An immuno-northern technique to measure the size of dsRNA byproducts in in vitro transcribed RNA.

Double-stranded RNA is an immunogenic byproduct present in RNA synthesized with in vitro transcription. dsRNA byproducts engage virus-sensing innate immunity receptors and cause inflammation. Removing dsRNA from in vitro transcribed messenger RNA (mRNA) reduces immunogenicity and improves protein translation. Levels of dsRNA are typically 0.1%-0.5% of total transcribed RNA. Because they form such a minor fraction of the total RNA in transcription reactions, it is difficult to confidently identify discrete bands on agarose gels that correspond to the dsRNA byproducts. Thus, the sizes of dsRNA byproducts are largely unknown. Total levels of dsRNA are typically assayed with dsRNA-specific antibodies in ELISA and immuno dot-blot assays. Here we report a dsRNA-specific immuno-northern blot technique that provides a clear picture of the dsRNA size distributions in transcribed RNA. This technique could complement existing dsRNA analytical methods in studies of dsRNA byproduct synthesis, dsRNA removal, and characterization of therapeutic RNA drug substances.

A mortalidade entre adolescentes egressos do sistema penal / Mortality among former youth offenders

Resumo O objetivo deste artigo é analisar as causas de internações da FASE do Estado do Rio Grande do Sul e a sua relação com a mortalidade de adolescentes egressos entre os anos de 2002 a 2014. Estudo observacional realizado com o banco de dados de adolescentes privados de liberdade nas unidades da FASE-RS de Porto Alegre, desligados nos anos de 2002 a 2012 (n = 8290). Informações sobre data de desligamento, ato infracional, tempo de internação e variáveis biológicas foram obtidas de banco de dados. Essa amostra foi comparada com o Sistema de Verificação de Óbitos da Secretaria Estadual de Saúde para analisar a mortalidade entre os anos de 2002 e 2014. Os adolescentes foram internados predominantemente por atos infracionais de cunho patrimonial e ligados a entorpecentes, os quais sofreram um aumento de aproximadamente 700% no período. O desfecho óbito associou-se (p < 0,001) às variáveis gênero masculino e número de entradas (≥ 3). Essa amostra apresentou alta taxa de mortalidade sendo a principal causa homicídio. Os achados evidenciam o alto grau de vulnerabilidade psicossocial dos egressos do sistema penal juvenil de internação. Nota-se uma associação entre crimes de baixo poder ofensivo e altas taxas de mortalidade pós-liberdade.

Abstract The objective of this article is to analyze the detention of youth offenders involved in the juvenile justice system in the State of Rio Grande do Sul (FASE-RS), the reason for detention, and mortality among former young offenders. We conducted an observational study with youth offenders discharged from facilities run by FASE-RS in Porto Alegre between 2002 and 2012 (n = 8,290). We collected the following information: date of discharge, offence committed, skin color, gender, and duration of detention. The data was crosschecked with data from the state's Mortality Information System to identify deaths among former young offenders up to December 2014. The predominant offences were crimes against property and drug-related crimes. The large majority of youth detained for drug-related offences were admitted for offences related to drug trafficking. There was a seven-fold increase in drug-related offences over the period. Death was associated (p<0.001) with being male and number of reentries (>3). The sample's mortality rate was high and the main cause of death was homicide. The findings suggest that young offenders face high levels of psychosocial vulnerability. There was an association between minor crimes and high rates of mortality among former young offenders.

Mortality among former youth offenders. / A mortalidade entre adolescentes egressos do sistema penal.

The objective of this article is to analyze the detention of youth offenders involved in the juvenile justice system in the State of Rio Grande do Sul (FASE-RS), the reason for detention, and mortality among former young offenders. We conducted an observational study with youth offenders discharged from facilities run by FASE-RS in Porto Alegre between 2002 and 2012 (n = 8,290). We collected the following information: date of discharge, offence committed, skin color, gender, and duration of detention. The data was crosschecked with data from the state's Mortality Information System to identify deaths among former young offenders up to December 2014. The predominant offences were crimes against property and drug-related crimes. The large majority of youth detained for drug-related offences were admitted for offences related to drug trafficking. There was a seven-fold increase in drug-related offences over the period. Death was associated (p<0.001) with being male and number of reentries (>3). The sample's mortality rate was high and the main cause of death was homicide. The findings suggest that young offenders face high levels of psychosocial vulnerability. There was an association between minor crimes and high rates of mortality among former young offenders.

O objetivo deste artigo é analisar as causas de internações da FASE do Estado do Rio Grande do Sul e a sua relação com a mortalidade de adolescentes egressos entre os anos de 2002 a 2014. Estudo observacional realizado com o banco de dados de adolescentes privados de liberdade nas unidades da FASE-RS de Porto Alegre, desligados nos anos de 2002 a 2012 (n = 8290). Informações sobre data de desligamento, ato infracional, tempo de internação e variáveis biológicas foram obtidas de banco de dados. Essa amostra foi comparada com o Sistema de Verificação de Óbitos da Secretaria Estadual de Saúde para analisar a mortalidade entre os anos de 2002 e 2014. Os adolescentes foram internados predominantemente por atos infracionais de cunho patrimonial e ligados a entorpecentes, os quais sofreram um aumento de aproximadamente 700% no período. O desfecho óbito associou-se (p < 0,001) às variáveis gênero masculino e número de entradas (≥ 3). Essa amostra apresentou alta taxa de mortalidade sendo a principal causa homicídio. Os achados evidenciam o alto grau de vulnerabilidade psicossocial dos egressos do sistema penal juvenil de internação. Nota-se uma associação entre crimes de baixo poder ofensivo e altas taxas de mortalidade pós-liberdade.

Perceived barriers to accessing mental health services among black and minority ethnic (BME) communities: a qualitative study in Southeast England.

OBJECTIVE: In most developed countries, substantial disparities exist in access to mental health services for black and minority ethnic (BME) populations. We sought to determine perceived barriers to accessing mental health services among people from these backgrounds to inform the development of effective and culturally acceptable services to improve equity in healthcare. DESIGN AND SETTING: Qualitative study in Southeast England. PARTICIPANTS: 26 adults from BME backgrounds (13 men, 13 women; aged >18 years) were recruited to 2 focus groups. Participants were identified through the registers of the Black and Minority Ethnic Community Partnership centre and by visits to local community gatherings and were invited to take part by community development workers. Thematic analysis was conducted to identify key themes about perceived barriers to accessing mental health services. RESULTS: Participants identified 2 broad themes that influenced access to mental health services. First, personal and environmental factors included inability to recognise and accept mental health problems, positive impact of social networks, reluctance to discuss psychological distress and seek help among men, cultural identity, negative perception of and social stigma against mental health and financial factors. Second, factors affecting the relationship between service user and healthcare provider included the impact of long waiting times for initial assessment, language barriers, poor communication between service users and providers, inadequate recognition or response to mental health needs, imbalance of power and authority between service users and providers, cultural naivety, insensitivity and discrimination towards the needs of BME service users and lack of awareness of different services among service users and providers. CONCLUSIONS: People from BME backgrounds require considerable mental health literacy and practical support to raise awareness of mental health conditions and combat stigma. There is a need for improving information about services and access pathways. Healthcare providers need relevant training and support in developing effective communication strategies to deliver individually tailored and culturally sensitive care. Improved engagement with people from BME backgrounds in the development and delivery of culturally appropriate mental health services could facilitate better understanding of mental health conditions and improve access.

Genetically enhanced lysozyme evades a pathogen derived inhibitory protein.

The accelerating spread of drug-resistant bacteria is creating demand for novel antibiotics. Bactericidal enzymes, such as human lysozyme (hLYZ), are interesting drug candidates due to their inherent catalytic nature and lack of susceptibility to the resistance mechanisms typically directed toward chemotherapeutics. However, natural antibacterial enzymes have their own limitations. For example, hLYZ is susceptible to pathogen derived inhibitory proteins, such as Escherichia coli Ivy. Here, we describe proof of concept studies demonstrating that hLYZ can be effectively redesigned to evade this potent lysozyme inhibitor. Large combinatorial libraries of hLYZ were analyzed using an innovative screening platform based on microbial coculture in hydrogel microdroplets. Isolated hLYZ variants were orders of magnitude less susceptible to E. coli Ivy yet retained high catalytic proficiency and inherent antibacterial activity. Interestingly, the engineered escape variants showed a disadvantageous increase in susceptibility to the related Ivy ortholog from Pseudomonas aeruginosa as well as an unrelated E. coli inhibitory protein, MliC. Thus, while we have achieved our original objective with respect to escaping E. coli Ivy, engineering hLYZ for broad-spectrum evasion of proteinaceous inhibitors will require consideration of the complex and varied determinants that underlie molecular recognition by these emerging virulence factors.

Bioengineered lysozyme in combination therapies for Pseudomonas aeruginosa lung infections.

There is increasing urgency in the battle against drug-resistant bacterial pathogens, and this public health crisis has created a desperate need for novel antimicrobial agents. Recombinant human lysozyme represents one interesting candidate for treating pulmonary infections, but the wild type enzyme is subject to electrostatic mediated inhibition by anionic biopolymers that accumulate in the infected lung. We have redesigned lysozyme's electrostatic potential field, creating a genetically engineered variant that is less susceptible to polyanion inhibition, yet retains potent bactericidal activity. A recent publication demonstrated that the engineered enzyme outperforms wild type lysozyme in a murine model of Pseudomonas aeruginosa lung infection. Here, we expand upon our initial studies and consider dual therapies that combine lysozymes with an antimicrobial peptide. Consistent with our earlier results, the charge modified lysozyme combination outperformed its wild type counterpart, yielding more than an order-of-magnitude reduction in bacterial burden following treatment with a single dose.

A high-throughput screen for antibiotic drug discovery.

We describe an ultra-high-throughput screening platform enabling discovery and/or engineering of natural product antibiotics. The methodology involves creation of hydrogel-in-oil emulsions in which recombinant microorganisms are co-emulsified with bacterial pathogens; antibiotic activity is assayed by use of a fluorescent viability dye. We have successfully utilized both bulk emulsification and microfluidic technology for the generation of hydrogel microdroplets that are size-compatible with conventional flow cytometry. Hydrogel droplets are ∼25 pL in volume, and can be synthesized and sorted at rates exceeding 3,000 drops/s. Using this technique, we have achieved screening throughputs exceeding 5 million clones/day. Proof-of-concept experiments demonstrate efficient selection of antibiotic-secreting yeast from a vast excess of negative controls. In addition, we have successfully used this technique to screen a metagenomic library for secreted antibiotics that kill the human pathogen Staphylococcus aureus. Our results establish the practical utility of the screening platform, and we anticipate that the accessible nature of our methods will enable others seeking to identify and engineer the next generation of antibacterial biomolecules.

Bioengineered lysozyme reduces bacterial burden and inflammation in a murine model of mucoid Pseudomonas aeruginosa lung infection.

The spread of drug-resistant bacterial pathogens is a growing global concern and has prompted an effort to explore potential adjuvant and alternative therapies derived from nature's repertoire of bactericidal proteins and peptides. In humans, the airway surface liquid layer is a rich source of antibiotics, and lysozyme represents one of the most abundant and effective antimicrobial components of airway secretions. Human lysozyme is active against both Gram-positive and Gram-negative bacteria, acting through several mechanisms, including catalytic degradation of cell wall peptidoglycan and subsequent bacterial lysis. In the infected lung, however, lysozyme's dense cationic character can result in sequestration and inhibition by polyanions associated with airway inflammation. As a result, the efficacy of the native enzyme may be compromised in the infected and inflamed lung. To address this limitation, we previously constructed a charge-engineered variant of human lysozyme that was less prone to electrostatic-mediated inhibition in vitro. Here, we employ a murine model to show that this engineered enzyme is superior to wild-type human lysozyme as a treatment for mucoid Pseudomonas aeruginosa lung infections. The engineered enzyme effectively decreases the bacterial burden and reduces markers of inflammation and lung injury. Importantly, we found no evidence of acute toxicity or allergic hypersensitivity upon repeated administration of the engineered biotherapeutic. Thus, the charge-engineered lysozyme represents an interesting therapeutic candidate for P. aeruginosa lung infections.

Crystal structure of a charge engineered human lysozyme having enhanced bactericidal activity.

Human lysozyme is a key component of the innate immune system, and recombinant forms of the enzyme represent promising leads in the search for therapeutic agents able to treat drug-resistant infections. The wild type protein, however, fails to participate effectively in clearance of certain infections due to inherent functional limitations. For example, wild type lysozymes are subject to electrostatic sequestration and inactivation by anionic biopolymers in the infected airway. A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules. Here, the 2.04 Å crystal structure of this variant is presented along with an analysis that provides molecular level insights into the origins of the protein's enhanced performance. The charge engineered variant's two mutated amino acids exhibit stabilizing interactions with adjacent native residues, and from a global perspective, the mutations cause no gross structural perturbations or loss of stability. Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues. The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers.

Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

Enhanced antimicrobial activity of engineered human lysozyme.

Lysozymes contain a disproportionately large fraction of cationic residues, and are thereby attracted toward the negatively charged surface of bacterial targets. Importantly, this conserved biophysical property may inhibit lysozyme antibacterial function during acute and chronic infections. A mouse model of acute pulmonary Pseudomonas aeruginosa infection demonstrated that anionic biopolymers accumulate to high concentrations in the infected lung, and the presence of these species correlates with decreased endogenous lysozyme activity. To develop antibacterial enzymes designed specifically to be used as antimicrobial agents in the infected airway, the electrostatic potential of human lysozyme (hLYS) was remodeled by protein engineering. A novel, high-throughput screen was implemented to functionally interrogate combinatorial libraries of charge-engineered hLYS proteins, and variants with improved bactericidal activity were isolated and characterized in detail. These studies illustrate a general mechanism by which polyanions inhibit lysozyme function, and they are the first direct demonstration that decreasing hLYS's net cationic character improves its antibacterial activity in the presence of disease-associated biopolymers. In addition to avoiding electrostatic sequestration, at least one charge-engineered variant also kills bacteria more rapidly in the absence of inhibitory biopolymers; this observation supports a novel hypothesis that tuning the cellular affinity of peptidoglycan hydrolases may be a general strategy for improving kinetics of bacterial killing.

The monoclonal myth.

Most researchers confidently assume that transformation of recombinant plasmid libraries into microbial hosts followed by outgrowth of isolated colonies results in a "one cell-one mutant gene-one protein variant" paradigm. Indeed, this assumption is supported by the overwhelming majority of published studies employing bacterial expression hosts. In stark contrast, we recently reported on Saccharomyces cerevisiae libraries containing unexpectedly high frequencies of cells harboring heterogeneous mixtures of plasmids, so called Multiple Vector Transformants (MVT). Intriguingly, we observed that yeast MVT persist as a significant proportion of populations for multiple generations. MVT can lead to misidentification of isolated mutants loss of functionally enhanced clones, and unwitting propagation of false positives derived from contaminating control sequences. Such experimental complications can have devastating outcomes in the context of protein engineering by combinatorial library screening. Herein, we demonstrate that the phenomenon of MVT is not restricted to vectors bearing the CEN/ARSH origin of replication, but may be an even greater concern when using high copy 2 µm plasmids. To mitigate the risks associated with MVT, we have developed an optimized sequencing procedure that facilitates rapid and reliable identification of MVT among clones of interest. In our experience, MVT and their associated risks can be virtually eliminated by employing extended liquid outgrowths of transformed populations and archiving sequence-verified, monoclonal, mutant genes from cell-templated PCR amplicons.

Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries.

BACKGROUND: In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. RESULTS: It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. CONCLUSION: These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. The quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid.

Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method.

The proteasome is the primary subcellular organelle responsible for protein degradation. It is a dynamic assemblage of 34 core subunits and many differentially expressed, transiently interacting, modulatory proteins. This paper describes a novel affinity chromatography method for the purification of functional human holoproteasome complexes using mild conditions. Human proteasomes purified by this simple procedure maintained the ability to proteolytically process synthetic peptide substrates and degrade ubiquitinated parkin. Furthermore, the entire purification fraction was analyzed by mass spectrometry in order to identify proteasomal proteins and putative proteasome-interacting proteins. The mild purification conditions maintained transient physical interactions between holoproteasomes and a number of known modulatory proteins. In addition, several classes of putative interacting proteins co-purified with the proteasomes, including proteins with a role in the ubiquitin proteasome system for protein degradation or DNA repair. These results demonstrate the efficacy of using this affinity purification strategy for isolating functional human proteasomes and identifying proteins that may physically interact with human proteasomes.

A programme for Health Impact Assessment in Brighton and Hove.

HIA is based on the theory of health determinants, which recognizes that well-being is determined by a wide range of economic, social and environmental factors, by heredity and medical intervention. The intended HIA procedure represents a new approach to the evaluation of all local authority policies in order to assess their potential health impacts and to improve the quality of governmental decisions, through recommendations to enhance predicted positive health impacts and minimize negative ones.