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Chemical modifications to protein encoding messenger RNA (mRNA) can modulate their localization, translation and stability within cells. Over 15 different types of mRNA modifications have been identified by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technologies. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. To overcome these challenges, we improved the mRNA purification and LC-MS/MS pipelines to identify new S. cerevisiae mRNA modifications and quantify 50 ribonucleosides in a single analysis. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and revealed four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2, N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells and used a reconstituted translation system to discern the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

Bacteriophages (phages) are naturally occurring viruses of bacteria that have a long history of use as antimicrobials, known as phage therapy. The antibiotic resistance crisis has driven renewed interest in phage therapy, which has been used on an unlicensed compassionate basis in various Western contexts. The option to use unlicensed medicines exists to allow clinicians to respond to genuine clinical needs arising in their own patients. However, in the UK some clinicians may, in the absence of suitable patients of their own, seek to transfer patients from other NHS trusts into their own Trust. This article sets out why patient transfer is not necessary and the practical, ethical and legal reasons why patients should not be transferred between NHS Trusts for phage therapy. Phage preparations should always be transported to the patient and the patient treated in the Trust in which they would have received care in the absence of phage. We enclose suggested best practice guidelines for adoption across the UK that will protect patient safety and safeguard clinicians and Trusts from potential litigation.

Objective: We evaluated the potential population health impact of launching heated tobacco products (HTP) in Japan. Method: We use a modeling approach to project the effects of HTP use in overall mortality up to 2100 and compare those projections against a baseline scenario based on smoking rates pre-HTP launch, ie, smoking only. The model was informed using data from publicly available sources and the literature, including population size, yearly deaths, and smoking prevalence with the initial year of 2004, and births and migration from 2004 to 2065. Transitions between products were estimated from cross-sectional population surveys in Japan. Result: In a worst-case scenario, population health gains would be seen with HTPs risk about 50% lower risk than smoking. Assuming equal risk for dual use and smoking, HTP risk would need to be at least 10% lower than smoking to achieve a population health benefit by 2100. Potential reduction in life-years lost with the introduction of HTPs was 13 million by 2100 compared with smoking only. Conclusions: In credible scenarios, substantial population harm reduction will follow the introduction of HTPs in Japan.

Objective: In this study, we obtained key population-level data on use patterns and behavior relating to tobacco and nicotine products in Japan. Methods: We performed a nationwide cross-sectional survey of the general population in Japan in 2019 to assess use patterns after the introduction of tobacco heating products (THPs). Eligible participants were Japanese residents, aged 20 years or older who consented to complete the survey. Individuals living in institutions were excluded. A 3-stage probability sampling method was applied that was geographically stratified by street blocks proportionate to population density. Respondents self-reported patterns of product use and reasons for THP use. Results: Complete responses were available from 5306 individuals, of whom 933.5 (17.6%) were current users, 984.2 (18.5%) were former users, and 3388.4 (63.9%) were never users of tobacco products (weighted respondent totals). Cigarettes were used by 14.6% of current tobacco product users and THPs by 5.3%. Cigarettes and THPs were used exclusively by 64.5% and 12.2%, respectively, and both were used by 12.7%. The most common reasons reported for THP use were perceived reduction in harm to self and others compared to cigarettes. Conclusions: Whereas the prevalence of cigarette use in Japan is decreasing, THPs seem to be increasingly used as long-term alternatives to cigarette smoking.

Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant post-transcriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence S. cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs.

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNA Phe bound to a ΨUU codon in the A site supports these findings. We also find that the presence of Ψ can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNA Val reacting on a ΨUU codon. The vast majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ can be to modestly alter both translation speed and mRNA decoding.

Post-transcriptional modifications to messenger RNAs (mRNAs) have the potential to alter the biological function of this important class of biomolecules. The study of mRNA modifications is a rapidly emerging field, and the full complement of chemical modifications in mRNAs is not yet established. We sought to identify and quantify the modifications present in yeast mRNAs using an ultra-high performance liquid chromatography tandem mass spectrometry method to detect 40 nucleoside variations in parallel. We observe six modified nucleosides with high confidence in highly purified mRNA samples (N7-methylguanosine, N6-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine, N4-acetylcytidine, and 5-formylcytidine) and identify the yeast protein responsible for N4-acetylcytidine incorporation in mRNAs (Rra1). In addition, we find that mRNA modification levels change in response to heat shock, glucose starvation, and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs and highlights the value of integrating mass spectrometry tools in the mRNA modification discovery and characterization pipeline.

Few data are available on the health impact of tobacco heating products (THPs) at the population level. We used systems dynamics modelling to estimate effects in the established THP market in Japan. We projected effects of THP use in overall mortality up to 2100 and compare those projections against a baseline scenario based on smoking rates pre-THP launch, i.e., smoking only. The model was informed using data from publicly available sources and the literature, including population size, yearly deaths and smoking prevalence with initialisation year (2004) and, births and migration from 2004 to 2065. Transitions between products were estimated from cross-sectional population surveys in Japan. Potential life-years saved with the introduction of THPs was 13 million by 2100 compared with smoking only. In worst-case scenario, population health gains would be seen with THPs risk 10–50% lower risk than smoking. Assuming equal risk for dual use and smoking, THP risk would need to be at least 10% lower than smoking to achieve a population health benefit by 2100. In credible scenarios, substantial population health gains will follow the introduction of THPs in Japan in a relatively short time frame.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, as viral polymerases cleave 5’-m7G-capped host transcripts to primeviral mRNA synthesis (‘cap-snatching’). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, that we named ‘start-snatching’. Depending on the reading frame, start-snatching allows the translation of host and viral “untranslated regions” (UTRs) to create Nterminallyextended viral proteins or entirely novel polypeptides by geneticoverprinting. We show that both types of chimeric proteins are made in IAVinfectedcells, generate T cell responses and contribute to virulence. Our resultsindicate that during infection with IAV, and likely a multitude of other human-,animal- and plant-viruses, a host-dependent mechanism allows the genesis ofhybrid genes.

Background This study represents the first Scottish retrospective analysis of the microbiology of diabetic foot infections (DFIs). The aims were to compare the microbiological profile of DFIs treated at a Scottish tertiary hospital to that in the literature, gather data regarding antimicrobial resistance and investigate potential trends between the microbiological results and nature or site of the clinical sample taken and age or gender of the patients. Methods A retrospective analysis of wound microbiology results was performed, data were obtained from one multidisciplinary outpatient foot clinic during the 12 months of the year 2017. Seventy-three patients and 200 microbiological investigations were included. In cases of soft tissue infection, the deepest part of a cleansed and debrided wound was sampled. In cases of osteomyelitis a bone biopsy was obtained. Factors influencing the pattern of microbial growth or prevalence of Staphylococcus aureus were investigated. Results Of the 200 microbiological investigations, 62% were culture positive, of which 37.9% were polymicrobial and 62.1% monomicrobial. Among the monomicrobial results (n = 77), most were Gram positive isolates (96.1%) and the most frequently isolated bacteria was S. aureus (84.4%). No methicillin-resistant S. aureus was reported. The prevalence of S. aureus in DFIs was associated with increasing age (p = 0.021), but no evidence of association with gender, anatomical sample site or sample material was found. Conclusion The microbiological profile of DFIs in Scotland resembles that reported elsewhere in the UK. In this context, Gram positive organisms, primarily S. aureus, are most frequently isolated from DFIs. The S. aureus isolates identified were largely susceptible to antibiotic therapy. An association between increasing patient age and the prevalence of S. aureus in DFIs was observed.

Cells use chemical modifications to alter the sterics, charge, and conformations of large biomolecules, modulating their biogenesis, function, and stability. Until recently post-transcriptional RNA modifications were thought to be largely limited to nonprotein coding RNA species. However, this dogma has rapidly transformed with the discovery of a host of modifications in protein coding messenger RNAs (mRNAs). Recent advancements in genome-wide sequencing technologies have enabled the identification of mRNA modifications as a potential new frontier in gene regulation-leading to the development of the epitranscriptome field. As a result, there has been a flurry of multiple groundbreaking discoveries, including new modifications, nucleoside modifying enzymes ("writers" and "erasers"), and RNA binding proteins that recognize chemical modifications ("readers"). These discoveries opened the door to understanding how post-transcriptional mRNA modifications can modulate the mRNA lifecycle, and established a link between the epitranscriptome and human health and disease. Despite a rapidly growing recognition of their importance, fundamental questions regarding the identity, prevalence, and functional consequences of mRNA modifications remain to be answered. Here, we highlight quantitative studies that characterize mRNA modification abundance, frequency, and interactions with cellular machinery. As the field progresses, we see a need for the further integration of quantitative and reductionist approaches to complement transcriptome wide studies in order to establish a molecular-level framework for understanding the consequences of mRNA chemical modifications on biological processes. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Processing > RNA Editing and Modification.

Actual use studies play a key part in evaluating the reduced risk potential of tobacco and nicotine products. This study was undertaken to determine the puffing topography, mouth level exposure (MLE) and average daily consumption (ADC) relating to two commercially available tobacco heating products (THPs) and a prototype electronic cigarette (or e-cigarette) among Italian non-mentholated 7 mg ISO tar cigarette smokers. The study was conducted in Milan, Italy, with three groups of approximately 50 participants. Groups 1 and 3 included adult smokers of 7 mg ISO tar tobacco cigarettes, and Group 2 consisted of both solus vapers and dual users of vapour and tobacco products. Amongst smokers, e-cigarette mean puff volumes (41.6 mL vs 41.3 mL) and mean puff durations (1.4 s vs 1.5 s) were similar to that of the cigarette, although the average usage session was significantly longer (1064.8 s vs 289.5 s) with a higher total number of puffs (58.6 vs 17.3), however this may be attributable to differences in product operation. There were no significant differences across puffing topography measurements observed between smokers (Group 1) and regular vapers/dual users (Group 2) when using the e-cigarette. As consistent with previous research, users took, on average, larger mean puff volumes when using a THP compared to the reference cigarette (C651), although puff numbers and puff durations remained similar. The average interval between puffs was considerably shorter for THP1.0(T) compared to THS2.4(T) (11.0 s vs 17.1 s). MLE to nicotine-free dry particulate matter and nicotine was significantly reduced for THP1.0(T) and THS2.4(T) compared to the tobacco cigarette (C651). MLE to nicotine was also significantly reduced for the e-cigarette (IS1.0(T)) compared to C651. The average daily consumption (ADC) of cigarettes by groups 1 and 3 were higher than the respective ADCs of both THP consumables. There were no significant differences in ADC when comparing the same product between different groups. Differences seen between sensory scores for each of the product categories may be attributed to fundamental differences in design and mode of operation resulting in very different characteristics of the aerosol generated.

Background Nodding syndrome (NS) is a childhood epileptic disorder of unknown aetiology confined to parts of South Sudan, Uganda and the United Republic of Tanzania. Patients suffer from atonic seizures that result in characteristic head nodding, and may develop other neurological, endocrinological and psychiatric manifestations. The objective of this study was to adopt a non-judgemental approach to review the hypothesised aetiologies and therapeutic options available for NS. To explore the reported association between NS and *Onchocerca volvulus* (OV) a meta-analysis of the prevalence of OV among NS cases and controls was performed. # Methods Systematic searching of five electronic databases yielded 26 articles that addressed potential aetiologies of NS and 12 that explored the treatment of NS. # Results Nine articles contained data about the prevalence of OV in NS cases that was suitable for inclusion in the meta-analysis. Four broad aetiological themes were identified. 1) Nutritional associations included biomarkers of malnutrition and conflicting reports regarding the consumption of some foodstuffs. 2) Regional conflict and the associated psychological trauma have led to consideration of psychiatric aetiologies. 3) There is a strong epidemiological association between NS and *Onchocerca volvulus*. The meta-analysis in this study revealed that 68.0% (95% confidence interval (CI)=45.2-87.0) of 416 NS patients were OV+, compared to 46.6% (95% CI=16.4-78.1) of 321 controls. Heterogeneity among both cases (*I^2^*=95.5%, 95% CI=93.3-97.0) and controls (*I^2^*=97.1%, 95% CI=95.5-98.2) was high. The difference of 21.4% in OV prevalence between the pooled populations was significant (*P*\

Background Visceral leishmaniasis is found primarily in developing countries and is a neglected tropical disease that causes approximately 300,000 new cases and 20,000 deaths annually and is often fatal if untreated. Monotherapy with pentavalent antimonials (SSG or MA) is a second-line treatment option in many endemic areas. This meta-analysis provides regional and national analyses of the proportions of antimonial treatment failures at the end-of-treatment and at follow-up according to the drug used. # Methods This meta-analysis was conducted in accordance with the PRISMA guidelines. Studies were identified by systematic searches of five electronic databases. Studies in English that reported the outcomes of 10 or more patients treated according to the World Health Organization (WHO) pentavalent antimonial regimen (20 mg/kg/day for 28-30 days) were included. Random-effects meta-analyses and funnel plots were performed, and bias was assessed using tools from the Cochrane collaboration. # Results 33 articles were included in the final per protocol analyses, 16 from the Indian subcontinent (India and Nepal), 15 from East Africa and one from each of Iran and Turkey. Data were analysed by drug type regionally, nationally and temporally. Six-month follow-up data from 13 studies (n=797) on the Indian subcontinent gave a sodium stibogluconate (SSG) failure rate of 11.34% (95% confidence interval (CI)=7.05-16.48%). Nationally, the SSG failure rate across 11 studies from northern India greatly exceeded that identified from two Nepalese studies. Six-month follow-up data from three studies (n=638) from East Africa gave an SSG failure rate of 6.25% (95% CI=4.51-8.25%). End-of-SSG-treatment failure rates across the Indian subcontinent (n=1,135), northern India (n=990) and Nepal (n=145) were 39.82% (95% CI=31.67-48.27%), 45.21% (95% CI=39.31-51.17%) and 12.17% (95% CI=7.40-17.93%) respectively. A lower end-of-SSG-treatment failure rate was recorded in East Africa (n=53,335), 6.60% (95% CI=2.94-11.58%). No studies contained six-month follow-up data regarding meglumine antimoniate (MA), and only four studies (n=257) contained end-of-MA-treatment data with a failure rate of 5.01% (95% CI=2.70-7.98%). # Conclusions Sb(V) remains an important second-line therapy in many areas. Knowledge of failure rates can inform policy decisions at all levels. The findings of this study are most applicable to SSG therapy, confirm the known patterns of resistance and reflect current policies.

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

Segmented negative-strand RNA viruses (sNSVs) include the influenza viruses, the bunyaviruses, and other major pathogens of humans, other animals and plants. The genomes of these viruses are extremely short. In response to this severe genetic constraint, sNSVs use a variety of strategies to maximise their coding potential. Because the eukaryotic hosts parasitized by sNSVs can regulate gene expression through low levels of translation initiation upstream of their canonical open reading frames (ORFs), we asked whether sNSVs could use upstream translation initiation to expand their own genetic repertoires. Consistent with this hypothesis, we showed that influenza A viruses (IAVs) and bunyaviruses were capable of upstream translation initiation. Using a combination of reporter assays and viral infections, we found that upstream translation in IAVs can initiate in two unusual ways: through non-AUG initiation in virally encoded ‘untranslated’ regions, and through the appropriation of an AUG-containing leader sequence from host mRNAs through viral cap-snatching, a process we termed ‘start-snatching.’ Finally, while upstream translation of cellular genes is mainly regulatory, for sNSVs it also has the potential to create novel viral gene products. If in frame with a viral ORF, this creates N-extensions of canonical viral proteins. If not, it allows the expression of cryptic overlapping ORFs, which we found were highly conserved in IAV and widely distributed in peribunyaviruses. Thus, by exploiting their host’s capacity for upstream translation initiation, sNSVs can expand still further the coding potential of their extremely compact RNA genomes.

Post-transcriptional modifications to messenger RNAs (mRNAs) have the potential to alter the biological function of this important class of biomolecules. The study of mRNA modifications is a rapidly emerging field, and the full complement of chemical modifications in mRNAs is not yet established. We sought to identify and quantify the modifications present in yeast mRNAs using an ultra-high performance liquid chromatography tandem mass spectrometry method to detect 40 nucleoside variations in parallel. We observe six modified nucleosides with high confidence in highly purified mRNA samples (N7-methylguanosine, N6-methyladenosine, 2′-O-methylguanosine, 2′-O-methylcytidine, N4-acetylcytidine, and 5-formylcytidine) and identify the yeast protein responsible for N4-acetylcytidine incorporation in mRNAs (Rra1). In addition, we find that mRNA modification levels change in response to heat shock, glucose starvation, and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs and highlights the value of integrating mass spectrometry tools in the mRNA modification discovery and characterization pipeline.

We tested for associations between socioeconomic status (SES) and adverse prostate cancer pathology in a population of African American (AA) men treated with radical prostatectomy (RP).We retrospectively reviewed data from 2 institutions for AA men who underwent RP between 2010 and 2015. Household incomes were estimated using census tract data, and patients were stratified into income groups relative to the study population median. Pathologic outcomes after RP were assessed, including the postsurgical Cancer of the Prostate Risk Assessment (CAPRA-S) score and a definition of adverse pathology (stage ≥ pT3, Gleason score ≥ 4+3, or positive lymph nodes), and compared between income groups.We analyzed data of 347 AA men. Median household income was $37,954. Low-SES men had significantly higher prostate-specific antigen values (mean 10.2 vs. 7.3; P .01) and CAPRA-S scores (mean 3.4 vs. 2.5; P .01), more advanced pathologic stage (T3-T4 31.8% vs. 21.5%; P = .03), and higher rates of seminal vesicle invasion (17.3% vs. 8.2%; P .01), positive surgical margins (35.3% vs. 22.1%; P .01), and adverse pathology (41.4% vs. 30.1%; P = .03). Linear and logistic regression showed significant inverse associations of SES with CAPRA-S score (P .01) and adverse pathology (P = .03).In a population of AA men who underwent RP, we observed an independent association of low SES with advanced stage or aggressive prostate cancer. By including only patients in a single racial demographic group, we eliminated the potential confounding effect of race on the association between SES and prostate cancer risk. These findings suggest that impoverished populations might benefit from more intensive screening and early, aggressive treatment of prostatic malignancies.

Bacterial resistance to antibiotics has catalysed interest in alternative antimicrobial strategies. Bacteriophages (phages) are viruses of bacteria with a long history of successful therapeutic use. Phage therapy is a promising antibacterial strategy for infections with a biofilm component, including recalcitrant bone and joint infections, which have significant social, financial and human impacts. Here, we report a systematic review of the safety and efficacy of phage therapy for the treatment of bone and joint infections. Three electronic databases were systematically searched for articles that reported primary data about human phage therapy for bone and joint infections. Two authors independently assessed study eligibility and performed data extraction. Seventeen reports were eligible for inclusion in this review, representing the treatment of 277 patients. A cautionary, crude, efficacy estimate revealed that 93.1% (n = 258/277) achieved clinical resolution, 3.3% (n = 9/277) had improvement and 3.6% (n = 10/277) showed no improvement. Seven of the nine reports that directly commented on the safety of phage therapy did not express safety concerns. The adverse effects reported in the remaining two were not severe and were linked to the presence of contaminating endotoxins and pre-existing liver pathology in a patient treated with high-titre intravenous phage therapy. Three other reports, from 1940–1987, offered general comments on the safety of phage therapy and documented adverse effects consistent with endotoxin co-administration concomitant with the use of raw phage lysates. Together, the reports identified by this review suggest that appropriately purified phages represent a safe and highly efficacious treatment option for complex and intractable bone and joint infections.

INTRODUCTION AND OBJECTIVES: Prior studies have suggested that poverty is associated with advanced stage or aggressive prostate cancer at presentation. However, due to strong associations between poverty and race, the independent contribution of poverty, irrespective of race, remains unclear. To eliminate potential confounding, we sought to test for an association between poverty and adverse pathology in a population comprised exclusively of African-American (AA) men treated with radical prostatectomy (RP). METHODS: African American men who underwent radical prostatectomy between September 2010 and September 2015 at Temple University Hospital and the University of Pennsylvania were identified through our institutional databases. Median household income for each patient’s residential zip code was determined using United States Census Bureau data. The median income for the study population was calculated, and patients were stratified as either lowor high-income relative to the median. Pathological outcomes, CAPRA-S score, and rates of adverse pathology (defined as pathological stage pT3, pathological Gleason score 4+3, or lymph node involvement) were compared between income groups. Linear and logistic regression were used to test for associations of income with CAPRA-S score and adverse pathology, respectively. RESULTS: 385 African American men met inclusion criteria. Median household income was $40,031 (range $14,297-$162,617), The table compares disease-specific and pathological outcomes between low and high income men. Low income men had significantly higher PSA values (p

Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.

On October 4, 2001, we confirmed the first bioterrorism-related anthrax case identified in the United States in a resident of Palm Beach County, Florida. Epidemiologic investigation indicated that exposure occurred at the workplace through intentionally contaminated mail. One additional case of inhalational anthrax was identified from the index patient’s workplace. Among 1,076 nasal cultures performed to assess exposure, Bacillus anthracis was isolated from a co-worker later confirmed as being infected, as well as from an asymptomatic mail-handler in the same workplace. Environmental cultures for B. anthracis showed contamination at the workplace and six county postal facilities. Environmental and nasal swab cultures were useful epidemiologic tools that helped direct the investigation towards the infection source and transmission vehicle. We identified 1,114 persons at risk and offered antimicrobial prophylaxis.

We collected data during postexposure antimicrobial prophylaxis campaigns and from a prophylaxis program evaluation 60 days after start of antimicrobial prophylaxis involving persons from six U.S. sites where Bacillus anthracis exposures occurred. Adverse events associated with antimicrobial prophylaxis to prevent anthrax were commonly reported, but hospitalizations and serious adverse events as defined by Food and Drug Administration criteria were rare. Overall adherence during 60 days of antimicrobial prophylaxis was poor (44%), ranging from 21% of persons exposed in the Morgan postal facility in New York City to 64% of persons exposed at the Brentwood postal facility in Washington, D.C. Adherence was highest among participants in an investigational new drug protocol to receive additional antibiotics with or without anthrax vaccine--a likely surrogate for anthrax risk perception. Adherence of

Background Monoclonal antibody therapy has an important role to play as a post-exposure prophylactic and therapeutic for the treatment of viral infections, including emerging infections. For example, several patients of the present Ebola virus outbreak in West Africa were treated with ZMapp, a cocktail of three monoclonal antibodies which are expressed in Nicotiana benthamiana. Discussion The majority of monoclonal antibodies in clinical use are expressed in mammalian cell lines which offer native folding and glycosylation of the expressed antibody. Monoclonal antibody expression in vegetal systems offers advantages over expression in mammalian cell lines, including improved potential for scale up and reduced costs. In this paper, I highlight the advantages of an upcoming protozoal system for the expression of recombinant antibody formats. Leishmania tarentolae offers a robust, economical expression of proteins with mammalian glycosylation patterns expressed in stable cell lines and grown in suspension culture. Several advantages of this system make it particularly suited for use in developing contexts. Summary Given the potential importance of monoclonal antibody therapy in the containment of emerging viral infections, novel and alternative strategies to improve production must be explored. Electronic supplementary material The online version of this article (doi:10.1186/2049-9957-4-8) contains supplementary material, which is available to authorized users.

Protein acetylation plays a critical regulatory role in eukaryotes but until recently its significance and function in bacteria and the archaea were obscure. It is now clear, however, that prokaryotes have the capacity to acetylate both the α-amino groups of N-terminal residues and the e-amino groups of lysine side chains. In this review, we bring together information indicating that such acetylation is widespread and that it is likely to regulate fundamental cellular processes. We particularly focus on lysine acetylation, which recent studies show can occur in proteins involved in transcription, translation, pathways associated with central metabolism and stress responses. Intriguingly, specific acetylated lysine residues map to critical regions in the three-dimensional structures of key proteins, e.g. to active sites or to surfaces that dock with other major cellular components. Like phosphorylation, acetylation appears to be an ancient reversible modification that can be present at multiple sites in proteins, thereby potentially producing epigenetic combinatorial complexity. It may be particularly important in regulating central metabolism in prokaryotes due to the requirement for acetyl-CoA and NAD(+) for protein acetyltransferases and Sir2-type deacetylases, respectively.

Background. The African meningitis belt undergoes recurrent epidemics caused by Neisseria meningitidis serogroup A. During 2002, Burkina Faso documented the first large serogroup W-135 (NmW-135) meningococcal disease epidemic. To understand the emergence of NmW-135, we investigated meningococcal carriage and immunity. Methods. Immediately after Burkina Faso's epidemic, we conducted a cross-sectional survey of meningococcal carriage and seroprevalence in an epidemic and a nonepidemic district. We identified predictors of elevated NmW-135 serum bactericidal activity (SBA), a functional correlate of protection, using multivariate logistic regression. Results. The NmW-135 carriage rate was 25.2% in the epidemic district and 3.4% in the nonepidemic district (P