Trematocine-derived antimicrobial peptides from the Antarctic fish Trematomus bernacchaii: potent antibacterial agents against ESKAPE pathogens.

Introduction: This study investigated the interaction with membrane mimetic systems (LUVs), bacterial membranes, the CD spectra, and the bactericidal activity of two designed trematocine mutants, named Trem-HK and Trem-HSK. Mutants were constructed from the scaffold of Trematocine (Trem), a natural 22-amino acid AMP from the Antarctic fish Trematomus bernacchii, aiming to increase their positive charge. Methods: The selectivity of the designed AMPs towards bacterial membranes was improved compared to Trematocine, verified by their interaction with different LUVs and their membranolytic activity. Additionally, their α-helical conformation was not influenced by the amino acid substitutions. Our findings revealed a significant enhancement in antibacterial efficacy against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae family) pathogens for both Trem-HK and Trem-HSK. Results: Firstly, we showed that the selectivity of the two new designed AMPs towards bacterial membranes was greatly improved compared to Trematocine, verifying their interaction with different LUVs and their membranolytic activity. We determined that their α-helical conformation was not influenced by the amino acid substitutions. We characterized the tested bacterial collection for resistance traits to different classes of antibiotics. The minimum inhibitory and bactericidal concentration (MIC and MBC) values of the ESKAPE collection were reduced by up to 80% compared to Trematocine. The bactericidal concentrations of Trematocine mutants showed important membranolytic action, evident by scanning electron microscopy, on all tested species. We further evaluated the cytotoxicity and hemolytic activity of the mutants. At 2.5 µM concentration, both mutants demonstrated low cytotoxicity and hemolysis, indicating selectivity towards bacterial cells. However, these effects increased at higher concentrations. Discussion: Assessment of in vivo toxicity using the Galleria mellonella model revealed no adverse effects in larvae treated with both mutants, even at concentrations up to 20 times higher than the lowest MIC observed for Acinetobacter baumannii, suggesting a high potential safety profile for the mutants. This study highlights the significant improvement in antibacterial efficacy achieved by increasing the positive charge of Trem-HK and Trem-HSK. This improvement was reached at the cost of reduced biocompatibility. Further research is necessary to optimize the balance between efficacy and safety for these promising AMPs.

Exploration of the Graphene Quantum Dots-Blue Light Combination: A Promising Treatment against Bacterial Infection.

Graphene Quantum Dots (GQDs) have shown the potential for antimicrobial photodynamic treatment, due to their particular physicochemical properties. Here, we investigated the activity of three differently functionalized GQDs-Blue Luminescent GQDs (L-GQDs), Aminated GQDs (NH2-GQDs), and Carboxylated GQDs (COOH-GQDs)-against E. coli. GQDs were administrated to bacterial suspensions that were treated with blue light. Antibacterial activity was evaluated by measuring colony forming units (CFUs) and metabolic activities, as well as reactive oxygen species stimulation (ROS). GQD cytotoxicity was then assessed on human colorectal adenocarcinoma cells (Caco-2), before setting in an in vitro infection model. Each GQD exhibits antibacterial activity inducing ROS and impairing bacterial metabolism without significantly affecting cell morphology. GQD activity was dependent on time of exposure to blue light. Finally, GQDs were able to reduce E. coli burden in infected Caco-2 cells, acting not only in the extracellular milieu but perturbating the eukaryotic cell membrane, enhancing antibiotic internalization. Our findings demonstrate that GQDs combined with blue light stimulation, due to photodynamic properties, have a promising antibacterial activity against E. coli. Nevertheless, we explored their action mechanism and toxicity on epithelial cells, fixing and standardizing these infection models.

Unraveling the potential of graphene quantum dots against Mycobacterium tuberculosis infection.

Introduction: The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains has underscored the urgent need for novel therapeutic approaches. Carbon-based nanomaterials, such as graphene oxide (GO), have shown potential in anti-TB activities but suffer from significant toxicity issues. Methods: This study explores the anti-TB potential of differently functionalized graphene quantum dots (GQDs) - non-functionalized, L-GQDs, aminated (NH2-GQDs), and carboxylated (COOH-GQDs) - alone and in combination with standard TB drugs (isoniazid, amikacin, and linezolid). Their effects were assessed in both axenic cultures and in vitro infection models. Results: GQDs alone did not demonstrate direct mycobactericidal effects nor trapping activity. However, the combination of NH2-GQDs with amikacin significantly reduced CFUs in in vitro models. NH2-GQDs and COOH-GQDs also enhanced the antimicrobial activity of amikacin in infected macrophages, although L-GQDs and COOH-GQDs alone showed no significant activity. Discussion: The results suggest that specific types of GQDs, particularly NH2-GQDs, can enhance the efficacy of existing anti-TB drugs. These nanoparticles might serve as effective adjuvants in anti-TB therapy by boosting drug performance and reducing bacterial counts in host cells, highlighting their potential as part of advanced drug delivery systems in tuberculosis treatment. Further investigations are needed to better understand their mechanisms and optimize their use in clinical settings.

Study of the Chemical Profile and Anti-Fungal Activity against Candida auris of Cinnamomum cassia Essential Oil and of Its Nano-Formulations Based on Polycaprolactone.

BACKGROUND: Candida auris represents an emerging pathogen that results in nosocomial infections and is considered a serious global health problem. The aim of this work was to evaluate the in vitro antifungal efficacy of Cinnamomum cassia essential oil (CC-EO) pure or formulated in polycaprolactone (PCL) nanoparticles against ten clinical strains of C. auris. METHODS: nanoparticles of PCL were produced using CC-EO (nano-CC-EO) and cinnamaldehyde (CIN) through the nanoprecipitation method. The chemical profile of both CC-EO and nano-CC-EO was evaluated using SPME sampling followed by GC-MS analysis. Micro-broth dilution tests were performed to evaluate both fungistatic and fungicidal effectiveness of CC-EO and CIN, pure and nano-formulated. Furthermore, checkerboard tests to evaluate the synergistic action of CC-EO or nano-CC-EO with micafungin or fluconazole were conducted. Finally, the biofilm disrupting activity of both formulations was evaluated. RESULTS: GC-MS analysis shows a different composition between CC-EO and nano-CC-EO. Moreover, the microbiological analyses do not show any variation in antifungal effectiveness either towards the planktonic form (MICCC-EO = 0.01 ± 0.01 and MICnano-CC-EO = 0.02 ± 0.01) or the biofilm form. No synergistic activity with the antifungal drugs tested was found. CONCLUSIONS: both CC-EO and nano-CC-EO show the same antimicrobial effectiveness and are potential assets in the fight against C. auris.

Accordion sign in COVID 19 related acute myocarditis, an old sign for a novel context? A cardiac magnetic resonance case series report study.

INTRODUCTION: The COVID-19 pandemic is related to a higher incidence of myocarditis; we present a case series of seven patients, admitted with COVID-19 related acute myocarditis, evaluated with cardiac magnetic resonance imaging, showing an altered profile of the free wall of the right ventricle, no longer present after six months follow-up. MATERIALS AND METHODS: Seven patients have been evaluated for COVID-19 related acute myocarditis, all patients have been evaluated with cardiac magnetic resonance imaging both in the acute setting and after six months follow-up. RESULTS: In the acute phase, myocarditis was confirmed in keeping with the current diagnostic criteria. In five out of seven cases, the presence of a crinkling profile of the free wall of the right ventricle was observed; at six months follow up, remission in four out of the five cases and a significant reduction in the remaining case, of the previously described findings, was observed. CONCLUSIONS: Crinkling appearance in the profile of the free wall of the right ventricle, detectable with cardiac magnetic resonance imaging, might represent a morphological feature present in the acute setting of COVID-19 related myocarditis; several underlying physiopathological mechanisms are conceivable. Further studies are needed to confirm this correlation, define the underlying mechanisms and the prognostic implication related to it. This is the first report in the literature that has considered such findings to the best of our knowledge.

Tricuspid annular disjunction can be isolated and even arrhythmogenic. A cardiac magnetic resonance study.

Mitral annular disjunction is related to increased arrhythmogenic risk; in a certain percentage of cases, mitral annular disjunction is associated with tricuspid annular disjunction. While the prognostic implications of mitral annular disjunction have been well established, there is still little data to define this aspect regarding the tricuspid annular disjunction. We present a case of a patient admitted for life-threatening ventricular arrhythmias that occurred during endurance sporting activity, who was found to have isolated tricuspid annular disjunction, not associated with mitral annular disjunction. Based on several factors, including the morphology and axis of QRS of the ventricular arrhythmic activity, and its behavior, including the response to antiarrhythmic treatment, and in keeping with the finding of edema and late gadolinium enhancement at the basal segment of the right ventricle free wall on cardiac magnetic resonance imaging, a direct relation between tricuspid annular disjunction and ventricular arrhythmias was highly conceivable. Control after three months showed almost complete remission of the previously described and persistence of LGE at the level of the basal segment of the free wall of the right ventricle, so giving strength to the hypothesis of an event related to increased acute RV free wall stress, secondary to high-intensity physical activity, established on a framework of chronic wall stress, as represented by LGE, similarly to what happens for mitral valve prolapse. To the best of our knowledge, this is the first case of a legitimately conceivable direct relation between tricuspid annular disjunction and ventricular arrhythmias.

Il Silenzio: The First Renaissance Oil Painting on Canvas from the Uffizi Museum Restored with a Safe, Green Antimicrobial Emulsion Based on Citrus aurantium var. amara Hydrolate and Cinnamomum zeylanicum Essential Oil.

Preserving artworks from the attacks of biodeteriogens is a primary duty of humanity. Nowadays, restorers use chemicals potentially dangerous for both artworks and human health. The purpose of this work was to find a green and safe formulation based on natural substances with fungicidal activity to restore ancient oil paintings, particularly "Il Silenzio" (by Jacopo Zucchi) preserved at the Uffizi Museum in Florence, Italy. The study was divided into two phases. First phase (in vitro study): three essential oils (EOs) and four hydrolates (Hys) were analysed by GC-mass spectrometry and in vitro tested against six ATCC strains of molds. An emulsion based on the more active natural compounds was tested on aged and unaged canvases samples to evaluate both their fungicidal activity and the impact on chemical-physical parameters. Finally, an in vivo toxicity test performed on the Galleria mellonella model assessed the safety for health. Second phase (in situ application): the emulsion was sprayed on the back of the painting and left to act for 24 h. Biodeteriogens present on the "Il Silenzio" painting were microbiologically identified before and after the treatment. The emulsion formulated with C. zeylanicum EO and C. aurantium var. amara Hy showed the best antifungal activity both in vitro and in situ without altering the chemical-physical characteristics of paintings. Furthermore, no in vivo toxicity was shown. For the first time, a green antimicrobial emulsion based on Hy and EO, safe for operators, was used to decontaminate an artwork colonised by fungi before the restoration practices.

Rapid detection of the widely circulating B.1.617.2 (Delta) SARS-CoV-2 variant.

The emergence of the B.1.617.2 (Delta) variant of the severe acute syndrome coronavirus (SARS-CoV-2) that emerged in 2019 (COVID-19), resulted in a surge of cases in India and has expanded and been detected across the world, including in the United States. The B.1.617.2 (Delta) variant has been seen to be twice more transmissible coupled with potential increases in disease severity and immune escape. As a result, case numbers and hospitalisations are once again on the rise in the USA. On 16 July 2021, the Centers for Disease Control and Prevention (CDC) reported a 7-day average 69.3% increase in new cases and a 35% increase in hospitalisations. Although the gold standard for SARS-CoV-2 variants identification remains genomic sequencing, this approach is not accessible to many clinical laboratories. The main goal of this study was to validate and implement the detection of the B.1.617.2 (Delta) variant utilising an open reverse transcription polymerase chain reaction (RT-PCR) platform by explicitly detecting the S-gene target failure (SGTF) corresponding to the deletion of two amino acids (ΔE156/ΔF157) characteristic of B.1.617.2 (Delta) variant. This approach was conceived as a rapid screening of B.1.617.2 (Delta) variant in conjunction with CDC's recommended N1 (nucleocapsid gene), N2, and RP (human RNase P) genes, as a pre-screening tool prior to viral genomic sequencing. We assessed 4,937 samples from 5 July to 5 September 2021. We identified the B.1.617.2 (Delta) variant in 435 of 495 positive samples (87.8%); the additional positive samples (7 samples, 1.4%) were found to belong to the B.1.1.7 (Alpha, UK) lineage and the remaining 53 samples (10.7%) were reported as 'other' lineages. Whole genome sequencing of 46 randomly selected samples validated the strains identified as positive and negative for the B.1.617.2 (Delta) variant and confirmed the S gene deletion in addition to B.1.617.2 characteristic mutations including L452R, T478K, P681R and D950N located in the spike protein. This modality has been used as routine testing at the Riverside University System Health (RUHS) Medical Center as a method for detection of B.1.617.2 (Delta) to pre-screen samples before genome sequencing. The assay can be easily implemented in clinical laboratories, most notably those with limited economic resources and access to genomic platforms.

A targetable LIFR-NF-κB-LCN2 axis controls liver tumorigenesis and vulnerability to ferroptosis.

The growing knowledge of ferroptosis has suggested the role and therapeutic potential of ferroptosis in cancer, but has not been translated into effective therapy. Liver cancer, primarily hepatocellular carcinoma (HCC), is highly lethal with limited treatment options. LIFR is frequently downregulated in HCC. Here, by studying hepatocyte-specific and inducible Lifr-knockout mice, we show that loss of Lifr promotes liver tumorigenesis and confers resistance to drug-induced ferroptosis. Mechanistically, loss of LIFR activates NF-κB signaling through SHP1, leading to upregulation of the iron-sequestering cytokine LCN2, which depletes iron and renders insensitivity to ferroptosis inducers. Notably, an LCN2-neutralizing antibody enhances the ferroptosis-inducing and anticancer effects of sorafenib on HCC patient-derived xenograft tumors with low LIFR expression and high LCN2 expression. Thus, anti-LCN2 therapy is a promising way to improve liver cancer treatment by targeting ferroptosis.

Staphylococcus pseudintermedius's PBP4 Is Directly Associated with the Dissociated Oxacillin and Cefoxitin Phenotype.

Staphylococcus pseudintermedius is an important pathogen responsible for infections in dogs and in humans. The emergence and dissemination of methicillin-resistant S. pseudintermedius (MRSP) and the multidrug resistance frequently seen in this species make difficult the treatment of these pathogens. The cefoxitin disk is widely used as a marker of methicillin resistance mediated by the mecA gene in Staphylococcus aureus and other staphylococcal species; however, it is not useful to detect ß-lactam resistance of MRSP in clinical microbiology laboratories. The purpose of this study was to elucidate the molecular bases of the dissociated phenotype between oxacillin and cefoxitin antibiotics. By using a combinatorial approach that included the Penicillin-Binding Proteins' (PBP) profile, their affinity for different ß-lactam antibiotics and the analyses of PBPs' sequence, we provide evidence that PBP4 showed still affinity for its target cefoxitin, impairing its phenotypic resistant detection in MRSP. Together, these findings provide evidence that S. pseudintermedius PBP4 is directly associated with the dissociated oxacillin and cefoxitin phenotype.

A comprehensive overview of metaplastic breast cancer: clinical features and molecular aberrations.

Metaplastic breast cancer (MpBC) is an exceedingly rare breast cancer variant that is therapeutically challenging and aggressive. MpBC is defined by the histological presence of at least two cellular types, typically epithelial and mesenchymal components. This variant harbors a triple-negative breast cancer (TNBC) phenotype, yet has a worse prognosis and decreased survival compared to TNBC. There are currently no standardized treatment guidelines specifically for MpBC. However, prior studies have found that MpBC typically has molecular alterations in epithelial-to-mesenchymal transition, amplification of epidermal growth factor receptor, PI3K/Akt signaling, nitric oxide signaling, Wnt/ß-catenin signaling, altered immune response, and cell cycle dysregulation. Some of these molecular alterations have been studied as therapeutic targets, in both the preclinical and clinical setting. This current review discusses the histological organization and cellular origins of MpBC, molecular alterations, the role of radiation therapy, and current clinical trials for MpBC.

Carbapenems drive the collateral resistance to ceftaroline in cystic fibrosis patients with MRSA.

Chronic airways infection with methicillin-resistant Staphylococcus aureus (MRSA) is associated with worse respiratory disease cystic fibrosis (CF) patients. Ceftaroline is a cephalosporin that inhibits the penicillin-binding protein (PBP2a) uniquely produced by MRSA. We analyzed 335 S. aureus isolates from CF sputum samples collected at three US centers between 2015-2018. Molecular relationships demonstrated that high-level resistance of preceding isolates to carbapenems were associated with subsequent isolation of ceftaroline resistant CF MRSA. In vitro evolution experiments showed that pre-exposure of CF MRSA to meropenem with further selection with ceftaroline implied mutations in mecA and additional mutations in pbp1 and pbp2, targets of carbapenems; no effects were achieved by other ß-lactams. An in vivo pneumonia mouse model showed the potential therapeutic efficacy of ceftaroline/meropenem combination against ceftaroline-resistant CF MRSA infections. Thus, the present findings highlight risk factors and potential therapeutic strategies offering an opportunity to both prevent and address antibiotic resistance in this patient population.

Simultaneous targeting of HER family pro-survival signaling with Pan-HER antibody mixture is highly effective in TNBC: a preclinical trial with PDXs.

BACKGROUND: The human epidermal growth factor receptor (HER) family, notably EGFR, is overexpressed in most triple-negative breast cancer (TNBC) cases and provides cancer cells with compensatory signals that greatly contribute to the survival and development of resistance in response to therapy. This study investigated the effects of Pan-HER (Symphogen, Ballerup, Denmark), a novel mixture of six monoclonal antibodies directed against members of the HER family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs). METHODS: Fifteen low passage TNBC PDX tumor samples were transferred into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 100-200 mm3, mice were randomized (n ≥ 6 per group) and treated following three 1-week cycles consisting of three times/week intraperitoneal (IP) injection of either formulation buffer (vehicle control) or Pan-HER (50 mg/kg). At the end of treatment, tumors were collected for Western blot, RNA, and immunohistochemistry analyses. RESULTS: All 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor downmodulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways AKT and ERK. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation. CONCLUSIONS: The marked effect of Pan-HER in numerous HER family-dependent TNBC PDX models justifies further studies of Pan-HER in TNBC clinical trials as a potential therapeutic option.

Correction to: A window-of-opportunity trial of the CXCR1/2 inhibitor reparixin in operable HER-2-negative breast cancer.

An amendment to this paper has been published and can be accessed via the original article.

A window-of-opportunity trial of the CXCR1/2 inhibitor reparixin in operable HER-2-negative breast cancer.

BACKGROUND: Cancer stem cells (CSCs) are purported to be responsible for tumor initiation, treatment resistance, disease recurrence, and metastasis. CXCR1, one of the receptors for CXCL8, was identified on breast cancer (BC) CSCs. Reparixin, an investigational allosteric inhibitor of CXCR1, reduced the CSC content of human BC xenograft in mice. METHODS: In this multicenter, single-arm trial, women with HER-2-negative operable BC received reparixin oral tablets 1000 mg three times daily for 21 days before surgery. Primary objectives evaluated the safety of reparixin and the effects of reparixin on CSC and tumor microenvironment in core biopsies taken at baseline and at treatment completion. Signal of activity was defined as a reduction of ≥ 20% in ALDH+ or CD24-/CD44+ CSC by flow cytometry, with consistent reduction by immunohistochemistry. RESULTS: Twenty patients were enrolled and completed the study. There were no serious adverse reactions. CSC markers ALDH+ and CD24-/CD44+ measured by flow cytometry decreased by ≥ 20% in 4/17 and 9/17 evaluable patients, respectively. However, these results could not be confirmed by immunofluorescence due to the very low number of CSC. CONCLUSIONS: Reparixin appeared safe and well-tolerated. CSCs were reduced in several patients as measured by flow cytometry, suggesting targeting of CXCR1 on CSC. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov, NCT01861054. Registered on April 18, 2013.

SNX27-retromer assembly recycles MT1-MMP to invadopodia and promotes breast cancer metastasis.

A variety of metastatic cancer cells use actin-rich membrane protrusions, known as invadopodia, for efficient ECM degradation, which involves trafficking of proteases from intracellular compartments to these structures. Here, we demonstrate that in the metastatic breast cancer cell line MDA-MB-231, retromer regulates the matrix invasion activity by recycling matrix metalloprotease, MT1-MMP. We further found that MT2-MMP, another abundantly expressed metalloprotease, is also invadopodia associated. MT1- and MT2-MMP showed a high degree of colocalization but were located on the distinct endosomal domains. Retromer and its associated sorting nexin, SNX27, phenocopied each other in matrix degradation via selectively recycling MT1-MMP but not MT2-MMP. ITC-based studies revealed that both SNX27 and retromer could directly interact with MT1-MMP. Analysis from a publicly available database showed SNX27 to be overexpressed or frequently altered in the patients having invasive breast cancer. In xenograft-based studies, SNX27-depleted cell lines showed prolonged survival of SCID mice, suggesting a possible implication for overexpression of the sorting nexin in tumor samples.

A randomized, controlled phase II trial of neoadjuvant ado-trastuzumab emtansine, lapatinib, and nab-paclitaxel versus trastuzumab, pertuzumab, and paclitaxel in HER2-positive breast cancer (TEAL study).

BACKGROUND: Neoadjuvant dual human epidermal growth factor receptor (HER2) blockade with trastuzumab and pertuzumab plus paclitaxel leads to an overall pathologic complete response (pCR) rate of 46%. Dual HER2 blockade with ado-trastuzumab emtansine (T-DM1) and lapatinib plus nab-paclitaxel has shown efficacy in patients with metastatic HER2-positive breast cancer. To test neoadjuvant effectiveness of this regimen, an open-label, multicenter, randomized, phase II trial was conducted comparing T-DM1, lapatinib, and nab-paclitaxel with trastuzumab, pertuzumab, and paclitaxel in patients with early-stage HER2-positive breast cancer. METHODS: Stratification by estrogen receptor (ER) status occurred prior to randomization. Patients in the experimental arm received 6 weeks of targeted therapies (T-DM1 and lapatinib) followed by T-DM1 every 3 weeks, lapatinib daily, and nab-paclitaxel weekly for 12 weeks. In the standard arm, patients received 6 weeks of trastuzumab and pertuzumab followed by trastuzumab weekly, pertuzumab every 3 weeks, and paclitaxel weekly for 12 weeks. The primary objective was to evaluate the proportion of patients with residual cancer burden (RCB) 0 or I. Key secondary objectives included pCR rate, safety, and change in tumor size at 6 weeks. Hypothesis-generating correlative assessments were also performed. RESULTS: The 30 evaluable patients were well-balanced in patient and tumor characteristics. The proportion of patients with RCB 0 or I was higher in the experimental arm (100% vs. 62.5% in the standard arm, p = 0.0035). In the ER-positive subset, all patients in the experimental arm achieved RCB 0-I versus 25% in the standard arm (p = 0.0035). Adverse events were similar between the two arms. CONCLUSION: In early-stage HER2-positive breast cancer, the neoadjuvant treatment with T-DM1, lapatinib, and nab-paclitaxel was more effective than the standard treatment, particularly in the ER-positive cohort. TRIAL REGISTRATION: Clinicaltrials.gov NCT02073487 , February 27, 2014.

VraSR and Virulence Trait Modulation during Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus Infection.

Methicillin-resistant Staphylococcus aureus (MRSA) threatens human health in hospital and community settings. The lipopeptide antibiotic daptomycin (DAP) is a frequently used treatment option for MRSA infection. DAP exposure can cause bacterial resistance because mutations are induced in genes implicated in cell membrane and cell wall metabolism. Adaptations aimed at surviving antimicrobial pressure can affect bacterial physiology and modify in vivo aptitude and pathogenesis. In this study, clinical DAP-susceptible (DAPs) and DAP-resistant (DAPr) MRSA isolates were used to investigate associations between DAP resistance and staphylococcal virulence. We previously found that VraSR is a critical sensor of cell membrane/wall homeostasis associated with DAP acquisition during MRSA infection. The present study found that DAPr CB1634 and CB5014 MRSA strains with vraSR upregulation were less virulent than their susceptible counterparts, CB1631 and CB5013. Differential gene-transcription profile analysis revealed that DAPr CB1634 had decreased agr two-component system expression, virulence factors, and highly suppressed hemolysis activity. Functional genetic analysis performed in DAPr CB1634 strains using vraSR inactivation followed by gene complementation found that vraSR acted as a transcriptional agrA regulator. These results indicated that VraSR has a broad range of regulatory functions. VraSR also appeared to affect DAPr adherence to epithelial cells, which would affect DAPr strain colonization and survival in the host. The correlation between DAP resistance and decreased virulence was also found in the CB5013 (DAPs) and CB5014 (DAPr) pair. Taken together, these findings are the first evidence that DAP resistance and MRSA virulence are tightly connected and involve compromised expression of regulatory and virulence determinants.IMPORTANCE Methicillin-resistant S. aureus continues to develop resistance to antimicrobials, including those in current clinical use as daptomycin (DAP). Resistance to DAP arises by mutations in cell membrane and cell wall genes and/or upregulation of the two-component VraSR system. However, less is known about the connection between the pathogen and virulence traits during DAP resistance development. We provide new insights into VraSR and its regulatory role for virulence factors during DAP resistance, highlighting coordinated interactions that favor the higher persistence of MRSA DAP-resistant strains in the infected host.

Cancer therapeutic targeting using mutant-p53-specific siRNAs.

Mutations in Tp53 compromise therapeutic response, due either to the dominant-negative effect over the functional wild-type allele; or as a result of the survival advantage conferred by mutant p53 to which cancer cells become addicted. Thus, targeting mutant p53 represents an effective therapeutic strategy to treat over half of all cancers. We have therefore generated a series of small-interfering-RNAs, capable of targeting four p53 hot-spot mutants which represent ~20% of all p53 mutations. These mutant-p53-specific siRNAs (MupSi) are highly specific in silencing the expression of the intended mutants without affecting wild-type p53. Functionally, these MupSis induce cell death by abrogating both the addiction to mutant p53 and the dominant-negative effect; and retard tumor growth in xenografts when administered in a therapeutic setting. These data together demonstrate the possibility of targeting mutant p53 specifically to improve clinical outcome.