Extracellular Vesicle Inhibitors Enhance Cholix-Induced Cell Death via Regulation of the JNK-Dependent Pathway.

Vibrio cholerae is an important foodborne pathogen. Cholix cytotoxin (Cholix), produced by V. cholerae, is a novel eukaryotic elongation factor 2 (eEF2) adenosine diphosphate ribosyltransferase that causes host cell death by inhibiting protein synthesis. However, the role of Cholix in the infectious diseases caused by V. cholerae remains unclear. Some bacterial cytotoxins are carried by host extracellular vesicles (EVs) and transferred to other cells. In this study, we investigated the effects of EV inhibitors and EV-regulating proteins on Cholix-induced hepatocyte death. We observed that Cholix-induced cell death was significantly enhanced in the presence of EV inhibitors (e.g., dimethyl amiloride, and desipramine) and Rab27a-knockdown cells, but it did not involve a sphingomyelin-dependent pathway. RNA sequencing analysis revealed that desipramine, imipramine, and EV inhibitors promoted the Cholix-activated c-Jun NH2-terminal kinase (JNK) pathway. Furthermore, JNK inhibition decreased desipramine-enhanced Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage. In addition, suppression of Apaf-1 by small interfering RNA further enhanced Cholix-induced PARP cleavage by desipramine. We identified a novel function of desipramine in which the stimulated JNK pathway promoted a mitochondria-independent cell death pathway by Cholix.

Unsupervised Exercise in Interstitial Lung Disease: A Delphi Study to Develop a Consensus Preparticipation Screening Tool for Lymphangioleiomyomatosis.

BACKGROUND: Little research is available to provide practical guidance to health care providers for exercise preparticipation screening and referral of patients with interstitial lung diseases (ILDs), including lymphangioleiomyomatosis (LAM), to participate in remote, unsupervised exercise programs. RESEARCH QUESTION: What exercise preparticipation screening steps are essential to determine whether a patient with LAM is medically appropriate to participate in a remote, unsupervised exercise program? STUDY DESIGN AND METHODS: Sixteen experts in LAM and ILD participated in a two-round modified Delphi study, ranking their level of agreement for 10 statements related to unsupervised exercise training in LAM, with an a priori definition of consensus. Additionally, 60 patients with LAM completed a survey of the perceived risks and benefits of remote exercise training in LAM. RESULTS: Seven of the 10 statements reached consensus among experts. Experts agreed that an in-person clinical exercise test is indicated to screen for exercise-induced hypoxemia and prescribe supplemental oxygen therapy as indicated prior to initiating a remote exercise program. Patients with recent pneumothorax should wait to start an exercise program for at least 4 weeks until after resolution of pneumothorax and clearance by a physician. Patients with high cardiovascular risk for event during exercise, severe resting pulmonary hypertension, or risk for falls may be more appropriate for referral to a rehabilitation center. A LAM-specific remote exercise preparticipation screening tool was developed from the consensus statements and agreed upon by the panelists. INTERPRETATION: A modified Delphi study approach was useful to develop disease-specific recommendations for safety and preparticipation screening prior to unsupervised, remotely administered exercise in LAM. The primary product of this study is a clinical decision aid for providers to use when medically screening patients prior to participation in the newly launched LAMFit remote exercise program.

Clinical Trial Validation of Automated Segmentation and Scoring of Pulmonary Cysts in Thoracic CT Scans.

In cystic lung diseases such as lymphangioleiomyomatosis (LAM), a CT-based cyst score that measures the percentage of the lung volume occupied by cysts is a common index of the cyst burden in the lungs. Although the current semi-automatic measurement of the cyst score is well established, it is susceptible to human operator variabilities. We recently developed a fully automatic method incorporating adaptive features in place of manual adjustments. In this clinical study, the automatic method is validated against the standard method in several aspects. These include the agreement between the cyst scores of the two methods, the agreement of each method with independent tests of pulmonary function, and the temporal consistency of the measurements in the consecutive visits of the same patients. We found that the automatic method agreed with the standard method as well as the agreement between two trained operators running the same standard method; both methods obtained the same level of correlation with laboratory pulmonary function tests; the automated method had better temporal consistency than the standard method (p < 0.0001). The study indicates that the automatic method could replace the standard method and provide better consistency in assessing the extent of cystic changes in the lungs of patients.

Glycoprotein non-metastatic melanoma protein B promotes tumor growth and is a biomarker for lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease affecting almost exclusively female-sexed individuals. The cysts represent regions of lung destruction caused by smooth muscle tumors containing mutations in one of the two tuberous sclerosis (TSC) genes. mTORC1 inhibition slows but does not stop LAM advancement. Furthermore, monitoring disease progression is hindered by insufficient biomarkers. Therefore, new treatment options and biomarkers are needed. LAM cells express melanocytic markers, including glycoprotein non-metastatic melanoma protein B (GPNMB). The function of GPNMB in LAM is currently unknown; however, GPNMB's unique cell surface expression on tumor versus benign cells makes GPNMB a potential therapeutic target, and persistent release of its extracellular ectodomain suggests potential as a serum biomarker. Here, we establish that GPNMB expression is dependent on mTORC1 signaling, and that GPNMB regulates TSC2-null tumor cell invasion in vitro. Further, we demonstrate that GPNMB enhances TSC2-null xenograft tumor growth in vivo, and that ectodomain release is required for this xenograft growth. We also show that GPNMB's ectodomain is released from the cell surface of TSC2-null cells by proteases ADAM10 and 17, and we identify the protease target sequence on GPNMB. Finally, we demonstrate that GPNMB's ectodomain is present at higher levels in LAM patient serum compared to healthy controls and that ectodomain levels decrease with mTORC1 inhibition, making it a potential LAM biomarker.

Synthetic Circular RNA for microRNA-1269a Suppresses Tumor Progression in Oral Squamous Cell Carcinoma.

microRNAs (miRs) function in cancer progression as post-transcriptional regulators. We previously reported that endogenous circular RNAs (circRNAs) function as efficient miR sponges and could act as novel gene regulators in oral squamous cell carcinoma (OSCC). In this study, we carried out cellular and luciferase reporter assays to examine competitive inhibition of miR-1269a, which is upregulated expression in several cancers, by circRNA-1269a, a synthetic circRNA that contains miR-1269a binding sequences. We also used data-independent acquisition (DIA) proteomics and in silico analyses to determine how circRNA-1269a treatment affects molecules downstream of miR-1269a. First, we confirmed the circularization of the linear miR-1269a binding site sequence using RT-PCR with divergent/convergent primers and direct sequencing of the head-to-tail circRNA junction point. In luciferase reporter and cellular functional assays, circRNA-1269a significantly reduced miR-1269a function, leading to a significant decrease in cell proliferation and migration. DIA proteomics and gene set enrichment analysis of OSCC cells treated with circRNA-1269a indicated high differential expression for 284 proteins that were mainly enriched in apoptosis pathways. In particular, phospholipase C gamma 2 (PLCG2), which is related to OSCC clinical stage and overall survival, was affected by the circRNA-1269a/miR-1269a axis. Taken together, synthetic circRNA-1269a inhibits tumor progression via miR-1269a and its downstream targets, indicating that artificial circRNAs could represent an effective OSCC therapeutic.

Ultra-low dose chest CT with silver filter and deep learning reconstruction significantly reduces radiation dose and retains quantitative information in the investigation and monitoring of lymphangioleiomyomatosis (LAM).

PURPOSE: Frequent CT scans to quantify lung involvement in cystic lung disease increases radiation exposure. Beam shaping energy filters can optimize imaging properties at lower radiation dosages. The aim of this study is to investigate whether use of SilverBeam filter and deep learning reconstruction algorithm allows for reduced radiation dose chest CT scanning in patients with lymphangioleiomyomatosis (LAM). MATERIAL AND METHODS: In a single-center prospective study, 60 consecutive patients with LAM underwent chest CT at standard and ultra-low radiation doses. Standard dose scan was performed with standard copper filter and ultra-low dose scan was performed with SilverBeam filter. Scans were reconstructed using a soft tissue kernel with deep learning reconstruction (AiCE) technique and using a soft tissue kernel with hybrid iterative reconstruction (AIDR3D). Cyst scores were quantified by semi-automated software. Signal-to-noise ratio (SNR) was calculated for each reconstruction. Data were analyzed by linear correlation, paired t-test, and Bland-Altman plots. RESULTS: Patients averaged 49.4 years and 100% were female with mean BMI 26.6 ± 6.1 kg/m2. Cyst score measured by AiCE reconstruction with SilverBeam filter correlated well with that of AIDR3D reconstruction with standard filter, with a 1.5% difference, and allowed for an 85.5% median radiation dosage reduction (0.33 mSv vs. 2.27 mSv, respectively, p < 0.001). Compared to standard filter with AIDR3D, SNR for SilverBeam AiCE images was slightly lower (3.2 vs. 3.1, respectively, p = 0.005). CONCLUSION: SilverBeam filter with deep learning reconstruction reduces radiation dosage of chest CT, while maintaining accuracy of cyst quantification as well as image quality in cystic lung disease. CLINICAL RELEVANCE STATEMENT: Radiation dosage from chest CT can be significantly reduced without sacrificing image quality by using silver filter in combination with a deep learning reconstructive algorithm. KEY POINTS: • Deep learning reconstruction in chest CT had no significant effect on cyst quantification when compared to conventional hybrid iterative reconstruction. • SilverBeam filter reduced radiation dosage by 85.5% compared to standard dose chest CT. • SilverBeam filter in coordination with deep learning reconstruction maintained image quality and diagnostic accuracy for cyst quantification when compared to standard dose CT with hybrid iterative reconstruction.

Automated Segmentation and Measurements of Pulmonary Cysts in Lymphangioleiomyomatosis across Multiple CT Scanner Platforms over a Period of Two Decades.

(1) Background: Lymphangioleiomyomatosis is a genetic disease that affects mostly women of childbearing age. In the lungs, it manifests as the progressive formation of air-filled cysts and is associated with a decline in lung function. With a median survival of 29 years after the onset of symptoms, computed-tomographic monitoring of cystic changes in the lungs is a key part of the management of the disease. However, the current standard method to measure cyst burdens from CT is semi-automatic and requires manual adjustments from trained operators to obtain consistent results due to variabilities in CT technology and imaging conditions over the long course of the disease. This can be impractical for longitudinal studies involving large numbers of scans and is susceptible to subjective biases. (2) Methods: We developed an automated method of pulmonary cyst segmentation for chest CT images incorporating novel graphics processing algorithms. We assessed its performance against the gold-standard semi-automated method performed by experienced operators who were blinded to the results of the automated method. (3) Results: the automated method had the same consistency over time as the gold-standard method, but its cyst scores were more strongly correlated with concurrent pulmonary function results from the physiology laboratory than those of the gold-standard method. (4) Conclusions: The automated cyst segmentation is a competent replacement for the gold-standard semi-automated process. It is a solution for saving time and labor in clinical studies of lymphangioleiomyomatosis that may involve large numbers of chest CT scans from diverse scanner platforms and protocols.

Unexpected sirolimus-stimulated airway hyperreactivity in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a multisystem disease affecting primarily women, characterised in the lung by proliferation of LAM cells, abnormal smooth muscle-like cells with dysfunctional tuberous sclerosis complex genes. This dysfunction results in activation of mechanistic target of rapamycin (mTOR), leading to LAM cell proliferation. Sirolimus (rapamycin) is the only United States Food and Drug Administration-approved treatment for pulmonary LAM, resulting in decreased LAM cell growth/size and stabilised lung function [1].

Comprehensive genetic and phenotype analysis of 95 individuals with mosaic tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder due to loss-of-function TSC1 or TSC2 variants, characterized by tumors affecting multiple organs, including skin, brain, heart, lung, and kidney. Mosaicism for TSC1 or TSC2 variants occurs in 10%-15% of individuals diagnosed with TSC. Here, we report comprehensive characterization of TSC mosaicism by using massively parallel sequencing (MPS) of 330 TSC samples from a variety of tissues and fluids from a cohort of 95 individuals with mosaic TSC. TSC1 variants in individuals with mosaic TSC are much less common (9%) than in germline TSC overall (26%) (p < 0.0001). The mosaic variant allele frequency (VAF) is significantly higher in TSC1 than in TSC2, in both blood and saliva (median VAF: TSC1, 4.91%; TSC2, 1.93%; p = 0.036) and facial angiofibromas (median VAF: TSC1, 7.7%; TSC2 3.7%; p = 0.004), while the number of TSC clinical features in individuals with TSC1 and TSC2 mosaicism was similar. The distribution of mosaic variants across TSC1 and TSC2 is similar to that for pathogenic germline variants in general TSC. The systemic mosaic variant was not present in blood in 14 of 76 (18%) individuals with TSC, highlighting the value of analysis of multiple samples from each individual. A detailed comparison revealed that nearly all TSC clinical features are less common in individuals with mosaic versus germline TSC. A large number of previously unreported TSC1 and TSC2 variants, including intronic and large rearrangements (n = 11), were also identified.

Cell survival pathways targeted in rare lung disease affecting women.

Novel drug targets are identified in lymphangioleiomyomatosis (LAM), a rare disease in women. These targets focus on uterine transcription factors necessary for LAM cell survival.

Mono-ADP-ribosyltransferase 1 ( Artc1 )-deficiency decreases tumorigenesis, increases inflammation, decreases cardiac contractility, and reduces survival.

Arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTCs) transfer ADP-ribose from NAD + to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to enhance tumorigenicity as does Arh1 deficiency. In this study, Artc1 -KO and Artc1/Arh1 -double-KO mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF- α . In a xenograft model using tumorigenic Arh1 -KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1 -KO and heterozygous recipient mice, with tumor infiltration by CD8 + T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1 -double-KO MEFs showed decreased tumorigenesis in nude mice, showing that tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1 -KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1 -KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1 -KO was less than their Artc1 -WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility.

Diagnosis of Mosaic Tuberous Sclerosis Complex Using Next-Generation Sequencing of Subtle or Unusual Cutaneous Findings.

Skin findings can be critical to determining whether a patient with lymphangioleiomyomatosis (LAM), a progressive pulmonary disease that predominantly affects adult women, has sporadic LAM or LAM in association with tuberous sclerosis complex (TSC). Three individuals with LAM underwent evaluation for TSC-associated mucocutaneous and internal findings. We used our previously published algorithm to confirm the clinical suspicion for mosaicism and guide the selection of tissue specimens and genetic workup. Next-generation sequencing of cutaneous findings was used to confirm clinical suspicion for mosaic TSC in individuals with LAM. Two individuals previously thought to have sporadic LAM were diagnosed with mosaic TSC-associated LAM upon next-generation sequencing of unilateral angiofibromas in one and an unusual cutaneous hamartoma in the other. A third individual, diagnosed with TSC in childhood, was found to have a mosaic pathogenic variant in TSC2 in cutaneous tissue from a digit with macrodactyly. Accurate diagnosis of mosaic TSC-associated LAM may require enhanced genetic testing and is important because of the implications regarding surveillance, prognosis, and risk of transmission to offspring.

ADP-ribose-acceptor hydrolase 2 ( Arh2 ) deficiency results in cardiac dysfunction, tumorigenesis, inflammation, and decreased survival.

ADP-ribosylation is a reversible reaction with ADP-ribosyltransferases catalyzing the forward reaction and ADP-ribose-acceptor hydrolases (ARHs) hydrolyzing the ADP-ribose acceptor bond. ARH2 is a member of the 39-kDa ARH family (ARH1-3), which is expressed in heart and skeletal muscle. ARH2 failed to exhibit any in vitro enzymatic activity. To determine its possible in vivo activities, Arh2 -knockout (KO) and - heterozygous (Het) mice were generated using CRISPR-Cas9. Arh2 -KO mice exhibited decreased cardiac contractility by MRI, echocardiography and dobutamine stress with cardiomegaly and abnormal motor function. Arh2 -Het mice showed results similar to those seen in Arh2 -KO mice except for cardiomegaly. Arh2 -KO and -Het mice and mouse embryonic fibroblasts (MEFs) developed spontaneous tumors and subcutaneous tumors in nude mice. We identified 13 mutations in Arh2 -Het MEFs and heterozygous tumors, corresponding to human ARH2 mutations in cancers obtained from COSMIC. Of interest, the L116R mutation in Arh2 gene plays a critical role in aggressive tumorigenesis in nude mice, corresponding to human ARH2 mutations in stomach adenocarcinoma. Both genders of Arh2 -KO and -Het mice showed increased unexpectedly deaths and decreased survival rate during a 24-month observation, caused by tumor, inflammation, non-inflammation (e.g., cardiomegaly, dental dysplasia), and congenital diseases. Thus, Arh2 plays a pivotal role in cardiac function, tumorigenesis, inflammation, and overall survival.

An Enzyme-Linked Immunosorbent Assay to Quantify Poly (ADP-Ribose) Level In Vivo.

PolyADP-ribosylation is a posttranslational modification of proteins that results from enzymatic synthesis of poly(ADP-ribose) with NAD+ as the substrate. A unique characteristic of polyADP-ribosylation is that the poly(ADP-ribose) chain can have 200 or more ADP-ribose residues in branched patterns, and the presence and variety of these chains can have substantive effects on protein function. To understand how polyADP-ribosylation affects biological processes, it is important to know the physiological level of poly(ADP-ribose) in cells. Under normal cell physiological conditions and in the absence of any exogenous DNA damaging agents, we found that the concentration of poly(ADP-ribose) in HeLa cells is approximately 0.04 pmol (25 pg)/106 cells, as measured with a double-antibody sandwich, enzyme-linked immunosorbent assay protocol that avoids artificial activation of PARP1 during cell lysis. Notably, this system demonstrated that the poly(ADP-ribose) level peaks in S phase and that the average cellular turnover of a single poly(ADP-ribose) is less than 40 s.

ARH Family of ADP-Ribose-Acceptor Hydrolases.

The ARH family of ADP-ribose-acceptor hydrolases consists of three 39-kDa members (ARH1-3), with similarities in amino acid sequence. ARH1 was identified based on its ability to cleave ADP-ribosyl-arginine synthesized by cholera toxin. Mammalian ADP-ribosyltransferases (ARTCs) mimicked the toxin reaction, with ARTC1 catalyzing the synthesis of ADP-ribosyl-arginine. ADP-ribosylation of arginine was stereospecific, with ß-NAD+ as substrate and, α-anomeric ADP-ribose-arginine the reaction product. ARH1 hydrolyzed α-ADP-ribose-arginine, in addition to α-NAD+ and O-acetyl-ADP-ribose. Thus, ADP-ribose attached to oxygen-containing or nitrogen-containing functional groups was a substrate. Arh1 heterozygous and knockout (KO) mice developed tumors. Arh1-KO mice showed decreased cardiac contractility and developed myocardial fibrosis. In addition to Arh1-KO mice showed increased ADP-ribosylation of tripartite motif-containing protein 72 (TRIM72), a membrane-repair protein. ARH3 cleaved ADP-ribose from ends of the poly(ADP-ribose) (PAR) chain and released the terminal ADP-ribose attached to (serine)protein. ARH3 also hydrolyzed α-NAD+ and O-acetyl-ADP-ribose. Incubation of Arh3-KO cells with H2O2 resulted in activation of poly-ADP-ribose polymerase (PARP)-1, followed by increased nuclear PAR, increased cytoplasmic PAR, leading to release of Apoptosis Inducing Factor (AIF) from mitochondria. AIF, following nuclear translocation, stimulated endonucleases, resulting in cell death by Parthanatos. Human ARH3-deficiency is autosomal recessive, rare, and characterized by neurodegeneration and early death. Arh3-KO mice developed increased brain infarction following ischemia-reperfusion injury, which was reduced by PARP inhibitors. Similarly, PARP inhibitors improved survival of Arh3-KO cells treated with H2O2. ARH2 protein did not show activity in the in vitro assays described above for ARH1 and ARH3. ARH2 has a restricted tissue distribution, with primary involvement of cardiac and skeletal muscle. Overall, the ARH family has unique functions in biological processes and different enzymatic activities.

Computer grading of lung disease severity in patients with lymphangioleiomyomatosis referred for transplantation.

OBJECTIVES: Lymphangioleiomyomatosis (LAM) patients with severe lung disease may be considered for lung transplantation. Clinical, physiologic, and quality of life data are usually employed for referral. The aim of this study was to determine whether computed tomographic measurement of lung volume occupied by cysts (cyst score) complemented clinical and physiologic data in supporting referral for transplantation. METHODS: Forty-one patients were studied. Pre-referral clinical data, pulmonary function tests, exercise testing, and high-resolution computed tomography (HRCT) scans were obtained. From HRCT, a computer-aided diagnostic program was employed to calculate cyst scores. These data were compared to those of 41 age-matched LAM patients not referred for lung transplantation. RESULTS: Cyst score, and % predicted FEV1 and DLCO were respectively, 48.1 ± 9.4%, 36.5 ± 9.1%, and 35.0 ± 10.7%. For the control group, cyst score, FEV1, and DLCO were respectively, 14.8 ± 8.3%, 77.2 ± 20.3%, and 66.7 ± 19.3%. Cyst score values showed a normal distribution. However, the frequency distribution of FEV1 was skewed to the right while the distribution of DLCO was bimodal. Correlations between cyst score and FEV1 and DLCO for the study group were respectively, r = - 0.319 and r = - 0.421. CONCLUSIONS: LAM patients referred for lung transplantation had nearly 50% of lungs occupied by cysts. Correlations between cyst score and FEV1 or DLCO were weak; as shown previously, DLCO was better related to cyst number while FEV1 had a better association with cyst size. Given its normal distribution, cyst score measurements may assist in evaluation of pre-transplant severity of lung disease before referral for transplantation.

Poly(ADP-ribose) Polymerase 1 Mediates Rab5 Inactivation after DNA Damage.

Parthanatos is programmed cell death mediated by poly(ADP-ribose) polymerase 1 (PARP1) after DNA damage. PARP1 acts by catalyzing the transfer of poly(ADP-ribose) (PAR) polymers to various nuclear proteins. PAR is subsequently cleaved, generating protein-free PAR polymers, which are translocated to the cytoplasm where they associate with cytoplasmic and mitochondrial proteins, altering their functions and leading to cell death. Proteomic studies revealed that several proteins involved in endocytosis bind PAR after PARP1 activation, suggesting endocytosis may be affected by the parthanatos process. Endocytosis is a mechanism for cellular uptake of membrane-impermeant nutrients. Rab5, a small G-protein, is associated with the plasma membrane and early endosomes. Once activated by binding GTP, Rab5 recruits its effectors to early endosomes and regulates their fusion. Here, we report that after DNA damage, PARP1-generated PAR binds to Rab5, suppressing its activity. As a result, Rab5 is dissociated from endosomal vesicles, inhibiting the uptake of membrane-impermeant nutrients. This PARP1-dependent inhibition of nutrient uptake leads to cell starvation and death. It thus appears that this mechanism may represent a novel parthanatos pathway.