spastin, which is most frequently mutated in hereditary spastic paraplegia. ZFYVE27 is a novel member of FYVE family, which is implicated in the formation of neurite extensions by promoting directional membrane trafficking in neurons. Now, through a yeast two-hybrid screen, we have identified that ZFYVE27 interacts with itself and the core interaction region resides within the third hydrophobic region (HR3) of the protein. We confirmed the ZFYVE27's self-interaction in the mammalian cells by co-immunoprecipitation and co-localization studies. To decipher the oligomeric nature of ZFYVE27, we performed sucrose gradient centrifugation and showed that ZFYVE27 oligomerizes into dimer/tetramer forms. Sub-cellular fractionation and Triton X-114 membrane phase separation analysis indicated that ZFYVE27 is a peripheral membrane protein. Furthermore, ZFYVE27 also binds to phosphatidylinositol 3-phosphate lipid moiety. Interestingly, cells expressing ZFYVE27ΔHR3 failed to produce protrusions instead caused swelling of cell soma. When ZFYVE27ΔHR3 was co-expressed with wild-type ZFYVE27 (ZFYVE27WT), it exerted a dominant negative effect on ZFYVE27WT as the cells co-expressing both proteins were also unable to induce protrusions and showed cytoplasmic swelling. Altogether, it is evident that a functionally active form of oligomer is crucial for ZFYVE27 ability to promote neurite extensions

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. Video Abstract [Figure presented] Development of the bioinformatics tool LOHHLA allows precise measurement of allele-specific HLA copy number, improves the accuracy in neoantigen prediction, and uncovers insights into how immune escape contributes to tumor evolution in non-small-cell lung cancer

Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies

Expression of tissue inhibitors of metalloproteinase 1 (TIMP-1) in gastric cancer tissue.

Degradation of extracellular matrix (ECM) is an essential step of invasion and metastasis of gastric cancer. The proteolysis of basement membranes depends on the balance between activities of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The aim of the study was to assess the expression of TIMP-1 in gastric cancer (GC) and interstitial inflammatory infiltrate cells within GC tissue in relation to clinico-pathological features of tumor and to estimate the prognostic significance of TIMP-1 expression for patients' survival. The presence of TIMP-1 in 54 cases of gastric cancer samples was investigated by immunohistochemistry. The expression of TIMP-1 in cancer and interstitial inflammatory infiltrate cells was evaluated in semi-quantitative scale. The immunoreactivity of TIMP-1 in cancer and inflammatory cells was positive in 100% of cases and varied from weak to intense reaction. The intensity of TIMP-1 expression increased with more advanced tumor stages and in patients who died of cancer during 2-year observation. TIMP-1 expression in interstitial inflammatory infiltrate cells was the independent prognostic factor for patients' survival. The results suggest the role of TIMP-1 in gastric tumorigenesis, although this issue requires further investigtions