Generation of Liposomes to Study the Effect of Mycobacterium Tuberculosis Lipids on HIV-1 cis- and trans-Infections

Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.     

The Therapeutic Effect of Intranasal Administration of Dexamethasone in Neuroinflammation Induced by Experimental Pulmonary Tuberculosis

Tuberculosis (TB) is an important infectious disease and a public health problem. The organs most frequently affected by TB are the lungs; despite this, it has been reported that TB patients suffer from depression and anxiety, which have been attributed to social factors. In previous experimental work, we observed that the extensive pulmonary inflammation characteristic of TB with high cytokine production induces neuroinflammation, neuronal death and behavioral abnormalities in the absence of brain infection. The objective of the present work was to reduce this neuroinflammation and avoid the psycho-affective disorders showed during pulmonary TB. Glucocorticoids (GCs) are the first-line treatment for neuroinflammation; however, their systemic administration generates various side effects, mostly aggravating pulmonary TB due to immunosuppression of cellular immunity. Intranasal administration is a route that allows drugs to be released directly in the brain through the olfactory nerve, reducing their doses and side effects. In the present work, dexamethasone’s (DEX) intranasal administration was evaluated in TB BALB /c mice comparing three different doses (0.05, 0.25 and 2.5 mg/kg BW) on lung disease evolution, neuroinflammation and behavioral alterations. Low doses of dexamethasone significantly decreased neuroinflammation, improving behavioral status without aggravating lung disease.     

The Antimicrobial Activity of Peripheral Blood Neutrophils Is Altered in Patients with Primary Ciliary Dyskinesia

The airways of patients with primary ciliary dyskinesia (PCD) contain persistently elevated neutrophil numbers and CXCL8 levels. Despite their abundance, neutrophils fail to clear the airways from bacterial infections. We investigated whether neutrophil functions are altered in patients with PCD. Neutrophils from patients and healthy controls (HC) were isolated from peripheral blood and exposed to various bacterial stimuli or cytokines. Neutrophils from patients with PCD were less responsive to low levels of fMLF in three different chemotaxis assays (p < 0.05), but expression of the fMLF receptors was unaltered. PCD neutrophils showed normal phagocytic function and expression of adhesion molecules. However, PCD neutrophils produced less reactive oxygen species upon stimulation with bacterial products or cytokines compared to HC neutrophils (p < 0.05). Finally, the capacity to release DNA, as observed during neutrophil extracellular trap formation, seemed to be reduced in patients with PCD compared to HC (p = 0.066). These results suggest that peripheral blood neutrophils from patients with PCD, in contrast to those of patients with cystic fibrosis or COPD, do not show features of over-activation, neither on baseline nor after stimulation. If these findings extend to lung-resident neutrophils, the reduced neutrophil activity could possibly contribute to the recurrent respiratory infections in patients with PCD.     

Bioinformatics Identification of Drug Resistance-Associated Gene Pairs in Mycobacterium tuberculosis

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Due to the extensive use of anti-tuberculosis drugs and the development of mutations, the emergence and spread of multidrug-resistant tuberculosis is recognized as one of the most dangerous threats to global tuberculosis control. Some single mutations have been identified to be significantly linked with drug resistance. However, the prior research did not take gene-gene interactions into account, and the emergence of transmissible drug resistance is connected with multiple genetic mutations. In this study we use the bioinformatics software GBOOST (The Hong Kong University, Clear Water Bay, Kowloon, Hong Kong, China) to calculate the interactions of Single Nucleotide Polymorphism (SNP) pairs and identify gene pairs associated with drug resistance. A large part of the non-synonymous mutations in the drug target genes that were included in the screened gene pairs were confirmed by previous reports, which lent sound solid credits to the effectiveness of our method. Notably, most of the identified gene pairs containing drug targets also comprise Pro-Pro-Glu (PPE) family proteins, suggesting that PPE family proteins play important roles in the drug resistance of Mtb. Therefore, this study provides deeper insights into the mechanisms underlying anti-tuberculosis drug resistance, and the present method is useful for exploring the drug resistance mechanisms for other microorganisms.     

In Vitro Activity of Copper(II) Complexes,Loaded or Unloaded into a Nanostructured Lipid System,against Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis (Mtb), presenting 9.5 million new cases and 1.5 million deaths in 2014. The aim of this study was to evaluate a nanostructured lipid system (NLS) composed of 10% phase oil (cholesterol), 10% surfactant (soy phosphatidylcholine, sodium oleate), and Eumulgin^® HRE 40 ([castor oil polyoxyl-40-hydrogenated] in a proportion of 3:6:8), and an 80% aqueous phase (phosphate buffer pH = 7.4) as a tactic to enhance the in vitro anti-Mtb activity of the copper(II) complexes [CuCl₂(INH)₂]·H₂O (1), [Cu(NCS)₂(INH)₂]·5H₂O (2) and [Cu(NCO)₂(INH)₂]·4H₂O (3). The Cu(II) complex-loaded NLS displayed sizes ranging from 169.5 ± 0.7095 to 211.1 ± 0.8963 nm, polydispersity index (PDI) varying from 0.135 ± 0.0130 to 0.236 ± 0.00100, and zeta potential ranging from −0.00690 ± 0.0896 to −8.43 ± 1.63 mV. Rheological analysis showed that the formulations behave as non-Newtonian fluids of the pseudoplastic and viscoelastic type. Antimycobacterial activities of the free complexes and NLS-loaded complexes against Mtb H₃₇Rv ATCC 27294 were evaluated by the REMA methodology, and the selectivity index (SI) was calculated using the cytotoxicity index (IC₅₀) against Vero (ATCC^® CCL-81), J774A.1 (ATCC^® TIB-67), and MRC-5 (ATCC^® CCL-171) cell lines. The data suggest that the incorporation of the complexes into NLS improved the inhibitory action against Mtb by 52-, 27-, and 4.7-fold and the SI values by 173-, 43-, and 7-fold for the compounds 1, 2 and 3, respectively. The incorporation of the complexes 1, 2 and 3 into the NLS also resulted in a significant decrease of toxicity towards an alternative model (Artemia salina L.). These findings suggest that the NLS may be considered as a platform for incorporation of metallic complexes aimed at the treatment of TB.     

Preliminary Evaluation of Artemisinin–Cholesterol Conjugates as Potential Drugs for the Treatment of Intractable Forms of Malaria and Tuberculosis

To evaluate the feasibility of developing drugs that may be active against both malaria and tuberculosis (TB) by using in part putative cholesterol transporters in the causative pathogens and through enhancement of passive diffusion in granulomatous TB, artemisinin–cholesterol conjugates were synthesized by connecting the component molecules through various linkers. The compounds were screened in vitro against Plasmodium falciparum (Pf) and Mycobacterium tuberculosis (Mtb). Antimalarial activities (IC₅₀) against Pf drug‐sensitive NF54, and drug‐resistant K1 and W2 strains ranged from 0.03–2.6, 0.03–1.9, and 0.02–1.7 μm . Although the compounds are less active than the precursor artemisinin derivatives, the cholesterol moiety renders the compounds relatively insoluble in the culture medium, and variation in solubilities among the different compounds may reflect in the range of efficacies observed. Activities against Mtb H37Rv were assessed using a standardized colony‐forming unit (CFU) assay after 24 h pretreatment of cultures with each of the compounds. Percentage inhibition ranged from 3–38 % and 18–52 % at 10 and 80 μm , respectively. Thus, in contrast to the comparator drug artemether, the conjugates display enhanced activities. The immediate aims include the preparation of conjugates with enhanced aqueous solubilities, assays against malaria and TB in vivo, and for TB, assays using an infected macrophage model and assessment of granuloma influx.     

Experimental Pulmonary Tuberculosis in the Absence of Detectable Brain Infection Induces Neuroinflammation and Behavioural Abnormalities in Male BALB/c Mice

Tuberculosis (TB) is a chronic infectious disease in which prolonged, non-resolutive inflammation of the lung may lead to metabolic and neuroendocrine dysfunction. Previous studies have reported that individuals coursing pulmonary TB experience cognitive or behavioural changes; however, the pathogenic substrate of such manifestations have remained unknown. Here, using a mouse model of progressive pulmonary TB, we report that, even in the absence of brain infection, TB is associated with marked increased synthesis of both inflammatory and anti-inflammatory cytokines in discrete brain areas such as the hypothalamus, the hippocampal formation and cerebellum accompanied by substantial changes in the synthesis of neurotransmitters. Moreover, histopathological findings of neurodegeneration and neuronal death were found as infection progressed with activation of p38, JNK and reduction in the BDNF levels. Finally, we perform behavioural analysis in infected mice throughout the infection, and our data show that the cytokine and neurochemical changes were associated with a marked onset of cognitive impairment as well as depressive- and anxiety-like behaviour. Altogether, our results suggest that besides pulmonary damage, TB is accompanied by an extensive neuroinflammatory and neurodegenerative state which explains some of the behavioural abnormalities found in TB patients.     

Biofragments: An Approach towards Predicting Protein Function Using Biologically Related Fragments and its Application to Mycobacterium tuberculosis CYP126

We present a novel fragment‐based approach that tackles some of the challenges for chemical biology of predicting protein function. The general approach, which we have termed biofragments, comprises two key stages. First, a biologically relevant fragment library (biofragment library) can be designed and constructed from known sets of substrate‐like ligands for a protein class of interest. Second, the library can be screened for binding to a novel putative ligand‐binding protein from the same or similar class, and the characterization of hits provides insight into the basis of ligand recognition, selectivity, and function at the substrate level. As a proof‐of‐concept, we applied the biofragments approach to the functionally uncharacterized Mycobacterium tuberculosis (Mtb) cytochrome P450 isoform, CYP126. This led to the development of a tailored CYP biofragment library with notable 3D characteristics and a significantly higher screening hit rate (14 %) than standard drug‐like fragment libraries screened previously against Mtb CYP121 and 125 (4 % and 1 %, respectively). Biofragment hits were identified that make both substrate‐like type‐I and inhibitor‐like type‐II interactions with CYP126. A chemical‐fingerprint‐based substrate model was built from the hits and used to search a virtual TB metabolome, which led to the discovery that CYP126 has a strong preference for the recognition of aromatics and substrate‐like type‐I binding of chlorophenol moieties within the active site near the heme. Future catalytic analyses will be focused on assessing CYP126 for potential substrate oxidative dehalogenation.     

Corticosteroids for COVID-19 Therapy: Potential Implications on Tuberculosis

On 11 March 2020, the World Health Organization announced the Corona Virus Disease-2019 (COVID-19) as a global pandemic, which originated in China. At the host level, COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), affects the respiratory system, with the clinical symptoms ranging from mild to severe or critical illness that often requires hospitalization and oxygen support. There is no specific therapy for COVID-19, as is the case for any common viral disease except drugs to reduce the viral load and alleviate the inflammatory symptoms. Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb), also primarily affects the lungs and has clinical signs similar to pulmonary SARS-CoV-2 infection. Active TB is a leading killer among infectious diseases and adds to the burden of the COVID-19 pandemic worldwide. In immunocompetent individuals, primary Mtb infection can also lead to a non-progressive, asymptomatic latency. However, latent Mtb infection (LTBI) can reactivate symptomatic TB disease upon host immune-suppressing conditions. Importantly, the diagnosis and treatment of TB are hampered and admixed with COVID-19 control measures. The US-Center for Disease Control (US-CDC) recommends using antiviral drugs, Remdesivir or corticosteroid (CST), such as dexamethasone either alone or in-combination with specific recommendations for COVID-19 patients requiring hospitalization or oxygen support. However, CSTs can cause immunosuppression, besides their anti-inflammatory properties. The altered host immunity during COVID-19, combined with CST therapy, poses a significant risk for new secondary infections and/or reactivation of existing quiescent infections, such as LTBI. This review highlights CST therapy recommendations for COVID-19, various types and mechanisms of action of CSTs, the deadly combination of two respiratory infectious diseases COVID-19 and TB. It also discusses the importance of screening for LTBI to prevent TB reactivation during corticosteroid therapy for COVID-19.     

Synthesis and in Vitro Characterization of Trehalose-Based Inhibitors of Mycobacterial Trehalose 6-Phosphate Phosphatases

α,α′-Trehalose plays roles in the synthesis of several cell wall components involved in pathogenic mycobacteria virulence. Its absence in mammalian biochemistry makes trehalose-related biochemical processes potential targets for chemotherapy. The trehalose 6-phosphate synthase (TPS)/trehalose 6-phosphate phosphatase (TPP) pathway, also known as the OtsA/OtsB2 pathway, is the major pathway involved in the production of trehalose in Mycobacterium tuberculosis (Mtb). In addition, TPP is essential for Mtb survival. We describe the synthesis of α,α′-trehalose derivatives in the forms of the 6-phosphonic acid 4 (TMP), the 6-methylenephosphonic acid 5 (TEP), and the 6-N-phosphonamide 6 (TNP). These non-hydrolyzable substrate analogues of TPP were examined as inhibitors of Mtb, Mycobacterium lentiflavum (Mlt), and Mycobacterium triplex (Mtx) TPP. In all cases the compounds were most effective in inhibiting Mtx TPP, with TMP [IC₅₀=(288±32) μm ] acting most strongly, followed by TNP [IC₅₀=(421±24) μm ] and TEP [IC₅₀=(1959±261) μm ]. The results also indicate significant differences in the analogue binding profile when comparing Mtb TPP, Mlt TPP, and Mtx TPP homologues.     

The Emerging Role of Neutrophils in the Pathogenesis of Thrombosis in COVID-19

Previous studies have shown that COVID-19 leads to thrombotic complications, which have been associated with high morbidity and mortality rates. Neutrophils are the largest population of white blood cells and play a pivotal role in innate immunity. During an infection, neutrophils migrate from circulation to the infection site, contributing to killing pathogens. This mechanism is regulated by chemokines such as IL-8. Moreover, it was shown that neutrophils play an important role in thromboinflammation. Through a diverse repertoire of mechanisms, neutrophils, apart from directly killing pathogens, are able to activate the formation of thrombi. In COVID-19 patients, neutrophil activation promotes neutrophil extracellular trap (NET) formation, platelet aggregation, and cell damage. Furthermore, neutrophils participate in the pathogenesis of endothelitis. Overall, this review summarizes recent progress in research on the pathogenesis of COVID-19, highlighting the role of the prothrombotic action of neutrophils in NET formation.     

Polyester functionalization for mycobacterial capture

Tuberculosis (TB) is one of the deadliest diseases known to man and ranks among the top ten global causes of fatalities. Children often develop a paucibacillary form of this disease which makes diagnosis arduous even with the aid of sophisticated techniques. In these cases, the concentration of bacteria in their sputum falls below the lowest limit of detection for sputum smear microscopy, the technique predominantly used in developing countries. In this study we aim to test the hypothesis that modifying a polymer, often used in buccal swabs, could assist in creating a capturing and concentrating oral swab for Mycobacterium tuberculosis (Mtb), the pathogenic bacterial species responsible for TB. Such a device will assist in meeting the detection limit and allow for rapid detection of the disease. The polymer used was a micro fibrous form of poly(ethylene terephthalate) (PET) which was surface functionalized with Concanavalin A (Con A), a lectin based adhesin with an affinity for the mannose groups on the Mtb cell wall. The functionalization was mediated with glutaraldehyde as bioconjugate molecule and found to produce a surface uniformly covered with Con A. Affinity studies between the modified fibers and the Mycobacterium bovis bacillus Calmette-Guérin (BCG), as Mtb mimic, were conducted to evaluate the capturing abilities of the substrate. The results indicate that the fibers avidly captured BCG and that the fibrous matrix aided in its function. Dilution studies showed successful capturing of the bacterial species at concentrations characteristic of paucibacillary cases. In this study a commercial polymeric material was successfully surface modified with a biological entity to create a substrate to which Mtb could adhere and accordingly be captured.     

Phage as a diagnostic – the use of phage in TB diagnosis

Rapid TB diagnosis and drug susceptibility testing is limited by the slow growth of the causative organism, Mycobacterium tuberculosis (MTB). Effective and rapid methods for the detection and drug susceptibility testing of TB, that are suitable for implementation in low income countries, which bear the highest TB burden, are urgently needed for effective identification, treatment and control of the disease. Mycobacteriophages have the potential to become useful tools in the diagnosis of TB, as they are specific for mycobacteria and only replicate in, and hence detect, viable cells. Phage‐based techniques involve simple manual manipulations that typically require little capital expense and yield results rapidly. Two techniques have been developed recently which use mycobacteriophage for the antibiotic susceptibility testing and direct diagnosis of TB from sputum. Luciferase Reporter Phages (LRP) are recombinant phages that contain the gene for the fire‐fly luciferase enzyme. Upon infection of mycobacteria by these phages, this enzyme is produced within a cell. In the presence of this enzyme, light is produced by viable cells that also contain the substrates for the enzyme: ATP and luciferin (added exogenously). The ‘Bronx Box’ has been developed as a low cost means of detecting light signal from mycobacteria infected with LRP using photographic film. A MTB strain's susceptibility to an antibiotic can be assessed by comparing light production, as an indication of the presence of viable cells, in samples pre‐incubated in the presence and absence of the test antibiotic. Susceptibility testing of some of the front line drugs for TB chemotherapy has given excellent results (detection of all resistant isolates), using LRP and the Bronx Box. Phage amplification uses the production of progeny phage, instead of a reporter gene product, to reflect the presence of target bacteria. The technique has been successfully applied to drug susceptibility testing and for diagnosis to the TB from sputum specimens. Viable MTB cells within a specimen are uniquely infected by a bacteriophage reagent. A potent selective virucide destroys all the phage particles that remain outside the cells while remaining inactive against the MTB bacilli (and the phages that have infected them). After the virucide has been neutralised, and phages that are released from infected cells are indicative of TB cells being present in the original specimen. These phages can be easily detected by propagation on lawns of non‐pathogenic rapidly growing mycobacteria. A commercially available test, termed FASTPlaqueTB‐RIF, determines the rifampicin susceptibility of isolates by comparing the viability of cells pre‐treated with and without rifampicin. Results give excellent agreement (97–98% overall) with conventional ‘gold standard’ methods, but in 2 days rather than 3 weeks. Preliminary work has shown that it should be possible to reduce the assay time to 24 h. A variation to this method, FASTPlaqueTB, detects TB directly from sputum specimens and is capable of rapidly reporting the presence of viable MTB cells within 48 h. This technique has achieved excellent specificity (close to 100%) when evaluated in the clinical setting. © 2001 Society of Chemical Industry     

Mycobacterium tuberculosis Rv1916 is an Acetyl-CoA-Binding Protein

Isocitrate lyase (ICL) isoform 2 is an essential enzyme for some clinical Mycobacterium tuberculosis (Mtb) strains during infection. In the laboratory Mtb strain H37Rv, the icl2 gene encodes two distinct gene products – Rv1915 and Rv1916 – due to a frameshift mutation. This study aims to characterise these two gene products to understand their structure and function. While we were unable to produce Rv1915 recombinantly, soluble Rv1916 was obtained with sufficient yield for characterisation. Kinetic studies using UV-visible spectrophotometry and ¹H-NMR spectroscopy showed that recombinant Rv1916 does not possess isocitrate lyase activity, while waterLOGSY binding experiments demonstrated that it could bind acetyl-CoA. Finally, X-ray crystallography revealed structural similarities between Rv1916 and the C-terminal domain of ICL2. Considering the probable differences between full-length ICL2 and the gene products Rv1915 and Rv1916, care must be taken when using Mtb H37Rv as a model organism to study central carbon metabolism.     

Exploring the Use of Medicinal Plants and Their Bioactive Derivatives as Alveolar NLRP3 Inflammasome Regulators during Mycobacterium tuberculosis Infection

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a successful intracellular pathogen that is responsible for the highest mortality rate among diseases caused by bacterial infections. During early interaction with the host innate cells, M. tuberculosis cell surface antigens interact with Toll like receptor 4 (TLR4) to activate the nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3 (NLRP3) canonical, and non-canonical inflammasome pathways. NLRP3 inflammasome activation in the alveoli has been reported to contribute to the early inflammatory response that is needed for an effective anti-TB response through production of pro-inflammatory cytokines, including those of the Interleukin 1 (IL1) family. However, overstimulation of the alveolar NLRP3 inflammasomes can induce excessive inflammation that is pathological to the host. Several studies have explored the use of medicinal plants and/or their active derivatives to inhibit excessive stimulation of the inflammasomes and its associated factors, thus reducing immunopathological response in the host. This review describes the molecular mechanism of the NLRP3 inflammasome activation in the alveoli during M. tuberculosis infection. Furthermore, the mechanisms of inflammasome inhibition using medicinal plant and their derivatives will also be explored, thus offering a novel perspective on the alternative control strategies of M. tuberculosis-induced immunopathology.     

Structure‐Guided Design of Thiazolidine Derivatives as Mycobacterium tuberculosis Pantothenate Synthetase Inhibitors

The pantothenate biosynthetic pathway is essential for the persistent growth and virulence of Mycobacterium tuberculosis (Mtb) and one of the enzymes in the pathway, pantothenate synthetase (PS, EC: 6.3.2.1), encoded by the panC gene, has become an appropriate target for new therapeutics to treat tuberculosis. Herein, we report nanomolar thiazolidine inhibitors of Mtb PS developed by a rational inhibitor design approach. The thiazolidine compounds were discovered by using energy‐based pharmacophore modelling and subsequent in vitro screening, which resulted in compounds with a half maximal inhibitory concentration (IC₅₀) value of (1.12±0.12) μM . These compounds were subsequently optimised by a combination of modelling and synthetic chemistry. Hit expansion of the lead by chemical synthesis led to an improved inhibitor with an IC₅₀ value of 350 nM and an Mtb minimum inhibitory concentration (MIC) of 1.55 μM . Some of these compounds also showed good activity against dormant Mtb cells.     

Biochemical Analysis of Leukocytes after In Vitro and In Vivo Activation with Bacterial and Fungal Pathogens Using Raman Spectroscopy

Biochemical information from activated leukocytes provide valuable diagnostic information. In this study, Raman spectroscopy was applied as a label-free analytical technique to characterize the activation pattern of leukocyte subpopulations in an in vitro infection model. Neutrophils, monocytes, and lymphocytes were isolated from healthy volunteers and stimulated with heat-inactivated clinical isolates of Candida albicans, Staphylococcus aureus, and Klebsiella pneumoniae. Binary classification models could identify the presence of infection for monocytes and lymphocytes, classify the type of infection as bacterial or fungal for neutrophils, monocytes, and lymphocytes and distinguish the cause of infection as Gram-negative or Gram-positive bacteria in the monocyte subpopulation. Changes in single-cell Raman spectra, upon leukocyte stimulation, can be explained with biochemical changes due to the leukocyte’s specific reaction to each type of pathogen. Raman spectra of leukocytes from the in vitro infection model were compared with spectra from leukocytes of patients with infection (DRKS-ID: DRKS00006265) with the same pathogen groups, and a good agreement was revealed. Our study elucidates the potential of Raman spectroscopy-based single-cell analysis for the differentiation of circulating leukocyte subtypes and identification of the infection by probing the molecular phenotype of those cells.     

Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.     

Substrate Fragmentation for the Design of M. tuberculosis CYP121 Inhibitors

The cyclo‐dipeptide substrates of the essential M. tuberculosis (Mtb) enzyme CYP121 were deconstructed into their component fragments and screened against the enzyme. A number of hits were identified, one of which exhibited an unexpected inhibitor‐like binding mode. The inhibitory pharmacophore was elucidated, and fragment binding affinity was rapidly improved by synthetic elaboration guided by the structures of CYP121 substrates. The resulting inhibitors have low micromolar affinity, good predicted physicochemical properties and selectivity for CYP121 over other Mtb P450s. Spectroscopic characterisation of the inhibitors′ binding mode provides insight into the effect of weak nitrogen‐donor ligands on the P450 heme, an improved understanding of factors governing CYP121–ligand recognition and speculation into the biological role of the enzyme for Mtb.