Host Sorbitol and Bacterial Sorbitol Utilization Promote Clostridioides difficile Infection in Inflammatory Bowel Disease.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a widespread gastrointestinal inflammatory disorder with globally increasing incidence. Clostridioides difficile infection (CDI) often occurs in patients with intestinal dysbiosis, such as after antibiotic therapy. Patients with IBD have increased incidence of CDI and the clinical outcome of IBD is reportedly worsened by CDI. However, the underlying reasons remain poorly understood. METHODS: We performed a retrospective single-center and a prospective multicenter analysis of CDI in patients with IBD, including genetic typing of C difficile isolates. Furthermore, we performed a CDI mouse model to analyze the role of the sorbitol metabolization locus that we found distinguished the main IBD- and non-IBD-associated sequence types (STs). Moreover, we analyzed sorbitol concentration in the feces of patients with IBD and healthy individuals. RESULTS: We detected a significant association of specific lineages with IBD, particularly increased abundance of ST54. We found that in contrast to the otherwise clinically predominant ST81, ST54 harbors a sorbitol metabolization locus and was able to metabolize sorbitol in vitro and in vivo. Notably, in the mouse model, ST54 pathogenesis was dependent on intestinal inflammation-induced conditions and the presence of sorbitol. Furthermore, we detected significantly increased sorbitol concentrations in the feces of patients with active IBD vs patients in remission or healthy controls. CONCLUSIONS: Sorbitol and sorbitol utilization in the infecting C difficile strain play major roles for the pathogenesis and epidemiology of CDI in patients with IBD. CDI in patients with IBD may thus be avoided or improved by elimination of dietary sorbitol or suppression of host-derived sorbitol production.

Transcriptomic and free monoterpene analyses of aroma reveal that isopentenyl diphosphate isomerase inhibits monoterpene biosynthesis in grape (Vitis vinifera L.).

BACKGROUND: Monoterpenes are among the most important volatile aromatic compounds contributing to the flavor and aroma of grapes and wine. However, the molecular basis of monoterpene biosynthesis has not yet been fully elucidated. RESULTS: In our study, transcriptomics and gas chromatography-mass spectrometry (GC-MS) were used to mine candidate genes and transcription factors involved in monoterpene biosynthesis between high-monoterpene and zero-monoterpene table grape cultivars. We found that monoterpene biosynthesis was positively correlated by the expression of five genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (VvDXSs), one encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (VvHDR), three hydroxy-3-methylglutaryl-CoA synthases (VvHMGSs) and one mevalonate kinase (VvMVK), whereas the expression of one isopentenyl diphosphate isomerase (VvIDI) and one 3-hydroxy-3-methylglutaryl-CoA reductase (VvHMGR) negatively correlated monoterpene biosynthesis. Of these genes, VvIDI was selected to validate its function in monoterpene accumulation through a transient overexpression experiment, and was shown to inhibit the biosynthesis of grape linalool and α-terpineol. Meanwhile, we found that a 64-amino acid extension sequence at the N-terminus can guide the VvIDI protein to target the chloroplast. CONCLUSIONS: The findings of this study should help to guide future functional analysis of key genes as well as mining the potential regulatory mechanism of monoterpene biosynthesis in grapes and grape products.

Targeting neuropilin-1 abolishes anti-PD-1-upregulated regulatory T cells and synergizes with 4-1BB agonist for liver cancer treatment.

BACKGROUND AIMS: Regulatory T cells (Tregs) are an obstacle to PD-1 blockade-mediated antitumor efficacy. However, the behaviors of Tregs response to anti-PD-1 in HCC and the characteristics of Tregs tissue adaptation from peripheral lymphoid tissues to the tumor are still unclear. APPROACH RESULTS: Here, we determine that PD-1 monotherapy potentially augments the accumulation of tumor CD4 + Tregs. Mechanistically, anti-PD-1 mediates Tregs proliferation in lymphoid tissues rather than in the tumor. Increased peripheral Tregs burden replenishes intratumoral Tregs, raising the ratio of intratumoral CD4 + Tregs to CD8 + T cells. Subsequently, single-cell transcriptomics revealed that neuropilin-1 (Nrp-1) supports Tregs migration behavior, and the genes of Crem and Tnfrsf9 regulate the behaviors of the terminal suppressive Tregs. Nrp-1 + 4-1BB - Tregs stepwise develop to the Nrp-1 - 4-1BB + Tregs from lymphoid tissues into the tumor. Moreover, Treg-restricted Nrp1 depletion abolishes anti-PD-1-upregulated intratumoral Tregs burden and synergizes with the 4-1BB agonist to enhance the antitumor response. Finally, a combination of the Nrp-1 inhibitor and the 4-1BB agonist in humanized HCC models showed a favorable and safe outcome and evoked the antitumor effect of the PD-1 blockade. CONCLUSION: Our findings elucidate the potential mechanism of anti-PD-1-mediated intratumoral Tregs accumulation in HCC and uncover the tissue adaptation characteristics of Tregs and identify the therapeutic potential of targeting Nrp-1 and 4-1BB for reprogramming the HCC microenvironment.

Joint intra and inter-channel nonlinear compensation scheme based on improved learned digital back propagation for WDM systems.

In this paper, we improve the learned digital back propagation (LDBP) and propose a novel joint intra and inter-channel nonlinearity compensation scheme for polarization division multiplexing wavelength-division multiplexed (PDM-WDM) systems. From the perspective of interpretable neural network, the scheme realizes the alternating compensation of chromatic dispersion (CD) and nonlinearity based on physical models. The chromatic dispersion compensation (CDC) adopts one-dimensional convolution operation in the time domain. Moreover, the pulse-broadening effect is introduced into the overlap-and-save method. For nonlinear compensation, the improved joint model is applied, and the impact of the intra-channel pulse broadening and the walk-off effect between different channels caused by CD on the nonlinear effect is considered. To validate the effectiveness of the proposed scheme, we construct an 11-channel simulation system of 36 GBaud PDM uniform 16 quadrature amplitude modulation (PDM-16QAM) 1600 km and 64 GBaud PDM-64QAM 400 km, as well as a 5-channel experimental system of 28 GBaud PDM-16QAM 806.4 km. The simulation results show that the performance of PDM-16QAM with 0.5 steps per span and PDM-64QAM with 2 steps per span improve the Q-factor by approximately 0.75 dB and 0.54 dB at the optimal launch power, compared with the linear compensation scheme. The transmission performance of PDM-16QAM is higher than that of digital back propagation with 5 steps per span (DBP-5StPS), and the complexity is only 31.36% of that of DBP-5StPS. The performance of PDM-64QAM is higher than that of DBP-10StPS, with a complexity of 62.72%. The experimental results show that the performance of PDM-16QAM with 0.5 steps per span is improved with 0.86 dB Q-factor improvement compared with the linear compensation scheme at the optimal launch power, and the performance of the proposed scheme is higher than that of DBP-5StPS with a complexity of only 23.68%.

A systematic review and meta-analysis of primary bypass surgery compared with bypass surgery after endovascular treatment in peripheral artery disease patients.

OBJECTIVE: Both bypass surgery and endovascular treatment are well-recognized interventions for the treatment of peripheral artery disease; however, the effect of failed endovascular treatment on subsequent surgeries remains controversial. A systematic review was conducted to compare the outcomes of primary bypass and bypass surgery after endovascular treatment. METHODS: Three academic databases (Embase, PubMed, and Scopus) were searched from their inception to August 2022. Two independent investigators searched for studies that reported the outcomes of primary bypass surgery and bypass surgery after endovascular treatment in patients with peripheral artery disease. Abstracts and full-text studies were screened independently using duplicate data abstraction. Dichotomous outcome measures were reported using a random-effects model to generate a summary odds ratio (OR) and 95% confidence interval (CI). The risk of bias was assessed using the Newcastle-Ottawa Scale. RESULTS: Seventeen retrospective observational studies were selected from 3911 articles and included 8064 patients, 6252 of whom underwent primary bypass surgery and 1812 underwent bypass surgery after endovascular treatment. The mean age was 69.0 years and 61.2% (n = 4938) were male. For perioperative outcomes, the 30-day results showed no difference in mortality (OR, 0.76; 95% CI, 0.53-1.10), or amputation (OR, 0.89; 95% CI, 0.67-1.20). For short- to mid-term outcomes, primary patency did not differ at 6 months (OR, 0.98; 95% CI, 0.81-1.19), 1 year (OR, 1.12; 95% CI, 0.97-1.30), or 2 years (OR, 1.17; 95% CI, 0.85-1.61) follow-up. Amputation-free survival did not differ at 6 months (OR, 1.03; 95% CI, 0.82-1.30), 1 year (OR, 1.09; 95% CI, 0.89-1.32), 2 years (OR, 1.18; 95% CI, 0.93-1.50), or 3 years (OR, 1.09; 95% CI, 0.84-1.40) of follow-up. No significant difference was found in overall survival or second patency. CONCLUSIONS: This meta-analysis of retrospective, nonrandomized, observational studies suggests that prior endovascular treatment of lower extremity arterial disease does not result in worse perioperative, short-term, or mid-term clinical outcomes of subsequent infrainguinal bypass surgery compared with patients without prior endovascular treatment.

Acute Renal Infarction Due to Symptomatic Isolated Spontaneous Renal Artery Dissection: A Rare and Fatal Disease.

OBJECTIVE: To report demographics and clinical, laboratory, and imaging features of acute renal infarction (ARI) due to symptomatic isolated spontaneous renal artery dissection (SISRAD) and to analyze outcomes after the initial therapy for SISRAD. METHODS: Thirteen patients with ARI due to SISRAD between January 2016 and March 2021 were enrolled in this retrospective study. We reviewed the demographics, clinical, laboratory, and imaging features (location of the infarct kidney, the branch artery involved by dissection, true lumen stenosis, false lumen thrombosis, and aneurysm), treatment modalities, and follow-up results; analyzed the difference between SISRAD and other causes of ARI; and propose an appropriate therapy strategy for SISRAD based on our data and literature. RESULT: Patients with ARI due to SISRAD were mostly young men (43 [24-53] years; 12/13 [92%]). No patients had atrial fibrillation or acute kidney injury at admission (0/13). All 13 patients received conservative treatment as the initial treatment. Sixty-two percent (8/13) of patients progressed, and 88% (7/8) of them had dissection aneurysm on the admission computed tomographic angiography (CTA) image. Seventy-five percent (6/8) of patients underwent endovascular intervention as follows, stent placement in 1 patient, renal artery embolization in 1, and stent placement with embolization in 4. Two patients with disease progression died: 1 during the conservative treatment period and 1 after the stent placement. Thirty-eight percent (5/13) of patients in remission continued to receive conservative treatment, none of whom had dissection aneurysm on the admission CTA. CONCLUSION: Symptomatic isolated spontaneous renal artery dissection is a rare and fatal disease. For young ARI patients with no previous history of tumors and cardiogenic diseases, CTA examination is recommended to exclude SISRAD. Dissection aneurysm seems to be a risk of progression for SISRAD in this series. Conservative treatment, a recognized initial treatment, has a good effect on patients without dissection aneurysm, and we recommend endovascular intervention as the initial treatment for the patient with dissection aneurysm at admission. Multicenter clinical studies are needed to explore a more-appropriate treatment for patients with SISRAD. CLINICAL IMPACT: This article report the related factors, risks, demographics and laboratory data of Acute renal infarction (ARI) due to Symptomatic isolated spontaneous renal artery dissection (SISRAD) and explore a better initial therapy strategy for SISRAD. It will help improve the effectiveness of SISRAD treatment and reduce the mortality rate from this rare but lethal disease.

CircRNA-SCAF8 promotes vascular endothelial cell pyroptosis by regulating the miR-93-5p/TXNIP axis. / 环状RNA-SCAF8通过miR-93-5p/TXNIPé€šè·¯ä¿ƒè¿›è¡€ç®¡å† çš®ç»†èƒžç„¦äº¡.

OBJECTIVES: To investigate the role and mechanism of circRNA-SR-related CTD associated factor 8 (SCAF8) in regulating endothelial cell pyroptosis in high glucose environment. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured and divided into six groups. The normal control group and high glucose control group were cultured in cell culture medium with 5 and 33 mmol/L glucose, respectively. The RNA control group, circRNA-SCAF8 inhibition group, miR-93-5p overexpression group and miR-93-5p inhibition group were added with non-functional siRNA, circRNA-SCAF8 inhibitor, miR-93-5p overexpression molecule and miR-93-5p inhibitor in high glucose environment, respectively. Cell viability and pyroptosis were detected by cell counting kit-8 (CCK-8) assay, flow cytometry and Hoechst 33342/propidium iodide fluorescence double staining. Western blotting and enzyme-linked immunosorbent assay were used to detect the expression of pyroptosis-related factors including apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartic acid specific protease-1 (caspase-1) and Gasdermin D (GSDMD), NOD like receptor protein 3 (NLRP-3), thioredoxin interacting proteins (TXNIP), IL-18 and IL-1ß. The expression of circRNA-SCAF8, miR-93-5p and TXNIP was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Fluorescence in situ hybridization (FISH) was used to locate circRNA-SCAF8 and miR-93-5p. Dual luciferase assay was used to verify the targeted regulatory relationship between miR-93-5p and upstream and downstream molecules. RESULTS: Compared with the RNA control group, the cell survival rate of circRNA-SCAF8 inhibition group and miR-93-5p overexpression group increased (both P<0.01), the pyroptosis decreased (both P<0.01), and the expressions of pyroptosis-related factors such as TXNIP, NLRP-3, caspase-1, GSDMD, ASC, IL-18 and IL-1ß were significantly decreased (all P<0.05). The expression of miR-93-5p was significantly increased after inhibition of circRNA-SCAF8 (P<0.01), and the expression of circRNA-SCAF8 tended to decrease after overexpression of miR-93-5p, but with no statistical significance (P>0.05). Dual luciferase assay showed that miR-93-5p downre-gulated circRNA-SCAF8 expression by binding to the 3 ´ UTR region of circRNA-SCAF8, and miR-93-5p downregulated TXNIP expression by binding to the 3 ´ UTR region of TXNIP. FISH showed that circRNA-SCAF8 and miR-93-5p were both located in the cytoplasm and were highly associated in the cells. qRT-PCR showed that the relative expression of TXNIP increased or decreased after overexpression or inhibition of miR-93-5p compared with the RNA control group, respectively (both P<0.05), suggesting that miR-93-5p could regulate TXNIP gene expression. CONCLUSIONS: CircRNA-SCAF8/miR-93-5p/TXNIP axis is involved in the regulation of pyroptosis in HUVECs under high glucose.

Blocking CD47 promotes antitumour immunity through CD103+ dendritic cell-NK cell axis in murine hepatocellular carcinoma model.

BACKGROUND & AIMS: The CD47-signal regulatory protein α (SIRPα) axis inhibits dendritic cell (DC) phagocytosis and contributes to immune evasion. However, the behaviour of DCs and the potential crosstalk between DCs and natural killer (NK) cells in the hepatocellular carcinoma (HCC) microenvironment after CD47 blockade remain unclear. METHODS: The infiltration of CD103+ DCs and NK cells were analysed by immunohistochemistry and immunofluorescence in both human and murine HCC specimens. An orthotopic liver tumour model was used to evaluate the function of the CD103+ DC-NK cell axis after CD47 blockade in vivo in wild-type, Rag1-/-, Batf3-/-, and STING1-/- mice. Phagocytosis assays were performed in CD103+ DC and HCC cell lines. CD103+ DC-derived cytokines were analysed by chemokine array. Spleen-derived NK cells in C57BL/6J mice were used to evaluate cytotoxic functions in vitro. RESULTS: Higher CD47 expression was associated with worse prognosis in patients with HCC. CD47 blockade enhanced antitumour efficacy by stimulating the CD103+ DC-NK cell axis. The hypoxic microenvironment promoted CD47 blockade-induced tumour DNA phagocytosis by CD103+ DCs. By releasing IL-12 and CXCL9, activated CD103+ DCs induced the recruitment of NK cells with upregulated expression of granzyme B, NKG2D, interferon-γ, and tumour necrosis factor-α and downregulated expression of NKG2A. The antitumour effects of CD47 blockade could be abolished by cyclic GMP-AMP synthase (cGAS)-STING pathway inhibition. CONCLUSIONS: In addition to the classical DC-T cell axis, CD47 blockade significantly enhanced the ability of CD103+ DCs to take up tumour DNA, resulting in the stimulation of the cGAS-STING pathway, which promoted the infiltration and activation of NK cells in liver cancer. LAY SUMMARY: Hypoxia (low oxygen levels) is prevalent in the hepatocellular carcinoma microenvironment and promotes the phagocytosis (ingestion and elimination) of tumour DNA by CD103+ dendritic cells (a type of immune cell). Blockade of the cell surface protein CD47 resulted in activation of CD103+ dendritic cells which led to the recruitment and activation of natural killer cells (a different immune cell). When activated, these cells exhibit an antitumour effect.

Mechanisms of high-level fosfomycin resistance in Staphylococcus aureus epidemic lineage ST5.

OBJECTIVES: Fosfomycin resistance has become a clinical concern. In this study, we analysed the dynamic change of fosfomycin MIC in the epidemic Staphylococcus aureus lineages in a teaching hospital in Shanghai for 12 years and sought to elucidate the major underlying mechanisms. METHODS: MLST was conducted for 4580 S. aureus isolates recovered from 2008 to 2019. Fosfomycin MIC was determined by the agar dilution method. The genome data of 230 S. aureus epidemic lineage isolates were acquired from a next-generation sequencing (NGS) platform. Gene deletion and corresponding complementation mutants were constructed to confirm the mechanism of fosfomycin resistance. RESULTS: The predominant S. aureus lineages during the past 12 years were ST5 and ST239 (45.6%; 2090/4580). However, ST5 has been spreading clinically, while ST239 has gradually disappeared recently. Consistent with epidemic trends, fosfomycin-resistant ST5 increased from 19.5% to 67.3%. Most fosfomycin-resistant ST5 isolates (92.7%; 647/698) possessed high-level resistance (MIC > 1024 mg/L) with combined mutations mainly in glpT and uhpT. In contrast, fosfomycin-resistant ST239 isolates (76.8%; 149/194) mainly acquired low-level resistance (MIC = 64-128 mg/L) with mutation primarily in hptA. Deletion of a single resistant gene merely resulted in low-level fosfomycin resistance, while double-gene mutants ΔglpTΔuhpT, ΔglpTΔhptA and ΔglpTΔhptR acquired high-level fosfomycin resistance. CONCLUSIONS: The high-level fosfomycin resistance of S. aureus epidemic lineage ST5 is mainly due to the accumulation of mutations in the resistant genes related to membrane transporter systems, and partly contributes to its persistent prevalence under clinical antibiotic pressure.

Mid-term Results of the Treatment of Isolated Dissection of the Celiac Artery: A Comparative Analysis of Endovascular Versus Conservative Therapy.

OBJECTIVES: Endovascular treatment (EVT) is an alternative method used to treat isolated dissection of the celiac artery (IDCA). However, only a few mid-term results have been reported. This study aimed to analyze and compare the outcomes of endovascular and non-operative therapies for IDCA. METHODS: Data from a cohort of consecutive IDCA patients enrolled in the study hospital between April 2012 and September 2020 were retrospectively reviewed. Demographic information, imaging features, treatment modalities, and follow-up results of celiac artery remodeling and adverse events were collected and analyzed. RESULTS: A total of 87 patients were enrolled in the study. Stents were deployed in 68 patients, and non-operative treatment (blood pressure control and pain management) was continued in the remaining 19 patients who did not receive stenting; among these 19 patients, EVT failed in 6. The mean follow-up period was 37.3 (range, 10-85 months) and 44.0 (range, 9-80 months) months in the EVT and non-operative groups, respectively. During follow-up, the overall complete remodeling (absence of residual dissection with no false lumen or no intramural thrombus) rate was significantly higher in the EVT group than in the non-operative group (87.3% vs 7.1%, p<0.001). The incomplete remodeling (improved true lumen with malabsorption or partial thrombosis of the false lumen) rate was not significantly different between the EVT and non-operative groups (6.3% vs 14.3%; p=0.2984). Meanwhile, the adverse event-free survival rates were 89.0%, 67.0%, and 67.0% at 1, 3, and 5 years, respectively, in the EVT group compared with 39.7% and 29.8% at 1 and 3 years in the non-operative group (p<0.0001). CONCLUSIONS: EVT for IDCA may be considered an effective management option with a favorable clinical success rate, an encouraging complete remodeling rate, and a satisfactory adverse event-free survival rate. However, further evaluation with a long-term follow-up is required. CLINICAL IMPACT: Endovascular intervention for isolated dissection of the celiac artery has attracted inadequate attention. In this retrospective study with comparative analysis of endovascular versus conservative therapy for isolated dissection of the celiac artery patients, a better complete remodeling rate and a higher adverse event-free survival rate were observed in the endovascular treatment (EVT) group during follow-up, indicating that EVT could be an effective management option for isolated dissection of the celiac artery.

LncRNA SNHG15 relieves hyperglycemia-induced endothelial dysfunction via increased ubiquitination of thioredoxin-interacting protein.

Numerous studies have revealed that hyperglycemia is a pivotal driver of diabetic vascular complications. However, the mechanisms of hyperglycemia-induced endothelial dysfunction in diabetes remain incompletely understood. This study aims to expound on the underlying mechanism of the endothelial dysfunction induced by hyperglycemia from the perspective of long non-coding RNAs (lncRNA). In this study, a downregulation of SNHG15 was observed in the ischemic hind limb of diabetic mice and high glucose (HG)-treated HUVECs. Functionally, the overexpression of SNHG15 promoted cell proliferation, migration, and tube formation, and suppressed cell apoptosis in HG-treated HUVECs. Mechanistically, SNHG15 reduced thioredoxin-interacting protein (TXNIP) expression by enhancing ITCH-mediated ubiquitination of TXNIP. TXNIP overexpression abrogated the protective effect of lncRNA SNHG15 overexpression on HG-induced endothelial dysfunction. The following experiment further confirmed that SNHG15 overexpression promoted angiogenesis of the ischemic hind limb in diabetic mice. In conclusion, SNHG15 is a novel protector for hyperglycemia-induced endothelial dysfunction via decreasing TXNIP expression.

Nuclear Factor Kappa-B/Homeobox A9-Mediated Modulation of Leucine-Rich Repeat Flightless-Interacting Protein 1 Is Involved in Advanced Glycation End Product-Induced Endothelial Dysfunction.

BACKGROUND: Pathogenesis of cardiovascular diseases begins with endothelial dysfunction. Our previous study has shown that advanced glycation end products (AGE) could inhibit the expression of homeobox A9 (Hoxa9), thereby inducing endothelial dysfunction. Leucine-rich repeat flightless-interacting protein 1 (LRRFIP1) has been found to participate in a variety of pathological processes, but reports of its role in endothelial dysfunction are rare. OBJECTIVES: This study aims to investigate whether LRRFIP1 is involved in AGE-induced endothelial dysfunction through Hoxa9-mediated transcriptional activation. METHODS: Chromatin immunoprecipitation was used to detect the transcriptional regulation of Hoxa9 on LRRFIP1 promoters. Human umbilical vein endothelial cells were treated with AGE or pyrrolidinedithiocarbamate (nuclear factor kappa-B [NF-κB] inhibitor). Moreover, changes in apoptosis, proliferation, migration, release of nitric oxide, and angiogenesis were detected. RESULTS: Hoxa9 promotes LRRFIP1 expression by binding to the -LRRFIP1 promoter. Meanwhile, overexpression of LRRFIP1 inhibited phosphorylation of P65 and elevated expression of Hoxa9. Overexpression of LRRFIP1 or/and Hoxa9 reversed the effects of AGE on HUVEC. AGE-induced inhibition on the expression of LRRFIP1 and Hoxa9 could be reversed by the NF-κB inhibitor. CONCLUSION: LRRFIP1 is involved in AGE-induced endothelial dysfunction via being regulated by the NF-κB/Hoxa9 axis.

Quantifying Homologous Proteins and Proteoforms.

Many proteoforms-arising from alternative splicing, post-translational modifications (PTM), or paralogous genes-have distinct biological functions, such as histone PTM proteoforms. However, their quantification by existing bottom-up mass-spectrometry (MS) methods is undermined by peptide-specific biases. To avoid these biases, we developed and implemented a first-principles model (HIquant) for quantifying proteoform stoichiometries. We characterized when MS data allow inferring proteoform stoichiometries by HIquant and derived an algorithm for optimal inference. We applied this algorithm to infer proteoform stoichiometries in two experimental systems that supported rigorous bench-marking: alkylated proteoforms spiked-in at known ratios and endogenous histone 3 PTM proteoforms quantified relative to internal heavy standards. When compared with the benchmarks, the proteoform stoichiometries interfered by HIquant without using external standards had relative error of 5-15% for simple proteoforms and 20-30% for complex proteoforms. A HIquant server is implemented at: https://web.northeastern.edu/slavov/2014HIquant/.

Mechanical forces in cell monolayers.

In various physiological processes, the cell collective is organized in a monolayer, such as seen in a simple epithelium. The advances in the understanding of mechanical behavior of the monolayer and its underlying cellular and molecular mechanisms will help to elucidate the properties of cell collectives. In this Review, we discuss recent in vitro studies on monolayer mechanics and their implications on collective dynamics, regulation of monolayer mechanics by physical confinement and geometrical cues and the effect of tissue mechanics on biological processes, such as cell division and extrusion. In particular, we focus on the active nematic property of cell monolayers and the emerging approach to view biological systems in the light of liquid crystal theory. We also highlight the mechanosensing and mechanotransduction mechanisms at the sub-cellular and molecular level that are mediated by the contractile actomyosin cytoskeleton and cell-cell adhesion proteins, such as E-cadherin and α-catenin. To conclude, we argue that, in order to have a holistic understanding of the cellular response to biophysical environments, interdisciplinary approaches and multiple techniques - from large-scale traction force measurements to molecular force protein sensors - must be employed.

Blocking Triggering Receptor Expressed on Myeloid Cells-1-Positive Tumor-Associated Macrophages Induced by Hypoxia Reverses Immunosuppression and Anti-Programmed Cell Death Ligand 1 Resistance in Liver Cancer.

Tumor-associated macrophages (TAMs) are recognized as antitumor suppressors, but how TAMs behave in the hypoxic environment of hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrated that hypoxia inducible factor 1α induced increased expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in TAMs, resulting in immunosuppression. Specifically, TREM-1-positive (TREM-1+ ) TAMs abundant at advanced stages of HCC progression indirectly impaired the cytotoxic functions of CD8+ T cells and induced CD8+ T-cells apoptosis. Biological and functional assays showed that TREM-1+ TAMs had higher expression of programmed cell death ligand 1 (PD-L1) under hypoxic environment. However, TREM-1+ TAMs could abrogate spontaneous and PD-L1-blockade-mediated antitumor effects in vivo, suggesting that TREM-1+ TAM-induced immunosuppression was dependent on a pathway separate from PD-L1/programmed cell death 1 axis. Moreover, TREM-1+ TAM-associated regulatory T cells (Tregs) were crucial for HCC resistance to anti-PD-L1 therapy. Mechanistically, TREM-1+ TAMs elevated chemokine (C-C motif) ligand 20 expression through the extracellular signal-regulated kinase/NF-κß pathway in response to hypoxia and tumor metabolites leading to CCR6+ Foxp3+ Treg accumulation. Blocking the TREM-1 pathway could significantly inhibit tumor progression, reduce CCR6+ Foxp3+ Treg recruitment, and improve the therapeutic efficacy of PD-L1 blockade. Thus, these data demonstrated that CCR6+ Foxp3+ Treg recruitment was crucial for TREM-1+ TAM-mediated anti-PD-L1 resistance and immunosuppression in hypoxic tumor environment. Conclusion: This study highlighted that the hypoxic environment initiated the onset of tumor immunosuppression through TREM-1+ TAMs attracting CCR6+ Foxp3+ Tregs, and TREM-1+ TAMs endowed HCC with anti-PD-L1 therapy resistance.

Young's double-slit experiment with a partially coherent vortex beam.

We perform a Young's double-slit experiment with a partially coherent vortex beam (PCVB) and explore its cross-spectral density (CSD) at the focal plane after passing through a double-slit. Our results reveal that the phase of the CSD distribution with respect to an on-axis reference point can simultaneously quantitatively characterize the sign and magnitude of the topological charge (TC) carried by such a beam. In particular, the magnitude of the TC is half of the number of coherence singularities and the sign of the TC is determined by the phase winding of the coherence singularities (i.e., counterclockwise- and clockwise increases correspond to positive and negative, respectively). Based on this property, we present and demonstrate experimentally a simple technique to measure the sign and magnitude of the TC of a PCVB through its CSD distribution after a double-slit. Our method allows for easy measurement of the TC by being conceptually simpler than other methods.

YAP promotes multi-drug resistance and inhibits autophagy-related cell death in hepatocellular carcinoma via the RAC1-ROS-mTOR pathway.

BACKGROUND: Multi-drug resistance is the major cause of chemotherapy failure in hepatocellular carcinoma (HCC). YAP, a critical effector of the Hippo pathway, has been shown to contribute to the progression, metastasis and invasion of cancers. However, the potential role of YAP in mediating drug resistance remains obscure. METHODS: RT-qPCR and western blot were used to assess YAP expression in HCC cell lines. CCK-8 assays, flow cytometry, a xenograft tumour model, immunochemistry and GFP-mRFP-LC3 fusion proteins were utilized to evaluate the effect of YAP on multi-drug resistance, intracellular ROS production and the autophagy of HCC cells in vitro and in vivo. Autophagy inhibitor and rescue experiments were carried out to elucidate the mechanism by which YAP promotes chemoresistance in HCC cells. RESULTS: We found that BEL/FU, a typical HCC cell line with chemoresistance, exhibited overexpression of YAP. Moreover, the inhibition of YAP by shRNA or verteporfin conferred the sensitivity of BEL/FU cells to chemotherapeutic agents through autophagy-related cell death in vitro and in vivo. Mechanistically, YAP silencing significantly enhanced autophagic flux by increasing RAC1-driven ROS, which contributed to the inactivation of mTOR in HCC cells. In addition, the antagonist of autophagy reversed the enhanced effect of YAP silencing on cell death under treatment with chemotherapeutic agents. CONCLUSION: Our findings suggested that YAP upregulation endowed HCC cells with multi-drug resistance via the RAC1-ROS-mTOR pathway, resulting in the repression of autophagy-related cell death. The blockade of YAP may serve as a promising novel therapeutic strategy for overcoming chemoresistance in HCC.

Dual-function of Baicalin in nsPEFs-treated Hepatocytes and Hepatocellular Carcinoma cells for Different Death Pathway and Mitochondrial Response.

Nanosecond pulsed electric fields (nsPEFs) is emerged as a potential curative modality to ablate hepatocellular carcinoma (HCC). The application of local ablation is usually limited by insufficiency of liver function. While baicalin, a flavonoid isolated from Scutellaria baicalensis Georgi, has been proven to possess both anti-tumor and protective effects. Our study aimed to estimate different responses of hepatic cancer cells and hepatocytes to the combination of nsPEFs and baicalin. Cell viability, apoptosis and necrosis, mitochondrial transmembrane potential (MTP) and reactive oxygen species (ROS) were examined by CCK-8, FCM, JC-1 and fluorescent probe, respectively. After treatment by nsPEFs, most hepatocytes died by apoptosis, nevertheless, nearly all cancer cells were killed through necrosis. Low concentration of baicalin synergically enhanced nsPEFs-induced suppression and necrosis of HCC cells, nevertheless, the application of baicalin protected normal hepatocytes from the injury caused by nsPEFs, owing to elevating mitochondrial transmembrane potential and reducing ROS generation. Our work provided an advantageous therapy for HCC through the enhanced combination treatment of nsPEFs and baicalin, with which could improve the tumor-ablation effect and alleviate the injury of hepatic tissues simultaneously.

RIPK1 Inhibition Enhances Pirarubicin Cytotoxic Efficacy through AKT-P21-dependent Pathway in Hepatocellular Carcinoma.

Pirarubicin (THP) is a new generation cell cycle nonspecific anthracycline anticancer drug. Pirarubicin and pirarubicin-based combination therapies have been demonstrated to be effective against HCC in TACE. However, the drug resistance limits its therapeutic efficacy. Receptor-interacting protein kinase 1 (RIPK1) displays a critical role in cell death. Here we found that RIPK1 and p21 may participate in the resistance to pirarubicin. In this study, we first found that inhibition of RIPK1 significantly decreased pAKT and increased p21, accompanied by G0/G1 phase cell cycle arrest and cell anti-proliferation in pirarubicin-treated hepatocellular carcinoma cells. Moreover, phosphorylation of AKT reversed the anti-proliferative effect of RIPK1 inhibitor in HCC, which proved that RIPK1-AKT-P21-dependent pathway played a key role in pirarubicin resistance. Using a mouse xenograft model, we further found that RIPK1 inhibitor combined with pirarubicin exerted synergistic anti-tumor effect in vivo. Upon exposure to pirarubicin treatment, xenografts under RIPK1 inhibition maintained higher levels of p21 than control xenografts. In conclusion, the results in our study demonstrated that RIPK1 inhibition enhances the anti-tumor effect of pirarubicin by overcoming drug resistance. RIPK1 inhibitor might be used as an adjuvant to potentiate the inhibitory effect of pirarubicin against primary hepatocellular carcinoma.

Single molecule imaging unveils cellular architecture, dynamics and mechanobiology.

The biomechanical regulation of the cytoskeleton and cell adhesions underlies various essential cellular functions. Studying them requires visualizing their nanostructure and molecular dynamics with evermore precise spatio-temporal resolution. In this review we will focus on the recent advances in single molecule fluorescence imaging techniques and discuss how they improve our understanding of mechanically sensitive cellular structures such as adhesions and the cytoskeleton. We will also discuss future directions for research, emphasizing on the 3D nature of cellular structures and tissues, their mechanical regulation at the molecule level, as well as how super-resolution microscopy will enhance our knowledge on protein structure and conformational changes in the cellular context.