Entangling roles of cholesterol-dependent interaction and cholesterol-mediated lipid phase heterogeneity in regulating listeriolysin O pore-formation.

Cholesterol-dependent cytolysins (CDCs) are the distinct class of ß-barrel pore-forming toxins (ß-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane ß-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membrane-binding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far.

Vibrio parahaemolyticus thermostable direct haemolysin induces non-classical programmed cell death despite caspase activation.

Thermostable direct haemolysin (TDH) is the key virulence factor secreted by the human gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin. It evokes potent cytotoxicity, the mechanism of which still remains under-explored. Here, we have elucidated the mechanistic details of cell death response elicited by TDH. Employing Caco-2 intestinal epithelial cells and THP-1 monocytic cells, we show that TDH induces some of the hallmark features of apoptosis-like programmed cell death. TDH triggers caspase-3 and 7 activations in the THP-1 cells, while caspase-7 activation is observed in the Caco-2 cells. Interestingly, TDH appears to induce caspase-independent cell death. Higher XIAP level and lower Smac/Diablo level upon TDH intoxication provide plausible explanation for the functional inability of caspases in the THP-1 cells, in particular. Further exploration reveals that mitochondria play a central role in the TDH-induced cell death. TDH triggers mitochondrial damage, resulting in the release of AIF and endonuclease G, responsible for the execution of caspase-independent cell death. Among the other critical mediators of cell death, ROS is found to play an important role in the THP-1 cells, while PARP-1 appears to play a critical role in the Caco-2 cells. Altogether, our work provides critical new insights into the mechanism of cell death induction by TDH, showing a common central theme of non-classical programmed cell death. Our study also unravels the interplay of crucial molecules in the underlying signalling processes. Our findings add valuable insights into the role of TDH in the context of the host-pathogen interaction processes.

Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient ß-barrel pore formation.

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging ß-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane ß-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane ß-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric ß-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional ß-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.

Pore formation-independent cell death induced by a ß-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a ß-barrel pore-forming toxin (ß-PFT). It exhibits potent hemolytic activity against erythrocytes that appears to be a direct outcome of its pore-forming functionality. However, VCC-mediated cell-killing mechanism is more complicated in the case of nucleated mammalian cells. It induces apoptosis in the target nucleated cells, mechanistic details of which are still unclear. Furthermore, it has never been explored whether the ability of VCC to trigger programmed cell death is stringently dependent on its pore-forming activity. Here, we show that VCC can evoke hallmark features of the caspase-dependent apoptotic cell death even in the absence of the pore-forming ability. Our study demonstrates that VCC mutants with abortive pore-forming hemolytic activity can trigger apoptotic cell death responses and cytotoxicity, similar to those elicited by the wild-type toxin. VCC as well as its pore formation-deficient mutants display prominent propensity to translocate to the target cell mitochondria and cause mitochondrial membrane damage. Therefore, our results for the first time reveal that VCC, despite being an archetypical ß-PFT, can kill target nucleated cells independent of its pore-forming functionality. These findings are intriguing for a ß-PFT, whose destination is generally expected to remain limited on the target cell membranes, and whose mode of action is commonly attributed to the membrane-damaging pore-forming ability. Taken together, our study provides critical new insights regarding distinct implications of the two important virulence functionalities of VCC for the V. cholerae pathogenesis process: hemolytic activity for iron acquisition and cytotoxicity for tissue damage by the bacteria.

The changing landscape of interventional cardiology: A survival guide in the era of health system consolidation: The Society of Cardiovascular Angiography and Interventions affirms the value of this manuscript.

Practice environments for interventional cardiologists have evolved dramatically and now include small independent practices, large cardiology groups, multispecialty groups, and large integrated health systems. Increasingly, cardiologists are employed by hospitals or health systems. Data from MedAxiom and the American College of Cardiology (ACC) demonstrate an exponential increase in the percentage of cardiologists in employed positions from 10% in 2009 to 87% in 2020. This white paper explores these profound changes, considers their impact on interventional cardiologists, and offers guidance on how interventional cardiologists can best navigate this challenging environment. Finally, the paper offers a potential model to improve the employed physician experience through greater physician involvement in decision making, which may increase jobs satisfaction.

Membrane Dynamics and Remodelling in Response to the Action of the Membrane-Damaging Pore-Forming Toxins.

Pore-forming protein toxins (PFTs) represent a diverse class of membrane-damaging proteins that are produced by a wide variety of organisms. PFT-mediated membrane perforation is largely governed by the chemical composition and the physical properties of the plasma membranes. The interaction between the PFTs with the target membranes is critical for the initiation of the pore-formation process, and can lead to discrete membrane reorganization events that further aids in the process of pore-formation. Punching holes on the plasma membranes by the PFTs interferes with the cellular homeostasis by disrupting the ion-balance inside the cells that in turn can turn on multiple signalling cascades required to restore membrane integrity and cellular homeostasis. In this review, we discuss the physicochemical attributes of the plasma membranes associated with the pore-formation processes by the PFTs, and the subsequent membrane remodelling events that may start off the membrane-repair mechanisms.

Pore-forming toxins in infection and immunity.

The integrity of the plasma membranes is extremely crucial for the survival and proper functioning of the cells. Organisms from all kingdoms of life employ specialized pore-forming proteins and toxins (PFPs and PFTs) that perforate cell membranes, and cause detrimental effects. PFPs/PFTs exert their damaging actions by forming oligomeric pores in the membrane lipid bilayer. PFPs/PFTs play important roles in diverse biological processes. Many pathogenic bacteria secrete PFTs for executing their virulence mechanisms. The immune system of the higher vertebrates employs PFPs to kill pathogen-infected cells and transformed cancer cells. The most obvious consequence of membrane pore-formation by the PFPs/PFTs is the killing of the target cells due to the disruption of the permeability barrier function of the plasma membranes. PFPs/PFTs can also activate diverse cellular processes that include activation of the stress-response pathways, induction of programmed cell death, and inflammation. Upon attack by the PFTs, host cells may also activate pathways to repair the injured membranes, restore cellular homeostasis, and trigger inflammatory immune responses. In this article, we present an overview of the diverse cellular responses that are triggered by the PFPs/PFTs, and their implications in the process of pathogen infection and immunity.

Sequence Diversity in the Pore-Forming Motifs of the Membrane-Damaging Protein Toxins.

Pore-forming proteins/toxins (PFPs/PFTs) are the distinct class of membrane-damaging proteins. They act by forming oligomeric pores in the plasma membranes. PFTs and PFPs from diverse organisms share a common mechanism of action, in which the designated pore-forming motifs of the membrane-bound protein molecules insert into the membrane lipid bilayer to create the water-filled pores. One common characteristic of these pore-forming motifs is that they are amphipathic in nature. In general, the hydrophobic sidechains of the pore-forming motifs face toward the hydrophobic core of the membranes, while the hydrophilic residues create the lining of the water-filled pore lumen. Interestingly, pore-forming motifs of the distinct subclass of PFPs/PFTs share very little sequence similarity with each other. Therefore, the common guiding principle that governs the sequence-to-structure paradigm in the mechanism of action of these PFPs/PFTs still remains an enigma. In this article, we discuss this notion using the examples of diverse groups of membrane-damaging PFPs/PFTs.

SCAI expert consensus statement update on best practices for transradial angiography and intervention.

Transradial angiography and intervention continues to become increasingly common as an access site for coronary procedures. Since the first "Best Practices" paper in 2013, ongoing trials have shed further light onto the safest and most efficient methods to perform these procedures. Specifically, this document comments on the use of ultrasound to facilitate radial access, the role of ulnar artery access, the utility of non-invasive testing of collateral flow, strategies to prevent radial artery occlusion, radial access for primary PCI and topics that require further study.

Role of Gender in Dual Antiplatelet Therapy After Acute Coronary Syndrome.

PURPOSE OF REVIEW: The effect of gender on use of dual antiplatelet therapy (DAPT) in treatment of acute coronary syndrome (ACS) is not well established. The purpose of this review is to understand gender-based differences in response to DAPT, so that treatment of ACS can be optimized in women to prevent ischemic events while minimizing bleeding risk. RECENT FINDINGS: There are innate gender differences in platelet reactivity and response. However, it is unknown if this translates into differences in clinical outcomes. In all major studies evaluating the effect of DAPT in ACS, women are underrepresented. Hence, the results from the existing trials cannot be generalizable to women. There is a significant knowledge gap regarding how to balance the bleeding and ischemic risk profile among women with ACS. Currently, there is no recommendation to consider gender as covariate in choosing the type of antiplatelet drug or duration. The existing clinical evidence is limited by under representation of women in DAPT trials. The current literature does not strongly support considering gender in decision making regarding type or duration of DAPT after ACS. Future dedicated trial designs with adequate representation from women and gender specific analysis from large registry data are warranted to enhance our understanding of the interaction of gender with DAPT after ACS.

Length of stay following percutaneous coronary intervention: An expert consensus document update from the society for cardiovascular angiography and interventions.

Since the publication of the 2009 SCAI Expert Consensus Document on Length of Stay Following percutaneous coronary intervention (PCI), advances in vascular access techniques, stent technology, and antiplatelet pharmacology have facilitated changes in discharge patterns following PCI. Additional clinical studies have demonstrated the safety of early and same day discharge in selected patients with uncomplicated PCI, while reimbursement policies have discouraged unnecessary hospitalization. This consensus update: (1) clarifies clinical and reimbursement definitions of discharge strategies, (2) reviews the technological advances and literature supporting reduced hospitalization duration and risk assessment, and (3) describes changes to the consensus recommendations on length of stay following PCI (Supporting Information Table S1). These recommendations are intended to support reasonable clinical decision making regarding postprocedure length of stay for a broad spectrum of patients undergoing PCI, rather than prescribing a specific period of observation for individual patients.

Pre-close technique of percutaneous closure for delayed hemostasis of large-bore femoral sheaths.

Advancement of coronary interventions and portable hemodynamic device requires placement of large bore sheaths. Access for large caliber sheaths, its placement, maintenance, and hemostasis is very challenging and one of the key ailments for successful procedures. Traditional hemostasis method is manual compression, which is unattractive due to its own limitations and subsequent complications. Single closure device for sheath size larger than 8 French (Fr) is not available. We performed retrospective analysis of large cohort of patients with 13, 14 Fr sheaths (Impella device [ABIOMED]) percutaneous closure with the use of two Perclose devices. Two perclose devices were placed in a "Preclose" fashion and hemostasis was obtained few days later once hemodynamic support was weaned off by deployment of perclose sutures.

An integrated bio-process for production of functional biomolecules utilizing raw and by-products from dairy and sugarcane industries.

The study investigated an integrated bioprocessing of raw and by-products from sugarcane and dairy industries for production of non-digestible prebiotic and functional ingredients. The low-priced feedstock, whey, molasses, table sugar, jaggery, etc., were subjected to transglucosylation reactions catalyzed by dextransucrase from Leuconostoc mesenteroides MTCC 10508. HPLC analysis approximated production of about 11-14 g L-1 trisaccharide i.e. 2-α-D-glucopyranosyl-lactose (4-galactosyl-kojibiose) from the feedstock prepared from table sugar, jaggery, cane molasses and liquid whey, containing about 30 g L-1 sucrose and lactose each. The trisaccharide was hydrolysed into the prebiotic disaccharide, kojibiose, by employing recombinant ß-galactosidase from Escherichia coli. The enzyme ß-galactosidase achieved about 90% conversion of 2-α-D-glucopyranosyl-lactose into kojibiose. The D-fructose generated by catalytic reactions of dextransucrase was targeted for catalytic transformation into rare sugar, D-allulose (or D-psicose), by treating the samples with Smt3-D-psicose 3-epimerase. The catalytic reactions resulted in the conversion of ~ 25% D-fructose to D-allulose. These bioactive compounds are known to exert a plethora of benefits to human health, and therefore, are preferred ingredients for making functional foods.

Helicobacter pylori TlyA Forms Amyloid-like Aggregates with Potent Cytotoxic Activity.

Helicobacter pylori is a potent human gastric pathogen. It is known to be associated with several gastroenteric disorders, including gastritis, peptic ulcer, and gastric cancer. The H. pylori genome encodes a gene product TlyA that has been shown to display potent membrane damaging properties and cytotoxic activity. On the basis of such properties, TlyA is considered as a potential virulence factor of H. pylori. In this study, we show that the H. pylori TlyA protein has a strong propensity to convert into the amyloid-like aggregated assemblies, upon exposure to elevated temperatures. Even at the physiological temperature of 37 °C, TlyA shows a strong amyloidogenic property. TlyA aggregates that are generated upon exposure at temperatures of ≥37 °C show prominent binding to dyes like thioflavin T and Nile Red. Transmission electron microscopy also demonstrates the presence of typical amyloid-like fibrils in the TlyA aggregates generated at 37 °C. Conversion of TlyA into the amyloid-like aggregates is found to be associated with major alterations in the secondary and tertiary structural organization of the protein. Finally, our study shows that the preformed amyloid-like aggregates of TlyA are capable of exhibiting potent cytotoxic activities against human gastric adenocarcinoma cells. Altogether, such a propensity of H. pylori TlyA to convert into the amyloid-like aggregated assemblies with cytotoxic activity suggests potential implications for the virulence functionality of the protein.

Knockdown of receptor for advanced glycation end products attenuate 17α-ethinyl-estradiol dependent proliferation and survival of MCF-7 breast cancer cells.

BACKGROUND: 17α-ethinyl-estradiol (17α-EE), a synthetic estrogen is the world's most widely and commonly used orally bioactive estrogen. Currently, 17α-EE is in use in all formulations of contraceptive pills and is implicated in the complication of breast cancer. Receptor for advanced glycation end products (RAGE) is a cell surface immunoglobulin class of molecule. RAGE is involved in the complication of various cancers. METHODS AND RESULTS: This study indicates that treatment of MCF-7 breast cancer cells with 17α-EE enhances the expression of estrogen receptor related receptor gamma (ERRγ), followed by enhanced level of oxidative stress and subsequent activation of the transcription factor, nuclear factor kappa-B (NF-кB), leading to increase in RAGE expression. RAGE thus expressed by 17α-EE treatment causes further enhancement of the oxidative stress which, in turn, activates expression of cell cycle protein cyclin D1 and subsequent induction of MCF-7 breast cancer cell proliferation. RAGE also enhanced phosphorylation of prosurvival protein AKT and increased expression of Bcl2, an antiapoptotic protein. CONCLUSION: In MCF-7 breast cancer cells, 17α-EE-ERRγ interaction induces the expression of RAGE, which in turn, enhances the number of MCF-7 breast cancer cells through a multiprong action on the divergent molecules like cyclin D1, AKT and Bcl2. GENERAL SIGNIFICANCE: This is the first report which explains the intermediate role of ERRγ in the 17α-EE dependent RAGE expression in MCF-7 breast cancer cells. This report for the first time explains that RAGE is important not only for MCF-7 breast cancer cell proliferation but also for its survival and anti-apoptotic activities.

Helicobacter pylori TlyA agglutinates liposomes and induces fusion and permeabilization of the liposome membranes.

Helicobacter pylori TlyA is a pore-forming hemolysin with potent cytotoxic activity. To explore the potential membrane-damaging activity of H. pylori TlyA, we have studied its interaction with the synthetic liposome vesicles. In our study, H. pylori TlyA shows a prominent ability to associate with the liposome vesicles without displaying an obligatory requirement for any protein receptor on the liposome membranes. Interaction of TlyA triggers agglutination of the liposome vesicles. Such agglutinating activity of TlyA could also be observed with erythrocytes before the induction of its pore-forming hemolytic activity. In addition to its agglutinating activity against liposomes, TlyA also induces fusion and disruption of the liposome membranes. Altogether, our study highlights novel membrane-damaging properties of H. pylori TlyA that have not been documented previously with any other TlyA family protein.

Functional characterization of Helicobacter pylori TlyA: pore-forming hemolytic activity and cytotoxic property of the protein.

Helicobacter pylori is a human specific gastric pathogen. H. pylori pathogenesis process involves a number of well-studied virulence factors that include the 'vacuolating cytotoxin' and the 'cytotoxin associated gene A'. Analysis of the H. pylori genome, however, indicates presence of additional virulence factors that are yet to be characterized in molecular detail. For example, H. pylori genome harbors a gene that has potential to encode a protein with sequence similarity to those of the TlyA-like proteins of several pathogenic bacteria. Earlier studies have indicated potential association of this H. pylori tlyA gene in the virulence mechanism of the organism. Despite such notions, however, the TlyA-like protein of H. pylori has not been studied previously in molecular detail. In particular, purified form of H. pylori TlyA has never been studied before toward exploring its functional properties. Here, we report characterization of the H. pylori TlyA protein purified from the recombinant over-expression system in Escherichia coli. Purified form of the recombinant TlyA exhibits prominent hemolytic activity against human erythrocytes, presumably via formation of pores of specific diameter in the cell membrane. Purified TlyA also triggers prominent cytotoxic responses in human gastric adenocarcinoma cells. Altogether, our study establishes H. pylori TlyA as a potential virulence factor of the organism.

Virilising Adrenal Adenoma in Children.

ABSTRACT: Tumours of the adrenal cortex are most commonly seen in adults but rare in children. The clinical manifestations depend on the age and sex of patients; about two-thirds of the cases virilisation is the predominant presentation, whereas many presents with both virilisation and cushingoid features. Diagnosis is confirmed by hormonal evaluation and radiological investigations. Surgical removal is the mainstay of treatment. Usually, there is a good prognosis in paediatric patients, whereas the high mortality rate reported in older literature may have been due to big tumour size, post-operative complications and inadequate steroid replacement. Here, we are presenting a series of three cases during childhood with virilising features due to adrenocortical tumours. All of them presented predominantly with features suggestive of virilisation. All patients underwent surgery and had a good outcome despite big tumour size in one of the patients. Histology revealed a benign lesion in the form of adrenal adenoma. Most virilisation and cushingoid features disappeared in the follow-up (median - 1 year). Although these tumours are rare, a high index of suspicion should be kept in children with cushingoid features, virilisation or a combination of both of them. Even if the tumour size is big, adequate steroid replacement and supportive management postoperatively have led to good prognosis in our patients.

Advocacy 101 for Interventional Cardiologists: A Society for Cardiovascular Angiography & Interventions Policy Statement.

Advocacy is a core mission of the Society for Cardiovascular Angiography & Interventions (SCAI). SCAI advocates on behalf of interventional cardiologists and our patients. This document provides foundational information and a toolkit for grassroots advocacy by interventional cardiologists. The first half of the document summarizes how health care laws are made, how medical devices are approved, and how procedure reimbursement is determined. The second half of the document is a playbook of advocacy strategies: legislative advocacy, judicial advocacy, advocacy with regulators and payors, advocacy in the media, and participation in SCAI advocacy initiatives, such as the Government Relations Committee and SCAI Political Action Committee. Equipped with this toolbox, interventional cardiologists must increase our advocacy activities with government, payors, and industry.