L-Thyroxine and L-thyroxine-based antimicrobials against Streptococcus pneumoniae and other Gram-positive bacteria.

Objectives: The rise of antibiotic-resistant Streptococcus pneumoniae (Sp) poses a significant global health threat, urging the quest for novel antimicrobial solutions. We have discovered that the human hormone l-thyroxine has antibacterial properties. In order to explore its drugability we perform here the characterization of a series of l-thyroxine analogues and describe the structural determinants influencing their antibacterial efficacy. Method: We performed a high-throughput screening of a library of compounds approved for use in humans, complemented with ITC assays on purified Sp-flavodoxin, to pinpoint molecules binding to this protein. Antimicrobial in vitro susceptibility assays of the hit compound (l-thyroxine) as well as of 13 l-thyroxine analogues were done against a panel of Gram-positive and Gram-negative bacteria. Toxicity of compounds on HepG2 cells was also assessed. A combined structure-activity and computational docking analysis was carried out to uncover functional groups crucial for the antimicrobial potency of these compounds. Results: Human l-thyroxine binds to Sp-flavodoxin, forming a 1:1 complex of low micromolar Kd. While l-thyroxine specifically inhibited Sp growth, some derivatives displayed activity against other Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, while remaining inactive against Gram-negative pathogens. Neither l-thyroxine nor some selected derivatives exhibited toxicity to HepG2 cells. Conclusions: l-thyroxine derivatives targeting bacterial flavodoxins represent a new and promising class of antimicrobials.

The Mla system and its role in maintaining outer membrane barrier function in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.

The influence of hindered rotation on electron transfer and exchange interaction in triarylamine-triptycene-perylene diimide triads.

Stretched electron-donor-bridge-acceptor triads that exhibit intramolecular twisting degrees of freedom are capable of modulating exchange interaction (J) as well as electronic couplings through variable π-overlap at the linear bond links, affecting the rate constants of photoinduced charge separation and recombination. Here we present an in-depth investigation of such effects induced by methyl substituents leading to controlled steric hindrance of intramolecular twisting around biaryl axes. Starting from the parent structure, consisting of a triphenyl amine donor, a triptycene (TTC) bridge and a phenylene-perylene diimide acceptor (Me0), one of the two phenylene linkers attached to the TTC was ortho-substituted by two methyl groups (Me2, Me3), or both such phenylene linkers by two pairs of methyl groups (Me23). Photoinduced charge separation (kCS) leading to a charge-separated (CS) state was studied by fs-laser spectroscopy, charge recombination to either singlet ground state (kS) or to the first excited local triplet state of the acceptor (kT) by ns-laser spectroscopy, whereby kinetic magnetic field effects in an external magnetic field were recorded and analysed using quantum dynamic simulations of the spin dependent kinetics of the CS state. Kinetic spectra of the initial first order rate constants of charge recombination (k(B)) exhibited characteristic J-resonances progressing to lower fields in the series Me0, Me2, Me3, Me23. From the quantum simulations, the values of the parameters J, kS, kT and kSTD, the singlet/triplet dephasing constant, were obtained. They were analysed in terms of molecular dynamics simulations of the intramolecular twisting dynamics based on potentials calculated by density functional theory. Apart from kT, all of the parameters exhibit a clear correlation with the averaged cosine square products of the biaryl angles.

Stenotrophomonas maltophilia affects the gene expression profiles of the major pathogens Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro multispecies biofilm model.

IMPORTANCE: In the past, studies have focused on bacterial pathogenicity in mono-species infections, in part ignoring the clinical relevance of diseases caused by more than one pathogen (i.e., polymicrobial infections). However, it is now common knowledge that multiple bacteria species are often involved in the course of an infection. For treatment of such infections, it is absolutely important to understand the dynamics of species interactions at possible infection sites and the molecular mechanisms behind these interactions. Here, we studied the impact of Stenotrophomonas maltophilia on its commensals Pseudomonas aeruginosa and Staphylococcus aureus in multispecies biofilms. We analyzed the 3D structural architectures of dual- and triple-species biofilms, niche formation within the biofilms, and the interspecies interactions on a molecular level. RNAseq data identified key genes involved in multispecies biofilm formation and interaction as potential drug targets for the clinical combat of multispecies infection with these major pathogens.

In vivo spectroscopy and machine learning for the early detection and classification of different stresses in apple trees.

The use of in vivo spectroscopy to detect plant stress in its early stages has the potential to enhance food safety and reduce the need for plant protection products. However, differentiating between various stress types before symptoms appear remains poorly studied. In this study, we investigated the potential of Vis-NIR spectroscopy to differentiate between stress types in apple trees (Malus x domestica Borkh.) exposed to apple scab, waterlogging, and herbicides in a greenhouse. Using a spectroradiometer, we collected spectral signatures of leaves still attached to the tree and utilized machine learning techniques to develop predictive models for detecting stress presence and classifying stress type as early as 1-5 days after exposure. Our findings suggest that changes in spectral reflectance at multiple regions accurately differentiate various types of plant stress on apple trees. Our models were highly accurate (accuracies between 0.94 and 1) when detecting the general presence of stress at an early stage. The wavelengths important for classification relate to photosynthesis via pigment functioning (684 nm) and leaf water (~ 1800-1900 nm), which may be associated with altered gas exchange as a short-term stress response. Overall, our study demonstrates the potential of spectral technology and machine learning for early diagnosis of plant stress, which could lead to reduced environmental burden through optimizing resource utilization in agriculture.

Identification of spectral ranges that contribute to phytoplasma detection in apple trees - A step towards an on-site method.

'Candidatus Phytoplasma mali' is the bacterial agent associated with Apple Proliferation, a disease that causes high economic losses in affected commercial apple growing regions. The identification of the disease is carried out by visual inspection performed by skilled professionals in the orchards. To confirm an infection, costly molecular laboratory methods must be applied. Furthermore, both methods are very time-consuming. Here, we analysed the potential of a non-destructive method using in-field measurements to differentiate infected from non-infected apple trees (Malus domestica) based on spectral signatures of fresh leaves. By using multivariate statistics, we were able to distinguish infected from non-infected trees and identified the wavelengths relevant for the differentiation. Factors affecting the differentiation performance were the sampling date and bacterial colonization behaviour.

Homology judgements of pre-evolutionary naturalists explained by general human shape matching abilities.

Many biological homologies were discovered before Darwin and without agreed criteria. Paradigmatic examples include the phylogenetic homology of mammalian forelimb bones and the serial homology of floral organs in waterlilies. It is generally assumed that perceiving similarities intuitively was the first step towards establishing morphological homologies. However, this assumption has never been tested. We hypothesize that pre-evolutionary naturalists relied on the well-established ability of humans to find visual correspondences between differently shaped objects. By using images of homologous organs and applying an experimental paradigm from cognitive psychology, we found that (1) naïve participants utilised this ability when identifying "corresponding" locations. In addition, (2) these locations were statistically indistinguishable from the locations that pre-evolutionary naturalists and contemporary experts considered homologous. Furthermore, (3) presenting naïve participants with images of intermediate organs influenced their correspondence judgements. This influence was in line with historical reports according to which intermediate organs facilitated the pre-evolutionary recognition of homologies.

tet-Dependent Gene Expression in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is increasingly recognized as an important nosocomial pathogen among the Gram-negative bacteria. Intrinsic resistance to different classes of antibiotics makes treatment of infections challenging. A deeper understanding of S. maltophilia physiology and virulence requires molecular genetic tools. Here, we describe the implementation of tetracycline-dependent gene regulation (tet regulation) in this bacterium. The exploited tet regulatory sequence of transposon Tn10 contained the tetR gene and three intertwined promoters, one of which was required for regulated expression of a target gene or operon. The episomal tet architecture was tested with a gfp variant as a quantifiable reporter. Fluorescence intensity was directly correlated with the concentration of the inducer anhydrotetracycline (ATc) applied and the duration of induction. Also, the expression of the rmlBACD operon of S. maltophilia K279a was subjected to tet control. These genes code for the synthesis of dTDP-l-rhamnose, an activated nucleotide sugar precursor of lipopolysaccharide (LPS) formation. A ΔrmlBACD mutant was complemented with a plasmid carrying this operon downstream of the tet sequence. In the presence of ATc, the LPS pattern was similar to that of wild-type S. maltophilia, whereas without the inducer, fewer and apparently shorter O-antigen chains were detected. This underscores the functionality and usefulness of the tet system for gene regulation and, prospectively, the validation of targets for new anti-S. maltophilia drugs. IMPORTANCE Stenotrophomonas maltophilia is an emerging pathogen in hospital settings and poses a threat to immunocompromised patients. Due to a high level of resistance to different types of antibiotics, treatment options are limited. We here adapted a tool for inducible expression of genes of interest, known as the tet system, to S. maltophilia. Genes relevant to producing surface carbohydrate structures (lipopolysaccharide [LPS]) were placed under the control of the tet system. In the presence of an inducer, the LPS pattern was similar to that of wild-type S. maltophilia, whereas in the "off" state of the system (without inducer), fewer and apparently shorter versions of LPS were detected. The tet system is functional in S. maltophilia and may be helpful to reveal gene-function relationships to gain a deeper understanding of the bacterium's physiology and virulence.

Amorphous Drug Nanoparticles for Inhalation Therapy of Multidrug-Resistant Tuberculosis.

Tuberculosis (TB) is one of the most prevalent infectious diseases. The global TB situation is further complicated by increasing patient numbers infected with Mycobacterium tuberculosis (M.tb.) strains resistant to either one or two of the first-line therapeutics, promoted by insufficient treatment length and/or drug levels due to adverse reactions and reduced patient compliance. An intriguing approach to improve anti-TB therapy relates to nanocarrier-based drug-delivery systems, which enhance local drug concentrations at infection sites without systemic toxicity. Recently developed anti-TB antibiotics, however, are lipophilic and difficult to transport in aqueous systems. Here, the very lipophilic TB-antibiotics bedaquiline (BDQ) and BTZ (1,3-benzothiazin-4-one 043) are prepared as high-dose, amorphous nanoparticles via a solvent-antisolvent technique. The nanoparticles exhibit mean diameters of 60 ± 13 nm (BDQ) and 62 ± 44 nm (BTZ) and have an extraordinarily high drug load with 69% BDQ and >99% BTZ of total nanoparticle mass plus a certain amount of surfactant (31% for BDQ, <1% for BTZ) to make the lipophilic drugs water-dispersible. Suspensions with high drug load (4.1 mg/mL BDQ, 4.2 mg/mL BTZ) are stable for several weeks. In vitro and in vivo studies employing M.tb.-infected macrophages and susceptible C3HeB/FeJ mice show promising activity, which outperforms conventional BDQ/BTZ solutions (in DMF or DMSO) with an up to 50% higher efficacy upon pulmonary delivery. In vitro, the BDQ/BTZ nanoparticles demonstrate their ability to cross the different biological barriers and to reach the site of the intracellular mycobacteria. In vivo, high amounts of the BDQ/BTZ nanoparticles are found in the lung and specifically inside granulomas, whereas only low BDQ/BTZ-nanoparticle levels are observed in spleen or liver. Thus, pulmonary delivered BDQ/BTZ nanoparticles are promising formulations to improve antituberculosis treatment.

Amikacin@SiO2 core@shell nanocarriers to treat pulmonal bacterial infections.

AMC@SiO2 core@shell nanocarriers (AMC: amikacin) are realized and contain an exceptionally high drug load of 0.8 mg mg-1 (i.e. 80% AMC of total nanocarrier mass). They are prepared via a solvent-antisolvent approach with AMC nanoparticles formed in a first step, which are then covered and stabilised by a thin silica shell in a one-pot synthesis. In total, the core@shell nanocarriers exhibit a mean diameter of 240 nm with an inner AMC core of 200 nm and an outer silica shell of 20 nm. Subsequent to synthesis, the nanocarriers can be stored in frozen dimethylsulfoxide (DMSO) and applied directly after warming to room temperature with particle contents of 5 mg mL-1. Size, structure, and composition of the AMC@SiO2 core@shell nanocarriers are evidenced by electron microscopy (SEM, TEM), spectroscopic methods (EDXS, FT-IR, UV-Vis), as well as X-ray powder diffraction and elemental analysis. As proof-of-concept, the AMC release and the activity of the novel nanocarriers are tested against two relevant, difficult-to-treat and notoriously multidrug resistant, bacterial pathogens: Mycobacterium tuberculosis (M.tb.) and Mycobacterium abscessus (M.abs.). Colloidal stability, storage stability, high drug load, and activity of the AMC@SiO2 core@shell nanocarriers are promising for, e.g., aerosol-type pulmonal application.

Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia.

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as Stenotrophomonas maltophilia. Using a Tn7-based genomic integration system, we report the construction of improved mini-Tn7 delivery plasmids for labeling of S. maltophilia with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different S. maltophilia wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in Galleria mellonella. It is also shown that the mini-Tn7 elements remained stably integrated in the genome of S. maltophilia over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn7 delivery plasmids are valuable tools for generating fluorescently labeled S. maltophilia strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium S. maltophilia is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of S. maltophilia infections worldwide. One important virulence-related trait of S. maltophilia is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn7-based labeling system for S. maltophilia to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions.

Fungicide-free management of Alternaria leaf blotch and fruit spot on apple indicates Alternaria spp. as secondary colonizer.

The fungal genus Alternaria is a pan-global pathogen of > 100 crops, and is associated with the globally expanding Alternaria leaf blotch in apple (Malus x domestica Borkh.) which leads to severe leaf necrosis, premature defoliation, and large economic losses. Up to date, the epidemiology of many Alternaria species is still not resolved as they can be saprophytic, parasitic or shift between both lifestyles and are also classified as primary pathogen able to infect healthy tissue. We argue that Alternaria spp. does not act as primary pathogen, but only as a necrosis-dependent opportunist. We studied the infection biology of Alternaria spp. under controlled conditions and monitored disease prevalence in real orchards and validated our ideas by applying fungicide-free treatments in 3-years field experiments. Alternaria spp. isolates were not able to induce necroses in healthy tissue, but only when prior induced damages existed. Next, leaf-applied fertilizers, without fungicidal effect, reduced Alternaria-associated symptoms (- 72.7%, SE: ± 2.5%) with the same efficacy as fungicides. Finally, low leaf magnesium, sulphur, and manganese concentrations were consistently linked with Alternaria-associated leaf blotch. Fruit spot incidence correlated positively with leaf blotch, was also reduced by fertilizer treatments, and did not expand during storage unlike other fungus-mediated diseases. Our findings suggest that Alternaria spp. may be a consequence of leaf blotch rather than its primary cause, as it appears to colonize the physiologically induced leaf blotch. Taking into account existing observations that Alternaria infection is connected to weakened hosts, the distinction may appear slight, but is of great significance, as we can now (a) explain the mechanism of how different stresses result in colonization with Alternaria spp. and (b) substitute fungicides for a basic leaf fertilizer. Therefore, our findings can result in significant decreases in environmental costs due to reduced fungicide use, especially if the same mechanism applies to other crops.

Development, Processing and Aging of Novel Zn-Ag-Cu Based Biodegradable Alloys.

The use of biodegradable materials for implants is a promising strategy to overcome known long-term clinical complications related to permanent implants. Ideally, biodegradable implants support the damaged tissue for a certain period and then degrade, while the physiological function of the surrounding tissue is restored. Although Mg-based alloys nearly ideally lend themselves to biodegradable implants, a few critical shortcomings promoted the development of alternative alloy systems. Due to their reasonably good biocompatibility, moderate corrosion rate without hydrogen evolution and adequate mechanical properties, increasing attention has been paid to Zn alloys. In this work, precipitation-hardening alloys in the system Zn-Ag-Cu were developed relying on thermodynamic calculations. After casting the alloys, their microstructures were refined by thermomechanical treatment. The processing was tracked and directed, respectively, by routine investigations of the microstructure, associated with hardness assessments. Although microstructure refinement increased the hardness, the material proved to be susceptible to aging as the homologous temperature of zinc is at 0.43 Tm. Besides mechanical performance and corrosion rate, long-term mechanical stability is another crucial factor that must be taken into consideration to ensure the safety of the implant and thus requires a profound understanding of the aging process.

Π-Π interactions stabilize PeptoMicelle-based formulations of Pretomanid derivatives leading to promising therapy against tuberculosis in zebrafish and mouse models.

Tuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and impede the development of drug resistance are desperately needed. Here, we used polymeric micelles to encapsulate four second-generation derivatives of the antitubercular drug pretomanid that had previously displayed much better in vivo activity against Mycobacterium tuberculosis than pretomanid itself. Because these compounds were relatively hydrophobic and had limited bioavailability, we expected that their micellar formulations would overcome these limitations, reduce toxicities, and improve therapeutic outcomes. The polymeric micelles were based on polypept(o)ides (PeptoMicelles) and were stabilized in their hydrophobic core by π-π interactions, allowing the efficient encapsulation of aromatic pretomanid derivatives. The stability of these π-π-stabilized PeptoMicelles was demonstrated in water, blood plasma, and lung surfactant by fluorescence cross-correlation spectroscopy and was further supported by prolonged circulation times of several days in the vasculature of zebrafish larvae. The most efficacious PeptoMicelle formulation tested in the zebrafish larvae infection model almost completely eradicated the bacteria at non-toxic doses. This lead formulation was further assessed against Mycobacterium tuberculosis in the susceptible C3HeB/FeJ mouse model, which develops human-like necrotic granulomas. Following intravenous administration, the drug-loaded PeptoMicelles significantly reduced bacterial burden and inflammatory responses in the lungs and spleens of infected mice.

Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis.

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼102 colony forming units (CFUs) and ∼103 CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity.

Evaluation of Myeloperoxidase as Target for Host-Directed Therapy in Tuberculosis In Vivo.

Due to the rise of tuberculosis cases infected with multi and extensively drug-resistant Mycobacterium tuberculosis strains and the emergence of isolates resistant to antibiotics newly in clinical use, host-directed therapies targeting pathogenesis-associated immune pathways adjunct to antibiotics may ameliorate disease and bacterial clearance. Active tuberculosis is characterized by neutrophil-mediated lung pathology and tissue destruction. Previously, we showed that preventing M. tuberculosis induced necrosis in human neutrophils by inhibition of myeloperoxidase (MPO) promoted default apoptosis and subsequent control of mycobacteria by macrophages taking up the mycobacteria-infected neutrophils. To translate our findings in an in vivo model, we tested the MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH) in C3HeB/FeJ mice, which are highly susceptible to M. tuberculosis infection manifesting in neutrophil-associated necrotic granulomas. MPO inhibition alone or as co-treatment with isoniazid, a first-line antibiotic in tuberculosis treatment, did not result in reduced bacterial burden, improved pathology, or altered infiltrating immune cell compositions. MPO inhibition failed to prevent M. tuberculosis induced neutrophil necrosis in C3Heb/FeJ mice in vivo as well as in murine neutrophils in vitro. In contrast to human neutrophils, murine neutrophils do not respond to M. tuberculosis infection in an MPO-dependent manner. Thus, the murine C3HeB/FeJ model does not fully resemble the pathomechanisms in active human tuberculosis. Consequently, murine infection models of tuberculosis are not necessarily adequate to evaluate host-directed therapies targeting neutrophils in vivo.

Mycobacterium tuberculosis Acetyltransferase Suppresses Oxidative Stress by Inducing Peroxisome Formation in Macrophages.

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal ß-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11ß, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.

Vitamin D3 and calcium carbonate supplementation for adolescents with HIV to reduce musculoskeletal morbidity and immunopathology (VITALITY trial): study protocol for a randomised placebo-controlled trial.

BACKGROUND: Of the 2 million children living with HIV globally, 90% live in sub-Saharan Africa. Despite antiretroviral therapy, longstanding HIV infection is associated with several chronic complications in children including growth failure, particularly stunting and delayed puberty. Vitamin D deficiency, which is highly prevalent among children living with HIV in sub-Saharan Africa, has a further adverse impact on bone health. This trial aims to establish whether supplementation with vitamin D3 and calcium carbonate improves musculoskeletal health among peripubertal children living with HIV. METHODS/DESIGN: We will conduct an individually randomised, double-blinded, placebo-controlled trial of weekly high-dose vitamin D3 (20,000 IU) plus daily calcium carbonate (500mg) supplementation for 48 weeks. Eight hundred and forty children living with HIV aged 11-19 years taking ART for ≥6 months will be enrolled and followed up for 96 weeks. The primary outcome is total body less-head bone mineral content for lean mass adjusted for height (TBLH-BMCLBM) Z-score at 48 weeks, measured by dual-energy X-ray absorptiometry (DEXA). Secondary outcomes are DEXA-measured lumbar spine bone mineral apparent density Z-score, number of respiratory infections, lean muscle mass and grip strength at 48 and 96 weeks and TBLH-BMCLBM Z-scores at 96 weeks. Sub-studies will investigate the effect of the intervention on vitamin D3 pathway metabolites and markers of bone turnover, intestinal microbiota, and innate and acquired immune function. DISCUSSION: This is the largest trial to date of vitamin D supplementation in children living with HIV. Intervening to address deficits in bone accrual in childhood is critical for optimising adolescent and early adult bone health and prevention of later adult osteoporotic fractures. Trial results will draw attention to the need to screen for and treat long-term comorbidities in children living with HIV in resource-limited settings. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR20200989766029 . Registered on 3 September 2020.