Protein Translocation Research Articles

Host-Driven Selection, Revealed by Comparative Analysis of Xanthomonas Type III Secretion Effectoromes, Unveils Novel Recognized Effectors.

Xanthomonas species are specialized plant pathogens, often exhibiting a narrow host range. They rely on the translocation of effector proteins through the type III secretion system to colonize their respective hosts. The effector arsenal varies among Xanthomonas spp., typically displaying species-specific compositions. This species-specific effector composition, collectively termed the effectorome, is thought to influence host specialization. We determined the plant host-derived effectoromes of more than 300 deposited genomes of Xanthomonas species associated with either Solanaceae or Brassicaceae hosts. Comparative analyses revealed clear species-specific effectorome signatures. However, Solanaceae or Brassicaceae host-associated effectorome signatures were not detected. Nevertheless, host biases in the presence or absence of specific effector classes were observed. To assess whether host-associated effector absence results from selective pressures, we introduced effectors unique to Solanaceae pathogens to X. campestris pv. campestris and effectors unique to Brassicaceae pathogens to X. euvesicatoria pv. euvesicatoria (Xeue) and evaluated if these introductions hindered virulence on their respective hosts. Introducing the effector XopI into X. campestris pv. campestris reduced virulence on white cabbage leaves without affecting localized or systemic colonization. Introducing the XopAC or XopJ5 effectors into Xeue reduced virulence and colonization on tomato but not on pepper. Additionally, XopAC and XopJ5 induced a hypersensitive response on tomato leaves when delivered by Xeue or through Agrobacterium-mediated transient expression, confirming recognition in tomato. This study demonstrates the role of host-derived selection in establishing species-specific effectoromes, identifying XopAC and XopJ5 as recognized effectors in tomato.

Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR: ABL1 signaling

BackgroundThe inability of biologics to pass the plasma membrane prevents their development as therapeutics for intracellular targets. To address the lack of methods for cytosolic protein delivery, we used the type III secretion system (T3SS) of Y. enterocolitica, which naturally injects bacterial proteins into eukaryotic host cells, to deliver monobody proteins into cancer cells. Monobodies are small synthetic binding proteins that can inhibit oncogene signaling in cancer cells with high selectivity upon intracellular expression. Here, we engineered monobodies targeting the BCR::ABL1 tyrosine kinase for efficient delivery by the T3SS, quantified cytosolic delivery and target engagement in cancer cells and monitored inhibition of BCR::ABL1 signaling.MethodsIn vitro assays were performed to characterize destabilized monobodies (thermal shift assay and isothermal titration calorimetry) and to assess their secretion by the T3SS. Immunoblot assays were used to study the translocation of monobodies into different cell lines and to determine the intracellular concentration after translocation. Split-Nanoluc assays were performed to understand translocation and degradation kinetics and to evaluate target engagement after translocation. Phospho flow cytometry and apoptosis assays were performed to assess the functional effects of monobody translocation into BCR:ABL1-expressing leukemia cells.ResultsTo enable efficient translocation of the stable monobody proteins by the T3SS, we engineered destabilized mutant monobodies that retained high affinity target binding and were efficiently injected into different cell lines. After injection, the cytosolic monobody concentrations reached mid-micromolar concentrations considerably exceeding their binding affinity. We found that injected monobodies targeting the BCR::ABL1 tyrosine kinase selectively engaged their target in the cytosol. The translocation resulted in inhibition of oncogenic signaling and specifically induced apoptosis in BCR::ABL1-dependent cells, consistent with the phenotype when the same monobody was intracellularly expressed.ConclusionHence, we establish the T3SS of Y. enterocolitica as a highly efficient protein translocation method for monobody delivery, enabling the selective targeting of different oncogenic signaling pathways and providing a foundation for future therapeutic application against intracellular targets.

Toward Understanding the Mechanism of Client-Selective Small Molecule Inhibitors of the Sec61 Translocon.

The Sec61 translocon mediates the translocation of numerous, newly synthesized precursor proteins into the lumen of the endoplasmic reticulum or their integration into its membrane. Recently, structural biology revealed conformations of idle or substrate-engaged Sec61, and likewise its interactions with the accessory membrane proteins Sec62, Sec63, and TRAP, respectively. Several natural and synthetic small molecules have been shown to block Sec61-mediated protein translocation. Since this is a key step in protein biogenesis, broad inhibition is generally cytotoxic, which may be problematic for a putative drug target. Interestingly, several compounds exhibit client-selective modes of action, such that only translocation of certain precursor proteins was affected. Here, we discuss recent advances of structural biology, molecular modelling, and molecular screening that aim to use Sec61 as feasible drug target.

Imaging the translocator protein 18 kDa within cognitive control and declarative memory circuits in virally-suppressed people with HIV.

Virally-suppressed people with HIV (VS-PWH) show heterogeneity in patterns of cognitive dysfunction. To better understand the relationship between the neuroimmune response and cognition, we used positron emission tomography (PET) to image the translocator protein 18 kDa (TSPO). The study examined HIV-serostatus differences in TSPO as well as associations between regional TSPO and select cognitive processes defined using the Research Domain Criteria (RDoC) framework. Cross-sectional investigation in VS-PWH (n = 25) versus HIV-uninfected individuals (n = 18) of cognitive control and declarative memory, as well as [11C]DPA-713 PET measures of TSPO within cognitive control and declarative memory regions of interest. Group differences in [11C]DPA-713 binding (VT) in cognitive control or declarative memory regions were examined using linear mixed models. Tests of associations between factor-derived cognitive system measures and PET measures were performed, controlling for TSPO genotype. There were no group differences in any of the four factor-derived cognitive system measures. VS-PWH had higher log [11C]DPA-713 VT across cognitive control regions(unstandardized beta coefficient reflecting mean difference [B] = 0.23, SE = 0.11, 95% confidence interval [CI] 0.01, 0.45, P = 0.04) and declarative memory regions (B = 0.24, SE = 0.11, 95%CI 0.02, 0.45, P = 0.03). Higher log [11C]DPA-713 VT in cognitive control regions related to poorer cognitive control in each group, and to worse self-reported cognitive performance in VS-PWH. Log [11C]DPA-713 VT in each declarative memory region did not associate with measured declarative memory. A localized neuroimmune response marked by high TSPO in brain regions that subserve cognitive control may contribute to poorer cognitive control in VS-PWH.

Single-molecule evidence of Entropic Pulling by Hsp70 chaperones.

Hsp70 chaperones are central components of the cellular network that ensures the structural quality of proteins. Despite crucial roles in processes such as protein disaggregation and protein translocation into organelles, their physical mechanism of action has remained hotly debated. To the best of our knowledge, no experimental data has directly proven any of the modelsproposed to date (Power Stroke, Brownian Ratchet, or Entropic Pulling) due to a lack of suitable methods. Here, we use nanopores, a powerful single-molecule tool, to investigate the mechanism of Hsp70s. We demonstrate that Hsp70s extract trapped polypeptide substrates from the nanopore by generating strong forces (equivalent to 46 pN over distances of 1 nm), that rely on the size of Hsp70. The findings provide unambiguous evidence of the Entropic Pulling mechanism, thus solving a long-standing debate, and proposing a potentially universal principle governing diverse cellular processes. Additionally, these results highlight the utility of biological nanopores for protein studies.

Single turnover transient state kinetics reveals processive protein unfolding catalyzed by Escherichia coli ClpB

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s−1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.

Single turnover transient state kinetics reveals processive protein unfolding catalyzed by Escherichia coli ClpB.

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s-1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.

Effect of mitochondrial translocator protein TSPO on LPS-induced cardiac dysfunction

IntroductionSepsis-induced cardiac dysfunction is one of the most serious complications of sepsis. The mitochondrial translocator protein (TSPO), a mitochondrial outer membrane protein, is widely used as a diagnostic marker of inflammation-related diseases and can also lead to the release of inflammatory components. However, whether TSPO has a therapeutic effect on sepsis-induced cardiac dysfunction is unclear. ObjectivesThe aim of this study is to investigate the involvement of TSPO in the pathogenesis of sepsis-induced cardiac dysfunction and elucidate its underlying mechanism, as well as develop therapeutic strategies targeting TSPO for the prevention and treatment of sepsis-induced cardiac dysfunction. MethodsThe sepsis-induced cardiac dysfunction model was established by intraperitoneal injection of lipopolysaccharide (LPS) in C57BL/6 mice (LPS-induced cardiac dysfunction, LICD). TSPO knockout mice were constructed,and the effects of TSPO was detected by survival rate, echocardiography, HE staining, mitochondrial electron microscopy, TUNEL staining. TSPO-binding proteins were identified by co-immunoprecipitation and mass spectrometry. The mechanisms underlying between TSPO and voltage-dependent anion channel (VDAC) was studied through western blot and immunofluorescence. Proteolysis-Targeting Chimeras (PROTAC) technology was used to construct TSPO-PROTAC molecules that can degrade TSPO. ResultsOur present study found that LPS increased cardiac TSPO expression. Knockout of TSPO in C57BL/6 mice with LICD attenuated the cardiac pathology, mitochondrial dysfunction, and apoptosis of cardiomyocytes and significantly improved cardiac function and survival rate. Co-immunoprecipitation and mass spectrometry identified VDAC as a TSPO binding protein.Down-regulation of TSPO reduced PKA-mediated VDAC phosphorylation and VDAC oligomerization, ameliorated mitochondrial function, and reduced cardiomyocyte apoptosis. The study has clinical translational potential, because administration of TSPO-PROTAC to degrade TSPO improved cardiac function in mice with LICD. ConclusionThis study elucidated the effect of TSPO in LICD, providing a new therapeutic strategy to down-regulate TSPO by administration of TSPO-PROTAC for the prevention and treatment of LICD.

NEUROINFLAMMATION IN PARKINSON’S DISEASE: A STUDY WITH [11C]PBR28 PET AND CEREBROSPINAL FLUID MARKERS

ObjectiveTo investigate neuroinflammation in Parkinson’s disease (PD) with [11C]PBR28 positron emission tomography (PET) and cerebrospinal fluid (CSF) biomarkers, and the relationship to dopaminergic functioning measured with 6-[18F]-fluoro-L-dopa ([18F]FDOPA) PET. MethodsThe clinical cohort consisted of 20 subjects with PD and 51 healthy controls (HC). All HC and 15 PD participants underwent [11C]PBR28 High Resolution Research Tomograph (HRRT) PET for the quantitative assessment of cerebral binding to the translocator protein (TSPO), a neuroinflammation marker. CSF samples were available from 17 subjects with PD and 21 HC and were examined for soluble triggering receptor expressed on myeloid cells 2 (sTREM2), chitinase 3-like 1 protein (YKL-40), neurogranin (NG), alpha-synuclein (aSyn) and oligo-alpha-synuclein. All subjects with PD underwent [18F]FDOPA HRRT PET. ResultsWhile the subjects with PD and HC did not differ in the total volume of distribution (VT) of [11C]PBR28 in any studied brain regions, higher levels of neuroinflammation and neurodegeneration CSF biomarkers sTREM2 and NG, respectively were associated with more severe motor symptoms evaluated by The Unified Parkinson’s Disease Rating Scale motor part (UPDRS-III) (r = 0.52, p=0.041 and r = 0.59, p=0.016 respectively). Additionally, in the PD group increased [11C]PBR28 VT in the basal ganglia and substantia nigra (SN) was related to higher levels of neuroinflammation biomarker YKL-40 (p <0.01). ConclusionAssociations between CSF biomarkers, motor disability and [11C]PBR28 VT in the striatum and SN may support a role for neuroinflammation in PD.

Knockdown of Translocator Protein (TSPO) Slows AKI to CKD Transition through Regulating Mitochondrial Dysfunction

Knockdown of Translocator Protein (TSPO) Slows AKI to CKD Transition through Regulating Mitochondrial Dysfunction

Isolation and Characterization of Heat Shock Protein 70-Ipek 1 from Toxoplasma gondii

Toxoplasma gondii is a common intracellular parasite that causes the toxoplasmosis. Heat shock proteins (Hsps) have a critical role in pathogenesis of toxoplasmosis. Hsps are highly conserved proteins in evolution among living organisms. This protein family responsible for a wide range of biological processes such as protein folding, protein translocation, protein aggregation. In the present study, Hsp70, a member of the Hsp family, was isolated from T. gondii and its sequence and motifs were determined by PCR, cloning, sequencing and homology modelling analysis. ATP hydrolysis, luciferase folding, and luciferase aggregation experiments were performed for determination of its chaperone activity while the stability and secondary structure of the Hsp70 were discovered by using biophysical experiments (FTIR, florescence and quenching experiment). In addition, in silico analysis were used to determine the physicochemical characteristics of Hsp70. The results revealed that Hsp70 protein obtained from T. gondii (Hsp70-IPEK1) is similar to Hsp70s from other organisms. Also, the chaperone activity, stability and secondary structure of Hsp70-IPEK1 were determined. Hsp70-IPEK1 together with other chaperones in the presence of nucleotide were dramatically increased protein folding and aggregation. According to these results, it is thought that Hsp70 has a potential to contribute many research areas such as pharmaceutical analysis

Dysregulation of lipid metabolism in the liver of Tspo knockout mice

The translocator protein, TSPO, has been implicated in a wide range of cellular processes exerted from its position in the outer mitochondrial membrane from where it influences lipid metabolism and mitochondrial oxidative activity. Understanding how this protein regulates a profusion of processes requires further elucidation and to that end we have examined lipid metabolism and used an RNAseq strategy to compare transcript abundance in wildtype and Tspo knockout (KO) mouse liver. The levels of cholesterol, triglyceride and phospholipid were significantly elevated in the KO mouse liver. The expression of cholesterol homeostasis genes was markedly downregulated. Determination of the differential expression revealed that many genes were either up- or downregulated in the KO animals. However, a striking observation within the results was a decrease of transcripts for protein degradation proteins in KO animals while protease inhibitors were enriched. When the entire abundance data-set was analysed with CEMiTool, and revealed a module of proteins that were under-represented in the KO animals. These could subsequently be formed into a network comprising three interlinked clusters at the centre of which were proteins of cytoplasmic ribosomes with gene ontology terms suggesting impairment to translation. The largest cluster was dominated by proteins of lipid metabolism but also contained disparate systems of iron metabolism and behaviour. The third cluster was dominated by proteins of the electron transport chain and oxidative phosphorylation. These findings suggest that TSPO contributes to lipid metabolism, detoxification of active oxygen species and oxidative phosphorylation, and regulates mitochondrial retrograde signalling.

NCLN as a potential prognosis biomarker in endometrial cancer

Endometrial cancer (ECs) stands as one of the three major malignancies impacting females globally, with its incidence steadily increasing. ECs can be categorized into two types based on their clinicopathological features. Type I ECs typically exhibit low stage, favorable histological types, and low histological grade, correlating with a more favorable prognosis. Conversely, Type II ECs present with advanced-stage disease, aggressive behavior, and poorer histological types, resulting in a worse prognosis.The expression level of progesterone receptors (PR) holds significant importance in determining the prognosis of patients with ECs. Elevated levels of PR are linked to a more favorable prognosis, primarily attributed to progesterone's inhibitory influence on cancer cell proliferation and invasion. Moreover, progesterone promotes cell cycle inhibition through its regulation of PR, further contributing to improved outcomes in ECs.Nicalin (NCLN) plays a crucial role in facilitating the translocation of multichannel membrane proteins to the endoplasmic reticulum membrane and is implicated in embryonic development. Structurally akin to NODAL Modulator (NOMO), NCLN antagonizes NOMO during embryogenesis, forming a complex that antagonizes the Nodal pathway, thereby influencing mesoderm development. However, the precise relationship between NCLN and ECs remains incompletely understood.Our research findings reveal that NCLN actively stimulates the proliferation of ECs cells and exhibits a positive correlation with PR, albeit without impacting ER. Moreover, the expression levels of NCLN in ECs demonstrate associations with distinct histological types. These observations suggest that NCLN could emerge as a promising marker in the histological classification of ECs.

Spatially resolved profiling of protein conformation and interactions by biocompatible chemical cross-linking in living cells

Unlocking the intricacies of protein structures and interactions within the dynamic landscape of subcellular organelles presents a significant challenge. To address this, we introduce SPACX, a method for spatially resolved protein complex profiling via biocompatible chemical cross(x)-linking with subcellular isolation, designed to monitor protein conformation, interactions, and translocation in living cells. By rapidly capturing protein complexes in their native physiological state and efficiently enriching cross-linked peptides, SPACX allows comprehensive analysis of the protein interactome within living cells. Leveraging structure refinement with cross-linking restraints, we identify subcellular-specific conformation heterogeneity of PTEN, revealing dynamic differences in its dual specificity domains between the nucleus and cytoplasm. Furthermore, by discerning conformational disparities, we identify 83 cytoplasm-exclusive and 109 nucleus-exclusive PTEN-interacting proteins, each associated with distinct biological functions. Upon induction of ubiquitin-proteasome system stress, we observe dynamic alterations in PTEN assembly and its interacting partners during translocation. These changes, including the identification of components and interaction sites, are characterized using the SPACX approach. Notably, SPACX enables identification of unique interacting proteins specific to PTEN isoforms, including PTEN and PTEN-Long, through the determination of sequence-specific cross-linking interfaces. These findings underscore the potential of SPACX to elucidate the functional diversity of proteins within distinct subcellular sociology.

Sonic hedgehog signaling facilitates pyroptosis in mouse heart following ischemia/reperfusion via enhancing the formation of CARD10-BCL10-MALT1 complex

Pyroptosis has been found to contribute to myocardial ischemia/reperfusion (I/R) injury, but the exact mechanisms that initiate myocardial pyroptosis are not fully elucidated. Sonic hedgehog (SHH) signaling is activated in heart suffered I/R, and intervention of SHH signaling has been demonstrated to protect heart from I/R injury. Caspase recruitment domain-containing protein 10 (CARD10)-B cell lymphoma 10 (BCL10)-mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) (CBM) complex could transduce signals from the membrane and induce inflammatory pathways in non-hematopoietic cells, which could be a downstream effector of SHH signaling pathway. This study aims to explore the role of SHH signaling in I/R-induced myocardial pyroptosis and its relationship with the CBM complex. C57BL/6J mice were subjected to 45 min-ischemia followed by 24 h-reperfusion to establish a myocardial I/R model, and H9c2 cells underwent hypoxia/reoxygenation (H/R) to mimic myocardial I/R model in vitro. Firstly, SHH signaling was significantly activated in heart suffered I/R in an autocrine- or paracrine-dependent manner via its receptor PTCH1, and inhibition of SHH signaling decreased myocardial injury via reducing caspase-11-dependent pyroptosis, concomitant with attenuating CBM complex formation. Secondly, suppression of SHH signaling decreased protein kinase C α (PKCα) level, but inhibition of PKCα attenuated CBM complex formation without impacting the protein levels of SHH and PTCH1. Finally, disruption of the CBM complex prevented MALT1 from recruiting of TRAF6, which was believed to trigger the caspase-11-dependent pyroptosis. Based on these results, we conclude that inhibition of SHH signaling suppresses pyroptosis via attenuating PKCα-mediated CARD10-BCL10-MALT1 complex formation in mouse heart suffered I/R.

TET1 overexpression affects cell proliferation and apoptosis in aging ovaries.

Along with the progress of society, human life expectancy has been increasing, and late marriage and late childbearing are the current trend. Since reproductive aging affects fertility, ovarian aging in women has become a major reproductive health issue in the current society. During ovarian aging, DNA methylation levels may change. The ten-eleven translocation (TET) protein family proteins TET1, TET2, and TET3 are important DNA demethylation enzymes, and differential expression of TET1, TET2, and TET3 may affect the proliferation and apoptosis of aging ovarian cells. The aim of this study was to investigate the role of TET1 in the regulation of ovarian aging. The expression of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) was analyzed by immunofluorescence (IF) in young and aging ovaries of six 6-8-week-old female mice and six 6-8-month-old female mice. Then, the expression pattern of the TET protein family in young and aging ovaries of mice was investigated. To determine the impact of TET1 on ovarian development, the aging of IOSE-80, KGN, and SKOV-3 cells was induced with D-galactosidase (D-gal). Cells were then transfected using the TET1 overexpression vector or si-TET1. We assessed the proliferation and apoptosis of aging cells after transfection and analyzed the regulatory effect of TET1 expression on aging cells. Additionally, we verified the Tet1 expression in Tet1-KO mice. The 5mC to 5hmC transition, oocyte maturation, and blastocyst rate were reduced in aging mice compared to young mice. In aging mice ovaries, the expression levels of Tet1, Tet2, and Tet3 were reduced significantly, with Tet1 being particularly pronounced. The overexpression of TET1 promoted proliferation and inhibited apoptosis in aging human ovarian cells. Furthermore, Tet1 expression was very low in Tet1-KO C57BL/6J mice ovaries. This study demonstrates that the expression levels of TET family proteins are low in aging ovaries, and the overexpression of TET1 can promote proliferation and inhibit apoptosis in aging ovarian cells.

Remimazolam attenuates lipopolysaccharide-induced neuroinflammation and cognitive dysfunction

ObjectiveRemimazolam, a novel benzodiazepine, is widely used as an anesthetic in endoscopic procedures; however, its effects on cognitive function remain unclear, limiting its broader application in general anaesthesia. Neuroinflammation is a well-established key factor in the etiology and progression of cognitive dysfunction, including conditions such as Alzheimer's disease, Parkinson's disease, postoperative delirium, and postoperative cognitive dysfunction. Preclinical studies have demonstrated that remimazolam exerts anti-inflammatory and neuroprotective effects, and clinical reports indicate a reduced incidence of postoperative delirium in patients treated with remimazolam. Nevertheless, whether remimazolam improves cognitive function through its anti-inflammatory properties remains uncertain. This study aimed to investigate the neuroprotective effects of remimazolam and its underlying mechanism in a lipopolysaccharide (LPS)-induced model of neuroinflammation, neuronal injury, and cognitive dysfunction MethodsC57BL/6 J male mice were administered LPS intraperitoneally to establish a model of neuroinflammation-induced cognitive impairment. A subset of mice received remimazolam via intraperitoneal injection 30 minutes prior to LPS administration. Cognitive performance was evaluated using behavioural tests, including the Morris Water Maze (MWM), Novel Object Recognition (NOR) test, and Open Field Test (OFT). Hippocampal tissues were analyzed by haematoxylin-eosin (HE) staining to assess structural changes. Inflammatory markers, including Interleukin (IL)-6, IL-1β, and tumor necrosis factor-α, were quantified using enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR. Immunofluorescence was used to detect translocator protein (TSPO) and markers of microglia activation (IBA-1, CD16/32, and CD206). Results(1) Remimazolam reversed LPS-induced cognitive deficits, as evidenced by shorter spatial exploration latency and increased platform crossings in the MWM, and an elevated recognition index in the NOR test. (2) Remimazolam improved hippocampal morphology, reducing LPS-induced neuronal damage. (3) Remimazolam significantly decreased levels of hippocampal inflammatory cytokines, inhibited microglial activation, promoted M2-type microglia polarization, and increased TSPO expression. ConclusionRemimazolam demonstrated neuroprotective and anti-neuroinflammatory effects in a mouse model of LPS-induced cognitive impairment. These effects are likely mediated through the regulation of TSPO, which inhibits microglial activation and promotes the polarization of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype.

Flow cytometric analysis of the interaction of monoclonal antibodies to various epitopes of the HSP70 molecule with intracellular and membrane-associated forms of this protein

Currently, a large amount of data has demonstrated that many types of tumor cells, in contrast to their non-transformed forms, are characterized by the translocation of intracellular heat shock proteins 70 kDa (HSP70) to the surface of the plasma membrane. This made it possible to classify HSP70 exposed on the cell surface as a tumor-associated antigen and was the basis for searching the opportunities for the practical use of this phenomenon in clinical oncology. A significant argument in favor of the prospects of such studies was the discovered ability of HSP70, present on the surface of target cells, to enhance the cytotoxic activity of NK cells. In this regard, the work of many research groups is devoted to developing approaches to increasing the level of membrane-associated HSP70 in tumor tissues. At the same time, given the presence of such proteins on the surface of many types of tumor cells, the use of monoclonal antibodies that interact with HSP70 molecules for antitumor therapy can be considered as one of the promising approaches. It is well known that antibody preparations that selectively interact with cancer cells can be used for targeted antitumor immunotherapy. Previously, we obtained a panel of six B cell hybridomas producing monoclonal antibodies to the inducible and constitutive forms of human HSP70, directed to various epitopes of this molecule. It is significant that three varieties of hybridomas produced antibodies with specificity for binding sites on the C-terminal domain of the HSP70 molecule, and the second trio of monoclonal antibodies were specific to epitopes on the N-terminal domain of HSP70, while almost all known commercial antibodies interact only with C-terminal fragments of HSP70. In this work, we conducted a comparative study of the binding of the obtained antibodies to these proteins localized in different types of cells, both in the intracellular space and on the cell surface.The results obtained allow us to consider the identified varieties of monoclonal antibodies that most effectively recognize surface HSP70 as a promising basis for the creation of new drugs for antitumor immunotherapy.

In silico study of some plant compounds as potential anticancer agents targeting MALT1 allosteric domain

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is the only human paracaspase, that serves as an adaptor protein and controls substantial genes expressed in the activation, proliferation of lymphocyte, and immune reactions by triggering the IKK/NF-kB signaling pathway. However, unusual MALT1-mediated NF-kB signaling pathway has been identified in multiple diseases like cancer, therefore making MALT1 a promising therapeutic target. There are scanty numbers of MALT1 inhibitors, thus the need to discover more compounds with less or no toxicity issue, that are cheap and pharmacologically efficient is of pertinence. Hence, our present study was to identify phyto-small molecules that could bind the allosteric interface of MALT1 using in silico methods. Total of 34 plant molecules were selected and screened for druglikeness, after which they were docked via Maestro 11.1 against the allosteric site of MALT1. The molecule with a binding score (kcal/mol) better than the control drug was subjected to molecular dynamics (MD) simulations of 100 ns via Desmond, free energy perturbations, principal component and Pearson correlation analyses. Our findings from this computational study presents cyanidin (–8.822 kcal/mol) as better binder to the allosteric site of MALT1 based on the molecular docking and pharmacokinetic profiling than thioridazine. Similarly, cyanidin-MALT1 complex showed significant stability and exhibiting contacts with critical amino acid residues in the site of interest than thioridazine-MALT1 complex. Hence, cyanidin is a potential allosteric inhibitor of MALT1. However, an urgent need for in vitro and in vivo validations is required to ascertain the efficacy of cyanidin in the fight against cancer and other MALT1-related diseases.

18F-DPA714 PET/MRI as a potential imaging tool for detecting possible antibody-negative autoimmune encephalitis: a prospective study.

Conventional magnetic resonance imaging (MRI) used for detecting possible antibody-negative autoimmune encephalitis (AIE) often fails to meet the diagnostic requirements of this disease. Positron emission tomography (PET) with a translocator protein radioligand can help visualize microglia distribution density in inflammation-related diseases, thereby offering potentially incremental value to conventional MRI for the in vivo assessment of possible antibody-negative AIE. In this prospective study, 15 participants diagnosed with possible antibody-negative AIE and 10 healthy controls were enrolled (ClinicalTrials.gov: NCT05293405, dated March 15, 2022). All participants underwent hybrid 18F-DPA714 PET/MRI and evaluation for modified Rankin scale (mRS) score, clinical assessment scale for AIE (CASE), and appropriate antibodies. A positive finding was defined as the intensity of 18F-DPA714 uptake that was above a threshold of mean standardized uptake value ratio (SUVR) + two standard deviations of SUVR within the corresponding brain regions of healthy controls. The positive detection rate of 18F-DPA714 PET for possible antibody-negative AIE was significantly higher than that of brain MRI (10/15 [67%] vs. 3/15 [20%]; P = 0.039). In addition, both the intensity and extent of 18F-DPA714 uptake were significantly associated with the CASE score (P = 0.002 and 0.001). Meanwhile, SUVR levels in the cerebellar region were significantly higher in patients with ataxia than in those without ataxia (P = 0.006). Furthermore, 18F-DPA714 uptake decreased in 5/10 [50%] patients who underwent follow-up PET/MRI, which mirrored their symptom relief. 18F-DPA714 PET demonstrated its potentially incremental value to conventional MRI for detecting possible antibody-negative AIE.