Protocol for reconstitution of oligomeric assembly of NAIP5-NLRC4 inflammasome in vitro.

Inflammasomes are multimeric protein complexes that have crucial functions in innate immunity. Here, we present a protocol to reconstitute the PELO-driven assembly of NAIP5-NLRC4 inflammasome in vitro. We describe steps for expression and purification of recombinant PELO and flagellin, preparation of native cell lysate containing NAIP5-NLRC4, and in vitro assembly of NAIP5-NLRC4 inflammasome. We then detail analysis of NAIP5-NLRC4 inflammasome by blue native polyacrylamide gel electrophoresis and immunoblotting. This protocol can be adapted to monitor the oligomeric assembly of other inflammasome types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2023).1.

Evaluation of DDA Library-Free Strategies for Phosphoproteomics and Ubiquitinomics Data-Independent Acquisition Data.

Phosphoproteomics and ubiquitinomics data-independent acquisition (DIA) mass spectrometry (MS) data is typically analyzed by using a data-dependent acquisition (DDA) spectral library. The performance of various library-free strategies for analyzing phosphoproteomics and ubiquitinomics DIA MS data has not been evaluated. In this study, we systematically compare four commonly used DDA library-free approaches including Spectronaut's directDIA, DIA-Umpire, DIA-MSFragger, and in silico-predicted library for analysis of phosphoproteomics SWATH, DIA, and diaPASEF data as well as ubiquitinomics diaPASEF data. Spectronaut's directDIA shows the highest sensitivity for phosphopeptide detection not only in synthetic phosphopeptide samples but also in phosphoproteomics SWATH-MS and DIA data from real biological samples, when compared to the other three library-free strategies. For phosphoproteomics diaPASEF data, Spectronaut's directDIA and the in silico-predicted library based on DIA-NN identify almost the same number of phosphopeptides as a project-specific DDA spectral library. However, only about 30% of the total phosphopeptides are commonly identified, suggesting that the library-free strategies for phospho-diaPASEF data need further improvement in terms of sensitivity. For ubiquitinomics diaPASEF data, the in silico-predicted library performs the best among the four workflows and detects ∼50% more K-GG peptides than a project-specific DDA spectral library. Our results demonstrate that Spectronaut's directDIA is suitable for the analysis of phosphoproteomics SWATH-MS and DIA MS data, while the in silico-predicted library based on DIA-NN shows substantial advantages for ubiquitinomics diaPASEF MS data.

Ribosome-rescuer PELO catalyzes the oligomeric assembly of NOD-like receptor family proteins via activating their ATPase enzymatic activity.

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.

A Novel Symbiotic Formulation Reduces Obesity and Concomitant Metabolic Syndrome in Rats by Raising the Relative Abundance of Blautia.

Obesity is regarded as an abnormal or excessive buildup of fat that may be bad for health and is influenced by a combination of intestinal flora, genetic background, physical activity level and environment. Symbiotic supplementation may be a realistic and easy therapy for the reversal of obesity and associated metabolic problems. In this study, we chose two Bifidobacterium species, three Lactobacilli species and four prebiotics to make a new symbiotic formulation. High or low doses of the symbiotic were administered to rats, and biochemical indicators were recorded to assess the biological effects in a high-fat-diet-induced rat model. The underlying mechanisms were explored by integrating 16S rRNA sequencing with an extensively targeted metabolome. High-dose symbiotic supplementation was effective in reducing obesity and concomitant metabolic syndrome. The high-dose symbiotic also significantly increased the abundance of Blautia, which was negatively correlated with taurocholic acid and the main differential metabolites involved in amino acid and bile acid metabolism. While the low-dose symbiotic had some therapeutic effects, they were not as strong as those at the high dose, demonstrating that the effects were dose-dependent. Overall, our novel symbiotic combination improved plasma glucose and lipid levels, shrunk adipocyte size, restored liver function, increased the abundance of Blautia and adjusted bile acid and amino acid metabolism.

Four new phenolic glycosides from Dobinea delavayi.

Three new benzophenone glycosides, 2-O-ß-D-glucopyranosyl-6,3',4'-trihydroxy-4-methoxybenzophenone (1), 4'-O-ß-D-glucopyranosyl-2,4,6,3'-tetramethoxybenzophenone (2) and 2-O-ß-D-glucopyranosyl-4'-hydroxy-6-methylbenzophenone (3), and a new flavonoid glycoside 5'-hydroxyombuoside (6), along with three known phenolic glycosides 4-O-ß-D-glucopyranosyl-2,6,4'-trihydroxybenzophenone (4), mangiferin (5), and ombuoside (7), were isolated from the 80% ethanol extract of roots of Dobinea delavayi. Their structures were determined by the comprehensive analyses of the physicochemical properties and spectroscopic data (1D NMR, 2D NMR, and HR-ESI-MS). Cytotoxicity and in vitro antimalarial activity of compounds 1, 3, 4, 6, and 7 were evaluated by MTT colorimetric assay and ß-hematin formation inhibition assay, respectively.

Flavonoids from Scutellaria likiangensis Diels and their antimalarial activities.

Two new flavonoid glycosides scutelikiosides A and B (13 and 23), along with twenty-one known compounds from the 75% ethanol extract of roots of Scutellaria likiangensis Diels. Their structures were determined by the comprehensive analyses of the spectroscopic data (1D NMR, 2D NMR, HRESIMS, and CD) and physicochemical properties. Compounds 4-14, 17-19, 21, and 22 were evaluated for their in vivo antimalarial activities against Plasmodium yoelii BY265RFP in mice. Compound 17 exhibited significant activity close to artemisinin with an inhibition ratio of 29.2%, and compounds 6, 9-12, 14, 18, 19, and 22 exhibited moderate antimalarial activities with inhibition ratios ranging from 10.2% to 20.0% at a dose of 25 mg/kg/day. In addition, a summary of preliminary structure-activity relationship of isolated flavonoids for in vivo antimalarial activity was described.

Dibenzofurans from Crataegus oresbia and their lipid-lowering activity.

Three new dibenzofurans, 2-hydroxy-3,4-dimethoxydibenzofuran (1), 2,8-dihydroxy-3,4,9-trimethoxydibenzofuran (5) and 2-hydroxy-3,4,6,8-tetramethoxydibenzofuran (6), together with three known dibenzofurans 1-hydroxy-2,3,4-trimethoxydibenzofuran (2), 2-hydroxy-3,4,6-trimethoxydibenzofuran (3) and 1,6-dihydroxy-2,3,4-trimethoxydibenzofuran (4), were isolated from the twigs and leaves of Crataegus oresbia. Their structures were identified by the comprehensive analyses of the physicochemical properties and spectroscopic data (NMR and HR-ESI-MS). Compounds 1-3 showed significant in vitro lipid-lowering activities, especially compound 3 was stronger than simvastatin.

An unusual colonic mass in a phlebosclerosis patient.

A 47-year-old man with idiopathic mesenteric phlebosclerosis presented to our hospital because of a 2-month history of diarrhea and edema of both lower limbs. Contrastenhanced abdominal computed tomography (CT) showed a 2-cm mass of mixed density in the ascending colon. On coloscopy, a solid, ulcerated, semi-pedunculated, lobulated protruding mass of 3.5×3.5×1.5 cm was observed and removed with hot snare polypectomy. Histologic examination demonstrated a hamartomatous polyp with normal epithelium and an inflammatory infiltrate with dilated, mucus-filled cystic glands in the lamina propria, indicating a juvenile polyp.

Sesquiterpenoids and their glycosides from Dobinea delavayi (Baill.) Baill.

Eight undescribed sesquiterpenoids, including two eudesmane glycosides dobinosides A and B, five eudesmane aglycones dobinins Q-U, and one germacrane dobinin P, were isolated from the 80% ethanol extract of roots of Dobinea delavayi. Their structures were elucidated by extensive spectroscopic data (1D and 2D NMR, HR-ESI-MS) and single-crystal X-ray diffraction analysis. Dobinoside A, dobinins Q and R, as well as six reported eudesmane sesquiterpenoids, were evaluated for their in vivo antimalarial activities against Plasmodium yoelii BY265RFP in mice. A summary of preliminary structure-activity relationship of eudesmane sesquiterpenoids for in vivo antimalarial activity was described.

Positron emission tomography and computed tomography with [68Ga]Ga-fibroblast activation protein inhibitors improves tumor detection and staging in patients with pancreatic cancer.

PURPOSE: This study aimed to investigate the diagnostic performance of [68Ga]Ga-FAPI PET/CT for primary and metastatic pancreatic carcinoma lesions and compare the results with those of [18F]-fluorodeoxyglucose ([18F]FDG) PET/CT. METHODS: Patients with suspected or diagnosed pancreatic malignancy, who underwent contemporaneous [18F]FDG and [68Ga]Ga-FAPI PET/CT between June 2020 and January 2021, were retrospectively analyzed. Routine contrast-enhanced CT (CE-CT) is performed in all patients as standardized care. Findings were confirmed by histopathology or radiographic follow-up. We compared radiotracer uptake, diagnostic performance, and TNM (tumor-node-metastasis) classifications. RESULTS: We evaluated 36 participants (25/36 men; median age, 60 years), including 26 patients with pancreatic malignancies and ten patients with pancreatic benign lesions. [68Ga]Ga-FAPI PET/CT showed higher radiotracer uptake and higher sensitivity than [18F]FDG PET/CT in evaluating primary tumors (SUVmax, 21.4 vs. 4.8; sensitivity, 100% vs. 73.1%), involved lymph nodes (SUVmax, 8.6 vs. 2.7; sensitivity, 81.8% vs. 59.1%), and metastases (SUVmax, 7.9 vs. 3.5; sensitivity, 91.5% vs. 44.0%); Compared with [18F]FDG, [68Ga]Ga-FAPI PET/CT upstaged six patients' TNM staging (6/23, 26.1%) and changed two patients' clinical management (2/23, 8.7%). Compared with CE-CT, [68Ga]Ga-FAPI PET/CT upgraded TNM staging in five patients (5/23, 21.7%) and changed the therapeutic regimen in only one patient (1/23, 4.3%). Intense [68Ga]Ga-FAPI uptake was observed throughout the pancreas in 12/26 pancreatic malignancies; dual-time point [68Ga]Ga-FAPI PET/CT may differentiate pancreatitis from malignancy. CONCLUSIONS: Compared with [18F]FDG PET/CT, [68Ga]Ga-FAPI PET/CT shows higher sensitivity in detecting primary pancreatic tumors, involved lymph nodes, and metastases and is superior in terms of TNM staging. Prospective trials with larger patient population are needed to evaluate whether [68Ga]Ga-FAPI PET/CT could elicit treatment modification in pancreatic cancer when compared with standard of care imaging.

Krüppel-like factor 5-induced overexpression of long non-coding RNA DANCR promotes the progression of cervical cancer via repressing microRNA-145-3p to target ZEB1.

Long non-coding RNA (lncRNA) differentiation antagonizing non-protein coding RNA (DANCR) participates in the development of diverse cancers. Nevertheless, the impact of DANCR on cervical cancer (CC) remains largely unknown. This study aims to explore the effects of DANCR sponging microRNA-145-3p (miR-145-3p) on CC. Expression of KLF5, DANCR, miR-145-3p, and zinc finger E-box binding homeobox 1 (ZEB1) in CC and adjacent normal tissues was determined. Human CC cell lines were, respectively, treated with silenced DANCR or miR145-3p mimic/inhibitor. Then, the viability, migration, invasion, and apoptosis of CC cells were measured. The cell growth in vivo was observed as well. Chromatin immunoprecipitation assay was performed to analyze the binding of KLF5 and DANCR promoter. Interaction among DANCR, miR-145-3p, and ZEB1 was assessed. KLF5, DANCR, and ZEB1 were upregulated but miR-145-3p was downregulated in CC tissues. KLF5 activated DANCR expression and the high DANCR expression was related to tumor staging, infiltrating muscle depth and lymphatic metastasis of CC patients. Reduced DANCR or elevated miR-145-3p repressed malignant behaviors of CC cells. The tumor diameter and weight were also repressed by DANCR silencing or miR-145-3p elevation. The effect of DANCR knockdown on CC cells could be reversed by miR-145-3p inhibitor. MiR-145-3p was targeted by DANCR and ZEB1 was targeted by miR-145-3p. KLF5-induced overexpression of DANCR promotes CC progression via suppressing miR-145-3p to target ZEB1. This study may provide potential targets for CC treatment.

Differential diagnosis of thalassemia and iron deficiency anemia in pregnant women using new formulas from multidimensional analysis of red blood cells.

BACKGROUND: Iron deficiency anemia (IDA) and thalassemia trait (TT) are the most common conditions of microcytic hypochromic anemia (MHA) in pregnant women. We used the BC-6800Plus analyzer to study the utility of erythrocyte and reticulocyte parameters for distinguishing TT from IDA in pregnant women. METHODS: A total of 454 anemic pregnant women, including 340 with IDA, 66 with ß-thalassemia trait (ß-TT) and 48 with α-thalassemia trait (α-TT), were included. Multiple comparisons among groups were performed, and diagnostic performance of parameters was determined using receiver operating characteristic (ROC) curve analysis, with P<0.05 indicating statistical significance. RESULTS: Reticulocyte production index (RPI) and the average volume of mature red blood cells (MCVm) in the IDA group were significantly higher than in the ß-TT and α-TT groups. Red blood cell (RBC), reticulocyte percentage (Ret%), and RPI in the IDA group were significantly lower than in the α-TT and ß-TT groups. We devised MHA 1=0.42× MCH -0.57× RPI -0.08× %MICROr -9.38 to distinguish IDA from α-TT. With a cut-off value of 0.61, the area under the receiver operating characteristic curve (AUC), sensitivity, and specificity were 0.868, 90.9%, and 68.5%, respectively. We devised MHA 2=0.04× %MICROr +0.12× MCVm -13.76× Ret# -6.29 to distinguish IDA from ß-TT. With a cut-off value of 0.55, the AUC, sensitivity, and specificity were 0.878, 81.3%, and 80.3%, respectively. CONCLUSIONS: Erythrocyte indices and formulas can be used as initial methods for the differential diagnosis of TT and IDA. MHA 1 and MHA 2 were the most useful indices in the differential diagnosis of α-TT from IDA and ß-TT from IDA in pregnant women.

RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes.

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

Usefulness of [68Ga]Ga-DOTA-FAPI-04 PET/CT in patients presenting with inconclusive [18F]FDG PET/CT findings.

PURPOSE: This prospective study aimed to evaluate the potential usefulness of [68Ga]Ga-DOTA-FAPI-04 positron emission tomography/computed tomography (PET/CT) in the oncological evaluation of patients presenting with inconclusive [18F]FDG PET/CT findings. METHODS: [68Ga]Ga-DOTA-FAPI-04 was performed in patients presenting with inconclusive [18F]FDG PET/CT findings. Tumour uptake was quantified by the maximum standard uptake value (SUV). Histopathology or follow-up imaging served as the standard for the final diagnosis. RESULTS: A total of 68 patients with inconclusive [18F]FDG PET/CT findings underwent additional [68Ga]Ga-DOTA-FAPI-04 PET/CT. Of them, 18 (26.5%) were for discrimination of mass lesions detected on conventional imaging, 6 (8.8%) for detection of the unknown primary site in biopsy-proven metastatic malignancy, 21 (30.9%) for the staging of cancer, and the other 23 (33.8%) for evaluation of suspected disease recurrence. Most of the primary and metastatic lesions demonstrated higher uptake of [68Ga]Ga-DOTA-FAPI-04 than did [18F]FDG, which resulted in favourable tumour-to-background contrast in various types of cancer. As a result, [68Ga]Ga-DOTA-FAPI-04 PET/CT identified suspicious mass lesions with an accuracy of 12/18 (66.7%), detected the primary site in 4/6 patients (66.7%) with unknown malignancy, upgraded tumour staging in 7/21 patients (33.3%), and detected disease recurrence in 20/23 patients (87.0%). CONCLUSIONS: In patients undergoing oncological evaluation with inconclusive [18F]FDG PET/CT findings, [68Ga]Ga-DOTA-FAPI-04 may have a complementary role in discriminating mass lesions on conventional imaging, locating the primary site of unknown malignancy, modifying tumour staging, and detecting suspected disease recurrence. Nevertheless, careful attention should be paid when reading the [68Ga]Ga-DOTA-FAPI-04 PET/CT images in tumours complicated with inflammation.

A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.

A New Antimalarial Noreudesmane Sesquiterpenoid from Dobinea delavayi.

One previously undescribed angeloylated noreudesmane sesquiterpenoid, dobinin O (1), along with four known eudesmane sesquiterpenoids (2-5) were isolated from the peeled roots of Dobinea delavayi. Their structures were elucidated by extensive spectroscopic data analyses. In addition, compound 1 exhibited moderate antimalarial activity against Plasmodium yoelii BY265RFP with the inhibition ratio of 17.8 ± 13.3% at the dose of 30 mg/kg/day.

Periprostatic Fat Thickness on MRI is an Independent Predictor of Time to Castration-resistant Prostate Cancer in Chinese Patients With Newly Diagnosed Prostate Cancer Treated With Androgen Deprivation Therapy.

BACKGROUND: The aim of this study was to evaluate the association between periprostatic fat thickness (PPFT) and time to castration-resistant prostate cancer (CRPC) in newly diagnosed patients with prostate cancer (PCa) treated with androgen deprivation therapy (ADT). PATIENTS AND METHODS: We retrospectively reviewed the medical records of 150 patients with PCa treated with ADT at our hospital between June 2011 and June 2017. PPFT measured on magnetic resonance imaging (MRI) and PPFT/periprostatic fat volume (PPFV) measured on computed tomography (CT) were evaluated. Kaplan-Meier curves and log-rank tests were used to assess significant differences in time to CRPC between the 2 groups (high PPFT vs. low PPFT, determined by PPFT > or < the median value, respectively). Univariable and multivariable Cox regression analyses were employed to identify the potential prognostic factors for survival. RESULTS: The median value of PPFT measured on MRI was 0.555 cm. PPFT was significantly associated with PPFV measured on CT images (with a correlation coefficient of 0.825; P < .001). A total of 66 patients (44%) progressed to CRPC during the follow-up period. Patients with high PPFT (measured on MRI) showed a significantly shorter PFS than patients with low PPFT. Multivariable Cox analysis demonstrated that T stage, presence of distant metastasis, shorter time to prostate-specific antigen nadir, higher prostate-specific antigen nadir, Gleason score (greater than 4 + 4), and high PPFT were significantly associated with shorter PFS. CONCLUSIONS: PPFT is significantly associated with PPFV measured on CT images. PPFT measured on MRI is a readily available and significant predictor of time to CRPC in patients with PCa receiving ADT as the primary treatment.