25 research outputs found
Molecular tools to unravel the mechanism of action of M. tuberculosis phenotypic hits
No full textUntil early 2020, tuberculosis (TB) was the deadliest disease caused by a single infectious agent. While Covid-19 has since gained the morbid accolade of the most lethal pathogen, TB remains a major threat to global health. The increasing rate of multi drug resistant (MDR) and extensively drug resistant (XDR) TB has led to the indisputable need for new anti-TB drugs. The primary aim of this research has been to investigate hits from drug screening programmes and, through the application of microbiological and molecular techniques, elucidate their targets. With a focus on re-examining existing therapeutics in the context of TB drug discovery, we tested three distinct groups of drugs to explore their activity against Mycobacterium tuberculosis (Mtb): BM212 series inhibitors of the transporter protein MmpL3; human diacylglycerol transferase (DGAT1) inhibitors and human kinase inhibitors. Inhibitors of MmpL3 are notoriously structurally diverse, and it is not uncommon for them to have alternative targets. Using chemoproteomic profiling the BM212 series of inhibitors were screened for alternative protein targets, revealing the binding of BM212 analogue compounds to the transcriptional regulator EthR2, which was confirmed using an in vitro tryptophan fluorescence assay. In the second chapter, we have explored the anti-TB activity of human DGAT1 inhibitors. Mtb is able to utilise host triacylglycerol as a source of energy and carbon and can store large amounts of TAG as intracellular lipid inclusion bodies (ILIs). Preliminary research showed that inhibiting host DGAT1 reduces TB infection in macrophages; we have demonstrated the activity of DGAT1 inhibitors against Mtb H37Rv and M. bovis BCG in vitro. Using a combination of whole cell assays, lipid profiling, and biochemical assays, we have observed the inhibition of bacterial triacylglycerol synthases by a DGAT1 inhibitor and have identified Tgs3 as the DGAT1 inhibitor protein target. Finally, we have selected phenotypic hits from a screen of human kinase inhibitors for their activity against Mtb and through the sequencing of spontaneous resistant mutants have identified a number of potential targets in Mtb
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Observations of the 57Fe+23 hyperfine transition in clusters of galaxies
No full textWe present a search for the hyperfine transition of the 57Fe+23 ion at 3.071 mm in clusters of galaxies with the ATNF Mopra telescope. The results are compared with a realistic estimate of the peak brightness temperature of the line in a cooling flow cluster A85, using the available X-ray data
The role of triacylglycerols and repurposing DGAT1 inhibitors for the treatment of Mycobacterium tuberculosis
No full textLatent tuberculosis poses a significant threat to global health through the incubation of undiagnosed infections within the community, and through its tolerance to antibiotics. This Special Features article explores the mechanisms by which the dormant Mycobacterium tuberculosis pathogen can store energy in the form of lipid inclusion bodies and triacylglycerols, which may be key in the development of novel therapeutics to treat TB
HIV drugs inhibit transfer of plasmids carrying extended-spectrum β-lactamase and carbapenemase genes
No full textAntimicrobial-resistant (AMR) infections pose a serious risk to human and animal health. A major factor contributing to this global crisis is the sharing of resistance genes between different bacteria via plasmids. The WHO lists Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae, producing extended-spectrum β-lactamases (ESBL) and carbapenemases as "critical" priorities for new drug development. These resistance genes are most often shared via plasmid transfer. However, finding methods to prevent resistance gene sharing has been hampered by the lack of screening systems for medium-/high-throughput approaches. Here, we have used an ESBL-producing plasmid, pCT, and a carbapenemase-producing plasmid, pKpQIL, in two different Gram-negative bacteria, E. coli and K. pneumoniae Using these critical resistance-pathogen combinations, we developed an assay using fluorescent proteins, flow cytometry, and confocal microscopy to assess plasmid transmission inhibition within bacterial populations in a medium-throughput manner. Three compounds with some reports of antiplasmid properties were tested; chlorpromazine reduced transmission of both plasmids and linoleic acid reduced transmission of pCT. We screened the Prestwick library of over 1,200 FDA-approved drugs/compounds. From this, we found two nucleoside analogue drugs used to treat HIV, abacavir and azidothymidine (AZT), which reduced plasmid transmission (AZT, e.g., at 0.25 μg/ml reduced pCT transmission in E. coli by 83.3% and pKpQIL transmission in K. pneumoniae by 80.8% compared to untreated controls). Plasmid transmission was reduced by concentrations of the drugs which are below peak serum concentrations and are achievable in the gastrointestinal tract. These drugs could be used to decolonize humans, animals, or the environment from AMR plasmids.Importance: More and more bacterial infections are becoming resistant to antibiotics. This has made treatment of many infections very difficult. One of the reasons this is such a large problem is that bacteria are able to share their genetic material with other bacteria, and these shared genes often include resistance to a variety of antibiotics, including some of our drugs of last resort. We are addressing this problem by using a fluorescence-based system to search for drugs that will stop bacteria from sharing resistance genes. We uncovered a new role for two drugs used to treat HIV and show that they are able to prevent the sharing of two different types of resistance genes in two unique bacterial strains. This work lays the foundation for future work to reduce the prevalence of resistant infections.</p
Memory, attention and fluency deficits in COPD may be a specific form of cognitive impairment
No full textThere is increasing evidence demonstrating an association between chronic obstructive pulmonary disease (COPD) and cognitive impairment. We present a narrative review of published studies on the subject and a cross-sectional study investigating domain-specific cognitive impairment in people with COPD compared to people with known Alzheimer's dementia, and controls without known COPD or cognitive impairment. The aim of the study was to compare prevalence and pattern of cognitive impairment between the three groups using the Addenbrooke's Cognitive Examination (ACE)-III tool. A total of 89 participants were recruited (44 with COPD, 17 with Alzheimer's and 28 controls). Patients with COPD had significantly lower total ACE-III scores than controls (p<0.001). When comparing the COPD group to the known Alzheimer's dementia group, overall ACE-III scores were significantly lower in the Alzheimer's dementia group than the COPD group (p=0.019). The domain-specific scores for attention (p<0.004), memory (p<0.004) and fluency (p<0.001) were significantly lower in the Alzheimer's dementia group than the COPD group. Our result suggest that the COPD group were significantly more likely to have cognitive impairment than the healthy control group. This was supported by the results of a narrative review of the published literature. Our results show that the pattern of impairment in the COPD group is different to the pattern of impairment shown in the known Alzheimer's dementia group, with significant differences in the cognitive domains affected. These results are in keeping with the findings of other previously published studies included in the narrative review
Antibiotics and resistance:the two-sided coin of the mycobacterial cell wall
No full textMycobacterium tuberculosis, the bacterium responsible for tuberculosis, is the global leading cause of mortality from an infectious agent. Part of this success relies on the unique cell wall, which consists of a thick waxy coat with tightly packed layers of complexed sugars, lipids and peptides. This coat provides a protective hydrophobic barrier to antibiotics and the host's defences, while enabling the bacterium to spread efficiently through sputum to infect and survive within the macrophages of new hosts. However, part of this success comes at a cost, with many of the current first- and second-line drugs targeting the enzymes involved in cell wall biosynthesis. The flip side of this coin is that resistance to these drugs develops either in the target enzymes or the activation pathways of the drugs, paving the way for new resistant clinical strains. This review provides a synopsis of the structure and synthesis of the cell wall and the major current drugs and targets, along with any mechanisms of resistance.</p
The singular Corynebacterium glutamicum Emb arabinofuranosyltransferase polymerises the α(1 → 5) arabinan backbone in the early stages of cell wall arabinan biosynthesis
No full textThe arabinan-containing polysaccharides, arabinogalactan (AG) and lipoarabinomannan (LAM), are key cell wall components of the Corynebacterineae, which include Corynebacteria, Norcadia and Mycobacteria. Both AG and LAM contain elaborate arabinan domains composed of distinct structural motifs. Mycobacterial EmbA, EmbB and EmbC, collectively known as the Emb proteins, have been identified as arabinosyltransferases (ArafTs), which are targeted by the front-line anti-tubercular drug ethambutol. Previous studies have established that EmbA and EmbB play a role in the synthesis of the characteristic terminal hexa-arabinosuranosyl motif, whilst EmbC is involved exclusively in the biosynthesis of LAM. Herein, we have investigated the role of the singular Emb protein from Corynebacterium glutamicum through the detailed biochemical and chemical analysis of a double ΔaftAΔemb mutant, where the priming Cg-AftA protein, which generates the substrate for Cg-Emb has been deleted. Analysis of its cell wall revealed a complete absence of arabinose resulting in a truncated cell wall containing only a galactan backbone accompanied with complete loss of cell wall bound mycolates. In vitro cell-free assays using C. glutamicumΔaftA, C. glutamicumΔemb, C. glutamicumΔaftAΔemb and C. glutamicumΔaftBΔaftD and two synthetic acceptors, which mimick the arabinofuranose (Araf) “primed” galactan chain, demonstrated that Cg-Emb is able to transfer an Araf residue to the C5 of the Araf positioned on the synthetic acceptor(s). These results indicate that Cg-Emb acts as an α(1 → 5) ArafT and elongates the arabinan core during the early stages of arabinan biosynthesis in C. glutamicum
The loss of a child: the long term impact upon the parent-child bond
No full textResearch has been carried out from a psychological perspective, to examine the effects of bereavement in families when they have experienced the loss of a baby, child, teenager or young adult. This has involved interviewing parents in Lebanon, Tanzania and Uganda. The results were then compared to previous research carried out by the author in England (For a M.Phil. in Theology titled, ‘Bereavement in Families who have lost Babies, Children and Teenagers; An Empirical and Theological Study’. Birmingham University , 1995). Using the collective data, the theory of Shadow Grief is investigated in terms of whether it is a genuine condition within bereaved parents, as compared to other grief reactions such as chronic grief, disenfranchised grief or pathological grief.
It was found that the bond between a parent and child was a particularly deep rooted affectionate bond. There are similarities between this bond and Bowlby’s concept of attachment theory. Parents from the English sample showed some signs of maintaining a bond with the deceased many years after the loss. This was seen to a lesser extent in the African context. This requires further research to clarify this effect both in the English culture and cross-culturally, looking at a broader section of communities where child loss had taken place. Grief therapists need to be more aware of the long lasting effects that the loss of a child has upon a parent, especially those who are bereaved of older children
The multi-target aspect of an MmpL3 inhibitor:the BM212 series of compounds bind EthR2, a transcriptional regulator of ethionamide activation
No full textThe emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) ensures that drug discovery efforts remain at the forefront of TB research. There are multiple different experimental approaches that can be employed in the discovery of anti-TB agents. Notably, inhibitors of MmpL3 are numerous and structurally diverse in Mtb and have been discovered through the generation of spontaneous resistant mutants and subsequent whole genome sequencing studies. However, this approach is not always reliable and can lead to incorrect target assignment and requires orthogonal confirmatory approaches. In fact, many of these inhibitors have also been shown to act as multi-target agents, with secondary targets in Mtb, as well as in other non-MmpL3-containing pathogens. Herein, we have investigated further the cellular targets of the MmpL3-inhibitor BM212 and a number of BM212 analogues. To determine the alternative targets of BM212, which may have been masked by MmpL3 mutations, we have applied a combination of chemo-proteomic profiling using bead-immobilised BM212 derivatives and protein extracts, along with whole-cell and biochemical assays. The study identified EthR2 (Rv0078) as a protein that binds BM212 analogues. We further demonstrated binding of BM212 to EthR2 through an in vitro tryptophan fluorescence assay, which showed significant quenching of tryptophan fluorescence upon addition of BM212. Our studies have demonstrated the value of revisiting drugs with ambiguous targets, such as MmpL3, in an attempt to find alternative targets and the study of off-target effects to understand more precisely target engagement of new hits emerging from drug screening campaigns.</p
Life beckoning. A thematic analysis of change in a deprived boy in long-term foster care, during intensive psychoanalytic psychotherapy
No full textThis research is based on a single case study of psychoanalytic therapy with a young adolescent boy in care. It is part of a growing movement to identify research methods for exploring the place of unconscious expectations, emotion and affect, in relationships. It experiments with methods for testing out psychoanalytic theory and contributing findings to evidence, modify or expand theory in new directions.
The patient Simon, had a history of deprivation and showed many features of ADHD and oppositional conduct disorder. The research locates him in a “family” of children who share histories of early traumas and serious behavioural difficulties. Therefore findings, while grounded in clinical material from a single case, and restricted in scope, are of relevance to work with a very needy and challenging population of children, who are often a major cause of concern to their carers, teachers, social workers and to mentalhealth professionals.
The research examines clinical material through the framework of Bion’s theoretical claim that identifies thinking as at bottom an emotional process, and relates symbolic capacity to early emotional experiences of communication and containment. The framework was selected because of its relevance to the particular features of the patient, which emerged through the detailed study of session records. The analysis of patient therapist
interaction follows Bion in looking at thinking and learning, side by side with the sort of internal objects active in the therapeutic relationship, and the emotions
connected to them.
Through a detailed focus on these aspects of clinical material, the author assesses some current ideas about what interferes with a deprived child’s capacity to think and learn from experience; and what are the factors in a therapeutic relationship that can help a child’s capacity in these areas to grow
