70-kDa Protein Research Articles

PDZD2 Is Required for Normal Erythropoiesis and Its 37-KDa Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP, and erythroid progenitors, however, it is almost universally lost through hypermethylation in acute myeloid leukemia (AML). PDZD2 encodes for a 300-kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37-kDa protein known as secreted PDZD2 (sPDZD2). sPDZD2 has been reported to function as a tumor suppressor in some solid tumors, including breast, prostate, and liver cancer. Thus, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. We first sought to determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment. Using an ELISA to quantify sPDZD2 levels in BM plasma isolated from healthy donors (n=6) and primary AMLs (n=20), we confirmed its secretion by normal CD34+ HSPCs (NBM) and downregulation in AMLs (NBM: 169 ± 17.53 vs AML: 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines (n=7) secreted significantly lower levels of sPDZD2 into the culture medium than normal HSPCs (n=2) (NBM: 28.71 ± 1.76 vs AML: 4.62 ± 1.19 ng/mL; p<0.0001). Next, to study the role of PDZD2 in normal hematopoiesis we performed in vitro myeloid and erythroid differentiation and colony-forming unit assays in CRISPR-Cas9-edited human HSPCs. CD34+ cells from healthy donors were electroporated with recombinant Cas9-sgRNA ribonucleoprotein complexes targeting PDZD2 and editing confirmed by T7I endonuclease assay and Sanger sequencing. PDZD2-edited cells (PDZD2 KO) were plated either on methylcellulose or liquid differentiation cultures (n=6). PDZD2 KO resulted in decreased colony number of all types (Control: 172.5 ± 34.37 vs PDZD2 KO: 137.0 ± 18.25, p<0.05), indicating reduced colony-forming potential. In addition, loss of PDZD2 resulted in a delay in terminal erythroid differentiation with an accumulation of pro-erythroblasts (CD71+CD235a lowCD105 high, p<0.05) and late basophilic erythroblasts (CD71+CD235a highCD105 int, p<0.01) as well as decreased orthochromatic erythroblasts (CD71+CD235a highCD105 neg, p<0.01) in a tri-phasic liquid culture system. By contrast, PDZD2 KO had no impact on myeloid differentiation. To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2 Gt/Gt gene-trap (GT) mouse model and performed peripheral blood (PB) and BM analysis compared to wild-type (WT) controls. PB counts at 2 months of age (n=6-9 per genotype) showed anemia, with a significant decrease in both red blood cell counts (WT: 9.04 ± 0.26 vs GT: 7.58 ± 0.47 x10 6/mL; p=0.026) and hemoglobin levels (WT: 14.88 ± 0.46 vs GT: 12.30 ± 0.81 g/dL; p=0.036), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decrease megakaryocyte-erythroid progenitors (MEPs) and short-term HSCs in the BM of 3 months-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. Next, we sought to evaluate the tumor suppressor role of sPDZD2 in the context of AML. We treated a panel of AML cell lines (n=9) and primary human AML specimens (n=4) with recombinant sPDZD2 ( r-sPDZD2; dose: 100nM-300nM/day) for a period of 8 days. AML cells showed dose-dependent growth inhibition (CellTiter-Glo, p<0.05 for all) and increased expression of either CD11b or CD15 differentiation markers. Cell cycle analysis on day 8 revealed that AML cells treated with r-sPDZD2 were arrested at G0/G1 phase compared to control. In summary, our study is the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for r-sPDZD2 as a novel therapeutic approach for AML.

11 C]-PBR28 positron emission tomography signal as an imaging marker of joint inflammation in knee osteoarthritis.

Although inflammation is known to play a role in knee osteoarthritis (KOA), inflammation-specific imaging is not routinely performed. In this article, we evaluate the role of joint inflammation, measured using [ 11 C]-PBR28, a radioligand for the inflammatory marker 18-kDa translocator protein (TSPO), in KOA. Twenty-one KOA patients and 11 healthy controls (HC) underwent positron emission tomography/magnetic resonance imaging (PET/MRI) knee imaging with the TSPO ligand [ 11 C]-PBR28. Standardized uptake values were extracted from regions-of-interest (ROIs) semiautomatically segmented from MRI data, and compared across groups (HC, KOA) and subgroups (unilateral/bilateral KOA symptoms), across knees (most vs least painful), and against clinical variables (eg, pain and Kellgren-Lawrence [KL] grades). Overall, KOA patients demonstrated elevated [ 11 C]-PBR28 binding across all knee ROIs, compared with HC (all P 's < 0.005). Specifically, PET signal was significantly elevated in both knees in patients with bilateral KOA symptoms (both P 's < 0.01), and in the symptomatic knee ( P < 0.05), but not the asymptomatic knee ( P = 0.95) of patients with unilateral KOA symptoms. Positron emission tomography signal was higher in the most vs least painful knee ( P < 0.001), and the difference in pain ratings across knees was proportional to the difference in PET signal ( r = 0.74, P < 0.001). Kellgren-Lawrence grades neither correlated with PET signal (left knee r = 0.32, P = 0.19; right knee r = 0.18, P = 0.45) nor pain ( r = 0.39, P = 0.07). The current results support further exploration of [ 11 C]-PBR28 PET signal as an imaging marker candidate for KOA and a link between joint inflammation and osteoarthritis-related pain severity.

TSPO is a novel biomarker for prognosis that regulates cell proliferation through influencing mitochondrial functions in HCC

The disorder of mitochondrial functions plays a key role in oncogenesis. It is known that TSPO (18-kDa translocator protein) lies in a peculiar location at the interface between the mitochondria and the cytosol. TSPO is found in many types of tissues and is associated with multiple cellular processes, including apoptosis, cell proliferation and the regulation of mitochondria. However, the involvement of TSPO in hepatocellular carcinoma (HCC) remains unclear. In this study, we found that TSPO is upregulated in HCC tissue and is associated with poor differentiation and poor survival. Multivariate analyses showed that TSPO was an independent predictive factor for poor prognosis in HCC patients. For the first time, we provided evidence that TSPO knockdown suppressed HCC cell proliferation in vitro. Hence, TSPO knockdown-induced apoptosis by disturbing mitochondrial function by enhancing the formation of reactive oxygen species (ROS) and decreasing the mitochondrial membrane potential (ΔΨm). An assay exploring the underlying mechanism revealed that TSPO knockdown modulated apoptotic regulatory proteins by regulating the ERK signaling pathway. Through a functional assay and an in vivo mouse model, the anti-cancer effect of PK11195, a specific ligand of TSPO, on HCC was revealed. In summary, TSPO may potentially serve as a prognostic biomarker, and TSPO might be a potential therapeutic target for HCC.

GAS6 attenuates sepsis-induced cardiac dysfunction through NLRP3 inflammasome-dependent mechanism

Sepsis is a major health threat and often results in heart failure. Growth arrest-specific gene 6 (GAS6), a 75-kDa vitamin K-dependent protein, participates in immune regulation and inflammation through binding to AXL (the TAM receptor family). This study was designed to examine the myocardial regulatory role of GAS6 in sepsis. Serum GAS6 levels were increased in septic patients and mice while myocardial GAS6 levels were decreased in septic mice. Single-cell RNA sequencing further revealed a decline in GAS6 levels of nearly all cell clusters including cardiomyocytes. GAS6 overexpression via adeno-associated virus 9 (AAV9) overtly improved cardiac dysfunction in cecum ligation and puncture (CLP)-challenged mice, along with alleviated mitochondrial injury, endoplasmic reticulum stress, oxidative stress, and apoptosis. However, GAS6-elicited beneficial effects were removed by GAS6 knockout. The in vitro study was similar to these findings. Our data also noted a downstream effector role for NLRP3 in GAS6-initiated myocardial response. GAS6 knockout led to elevated levels of NLRP3, the effect of which was reconciled by GAS6 overexpression. Taken together, these results revealed the therapeutical potential of targeting GAS6/AXL-NLRP3 signaling in the management of heart anomalies in sepsis.

Mycobacterium tuberculosis virulence protein ESAT-6 influences M1/M2 polarization and macrophage apoptosis to regulate tuberculosis progression.

Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium tuberculosis (Mtb), and it remains one of the major threats to human health worldwide. To our knowledge, the polarization of M1/M2 macrophages were critical innate immune cells which play important roles in regulating the immune response during TB progression. We aimed to explore the potential mechanisms of M1/M2 macrophage polarization in TB development. THP-1 macrophages were treated with early secreted antigenic target of 6kDa (ESAT-6) protein for an increasing time. The polarization profiles, apoptosis levels of M1 and M2 macrophages were detected by RT-qPCR, immunofluorescence, Western blot and flow cytometry. ESAT-6 initially promoted the generation of pro-inflammatory M1-polarized macrophages in THP-1 cells within 24h, which were suppressed by further ESAT-6 treatment at 30-42h. Interestingly, ESAT-6 continuously promoted M2 polarization of THP-1 cells, thereby maintaining the anti-inflammatory response in a time-dependent manner. In addition, ESAT-6 promoted apoptotic cell death in M1-polarized macrophages, which had little effects on apoptosis of M2-phenotype of macrophages. Then, the potential underlying mechanisms were uncovered, and we verified that ESAT-6 increased the protein levels of TLR4, MyD88 and NF-κB to activate the TLR4/MyD88/NF-κB pathway within 24h, and this signal pathway was significantly inactivated at 36h post-treatment. Interestingly, the following experiments confirmed that ESAT-6 TLR4/MyD88/NF-κB pathway-dependently regulated M1/M2 polarization and apoptosis of macrophage in THP-1 cells. Our study investigated the detailed effects and mechanisms of M1/M2 macrophages in regulating innate responses during TB development, which provided a new perspective on the development of treatment strategies for this disease.

Sensitive Blood-Based Detection of HIV-1 and Mycobacterium tuberculosis Peptides for Disease Diagnosis by Immuno-Affinity Liquid Chromatography-Tandem Mass Spectrometry: A Method Development and Proof-of-Concept Study.

Novel approaches that allow early diagnosis and treatment monitoring of both human immunodeficiency virus-1 (HIV-1) and tuberculosis disease (TB) are essential to improve patient outcomes. We developed and validated an immuno-affinity liquid chromatography-tandem mass spectrometry (ILM) assay that simultaneously quantifies single peptides derived from HIV-1 p24 and Mycobacterium tuberculosis (Mtb) 10-kDa culture filtrate protein (CFP10) in trypsin-digested serum derived from cryopreserved serum archives of cohorts of adults and children with/without HIV and TB. ILM p24 and CFP10 results demonstrated good intra-laboratory precision and accuracy, with recovery values of 96.7% to 104.6% and 88.2% to 111.0%, total within-laboratory precision (CV) values of 5.68% to 13.25% and 10.36% to 14.92%, and good linearity (r2 > 0.99) from 1.0 to 256.0 pmol/L and 0.016 to 16.000 pmol/L, respectively. In cohorts of adults (n = 34) and children (n = 17) with HIV and/or TB, ILM detected p24 and CFP10 demonstrated 85.7% to 88.9% and 88.9% to 100.0% diagnostic sensitivity for HIV-1 and TB, with 100% specificity for both, and detected HIV-1 infection earlier than 3 commercial p24 antigen/antibody immunoassays. Finally, p24 and CFP10 values measured in longitudinal serum samples from children with HIV-1 and TB distinguished individuals who responded to TB treatment from those who failed to respond or were untreated, and who developed TB immune reconstitution inflammatory syndrome. Simultaneous ILM evaluation of p24 and CFP10 results may allow for early TB and HIV detection and provide valuable information on treatment response to facilitate integration of TB and HIV diagnosis and management.

ITRAQ-based proteomics reveals potential markers and treatment pathways for acute Achilles tendon rupture

BackgroundDue to its limited blood supply and irregular mechanical loading, the Achilles tendon is the most frequently ruptured tendon. Despite the rising incidence of acute Achilles tendon rupture (AATR), the optimal treatment remains controversial. Missed diagnoses and delayed treatments lead to poor outcomes and limited treatment options. This study aimed to identify potential biomarkers for diagnosing and developing therapies for AATR.MethodsWe employed the coupled isobaric tag for relative and absolute quantitation-liquid chromatography–electrospray ionization-tandem mass spectrometry approach to investigate protein expression in tissues from AATR patients. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to identify differentially expressed proteins (DEPs) between AATR patients and healthy individuals. A protein–protein interaction (PPI) network of DEPs was constructed using the Search Tool for the Retrieval of Interacting Genes. The screened hub genes were selectively verified by immunohistochemical staining.ResultsWe identified 410 DEPs between AATR patients and controls. The DEPs were significantly enriched in GO terms such as the extracellular region, extracellular region part, and defense response, as well as KEGG pathways, including complement and coagulation cascades, focal adhesion, and regulation of actin cytoskeleton. The main hub nodes in the PPI network comprised fibronectin 1 (FN1), major histocompatibility complex, class I, B (HLA-B), filamin A (FLNA), heat shock 27-kDa protein 1 (HSPB1), heat shock protein family A member 5 (HSPA5), apolipoprotein A4 (APOA4), and myosin IC (MYO1C). Although APOA4 and collagens I, II, and III were detectable in healthy tendons, immunohistochemical staining confirmed higher expression of these proteins in the acutely ruptured Achilles tendon.ConclusionsOur findings lay a foundation for further molecular studies of AATR. Inflammation and age-related degeneration may contribute to the pathogenesis of AATR. Moreover, the identified DEPs could be potential biomarkers for AATR diagnosis and treatment.

20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis ISPC-12: Purification, characterization, solution scattering and structural analysis

The 20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis (Bti) has been identified as an essential molecular chaperone in the enhancement of Cry11Aa and Cyt1Aa toxins production and their bio-crystallization. Additionally, P20 plays a vital role in suppressing the toxic effect of Cyt toxin on the host bacterium and also enhances insecticidal activity of Cry1Ac protein. Thus, the function of P20 is more specific than that of the chaperones. However, P20 is poorly investigated and insufficiently characterized. In the present study, we recombinantly expressed p20 from local isolate Bti ISPC-12 in heterologous bacterium E. coli and P20 protein was purified to homogeneity. Detailed biochemical and biophysical characterization provides crucial insights about in-vitro behavior as well as spatial conformations of P20 protein. Further, structural modelling and analysis provides insights into three-dimensional organization of the protein and shows that P20 is a non-toxic member of cytolytic (Cyt) toxin family similar to Cyt1Ca, with presence of conserved cytolysin fold. Additionally, solution scattering reveals that P20 is present as a dimer in the solution and probable dimeric assembly of P20 is presented. The findings reported here reveal engaging facts about P20 thereby advancing our understanding about this protein, which will expedite future studies.

MiR‑3120/Hsc70 participates in forced swim stress‑induced mechanical hyperalgesia in rats in an inflammatory state.

The heat shock cognate 71kDa protein (Hsc70) is a stress‑inducible ATPase that can protect cells against harmful stimuli. Transient receptor potential vanilloid 1 (TRPV1) is a well‑documented nociceptor. Notably, Hsc70 can inhibit TRPV1 expression and function, suggesting that Hsc70 may have pain regulation potential. However, the role of Hsc70 in stress‑induced hyperalgesia remains unclear. In the present study, the participation of Hsc70 and its regulator microRNA (miR)‑3120 were investigated in forced swim (FS) stress‑induced mechanical hyperalgesia in rats in an inflammatory state. Complete Freund's adjuvant (CFA) hind paw injection was performed to induce inflammatory pain in rats (CFA rats). Furthermore, in FS+CFA rats, FS stress was performed for 3days before CFA injection. The levels of Hsc70, miR‑3120 and their downstream molecule TRPV1 were measured in the dorsal root ganglion (DRG) with western blotting, immunofluorescence, reverse transcription‑quantitative polymerase chain reaction and fluorescence insitu hybridization. The results revealed that FS stress significantly exacerbated CFA‑induced mechanical pain. Furthermore, CFA upregulated Hsc70 and TRPV1 expression, which was partially inhibited or further enhanced by FS stress, respectively. In FS+CFA rats, intrathecal injection of a lentiviral vector overexpressing Hsc70 (LV‑Hsc70) could decrease TRPV1 expression and improve the mechanical pain. Additionally, the expression levels of miR‑3120, a regulator of Hsc70, were markedly upregulated on day3 following FS stress. Finally, miR‑3120 was identified to be colocalized with Hsc70 and expressed in all sizes of DRG neurons. In CFA rats, DRG injection of miR‑3120 agomir to induce overexpression of miR‑3120 resulted in similar TRPV1 expression and behavioral changes as those caused by FS stress, which were abolished in the presence of LV‑Hsc70. These findings suggested that miR‑3120/Hsc70 may participate in FS stress‑induced mechanical hyperalgesia in rats in an inflammatory state, possibly via disinhibiting TRPV1 expression in the DRG neurons.

Targeting TNFAIP2 induces immunogenic cell death and sensitizes glioblastoma multiforme to anti-PD-1 therapy.

The efficacy of current immunotherapeutic strategies for patients with glioblastoma multiforme (GBM) remains unsatisfactory. The purpose of this study was to investigate the correlation between tumor necrosis factor alpha-induced protein 2 (TNFAIP2) and immunogenic cell death (ICD) in GBM, and to examine the effect of TNFAIP2 knockdown and anti-PD-1 combination treatment in a mouse glioma model. The CGGA and TCGA databases were used to explore the possible function of TNFAIP2 in GBM. Multiplex immunohistochemistry (mIHC) staining was performed to detect the immune infiltration of tissues. Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry, and enzyme linked immunosorbent assay (ELISA) were utilized to detect the release of damage-associated molecular patterns (DAMPs) and the activation of the immune response. A mouse glioma model was applied to examine the induction of immune response. In vitro and in vivo studies demonstrated that TNFAIP2 knockdown increased the surface exposure of calreticulin (CALR), heat shock protein 70kDa (HSP70), and heat shock protein 90kDa (HSP90) in GBM cell lines, thereby inducing immunogenic cell death (ICD). Importantly, the study found that TNFAIP2 knockdown in combination with anti-PD-1 therapy significantly improved the overall survival of glioma in a mouse model. TNFAIP2 knockdown induces ICD by downregulating TNFAIP2 in GBM. In addition, TNFAIP2 knockdown sensitized glioma to anti-PD-1 therapy. Hence, targeting TNFAIP2 alone or in combination with anti-PD-1 therapy may be a potential strategy for GBM treatment through ICD.

PIP5Kγ Mediates PI(4,5)P2/Merlin/LATS1 Signaling Activation and Interplays with Hsc70 in Hippo-YAP Pathway Regulation.

The type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family produces the critical lipid regulator phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM). Here, we investigated the potential role of PIP5Kγ, a PIP5K isoform, in the Hippo pathway. The ectopic expression of PIP5Kγ87 or PIP5Kγ90, two major PIP5Kγ splice variants, activated large tumor suppressor kinase 1 (LATS1) and inhibited Yes-associated protein (YAP), whereas PIP5Kγ knockdown yielded opposite effects. The regulatory effects of PIP5Kγ were dependent on its catalytic activity and the presence of Merlin and LATS1. PIP5Kγ knockdown weakened the restoration of YAP phosphorylation upon stimulation with epidermal growth factor or lysophosphatidic acid. We further found that PIP5Kγ90 bound to the Merlin's band 4.1/ezrin/radixin/moesin (FERM) domain, forming a complex with PI(4,5)P2 and LATS1 at the PM. Notably, PIP5Kγ90, but not its kinase-deficient mutant, potentiated Merlin-LATS1 interaction and recruited LATS1 to the PM. Consistently, PIP5Kγ knockdown or inhibitor (UNC3230) enhanced colony formation in carcinoma cell lines YAP-dependently. In addition, PIP5Kγ90 interacted with heat shock cognate 71-kDa protein (Hsc70), which also contributed to Hippo pathway activation. Collectively, our results suggest that PIP5Kγ regulates the Hippo-YAP pathway by forming a functional complex with Merlin and LATS1 at the PI(4,5)P2-rich PM and via interplay with Hsc70.

Seasonal expressions of the translocator protein (18 kDa), voltage-dependent anion channel, and steroidogenic acute regulatory protein in the scent glands of muskrats (Ondatra zibethicus)

Steroidogenesis machinery involves the steroidogenic acute regulatory protein (StAR), which regulates cholesterol transfer within the mitochondria, and the transport of cholesterol via a channel composed of 18-kDa translocator protein (TSPO), the voltage-dependent anion channel (VDAC) plus some accessory proteins. In this study, we investigated the immunolocalizations and expressions of StAR, TSPO, VDAC and cytochrome P450 side chain cleavage enzyme (P450scc, CYP11A1) in the scent glands of muskrats (Ondatra zibethicus) during the breeding and non-breeding periods. StAR, TSPO, VDAC and CYP11A1 were immunolocalized in the scent glandular, interstitial and epithelial cells in both breeding and non-breeding seasons with stronger immunostaining in the breeding season. The mRNA expression levels of StAR, TSPO, VDAC and CYP11A1 were higher in the scent glands of the breeding season than those of the non-breeding season. The circulating follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) as well as scent glandular T and dihydrotestosterone (DHT) concentrations were also significantly higher in the breeding season. Additionally, the transcriptomic study in the scent glands identified that differentially expressed genes might be related to the lipid metabolic process, integral component of membrane, and steroid hormone receptor activity and hormone activity using GO analysis. Further in vitro study verified that StAR, TSPO, VDAC and CYP11A1 expression levels increased significantly after human chorionic gonadotropin, hCG/FSH treatment compared with the control group. The KEGG pathway enriched by differentially expressed genes detected to be involved in endocrine system or amino acid metabolism. These findings suggested that the scent glands of the muskrats have ability to synthesize steroids de novo, and that the steroid hormones may have an important regulatory role in the scent glandular function via an autocrine/paracrine pathway.

TSPO exacerbates acute cerebral ischemia/reperfusion injury by inducing autophagy dysfunction

Autophagy is considered a double-edged sword, with a role in the regulation of the pathophysiological processes of the central nervous system (CNS) after cerebral ischemia-reperfusion injury (CIRI). The 18-kDa translocator protein (TSPO) is a highly conserved protein, with its expression level in the nervous system closely associated with the regulation of pathophysiological processes. In addition, the ligand of TSPO reduces neuroinflammation in brain diseases, but the potential role of TSPO in CIRI is largely undiscovered. On this basis, we investigated whether TSPO regulates neuroinflammatory response by affecting autophagy in microglia. In our study, increased expression of TSPO was detected in rat brain tissues with transient middle cerebral artery occlusion (tMCAO) and in BV2 microglial cells exposed to oxygen-glucose deprivation or reoxygenation (OGD/R) treatment, respectively. In addition, we confirmed that autophagy was over-activated during CIRI by increased expression of autophagy activation related proteins with Beclin-1 and LC3B, while the expression of p62 was decreased. The degradation process of autophagy was inhibited, while the expression levels of LAMP-1 and Cathepsin-D were significantly reduced. Results of confocal laser microscopy and transmission electron microscopy (TEM) indicated that autophagy flux was disordered. In contrast, inhibition of TSPO prevented autophagy over-activation both in vivo and in vitro. Interestingly, suppression of TSPO alleviated nerve cell damage by reducing reactive oxygen species (ROS) and pro-inflammatory factors, including TNF-α and IL-6 in microglia cells. In summary, these results indicated that TSPO might affect CIRI by mediating autophagy dysfunction and thus might serve as a potential target for ischemic stroke treatment.

A seminested recombinase polymerase amplification assay to detect rickettsial pathogens in clinical samples

Treatment at the early stage of onset is vital for the prognosis of rickettsioses. But the absence of specific clinical symptoms complicates the diagnosis of this condition. Herein we established a seminested recombinase polymerase amplification assay (snRPA-nfo) that enables quick detection and differentiation of rickettsial pathogens in clinical samples with high sensitivity and specificity. The conserved 17-kDa protein gene of Rickettsia sibirica and the 47-kDa protein gene of Orientia tsutsugamushi were targeted for the duplex RPA-nfo assay. The snRPA-nfo assay exhibited an increased LOD in spiked blood samples, up to 1000-fold in comparison to standard RPA-nfo, and a better detection rate (83.3%, 5/6) than TaqMan PCR (16.6%, 1/6, Ct ≤ 35) in clinically confirmed patient blood samples. Thus, snRPA-nfo assay represents a promising alternative to TaqMan PCR in the early diagnosis of rickettsioses for point-of-care testing as well as in resource-limited settings.

Serum inducible protein 10kDa/C-X-C motif chemokine10 levels predict regression of M2BPGi-based liver fibrosis after hepatitisC virus eradication by direct-acting antiviral agents.

It is desirable to identify predictors of regression of liver fibrosis after achieving sustained virological response by anti-hepatitisC virus (anti-HCV) therapy. We retrospectively investigated the serum interferon-γ inducible protein 10kDa (IP-10) level as a predictive indicator of regression of liver fibrosis after successful hepatitis C virus eradication by direct-acting antiviral agents (DAAs) therapy. The study participants were recruited from a historical cohort of 116 chronically hepatitisC virus-infected patients who had achieved sustained virological response by DAAs therapy and whose serum Mac-2 binding protein glycosylation isomer (M2BPGi) levels at baseline (before DAAs therapy) were ≥2.0 cut-off index. We defined patients with M2BPGi levels <1.76 and ≥1.76 cut-off index at 2years after the end of treatment (EOT) as the regression (n=71) and non-regression (n=45) groups, respectively. Multivariate analyses revealed that the albumin-bilirubin score at baseline, and albumin-bilirubin score, Fibrosis-4 index at 24weeks after the EOT, and serum IP-10 change from baseline to 24weeks after the EOT (IP-10 change) were significantly associated with regression of M2BPGi-based liver fibrosis. In addition, IP-10 change was significantly associated with regression of M2BPGi-based liver fibrosis by a multivariate analysis, even when the serum M2BPGi levels were aligned by propensity score matching and in patients with advanced M2BPGi-based liver fibrosis: M2BPGi levels ≥3.3 cut-off index at baseline. Serum IP-10 change from baseline to 24weeks after the EOT is a feasible predictor of regression of M2BPGi-based liver fibrosis after achieving sustained virological response with DAA therapy.

Roles of increased NUCKS1 expression in endometriosis

BackgroundEndometriosis is still a difficult problem for women. The Nuclear Ubiquitous Casein and cyclin-dependent Kinase Substrate 1 (NUCKS1) gene is located on human chromosome 1q32.1. It encodes the NUCKS1 protein, a 27 kDa nuclear DNA binding protein that plays an important role in cell growth and proliferation. NUCKS1 plays an important role in the development of many diseases. However, its role in endometriosis is unclear.MethodsEctopic endometrial tissues and normal tissue specimens were collected, and the expression of NUCKS1, NF-κB and PI3K was detected by RT-qPCR and immunohistochemistry. Inhibition of NUCKS1 in hEM15A cells, study the changes in cell viability, apoptosis, migration and protein expression by CCK8 assay, flow cytometry, wound-healing assay, western blot and ELISA techniques. The comparison of differences between the two groups was implemented using unpaired sample t test or Mann-whitney U test. One-way analysis of variance or Kruskal-wallis test was used for comparisons among the three groups.Results(1) NUCKS1 is highly expressed in endometriosis tissues. (2) Inhibition of NUCKS1 decreases cell viability and capability of migration, and increases apoptosis in endometriosis cells. (3) Expressions of NF-κB and PI3K are increased in endometriosis tissues, and inhibition of NUCKS1 decreases the expression levels of PI3K and NF-κB in endometriosis cells. (4) Inhibition of NUCKS1 decreases the expression of VEGF.Conclusion(1) NUCKS1 is overexpressed in endometriosis, and inhibition of NUCKS1 inhibits cell viability and capability of migration, and increases apoptosis. (2) NUCKS1 promotes the progress of endometriosis through activating PI3K and NF-κB pathways, and VEFG is also involved in this process.

Prognostic Value of TSPO PET Before Radiotherapy in Newly Diagnosed IDH-Wild-Type Glioblastoma.

The 18-kDa translocator protein (TSPO) is gaining recognition as a relevant target in glioblastoma imaging. However, data on the potential prognostic value of TSPO PET imaging in glioblastoma are lacking. Therefore, we investigated the association of TSPO PET imaging results with survival outcome in a homogeneous cohort of glioblastoma patients. Methods: Patients were included who had newly diagnosed, histologically confirmed isocitrate dehydrogenase (IDH)-wild-type glioblastoma with available TSPO PET before either normofractionated radiotherapy combined with temozolomide or hypofractionated radiotherapy. SUVmax on TSPO PET, TSPO binding affinity status, tumor volumes on MRI, and further clinical data, such as O 6-alkylguanine DNA methyltransferase (MGMT) and telomerase reverse transcriptase (TERT) gene promoter mutation status, were correlated with patient survival. Results: Forty-five patients (median age, 63.3 y) were included. Median SUVmax was 2.2 (range, 1.0-4.7). A TSPO PET signal was associated with survival: High uptake intensity (SUVmax > 2.2) was related to significantly shorter overall survival (OS; 8.3 vs. 17.8 mo, P = 0.037). Besides SUVmax, prognostic factors for OS were age (P = 0.046), MGMT promoter methylation status (P = 0.032), and T2-weighted MRI volume (P = 0.031). In the multivariate survival analysis, SUVmax in TSPO PET remained an independent prognostic factor for OS (P = 0.023), with a hazard ratio of 2.212 (95% CI, 1.115-4.386) for death in cases with a high TSPO PET signal (SUVmax > 2.2). Conclusion: A high TSPO PET signal before radiotherapy is associated with significantly shorter survival in patients with newly diagnosed IDH-wild-type glioblastoma. TSPO PET seems to add prognostic insights beyond established clinical parameters and might serve as an informative tool as clinicians make survival predictions for patients with glioblastoma.

TSPO PET brain inflammation imaging: A transdiagnostic systematic review and meta-analysis of 156 case-control studies

IntroductionThe 18-kDa translocator protein (TSPO) is increasingly recognized as a molecular target for PET imaging of inflammatory responses in various central nervous system (CNS) disorders. However, the reported sensitivity and specificity of TSPO PET to identify brain inflammatory processes appears to vary greatly across disorders, disease stages, and applied quantification methods. To advance TSPO PET as a potential biomarker to evaluate brain inflammation and anti-inflammatory therapies, a better understanding of its applicability across disorders is needed. We conducted a transdiagnostic systematic review and meta-analysis of all in vivo human TSPO PET imaging case-control studies in the CNS. Specifically, we investigated the direction, strength, and heterogeneity associated with the TSPO PET signal across disorders in pre-specified brain regions, and explored the demographic and methodological sources of heterogeneity. MethodsWe searched for English peer-reviewed articles that reported in vivo human case-control TSPO PET differences. We extracted the demographic details, TSPO PET outcomes, and technical variables of the PET procedure. A random-effects meta-analysis was applied to estimate case-control standardized mean differences (SMD) of the TSPO PET signal in the lobar/whole-brain cortical grey matter (cGM), thalamus, and cortico-limbic circuitry between different illness categories. Heterogeneity was evaluated with the I2 statistic and explored using subgroup and meta-regression analyses for radioligand generation, PET quantification method, age, sex, and publication year. Significance was set at the False Discovery Rate (FDR)-corrected P < 0.05. Results156 individual case-control studies were included in the systematic review, incorporating data for 2381 healthy controls and 2626 patients. 139 studies documented meta-analysable data and were grouped into 11 illness categories. Across all the illness categories, we observed a significantly higher TSPO PET signal in cases compared to controls for the cGM (n = 121 studies, SMD = 0.358, PFDR < 0.001, I2 = 68%), with a significant difference between the illness categories (P = 0.004). cGM increases were only significant for Alzheimer’s disease (SMD = 0.693, PFDR < 0.001, I2 = 64%) and other neurodegenerative disorders (SMD = 0.929, PFDR < 0.001, I2 = 73%). Cortico-limbic increases (n = 97 studies, SMD = 0.541, P < 0.001, I2 = 67%) were most prominent for Alzheimer’s disease, mild cognitive impairment, other neurodegenerative disorders, mood disorders and multiple sclerosis. Thalamic involvement (n = 79 studies, SMD = 0.393, P < 0.001, I2 = 71%) was observed for Alzheimer’s disease, other neurodegenerative disorders, multiple sclerosis, and chronic pain and functional disorders (all PFDR < 0.05). Main outcomes for systemic immunological disorders, viral infections, substance use disorders, schizophrenia and traumatic brain injury were not significant. We identified multiple sources of between-study variance to the TSPO PET signal including a strong transdiagnostic effect of the quantification method (explaining 25% of between-study variance; VT-based SMD = 0.000 versus reference tissue-based studies SMD = 0.630; F = 20.49, df = 1;103, P < 0.001), patient age (9% of variance), and radioligand generation (5% of variance). ConclusionThis study is the first overarching transdiagnostic meta-analysis of case-control TSPO PET findings in humans across several brain regions. We observed robust increases in the TSPO signal for specific types of disorders, which were widespread or focal depending on illness category. We also found a large and transdiagnostic horizontal (positive) shift of the effect estimates of reference tissue-based compared to VT-based studies. Our results can support future studies to optimize experimental design and power calculations, by taking into account the type of disorder, brain region-of-interest, radioligand, and quantification method.

Immunodiagnostic Detection of Angiostrongylus cantonensis Exposure on Hawaii Island Using Isogeographic 31-kDa Antigen.

Angiostrongylus cantonensis is the leading cause of neuroangiostrongyliasis worldwide, and east Hawaii Island is a hotspot for the disease in the United States. A combination of glycoproteins with molecular weight of 31 kDa has been used as antigen to evaluate antibody response in human serum samples in Thailand with high specificity and sensitivity. In a previous pilot study, the Thailand-isolated 31-kDa proteins showed efficacy in dot-blot tests using serum samples from 435 human volunteers on Hawaii Island. However, we hypothesized that native antigen isolated from Hawaii A. cantonensis may exhibit higher specificity than the Thailand-isolated 31-kDa antigen due to potential minor variation in epitopes between isolates. In this study, 31-kDa glycoproteins were isolated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis from adult A. cantonensis nematodes collected from rats captured on east Hawaii Island. The resultant proteins were purified by electroelution, pooled, bioanalyzed, and quantified. A subset of 148 samples from human participants of the original cohort of 435 was consented for this study, including 12 of the original 15 clinically diagnosed participants. Results of ELISA using the Hawaii-isolated 31-kDa antigen were compared with results of the same serum samples previously tested with both crude Hawaii antigen ELISA and Thailand 31-kDa antigen dot blot. This study shows a seroprevalence in the general population of East Hawaii Island of 25.0%, similar to previous findings of 23.8% seroprevalence in this cohort using crude antigen from Hawaii A. cantonensis and 26.5% using Thailand 31-kDa antigen.

Imaging the brain's immune response to alcohol with [11C]PBR28 TSPO Positron Emission Tomography.

In humans, the negative effects of alcohol are linked to immune dysfunction in both the periphery and the brain. Yet acute effects of alcohol on the neuroimmune system and its relationships with peripheral immune function are not fully understood. To address this gap, immune response to an alcohol challenge was measured with positron emission tomography (PET) using the radiotracer [11C]PBR28, which targets the 18-kDa translocator protein, a marker sensitive to immune challenges. Participants (n = 12; 5 F; 25-45 years) who reported consuming binge levels of alcohol (>3 drinks for females; >4 drinks for males) 1-3 months before scan day were enrolled. Imaging featured a baseline [11C]PBR28 scan followed by an oral laboratory alcohol challenge over 90 min. An hour later, a second [11C]PBR28 scan was acquired. Dynamic PET data were acquired for at least 90 min with arterial blood sampling to measure the metabolite-corrected input function. [11C]PBR28 volume of distributions (VT) was estimated in the brain using multilinear analysis 1. Subjective effects, blood alcohol levels (BAL), and plasma cytokines were measured during the paradigm. Full completion of the alcohol challenge and data acquisition occurred for n = 8 (2 F) participants. Mean peak BAL was 101 ± 15 mg/dL. Alcohol significantly increased brain [11C]PBR28 VT (n = 8; F(1,49) = 34.72, p > 0.0001; Cohen's d'=0.8-1.7) throughout brain by 9-16%. Alcohol significantly altered plasma cytokines TNF-α (F(2,22) = 17.49, p < 0.0001), IL-6 (F(2,22) = 18.00, p > 0.0001), and MCP-1 (F(2,22) = 7.02, p = 0.004). Exploratory analyses identified a negative association between the subjective degree of alcohol intoxication and changes in [11C]PBR28 VT. These findings provide, to our knowledge, the first in vivo human evidence for an acute brain immune response to alcohol.