A New Strategy for Persister Control

Eradicating persistent bacterial infections is an ever-growing problem in the medical field. Conventional antibiotics are becoming less effective against persistent infections in tuberculosis, cystic fibrosis, Lyme disease, device-associated, urinary tract, gastric and pneumonia still remains a problem. Left untreated, the persistent infections have the potential to increase mortality, morbidity, and the development of antibiotic resistant bacteria. One major mechanism that contributes to the failure of current antibiotic treatment is the formation of persister cells. Persisters are formed when bacterial cells undergo phenotypic changes and enter dormancy which enables their survival under lethal doses of antibiotics. As phenotypic variants, persister formation is transient and reversible. During antibiotic treatment, persisters remain growth arrested and repopulate after the antibiotic is withdrawn, causing the failure of treatment and relapse of many infections. Because most, if not all, antibiotics target growth-associated activities, eradication of persister cells has been challenging.Here, we report new findings that challenge the conventional view that bacterial persister cells are tolerant to all antibiotics and provide evidence that it is possible to achieve persister killing by leveraging dormancy-associated reduction of antibiotic efflux. We demonstrate that antibiotics capable of penetrating bacterial cells by energy- independent diffusion and binding to their target strongly can kill persister cells during wake-up. This was demonstrated using minocycline. At the concentration of 100 μg/mL, minocycline killed Escherichia coli persister cells by 70.8 ± 5.9% while it did not have significant killing of normal cells. Consistently, the results showed that persister cells had a higher intracellular minocycline concentration (~2.6 times more minocycline per cell) compared to normal cells. The results were corroborated with tests using efflux pump mutants and efflux pump inhibitors. Specifically, treatment with carbonyl cyanide m- chlorophenylhydrazone (CCCP), an efflux pump inhibitor, at 10μM led to 94.7 ± 2.5% killing of E. coli normal cells by minocycline. This is a 4-time increase compared to treatment without membrane depotentiation, which only led to 22.0 ± 3.3% killing. We demonstrated here that E. coli persister cells have reduced efflux and thus more accumulation of minocycline than normal cells, leading to an effective killing of this dormant population. Encouraged by these findings, we then sought out to determine if persister killing can be further enhanced by increasing the target binding affinity of antibiotics. Through literature research, we found eravacycline, which also targets the ribosome but has a stronger binding than minocycline. Our results demonstrated eravacycline can kill E. coli persisters by 3 logs when treated at 100 μg/mL. In addition, it has a strong synergy with ampicillin, eradicating both normal and persister cells of E. coli. With our proposed mechanism, we have proven thus far, that the increased in sensitivity of persister cells is the result of at least three factors which are low membrane potential, reduced efflux pump activity and the tight binding affinity of this antibiotic with the ribosome. To validate this new strategy, it is necessary to test if these antibiotics are also effective against persister cells of other pathogenic bacteria. This motivated us to test eravacycline on uropathogenic E. coli (UPEC) and Pseudomonas. aeruginosa. Our results demonstrated that eravacycline is effective in killing both UPEC normal and persister cells; by 97.8 ± 0.7% and 99.9 ± 0.1%, respectively at a concentration of 100 μg/mL. In addition, we tested the effects of eravacycline on P. aeruginosa PAO1 (wild-type) and PDO300 (mucoid mutant) persisters cells. Our results demonstrate that CCCP-isolated PAO1(wildtype) and PDO300 (mucoid) persisters can be effectively killed by eravacycline (by 99.7 ± 0.0% and 99.0 ± 0.1%, respectively) with a concentration of 100 μg/mL. Lastly, we tested the effects of eravacycline on biofilm associated persister cells given that biofilms play a major role in chronic infections. At the concentration of 100 μg/mL eravacycline reduced the number of viable biofilm cells of UPEC, P. aeruginosa PAO1, and P. aeruginosa PDO300 by 99.2 ± 0.5%, 99.9 ± 0.8%, and 99.6 ± 0.4%, respectively, compared to untreated controlFurthermore, the results from E. coli persister data were used to develop a set of criteria for persister control. The set of criteria outline that effective control agents need to (1) be positively charged under physiological condition to interact with the negatively charged lipopolysaccharides on bacterial outer membrane, (2) be able to penetrate persister cells via energy-independent diffusion, (3) be amphiphilic to have membrane activity for penetration, and (4) have strong binding to an intracellular target. Using this set of criteria, we optimized a chemoinformatic clustering algorithm to design and select persister control agents. To validate these criteria, we first extracted structural and physiochemical parameters of persister killing agents identified in this study (minocycline, rifamycin SV, and eravacycline) and those from a small chemical library (80 chemical compounds). Next, we used k-means clustering to assess the logP (octanol-water partition) and the number of halogen atoms that appear on the compounds. We selected the top ten compounds based on the clustering result to furhter test for persister killing. Among the tested top leads, we discovered four persister control agents that showed potent activity against E. coli persister cells. While most of the compounds had similar moderate effects on normal (~20% killing) and persister cells (~30% killing) when treated at 100 μg/mL, four compounds (161,171, 173 and 175) exhibited stronger effects against persister cells than normal cells. At a concentration of 100 μg/mL, they showed ~ 30% killing of normal cells; however, compounds 161, 171, 173, and 175 killed 95.5 ± 1.7% (p\u3c0.0001), 85.2 ± 2.6% (p=0.0003), 94.2 ± 1.4% (p\u3c0.0001), and 99.6 ± 0.1% (p\u3c0.0001) respectively of the persister cells. To further understand the stronger killing efficacy of persister cells than normal cells by these compounds, we quantified the intracellular concentrations and found persister cells accumulated more compared to normal cells. Since, we demonstrated that persister cells have reduced efflux activities, we further evaluated if both compounds are substrate of the multidrug AcrAB-TolC efflux pump. To test this, E. coli JW4364 (ΔacrA mutant), JW5536 (ΔacrB mutant), and JW5503 (ΔtolC mutant) were compared with their wild-type strain E. coli BW25113 for susceptibility to compounds. Increased killing of all three efflux mutants compared to the wild-type strain was observed for all three of the four compounds. This finding further demonstrates a correlation between the lack of efflux and increase in persister killing.While it is commonly stated that persister cells are tolerant to conventional antibiotics, our study reveals that these dormant cells can be killed by selecting the right antibiotics with appropriate treatment conditions. Specifically, we demonstrate that antimicrobial capable of penetrating bacterial cells by energy-independent diffusion and binding to their target strongly can kill persister cells during wake-up. These results can help develop better strategies to combat persistent infections

The Role of the District Public Health Nurses: A Study from Gujarat

The role of District Public Health Nurses (DPHN) and District Public Health Nurse Officers (DPHNOs) as supervisors of the Public Health nursing and midwifery staff in a district was investigated. Thirteen DPHNs and DPHNOs from six districts selected from six geographic zones of Gujarat were observed for one week using the time motion method. Their activities and time spent were noted and the DPHNs/DPHNOs and their supervisors were interviewed. The role of the DPHNs has reduced over the years because they have not been assigned new roles with change in programmes and policies. Most of the DPHNs have training for clinical work in hospitals. Their 10 month training to qualify for PHN is inadequate to develop knowledge and skills in public health. There is a gap between their training and posting due to delays in government procedures of promotion. The DPHN/DPHNOs spend majority of their time in the office (49%) where they have a limited role. Their supervisory role for nurses and midwives has lost its importance. They spend about 1/3rd of their time in field supervision mostly visiting centres accessible by public transport as they do not have an allotted government vehicle. As they do not submit any field report, there is no follow-up action from their visit. Nevertheless they seem to have an important role in solving problems of field workers as they are mediators between the district and peripheral facilities. To conclude the DPHNs are under utilized which affects the quality of maternal and child health services in the district.

Interrogating the development of Enteric Nervous System in zebrafish using transcriptomics

The enteric nervous system (ENS) is the set of neurons that control the activity of the gastrointestinal system. These activities include secretion of digestive juices, absorption of food, and motility of the gut. The enteric neurons are derived from the neural crest cells (NCC) which migrate to the gut during development. We have a sparse knowledge of the genes and the signaling pathways that are known to be involved in the migration, specification, and differentiation of the enteric neurons from neural crest precursors. Malfunction in any of these processes hampers normal ENS development and can result in a variety of diseases including Hirschsprung’s disease, a disorder in which the distal intestinal tract lacks enteric neurons. With the aid of transcriptomic study, we aimed to understand the molecular basis behind the different processes of development of a functional enteric nervous system using zebrafish as our model organism. In my research, we were able to generate the transcriptome of the neuronal cells as well as of the microenvironment that is known to provide external signals to these neurons in a normal developing ENS. We were able to identify previously linked genes and pathways associated with ENS development and also ascertain a large number of novel candidate genes that might be potential regulators in driving a normal ENS development. We also tried to elucidate the heterogeneity that exists between enteric neurons by performing a single cell transcriptomic study. Our findings from this project provided an insight into the different genetic and molecular factors that are specific to the different developmental stages. Along with shedding a light on the developmental timeline, this project also assisted in unraveling the factors distinctive of the subpopulations of the enteric neurons that reside in the gastrointestinal tract.</p

Advancements in Cancer Immunotherapies

Recent work has suggested involvement of the immune system in biological therapies specifically targeting tumor microenvironment. Substantial advancement in the treatment of malignant tumors utilizing immune cells, most importantly T cells that play a key role in cell-mediated immunity, have led to success in clinical trials. Therefore, this article focuses on the therapeutic approaches and developmental strategies to treat cancer. This review emphasizes the immunomodulatory response, the involvement of key tumor-infiltrating cells, the mechanistic aspects, and prognostic biomarkers. We also cover recent advancements in therapeutic strategies

Regioselective synthesis of 4-fluoro-1,5-substituted-1,2,3-triazoles from synthetic surrogates of 1-fluoroalkynes

Here, we show that 1-fluoronitroalkenes can serve as synthetic surrogates of 1-fluoroalkynes in [3+2] cycloaddition reactions with organic azides facilitated by a catalytic amount of trifloroacetic acid. This work provides the first regioselective method to access 4-fluoro-1,5-substituted-1,2,3-triazoles

A systematic review and lived experience synthesis of self-disclosure as an active ingredient in interventions for adolescents and young adults with anxiety and depression

Background: Self-disclosure, referring to the ability to communicate and share intimate personal feelings, has strong face validity for many young people as a way of improving anxiety and depression outcomes. The current review aimed to generate the first comprehensive evidence synthesis of self-disclosure interventions involving young people aged 14-24 years who are either disclosers or recipients of personal information about living with anxiety and/or depression. Methods: A systematic review of quantitative and qualitative data was combined with new insights from an adolescents and young adults lived-experience panel (n=7) with the intention to combine rigorous systematic review methods and experiential knowledge. Results: Six studies of variable quality were included in this review, five were quantitative and one was qualitative. Findings suggest that self-disclosure may be effective at reducing symptoms for adolescents and young adults with established depression; effects were not apparent when delivered as early prevention. No evidence for impacts on anxiety was found. The potential for negative effects like bullying or harassment was identified. Limitations: Findings were limited by a small number of studies; low representation of peerreviewed studies from low-or middle-income countries; and varied interventions in terms of format, participants' context, and nature of delivery. Conclusions: Self-disclosure may be of value in the context of interventions intended explicitly to reduce depression for those already showing symptoms. Delivery by nonspecialists (such as peers and teachers) in addition to mental health professionals can help build capacity in community health systems. Self-disclosure may also be helpful at reducing stigma and stimulating help-seeking at earlier stages of mental health problems

A Combination RNAi-Chemotherapy Layer-by-Layer Nanoparticle for Systemic Targeting of KRAS/P53 with Cisplatin to Treat Non–Small Cell Lung Cancer

Purpose: Mutation of the Kirsten ras sarcoma viral oncogene homolog (KRAS) and loss of p53 function are commonly seen in patients with non–small cell lung cancer (NSCLC). Combining therapeutics targeting these tumor-defensive pathways with cisplatin in a single-nanoparticle platform are rarely developed in clinic. Experimental Design: Cisplatin was encapsulated in liposomes, which multiple polyelectrolyte layers, including siKRAS and miR-34a were built on to generate multifunctional layer-by-layer nanoparticle. Structure, size, and surface charge were characterized, in addition to in vitro toxicity studies. In vivo tumor targeting and therapy was investigated in an orthotopic lung cancer model by microCT, fluorescence imaging, and immunohistochemistry. Results: The singular nanoscale formulation, incorporating oncogene siKRAS, tumor-suppressor stimulating miR-34a, and cisplatin, has shown enhanced toxicity against lung cancer cell line, KP cell. In vivo, systemic delivery of the nanoparticles indicated a preferential uptake in lung of the tumor-bearing mice. Efficacy studies indicated prolonged survival of mice from the combination treatment. Conclusions: The combination RNA-chemotherapy in an LbL formulation provides an enhanced treatment efficacy against NSCLC, indicating promising potential in clinic

A systematic review and lived experience synthesis of self-disclosure as an active ingredient in interventions for youth anxiety and depression

Background: Self-disclosure, referring to the ability to communicate and share intimate personal feelings, has strong face validity for many young people as a way of improving anxiety and depression outcomes. The current review aimed to generate the first comprehensive evidence synthesis of self-disclosure interventions involving young people aged 14-24 years who are either disclosers or recipients of personal information about living with anxiety and/or depression. Methods: A systematic review of quantitative and qualitative data was combined with new insights from a youth lived-experience panel (n=7) with the intention to combine rigorous systematic review methods and experiential knowledge. Results: Six studies of variable quality were included in this review, five were quantitative and one was qualitative. Findings suggest that self-disclosure may be effective at reducing symptoms for youth with established depression; effects were not apparent when delivered as early prevention. No evidence for impacts on anxiety was found. The potential for negative effects like bullying or harassment was identified. Limitations: Findings were limited by a small number of studies; low representation of peer-reviewed studies from low-or middle-income countries; and varied interventions in terms of format, participants' context, and nature of delivery. Conclusions: Self-disclosure may be of value in the context of interventions intended explicitly to reduce depression for those already showing symptoms. Delivery by non-specialists (such as peers and teachers) in addition to mental health professionals can help build capacity in community health systems. Self-disclosure may also be helpful at reducing stigma and stimulating help-seeking at earlier stages of mental health problems

Morpholine-mediated defluorinative cycloaddition of gem-difluoroalkenes and organic azides

Here, we report the first transition-metal free defluorinative cycloaddition of gem-difluoroalkenes with organic azides in morpholine as a solvent to construct fully decorated morpholine substituted-1,2,3-triazoles. Mechanistic studies revealed the formation of an addition-elimination intermediate containing a morpholine adduct of gem-difluoroalkenes prior to triazolization reaction via two plausible pathways. Attractive elements include regioselective and straightforward direct synthesis of fully substituted 1,2,3-triazoles, which are difficult to access, from readily available starting materials