A FtsZ Inhibitor That Can Utilize Siderophore-Ferric Iron Uptake Transporter Systems for Activity against Gram-Negative Bacterial Pathogens.

The global threat of multidrug-resistant Gram-negative bacterial pathogens necessitates the development of new and effective antibiotics. FtsZ is an essential and highly conserved cytoskeletal protein that is an appealing antibacterial target for new antimicrobial therapeutics. However, the effectiveness of FtsZ inhibitors against Gram-negative species has been limited due in part to poor intracellular accumulation. To address this limitation, we have designed a FtsZ inhibitor (RUP4) that incorporates a chlorocatechol siderophore functionality that can chelate ferric iron (Fe3+) and utilizes endogenous siderophore uptake pathways to facilitate entry into Gram-negative pathogens. We show that RUP4 is active against both Klebsiella pneumoniae and Acinetobacter baumannii, with this activity being dependent on direct Fe3+ chelation and enhanced under Fe3+-limiting conditions. Genetic deletion studies in K. pneumoniae reveal that RUP4 gains entry through the FepA and CirA outer membrane transporters and the FhuBC inner membrane transporter. We also show that RUP4 exhibits bactericidal synergy against K. pneumoniae when combined with select antibiotics, with the strongest synergy observed with PBP2-targeting ß-lactams or MreB inhibitors. In the aggregate, our studies indicate that incorporation of Fe3+-chelating moieties into FtsZ inhibitors is an appealing design strategy for enhancing activity against Gram-negative pathogens of global clinical significance.

User-Guided Enhancements to a Technology-Facilitated Resilience Program to Address Opioid Risks Following Traumatic Injury in Youth: Qualitative Interview Study.

BACKGROUND: Youth with traumatic injury experience elevated risk for behavioral health disorders, yet posthospital monitoring of patients' behavioral health is rare. The Telehealth Resilience and Recovery Program (TRRP), a technology-facilitated and stepped access-to-care program initiated in hospitals and designed to be integrated seamlessly into trauma center operations, is a program that can potentially address this treatment gap. However, the TRRP was originally developed to address this gap for mental health recovery but not substance use. Given the high rates of substance and opioid use disorders among youth with traumatic injury, there is a need to monitor substance use and related symptoms alongside other mental health concerns. OBJECTIVE: This study aimed to use an iterative, user-guided approach to inform substance use adaptations to TRRP content and procedures. METHODS: We conducted individual semistructured interviews with adolescents (aged 12-17 years) and young adults (aged 18-25 years) who were recently discharged from trauma centers (n=20) and health care providers from two level 1 trauma centers (n=15). Interviews inquired about reactions to and recommendations for expanding TRRP content, features, and functionality; factors related to TRRP implementation and acceptability; and current strategies for monitoring patients' postinjury physical and emotional recovery and opioid and substance use. Interview responses were transcribed and analyzed using thematic analysis to guide new TRRP substance use content and procedures. RESULTS: Themes identified in interviews included gaps in care, task automation, user personalization, privacy concerns, and in-person preferences. Based on these results, a multimedia, web-based mobile education app was developed that included 8 discrete interactive education modules and 6 videos on opioid use disorder, and TRRP procedures were adapted to target opioid and other substance use disorder risk. Substance use adaptations included the development of a set of SMS text messaging-delivered questions that monitor both mental health symptoms and substance use and related symptoms (eg, pain and sleep) and the identification of validated mental health and substance use screening tools to monitor patients' behavioral health in the months after discharge. CONCLUSIONS: Patients and health care providers found the TRRP and its expansion to address substance use acceptable. This iterative, user-guided approach yielded novel content and procedures that will be evaluated in a future trial.

Structural and Antibacterial Characterization of a New Benzamide FtsZ Inhibitor with Superior Bactericidal Activity and In Vivo Efficacy Against Multidrug-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant (MDR) bacterial pathogen of acute clinical significance. Resistance to current standard-of-care antibiotics, such as vancomycin and linezolid, among nosocomial and community-acquired MRSA clinical isolates is on the rise. This threat to global public health highlights the need to develop new antibiotics for the treatment of MRSA infections. Here, we describe a new benzamide FtsZ inhibitor (TXH9179) with superior antistaphylococcal activity relative to earlier-generation benzamides like PC190723 and TXA707. TXH9179 was found to be 4-fold more potent than TXA707 against a library of 55 methicillin-sensitive S. aureus (MSSA) and MRSA clinical isolates, including MRSA isolates resistant to vancomycin and linezolid. TXH9179 was also associated with a lower frequency of resistance relative to TXA707 in all but one of the MSSA and MRSA isolates examined, with the observed resistance being due to mutations in the ftsZ gene. TXH9179 induced changes in MRSA cell morphology, cell division, and FtsZ localization are fully consistent with its actions as a FtsZ inhibitor. Crystallographic studies demonstrate the direct interaction of TXH9179 with S. aureus FtsZ (SaFtsZ), while delineating the key molecular contacts that drive complex formation. TXH9179 was not associated with any mammalian cytotoxicity, even at a concentration 10-fold greater than that producing antistaphylococcal activity. In serum, the carboxamide prodrug of TXH9179 (TXH1033) is rapidly hydrolyzed to TXH9179 by serum acetylcholinesterases. Significantly, both intravenously and orally administered TXH1033 exhibited enhanced in vivo efficacy relative to the carboxamide prodrug of TXA707 (TXA709) in treating a mouse model of systemic (peritonitis) MRSA infection. Viewed as a whole, our results highlight TXH9179 as a promising new benzamide FtsZ inhibitor worthy of further development.

Characterizing Aerosol Generating Procedures with Background Oriented Schlieren

The potential for characterizing aerosol generating procedures (AGPs) using background oriented schlieren (BOS) flow visualization was investigated in two clinical situations. A human-scale BOS system was used on a manikin simulating jet ventilation and extubation. A novel approach to representation of the BOS images using line integral convolution allows direct evaluation of both magnitude and direction of the refractive index gradient field. Plumes issuing from the manikins mouth were clearly visualized and characterized in both experiments, and it is recommended that BOS be adapted into a clinical tool for risk evaluation in clinical environments.

Reporter system controlled by the involucrin promoter as a tool to follow epidermal differentiation.

Various approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSCs) from the umbilical cord can be safely and easily obtained; however, a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under the control of pINV to assess MSC transdifferentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, producing LeGO-GpINV. MSCs were transduced with LeGO-GpINV and induced to transdifferentiate into keratinocytes under cultivation with keratinocyte serum-free medium. MSC transdifferentiation was confirmed by morphological changes and by the expression of epidermal markers by flow cytometry, quantitative PCR, Western blot and the activity of epidermal kallikreins 5, 6 and 7. After 14 days of transdifferentiation, MSCs transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filaggrin. There was also an increase in kallikrein activity. This reporter system allowed us to temporally assess epidermal differentiation, simultaneously with involucrin expression, opening possibilities for the in vivo study of skin biology and in regenerative medicine.

TXH11106: A Third-Generation MreB Inhibitor with Enhanced Activity against a Broad Range of Gram-Negative Bacterial Pathogens.

The emergence of multi-drug-resistant Gram-negative pathogens highlights an urgent clinical need to explore and develop new antibiotics with novel antibacterial targets. MreB is a promising antibacterial target that functions as an essential elongasome protein in most Gram-negative bacterial rods. Here, we describe a third-generation MreB inhibitor (TXH11106) with enhanced bactericidal activity versus the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa compared to the first- and second-generation compounds A22 and CBR-4830, respectively. Large inocula of these four pathogens are associated with a low frequency of resistance (FOR) to TXH11106. The enhanced bactericidal activity of TXH11106 relative to A22 and CBR-4830 correlates with a correspondingly enhanced capacity to inhibit E. coli MreB ATPase activity via a noncompetitive mechanism. Morphological changes induced by TXH11106 in E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa provide further evidence supporting MreB as the bactericidal target of the compound. Taken together, our results highlight the potential of TXH11106 as an MreB inhibitor with activity against a broad spectrum of Gram-negative bacterial pathogens of acute clinical importance.

Novel MreB inhibitors with antibacterial activity against Gram (-) bacteria.

MreB is a cytoskeleton protein present in rod-shaped bacteria that is both essential for bacterial cell division and highly conserved. Because most Gram (-) bacteria require MreB for cell division, chromosome segregation, cell wall morphogenesis, and cell polarity, it is an attractive target for antibacterial drug discovery. As MreB modulation is not associated with the activity of antibiotics in clinical use, acquired resistance to MreB inhibitors is also unlikely. Compounds, such as A22 and CBR-4830, are known to disrupt MreB function by inhibition of ATPase activity. However, the toxicity of these compounds has hindered efforts to assess the in vivo efficacy of these MreB inhibitors. The present study further examines the structure-activity of analogs related to CBR-4830 as it relates to relative antibiotic activity and improved drug properties. These data reveal that certain analogs have enhanced antibiotic activity. In addition, we evaluated several representative analogs (9, 10, 14, 26, and 31) for their abilities to target purified E. coli MreB (EcMreB) and inhibit its ATPase activity. Except for 14, all these analogs were more potent than CBR-4830 as inhibitors of the ATPase activity of EcMreB with corresponding IC50 values ranging from 6 ± 2 to 29 ± 9 µM.

Metformin-induced chemosensitization to cisplatin depends on P53 status and is inhibited by Jarid1b overexpression in non-small cell lung cancer cells.

Metformin has been tested as an anti-cancer therapy with potential to improve conventional chemotherapy. However, in some cases, metformin fails to sensitize tumors to chemotherapy. Here we test if the presence of P53 could predict the activity of metformin as an adjuvant for cisplatin-based therapy in non-small cell lung cancer (NSCLC). A549, HCC 827 (TP53 WT), H1299, and H358 (TP53 null) cell lines were used in this study. A549 cells were pre-treated with a sub-lethal dose of cisplatin to induce chemoresistance. The effects of metformin were tested both in vitro and in vivo and related to the ability of cells to accumulate Jarid1b, a histone demethylase involved in cisplatin resistance in different cancers. Metformin sensitized A549 and HCC 827 cells (but not H1299 and H358 cells) to cisplatin in a P53-dependent manner, changing its subcellular localization to the mitochondria. Treatment with a sub-lethal dose of cisplatin increased Jarid1b expression, yet downregulated P53 levels, protecting A549Res cells from metformin-induced chemosensitization to cisplatin and favored a glycolytic phenotype. Treatment with FL3, a synthetic flavagline, sensitized A549Res cells to cisplatin. In conclusion, metformin could potentially be used as an adjuvant for cisplatin-based therapy in NSCLC cells if wild type P53 is present.

miR-138-5p induces aggressive traits by targeting Trp53 expression in murine melanoma cells, and correlates with poor prognosis of melanoma patients.

Deregulation of miRNAs contributes to the development of distinct cancer types, including melanoma, an aggressive form of skin cancer characterized by high metastatic potential and poor prognosis. The expression of a set of 580 miRNAs was investigated in a model of murine melanoma progression, comprising non-metastatic (4C11-) and metastatic melanoma (4C11+) cells. A significant increase in miR-138-5p expression was found in the metastatic 4C11+ melanoma cells compared to 4C11-, which prompted us to investigate its role in melanoma aggressiveness. Functional assays, including anoikis resistance, colony formation, collective migration, serum-deprived growth capacity, as well as in vivo tumor growth and experimental metastasis were performed in 4C11- cells stably overexpressing miR-138-5p. miR-138-5p induced an aggressive phenotype in mouse melanoma cell lines leading to increased proliferation, migration and cell viability under stress conditions. Moreover, by overexpressing miR-138-5p, low-growing and non-metastatic 4C11- cells became highly proliferative and metastatic in vivo, similar to the metastatic 4C11+ cells. Luciferase reporter analysis identified the tumor suppressor Trp53 as a direct target of miR-138-5p. Using data sets from independent melanoma cohorts, miR-138-5p and P53 expression were also found deregulated in human melanoma samples, with their levels negatively and positively correlated with prognosis, respectively. Our data shows that the overexpression of miR-138-5p contributes to melanoma metastasis through the direct suppression of Trp53.

ACUTE MACULAR EDEMA WITH SEROUS RETINAL DETACHMENT AFTER CATARACT SURGERY IN A VITRECTOMIZED EYE: A CASE REPORT.

PURPOSE: To present the first case of acute macular edema with serous retinal detachment after cataract surgery in a vitrectomized eye. METHODS: A 63-year-old female patient, with history of pars plana vitrectomy and epiretinal membrane removal, underwent uneventful phacoemulsification surgery with injection of standard intracameral dose of cefuroxime (1 mg/0.1 mL of solution) at the end of the procedure. RESULTS: First day after cataract surgery, visual acuity did not correlate with anterior segment findings, and funduscopic eye examination revealed acute macular edema with serous retinal detachment, which was confirmed by spectral domain optical coherence tomography. Fluorescein angiography showed no retinal or choroidal hyperpermeability. At 2-week follow-up visit, visual acuity had significantly improved, and there was complete resolution of macular edema and subretinal fluid. CONCLUSION: The current case suggests that acute macular edema with serous retinal detachment after cataract surgery with standard cefuroxime prophylaxis can occur even in vitrectomized eyes. A high level of suspicious is needed when visual acuity does not correlate with anterior segment findings immediately after cataract surgery. Similar to reports from nonvitrectomized eyes, visual prognosis was favorable.

HIV Vulnerability Among Survival Sex Workers Through Sexual Violence and Drug Taking in a Qualitative Study From Victoria, Canada, With Additional Implications for Pre-exposure Prophylaxis for Sex Workers.

Objective: This qualitative study investigates how social and structural forces mediate vulnerability to HIV infection and transmission among survival sex workers, their clients, and their non-commercial, intimate partners-with especial focus on sexual violence and drug taking. Method: I employed an adapted grounded theory approach to conducting and analyzing (n = 9) open-ended, in-depth interviews with a convenience sample of currently working (and recently exited) survival sex workers from a community setting in Victoria, Canada. Findings: Participants revealed important contexts and conditions under which they were vulnerable to HIV infection. At the behavioural level, participants were aware of how HIV could be transmitted (condomless sex and sharing drug equipment), yet participants voiced strongly how structural and systemic features (for instance, client violence, the need for drugs, and "bad date" referrals) could squeeze and constrain their agency to take up safer practices, mediating their optimal HIV health and safety. Some participants reported strained relationships with police because of previous drug involvement. Conclusion: Survival sex workers constitute a health population vulnerable to HIV infection, and ensuring there could be a supportive (outreach) community replete with HIV resources is paramount. The availability of safer sex and drug equipment play important roles in HIV behavioural prevention efforts. However, uptake of pre-exposure prophylaxis (PrEP) at no cost in the Canadian province of British Columbia could be an important and beneficial structural intervention for non-injection drug taking cis-female sex workers in this study who are presently ineligible for no cost PrEP.

Attorneys' Attitudes Toward Coaching Forensic Clients on the MMPI-2: Replication and Extension of Attorney Survey by Wetter and Corrigan (1995).

In this study we replicated and extended Wetter and Corrigan's (1995) commonly cited convenience survey of attorneys and law students regarding their attitudes toward coaching litigants prior to forensic psychological testing. We conducted a target survey of attorneys practicing in specialty areas of law where it is common to enlist psychological testing as part of legal proceedings (family law, juvenile law, personal injury, criminal law, social security/disability, workman's compensation). The estimated prevalence of attorneys who endorse providing their clients with information about the presence of MMPI-2 validity scales is 53%, with a 95% confidence interval of ±7%. Compared with Wetter and Corrigan's results of 63%, this represents a slightly lower estimate of attorneys who indicate a positive attitude toward coaching their clients on the presence and purpose of validity scales. More than 70% of attorneys, in both surveys, believe they should provide general advice and preparation for psychological testing to their clients. Survey results were further analyzed as a function of attorney age, years in practice, and specialty area. Implications are discussed in relation to future research and the practice of forensic psychological evaluations.

Proteasome and heat shock protein 70 (HSP70) inhibitors as therapeutic alternative in multiple myeloma.

HSP70 connects multiple signaling pathways that work synergistically to protect tumor cells from death by proteotoxic stress and represents a possible target to establish a new approach for multiple myeloma treatment. Therefore, bioluminescent cell lines RPMI8226-LUC-PURO and U266-LUC-PURO were treated with HSP70 (VER155008) and/or proteasome (bortezomib) inhibitors and immunodeficient mice were used for subcutaneous xenograft models to evaluate tumor growth reduction and tumor growth inhibition after treatment. Bioluminescence imaging was used to follow tumor response. Treatment with bortezomib showed ∼60% of late apoptosis in RPMI8226-LUC-PURO (without additional benefit of VER155008 in this cell line). However, U266-LUC-PURO showed ∼60% of cell death after treatment with VER155008 (alone or with bortezomib). RPMI8226-LUC-PURO xenograft presented tumor reduction by bioluminescence imaging after treatment with bortezomib, VER155008 or drug combination compared to controls. Treatment with bortezomib, alone or combined with VER155008, showed inhibition of tumor growth assessed by bioluminescence imaging after one week in both RPMI8226-LUC-PURO and U266-LUC-PURO cell lines when compared to controls. In conclusion, our study shows that the combination of proteasome and HSP70 inhibitors induced cell death in tumor cells in vitro (late apoptosis induction) and in vivo (inhibition of tumor growth) with special benefit in U266-LUC-PURO, bearing 17p deletion.

Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos.

Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

Knockdown of E2f1 by RNA interference impairs proliferation of rat cells in vitro.

E2F1 plays a key role in cell-cycle regulation in mammals, since its transcription factor activity controls genes required for DNA synthesis and apoptosis. E2F1 deregulation is a common feature among different tumor types and can be a major cause of cell proliferation. Thus, blocking E2F1 expression by RNA interference represents a promising therapeutic approach. In this study, the introduction of specific short hairpin RNAs (shRNAs) reduced E2f1 expression by up to 77%, and impaired rat glioma cell proliferation by approximately 70%, as compared to control cells. Furthermore, we investigated the expression of E2f1 target genes, Cyclin A and Cyclin E. Cyclin A was found to be down-regulated, whereas Cyclin E had similar expression to control cells, indicating that gene(s) other than E2f1 control its transcription. Other E2f family members, E2f2 and E2f3, which have been classified in the same subgroup of transcriptional activators, were also analyzed. Expression of both E2f2 and E2f3 was similar to control cells, showing no cross-inactivation or up-regulation to compensate for the absence of E2f1. Nevertheless, their expression was insufficient to maintain the initial proliferation potential. Taken together, our results suggest that shE2f1 is a promising therapy to control tumor cell proliferation.

Knockdown of E2f1 by RNA interference impairs proliferation of rat cells in vitro

E2F1 plays a key role in cell-cycle regulation in mammals, since its transcription factor activity controls genes required for DNA synthesis and apoptosis. E2F1 deregulation is a common feature among different tumor types and can be a major cause of cell proliferation. Thus, blocking E2F1 expression by RNA interference represents a promising therapeutic approach. In this study, the introduction of specific short hairpin RNAs (shRNAs) reduced E2f1 expression by up to 77 percent, and impaired rat glioma cell proliferation by approximately 70 percent, as compared to control cells. Furthermore, we investigated the expression of E2f1 target genes, Cyclin A and Cyclin E. Cyclin A was found to be down-regulated, whereas Cyclin E had similar expression to control cells, indicating that gene(s) other than E2f1 control its transcription. Other E2f family members, E2f2 and E2f3, which have been classified in the same subgroup of transcriptional activators, were also analyzed. Expression of both E2f2 and E2f3 was similar to control cells, showing no cross-inactivation or up-regulation to compensate for the absence of E2f1. Nevertheless, their expression was insufficient to maintain the initial proliferation potential. Taken together, our results suggest that shE2f1 is a promising therapy to control tumor cell proliferation.