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Objectives : The stimulation threshold across channels is an essential component of cochlear implant fitting. Thresholds can be obtained objectively by recording the electrically evoked compound action potential (eCAP). A commonly applied approach to determine the eCAP threshold is based on linear extrapolation of the eCAP amplitude growth function (AGF) to zero amplitude. The present study determines the precision of the eCAP threshold as a function of the number of waveform averages. Material and methods : AGFs were obtained in 7 Advanced Bionics cochlear implant users from 3 electrode contacts: one in the base, one in the middle of the array, and one in the apical region. We recorded 256 eCAP waveforms per stimulus level. All 256 waveforms were averaged to obtain the best-estimate eCAP threshold (the reference). The precision of the eCAP threshold was determined by generating probability distributions using a Monte Carlo approach. To this end, AGFs were reconstructed using only a random subset of the 256 eCAP waveforms available per stimulus level. The precision of eCAP thresholds was determined for 8 to 128 eCAP averages. From these probability distributions the standard deviation was determined and expressed as fractions of the reference threshold. Results and Conclusions : At the default of 32 averages, the median SD of the eCAP threshold was approximately 1% at all three electrode positions. However, the SD of individual subjects could be >10%. Doubling of the number of averages improved the SD of the eCAP threshold by a factor of approximately 1.4. This factor corresponds to the theoretical improvement in a noisy signal when the number averages is doubled, namely the square root of 2. We conclude that at default settings, the median standard deviation of the eCAP threshold in the AB system is about 1%. Individual SDs may exceed the median by an order of a magnitude. [ABSTRACT FROM AUTHOR]

Pulmonary actinomycosis is a rare infection caused by the bacterial species actinomyces. This paper aims to provide a comprehensive review of pulmonary actinomycosis to improve awareness and knowledge. The literature was analysed using databases including Pubmed, Medline and Embase from 1974 to 2021. After inclusion and exclusion, a total of 142 papers were reviewed. Pulmonary actinomycosis is a rare disease occurring in approximately 1 per 3,000,000 people annually. Historically, pulmonary actinomycosis was a common infection with high mortality; however, the infection has become rarer since the widespread use of penicillins. Actinomycosis is known as "the great masquerade"; however, it can be differentiated from other diseases with acid-fast negative ray-like bacilli and sulphur granules being pathognomonic. Complications of the infection include empyema, endocarditis, pericarditis, pericardial effusion, and sepsis. The mainstay of treatment is prolonged antibiotic therapy, with adjuvant surgery in severe cases. Future research should focus on multiple areas, including the potential risk secondary to immunosuppression from newer immunotherapies, the utility of newer diagnostic techniques and ongoing surveillance post-therapy., Competing Interests: Conflict of interest No conflict of interest., (Crown Copyright © 2023. Published by Elsevier Ltd. All rights reserved.)

Inpatient falls are one of the most frequent concerns to patient safety within the acute hospital environment, equating to 1700 falls per year in an 800-bed general hospital. They are predicted to cost approximately £2600 per patient, however, this estimate does not capture the costs and impact that inpatient falls have on the wider health and social care system. It also does not take into the account loss of confidence and delays in functional recovery.This report shares the learning from a quality improvement (QI) initiative that took place in a District General Hospital (DGH) in the UK. The initiative started in February 2020, was paused November 2020 due to wave 2 of the pandemic and restarted in March 2021. Improvement was achieved in January 2021.Data for falls within the Trust identified that falls were within common cause variation. A system change was needed to achieve an improvement.A QI project was commenced with the aim to achieve a 5% reduction in falls per 1000 bed days in a care of the elderly ward.Two primary drivers were identified: recognising patients at high risk of falls and preventing them from falling. Change ideas to address these primary drivers were tested using Plan Do Study Act (PDSA) cycles. Changes tested included: the development of an assessment tool to identify patients at high risk of falls, use of a wristband to identify patients at high risk of a fall, and increased observation.Change ideas achieved some success with the process measures but did not achieve an improvement with the outcome measures. A Trust wide change idea relating to the falls prevention service did lead to a sustained improvement in falls reduction.The barriers to the improvement included changing Trust priorities during the pandemic, and limited opportunities to fully engage the ward-based team with systems thinking and changing mental models., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Duodenal diverticulum perforation is a rare and life-threatening pathology. Perforation secondary to iatrogenic causes is rare, with only 14 cases previously reported. This paper explores a world-first case report on iatrogenic duodenal diverticulum perforation during right laparoscopic nephroureterectomy and a systematic review of all reported cases of duodenal diverticulum perforation in the literature., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Triglyceride (TG)-lowering LPL variants in combination with genetic LDL-C-lowering variants are associated with reduced risk of coronary artery disease (CAD). Genetic variation in the APOA5 gene encoding apolipoprotein A-V also strongly affects TG levels, but the potential clinical impact and underlying mechanisms are yet to be resolved. Here, we aimed to study the effects of APOA5 genetic variation on CAD risk and plasma lipoproteins through factorial genetic association analyses. Using data from 309,780 European-ancestry participants from the UK Biobank, we evaluated the effects of lower TG levels as a result of genetic variation in APOA5 and/or LPL on CAD risk with or without a background of reduced LDL-C. Next, we compared lower TG levels via APOA5 and LPL variation with over 100 lipoprotein measurements in a combined sample from the Netherlands Epidemiology of Obesity study (N = 4,838) and the Oxford Biobank (N = 6,999). We found that lower TG levels due to combined APOA5 and LPL variation and genetically-influenced lower LDL-C levels afforded the largest reduction in CAD risk (odds ratio: 0.78 (0.73-0.82)). Compared to patients with genetically-influenced lower TG via LPL, genetically-influenced lower TG via APOA5 had similar and independent, but notably larger, effects on the lipoprotein profile. Our results suggest that lower TG levels as a result of APOA5 variation have strong beneficial effects on CAD risk and the lipoprotein profile, which suggest apo A-V may be a potential novel therapeutic target for CAD prevention., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.

Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX-1 secretion system, which extends the role of ESX-1 secretion beyond the macrophage infection cycle., (© 2018 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

Due to the rise of drug-resistant forms of tuberculosis, there is an urgent need for novel antibiotics to effectively combat these cases and shorten treatment regimens. Recently, drug screens using whole-cell analyses have been shown to be successful. However, current high-throughput screens focus mostly on stricto sensu life/death screening that give little qualitative information. In doing so, promising compound scaffolds or nonoptimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early tuberculosis (TB) drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to subinhibitory concentrations of antibiotics with known targets, ciprofloxacin, ethambutol, isoniazid, streptomycin, and rifampin. The resulting data set comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint. Notably, this fingerprint was more distinctive in M. marinum We decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed to respond to DNA damage, cell wall damage, and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so, we identified the putative modes of action for three novel compounds, which confirms the utility of our approach., (Copyright © 2018 American Society for Microbiology.)

The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the iniBAC promoter. In this study, we set out to identify the regulator of the iniBAC operon in Mycobacterium marinum using an unbiased transposon mutagenesis screen in a constitutively iniBAC-expressing mutant background. We obtained multiple mutants in the mce1 locus as well as mutants in an uncharacterized putative transcriptional regulator (MMAR_0612). This latter gene was shown to function as the iniBAC regulator, as overexpression resulted in constitutive iniBAC induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the M. tuberculosis homologue (Rv0339c) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the iniBAC operon. We therefore propose to name this dedicated regulator iniBAC Regulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for iniBAC activation, which could also explain the effect of the mce1 mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose.

Cytochrome bd is a component of the oxidative phosphorylation pathway in many Gram-positive and Gram-negative bacteria. Next to its role as a terminal oxidase in the respiratory chain this enzyme plays an important role as a survival factor in the bacterial stress response. In Mycobacterium tuberculosis and related mycobacterial strains, cytochrome bd is an important component of the defense system against antibacterial drugs. In this report we describe and evaluate an mCherry-based fluorescent reporter for detection of cytochrome bd expression in Mycobacterium marinum. Cytochrome bd was induced by mycolic acid biosynthesis inhibitors such as isoniazid and most prominently by drugs targeting oxidative phosphorylation. We observed no induction by inhibitors of protein-, DNA- or RNA-synthesis. The constructed expression reporter was suitable for monitoring mycobacterial cytochrome bd expression during mouse macrophage infection and in a zebrafish embryo infection model when using Mycobacterium marinum. Interestingly, in both these infection models cytochrome bd levels were considerably higher than during in vitro culturing of M. marinum. The expression reporter described here can be a valuable tool for elucidating the role of cytochrome bd as a survival factor.

Mycobacterium tuberculosis is protected by an unusual and highly impermeable cell envelope that is critically important for the successful colonization of the host. The outermost surface of this cell envelope is formed by capsular polysaccharides that play an important role in modulating the initial interactions once the bacillus enters the body. Although the bioenzymatic steps involved in the production of the capsular polysaccharides are emerging, information regarding the ability of the bacterium to modulate the composition of the capsule is still unknown. Here, we study the mechanisms involved in regulation of mycobacterial capsule biosynthesis using a high throughput screen for gene products involved in capsular α-glucan production. Utilizing this approach we identified a group of mutants that all carried mutations in the ATP-binding cassette phosphate transport locus pst These mutants collectively exhibited a strong overproduction of capsular polysaccharides, including α-glucan and arabinomannan, suggestive of a role for inorganic phosphate (Pi) metabolism in modulating capsular polysaccharide production. These findings were corroborated by the observation that growth under low Pi conditions as well as chemical activation of the stringent response induces capsule production in a number of mycobacterial species. This induction is, in part, dependent on σ factor E. Finally, we show that Mycobacterium marinum, a model organism for M. tuberculosis, encounters Pi stress during infection, which shows the relevance of our findings in vivo., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Genes encoding Extended Spectrum Beta Lactamases are usually located on transferable plasmids. Each plasmid contains its own replication mechanism. Carattoli et al. developed an extended PCR-based replicon typing method to characterize and identify the replicons of the major plasmid incompatibility groups in Enterobacteriaceae. Based on this method, we designed a rapid approach with amplicon detection by real-time melting curve analysis. This method appeared to be fast, sensitive, less laborious, less prone to contamination and applicable in a routine laboratory environment., Results: We successfully integrated the post-PCR analysis of the replicon typing into a closed system, which leads to a 10-fold increase in sensitivity compared to agarose gel visualization. Moreover, the use of crude lysates and SYBR-green saves a considerable amount of hands-on time without decreasing the sensitivity or specificity., Conclusions: This real-time melting curve replicon typing method appears to be fast, sensitive, less laborious, less prone to contamination and applicable in a routine laboratory environment.

HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env(GM-CSF) chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized Env(GM-CSF) proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric Env(GM-CSF) should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins.

Background: Hodgkin's lymphoma (HL) is characterised by an ineffective immune response that is predominantly mediated by CD4+ T-cells., Aims: To analyse the expression of the key regulatory T-cell transcription factors (TFs) in the T-cells of HL involved tissues in order to assess the nature of the T(H) immune response in HL., Methods and Results: By immunohistochemistry, GATA3 was strongly and T-bet exclusively expressed in a subset of interfollicular lymphocytes in the reactive lymphoid tissues. In classical HL (CHL), which is generally located in the interfollicular zones, a predominance of T-bet+ T-cells and lesser amounts of GATA3+ and c-Maf+ T-cells was found, concordant with the pattern of the normal interfollicular compartment. In reactive lymphoid tissues, c-Maf was observed mostly in T-lymphocytes within the germinal centres (GCs). Nodular lymphocyte predominance type of Hodgkin's lymphoma (NLPHL) and progressively transformed germinal centres cases, showed a majority of c-Maf+ T-cells, consistent with the pattern in normal GCs. NLPHL cases uniformly showed c-Maf+/CD57+ T-cell rosettes around the neoplastic cells; these rosettes were absent in "paragranuloma-type" T-cell/histiocyte rich B-cell lymphoma., Conclusions: T-cell TF expression profiles of the reactive T-cells in both subtypes of HL are in accordance with the expression profile observed in the distinct lymphoid compartments.

Since Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) generally have immunoglobulin gene rearrangements, they are considered to be of B-cell origin. One of the characteristics of HRS cells is a prominent production of various cytokines and chemokines. Cytokine production is generally driven by expression of T-cell transcription factors (TFs). Only limited information is available on the expression of T-cell TFs in HL. Expression of four T-cell TFs and the target cytokine spectrum of these TFs were analyzed in six HL and three large B-cell lymphoma (LBCL) cell lines using quantitative PCR. ERM expression was observed in all HL and LBCL cell lines. Out of HL cell lines, T-bet was expressed in five, GATA-3 in four, and c-Maf in two cell lines. Immunohistochemistry in HL tissues revealed that in 11 of 12 (92%) of the classical HL cases HRS cells were GATA-3 and/or T-bet positive. In three of six cases of nodular lymphocyte predominance type of HL, the neoplastic cells were T-bet positive. Overall, the T-cell TF and cytokine profiles of the HL cell lines showed a considerable degree of consistency. The expression of T-cell TFs may explain the production of various cytokines by HL cell lines and HRS cells.

We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins alphaB-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of beta-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of alphaB-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock.