Manipulation of photosensory and circadian signaling restricts phenotypic plasticity in response to changing environmental conditions in Arabidopsis.

Plants exploit phenotypic plasticity to adapt their growth and development to prevailing environmental conditions. Interpretation of light and temperature signals is aided by the circadian system, which provides a temporal context. Phenotypic plasticity provides a selective and competitive advantage in nature but is obstructive during large-scale, intensive agricultural practices since economically important traits (including vegetative growth and flowering time) can vary widely depending on local environmental conditions. This prevents accurate prediction of harvesting times and produces a variable crop. In this study, we sought to restrict phenotypic plasticity and circadian regulation by manipulating signaling systems that govern plants' responses to environmental signals. Mathematical modeling of plant growth and development predicted reduced plant responses to changing environments when circadian and light signaling pathways were manipulated. We tested this prediction by utilizing a constitutively active allele of the plant photoreceptor phytochrome B, along with disruption of the circadian system via mutation of EARLY FLOWERING3. We found that these manipulations produced plants that are less responsive to light and temperature cues and thus fail to anticipate dawn. These engineered plants have uniform vegetative growth and flowering time, demonstrating how phenotypic plasticity can be limited while maintaining plant productivity. This has significant implications for future agriculture in both open fields and controlled environments.

Strathclyde minor groove binders (S-MGBs) with activity against Acanthamoeba castellanii.

BACKGROUND: Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms. OBJECTIVES: To synthesize and evaluate the anti-Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development. METHODS: A panel of 12 S-MGBs was synthesized and anti-Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei, Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. RESULTS AND DISCUSSION: S-MGB-241 has an IC50 of 6.6 µM against A. castellanii, comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50 > 100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a ΔTm = 1 ±â€Š0.1°C. CONCLUSIONS: Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii, providing a starting point from which to begin optimization of DNA binding and potency.

Spectroscopic, biochemical and computational studies of bioactive DNA minor groove binders targeting 5'-WGWWCW-3' motif.

Spectroscopic, biochemical, and computational modelling studies have been used to assess the binding capability of a set of minor groove binding (MGB) ligands against the self-complementary DNA sequences 5'-d(CGCACTAGTGCG)-3' and 5'-d(CGCAGTACTGCG)-3'. The ligands were carefully designed to target the DNA response element, 5'-WGWWCW-3', the binding site for several nuclear receptors. Basic 1D 1H NMR spectra of the DNA samples prepared with three MGB ligands show subtle variations suggestive of how each ligand associates with the double helical structure of both DNA sequences. The variations among the investigated ligands were reflected in the line shape and intensity of 1D 1H and 31P-{1H} NMR spectra. Rapid visual inspection of these 1D NMR spectra proves to be beneficial in providing valuable insights on MGB binding molecules. The NMR results were consistent with the findings from both UV DNA denaturation and molecular modelling studies. Both the NMR spectroscopic and computational analyses indicate that the investigated ligands bind to the minor grooves as antiparallel side-by-side dimers in a head-to-tail fashion. Moreover, comparisons with results from biochemical studies offered valuable insights into the mechanism of action, and antitumor activity of MGBs in relation to their structures, essential pre-requisites for future optimization of MGBs as therapeutic agents.

MptpB Inhibitor Improves the Action of Antibiotics against Mycobacterium tuberculosis and Nontuberculous Mycobacterium avium Infections.

Treatment of Mycobacterium tuberculosis and Mycobacterium avium infections requires multiple drugs for long time periods. Mycobacterium protein-tyrosine-phosphatase B (MptpB) is a key M. tuberculosis virulence factor that subverts host antimicrobial activity to promote intracellular survival. Inhibition of MptpB reduces the infection burden in vivo and offers new opportunities to improve current treatments. Here, we demonstrate that M. avium produces an MptpB orthologue and that the MptpB inhibitor C13 reduces the M. avium infection burden in macrophages. Combining C13 with the antibiotics rifampicin or bedaquiline showed an additive effect, reducing intracellular infection of both M. tuberculosis and M. avium by 50%, compared to monotreatment with antibiotics alone. This additive effect was not observed with pretomanid. Combining C13 with the minor groove-binding compounds S-MGB-362 and S-MGB-363 also reduced the M. tuberculosis intracellular burden. Similar additive effects of C13 and antibiotics were confirmed in vivo using Galleria mellonella infections. We demonstrate that the reduced mycobacterial burden in macrophages observed with C13 treatments is due to the increased trafficking to lysosomes.

Systematic review and meta-analysis of augmentation and combination treatments for early-stage treatment-resistant depression.

BACKGROUND: Major depressive disorder (MDD) is a highly burdensome health condition, for which there are numerous accepted pharmacological and psychological interventions. Adjunctive treatment (augmentation/combination) is recommended for the ~50% of MDD patients who do not adequately respond to first-line treatment. We aimed to evaluate the current evidence for concomitant approaches for people with early-stage treatment-resistant depression (TRD; defined below). METHODS: We systematically searched Medline and Institute for Scientific Information Web of Science to identify randomised controlled trials of adjunctive treatment of ⩾10 adults with MDD who had not responded to ⩾1 adequate antidepressant. The cochrane risk of bias (RoB) tool was used to assess study quality. Pre-post treatment meta-analyses were performed, allowing for comparison across heterogeneous study designs independent of comparator interventions. RESULTS: In total, 115 trials investigating 48 treatments were synthesised. The mean intervention duration was 9 weeks (range 5 days to 18 months) with most studies assessed to have low (n = 57) or moderate (n = 51) RoB. The highest effect sizes (ESs) were from cognitive behavioural therapy (ES = 1.58, 95% confidence interval (CI): 1.09-2.07), (es)ketamine (ES = 1.48, 95% CI: 1.23-1.73) and risperidone (ES = 1.42, 95% CI: 1.29-1.61). Only aripiprazole and lithium were examined in ⩾10 studies. Pill placebo (ES = 0.89, 95% CI: 0.81-0.98) had a not inconsiderable ES, and only six treatments' 95% CIs did not overlap with pill placebo's (aripiprazole, (es)ketamine, mirtazapine, olanzapine, quetiapine and risperidone). We report marked heterogeneity between studies for almost all analyses. CONCLUSIONS: Our findings support cautious optimism for several augmentation strategies; although considering the high prevalence of TRD, evidence remains inadequate for each treatment option.

Ratiometric imaging of minor groove binders in mammalian cells using Raman microscopy.

Quantitative drug imaging in live cells is a major challenge in drug discovery and development. Many drug screening techniques are performed in solution, and therefore do not consider the impact of the complex cellular environment in their result. As such, important features of drug-cell interactions may be overlooked. In this study, Raman microscopy is used as a powerful technique for semi-quantitative imaging of Strathclyde-minor groove binders (S-MGBs) in mammalian cells under biocompatible imaging conditions. Raman imaging determined the influence of the tail group of two novel minor groove binders (S-MGB-528 and S-MGB-529) in mammalian cell models. These novel S-MGBs contained alkyne moieties which enabled analysis in the cell-silent region of the Raman spectrum. The intracellular uptake concentration, distribution and mechanism were evaluated as a function of the pK a of the tail group, morpholine and amidine, for S-MGB-528 and S-MGB-529, respectively. Although S-MGB-529 had a higher binding affinity to the minor groove of DNA in solution-phase measurements, the Raman imaging data indicated that S-MGB-528 showed a greater degree of intracellular accumulation. Furthermore, using high resolution stimulated Raman scattering (SRS) microscopy, the initial localisation of S-MGB-528 was shown to be in the nucleus before accumulation in the lysosome, which was demonstrated using a multimodal imaging approach. This study highlights the potential of Raman spectroscopy for semi-quantitative drug imaging studies and highlights the importance of imaging techniques to investigate drug-cell interactions, to better inform the drug design process.

Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3.

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet-visible (UV-vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3's lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.

Selective Anti-Leishmanial Strathclyde Minor Groove Binders Using an N-Oxide Tail-Group Modification.

The neglected tropical disease leishmaniasis, caused by Leishmania spp., is becoming more problematic due to the emergence of drug-resistant strains. Therefore, new drugs to treat leishmaniasis, with novel mechanisms of action, are urgently required. Strathclyde minor groove binders (S-MGBs) are an emerging class of anti-infective agent that have been shown to have potent activity against various bacteria, viruses, fungi and parasites. Herein, it is shown that S-MGBs have potent activity against L. donovani, and that an N-oxide derivation of the tertiary amine tail of typical S-MGBs leads to selective anti-leishmanial activity. Additionally, using S-MGB-219, the N-oxide derivation is shown to retain strong binding to DNA as a 2:1 dimer. These findings support the further study of anti-leishmanial S-MGBs as novel therapeutics.

Multitargeted anti-infective drugs: resilience to resistance in the antimicrobial resistance era.

The standard drug discovery paradigm of single molecule - single biological target - single biological effect is perhaps particularly unsuitable for anti-infective drug discovery. This is due to the rapid evolution of resistance likely to be observed with single target drugs. Multitargeted anti-infective drugs are likely to be superior due to their lower susceptibility to target-related resistance mechanisms. Strathclyde minor groove binders are a class of compounds which have been developed by adopting the multitargeted anti-infective drugs paradigm, and their effectiveness against a wide range of pathogenic organisms is discussed. The renaming of this class to Strathclyde nucleic acid binders is also presented due to their likely targets including both DNA and RNA.

Intranasally administered S-MGB-364 displays antitubercular activity and modulates the host immune response to Mycobacterium tuberculosis infection.

BACKGROUND: Previously, we evaluated the intracellular mycobactericidal activity of the minor groove binder, S-MGB-364 against the clinical Mycobacterium tuberculosis (Mtb) strain HN878 in macrophages. OBJECTIVES: To assess the mycobactericidal activity of S-MGB-364 in Mtb-infected mice. Further, we investigated a plausible DNA binding mechanism of action of S-MGB-364. METHODS: The anti-TB and host immune effects of intranasal S-MGB-364 or S-MGB-364 encapsulated in non-ionic surfactant vesicles (NIV) were assessed in Mtb-infected mice by cfu enumeration, ELISA, histology, and flow cytometry. DNA binding was examined using native mass spectrometry and UV-vis thermal melt determination. S-MGB interference with DNA-centric biological events was assessed using a representative panel of Mtb and human topoisomerase I, and gyrase assays. RESULTS: S-MGB-364 bound strongly to DNA as a dimer, significantly increasing the stability of the DNA:S-MGB complex compared with DNA alone. Moreover, S-MGB-364 inhibited the relaxation of Mtb topoisomerase I but not the human form. In macrophages, S-MGB-364 or S-MGB-364-NIV did not cause DNA damage as shown by the low γ-H2AX expression. Importantly, in the lungs, the intranasal administration of S-MGB-364 or S-MGB-364-NIV formulation in Mtb-infected mice was non-toxic and resulted in a ∼1 log cfu reduction in mycobacterial burden, reduced the expression of proinflammatory cytokines/chemokines, altered immune cell recruitment, and importantly reduced recruitment of neutrophils. CONCLUSIONS: Together, these data provide proof of concept for S-MGBs as novel anti-TB therapeutics in vivo.

Catatonic Episodes Related to Substance Use: A Cross-Sectional Study Using Electronic Healthcare Records.

Objective: Substance use has increasingly been linked to the onset of catatonic episodes; however, no large observational studies have examined this association. This study aimed to identify catatonic episodes temporally associated with acute intoxication, withdrawal or chronic substance use, investigate which substances were involved, and compare clinical characteristics of substance-related and non-substance-related catatonic episodes. Methods: This study retrospectively identified all catatonic episodes recorded in an electronic case register hosted at a large secondary mental health trust in London, UK. Episodes were categorized as substance-related if the clinical record reported either a positive urine drug screen, an ICD-10 diagnosis of a mental or behavioral disorder due to substance use, or documented substance use between two weeks prior to the catatonic episode and the date of the catatonic episode. Results: 108 of 2130 catatonic episodes (5.1%) were deemed substance-related. The number of contemporaneously reported substance-related episodes increased between 2007 and 2016 [r = 0.72, p = 0.02]. Episodes in the context of acute intoxication (n = 54) were most frequently related to cannabis (n = 31) or cocaine (n = 5) use, whilst those in the context of drug withdrawal (n = 8) were most commonly related to alcohol, opioids and benzodiazepines. There were 50 episodes of catatonia associated with chronic substance use without intoxication or withdrawal, of which the majority were related to cannabis use (n = 37). 21 episodes had overlapping intoxication, withdrawal and chronic use of different substances within an episode. Compared to catatonic episodes not related to substance use, episodes of substance-related catatonia occurred in individuals who were younger (mean age 31.3 years [SD 12.2] vs 35.7 years [SD 16.3], p = 0.01) and more likely to be men (74.0% vs 54.3%, p < 0.001). The clinical features of catatonia were similar between the two groups. Conclusions: A relatively small proportion of catatonic episodes were temporally associated with reported substance use within their electronic records. Substance-related catatonic episodes were mostly related to cannabis use, but other substances including cocaine, alcohol, opioids and benzodiazepines were sometimes implicated. This is likely an underestimate of substance-related catatonia use due to issues with documentation and appropriate investigation.

Psychiatrists should investigate their patients less.

Psychiatrists often order investigations such as blood tests, neuroimaging and electroencephalograms for their patients. Rationales include ruling out 'organic' causes of psychiatric presentations, providing baseline parameters before starting psychotropic medications, and screening for general cardiometabolic health. Hospital protocols often recommend an extensive panel of blood tests on admission to a psychiatric ward. In this Against the Stream article, we argue that many of these investigations are at best useless and at worst harmful: the yield of positive findings that change clinical management is extremely low; special investigations are a poor substitute for a targeted history and examination; and incidental findings may cause anxiety and further unwarranted investigation. Cognitive and cultural reasons why over-investigation continues are discussed. We conclude by encouraging a more targeted approach guided by a thorough bedside clinical assessment.

Truncated S-MGBs: towards a parasite-specific and low aggregation chemotype.

This paper describes the design and synthesis of Strathclyde minor groove binders (S-MGBs) that have been truncated by the removal of a pyrrole ring in order to mimic the structure of the natural product, disgocidine. S-MGBs have been found to be active against many different organisms, however, selective antiparasitic activity is required. A panel of seven truncated S-MGBs was prepared and the activities examined against a number of clinically relevant organisms including several bacteria and parasites. The effect of the truncation strategy on S-MGB aggregation in aqueous environment was also investigated using 1H inspection and DOSY experiments. A lead compound, a truncated S-MGB, which possesses significant activity only against trypanosomes and Leishmania has been identified for further study and was also found to be less affected by aggregation compared to its full-length analogue.

Correction: Truncated S-MGBs: towards a parasite-specific and low aggregation chemotype.

[This corrects the article DOI: 10.1039/D1MD00110H.].

Cyrene™ is a green alternative to DMSO as a solvent for antibacterial drug discovery against ESKAPE pathogens.

Dimethyl sulfoxide (DMSO) is currently employed across the biomedical field, from cryopreservation to in vitro assays, despite the fact that it has been shown to have an assortment of biologically relevant effects. The amphiphilic nature of DMSO along with its relatively low toxicity at dilute concentrations make it a challenging solvent to replace. A possible alternative is Cyrene™ (dihydrolevoglucosenone), an aprotic dipolar solvent that is derived from waste biomass. In addition to being a green solvent, Cyrene™ has comparable solvation properties and is reported to have low toxicity. Herein the abilities of the two solvents to solubilize drug compounds and to act as non-participatory vehicles in drug discovery for antibacterials are compared. It was demonstrate that the results of standardised antimicrobial susceptibility testing do not differ between drugs prepared from either Cyrene™ or DMSO stock. Moreover, in contrast to DMSO, Cyrene™ does not offer protection from ROS mediated killing of bacteria and may therefore be an improvement over DMSO as a vehicle in antimicrobial drug discovery.

Inflammatory proteins are altered in chronic fatigue syndrome-A systematic review and meta-analysis.

Immune dysfunction has been posited as a key element in the aetiology of chronic fatigue syndrome (CFS) since the illness was first conceived. However, systematic reviews have yet to quantitatively synthesise inflammatory biomarkers across the literature. We undertook a systematic review and meta-analysis to quantify available data on circulating inflammatory proteins, examining studies recruiting patients with a CFS diagnosis and a non-affected control group. Results were meta-analysed from 42 studies. Patients with CFS had significantly elevated tumour necrosis factor (ES = 0.274, p < 0.001), interleukin-2 (ES = 0.203, p = 0.006), interleukin-4 (ES = 0.373, p = 0.004), transforming growth factor-ß (ES = 0.967, p < 0.001) and c-reactive protein (ES = 0.622, p = 0.019). 12 proteins did not differ between groups. These data provide some support for an inflammatory component in CFS, although inconsistency of results indicates that inflammation is unlikely to be a primary feature in all those suffering from this disorder. It is hoped that further work will elucidate whether there are subgroups of patients with clinically-relevant inflammatory dysfunction, and whether inflammatory cytokines may provide a prognostic biomarker or moderate treatment effects.

Novel Minor Groove Binders Cure Animal African Trypanosomiasis in an in Vivo Mouse Model.

Animal African trypanosomiasis (AAT) is a significant socioeconomic burden for sub-Saharan Africa because of its huge impact on livestock health. Existing therapies including those based on minor groove binders (MGBs), such as the diamidines, which have been used for decades, have now lost efficacy in some places because of the emergence of resistant parasites. Consequently, the need for new chemotherapies is urgent. Here, we describe a structurally distinct class of MGBs, Strathclyde MGBs (S-MGBs), which display excellent in vitro activities against the principal causative organisms of AAT: Trypanosoma congolense, and Trypanosoma vivax. We also show the cure of T. congolense-infected mice by a number of these compounds. In particular, we identify S-MGB-234, compound 7, as curative by using two applications of 50 mg/kg intraperitoneally. Crucially, we demonstrate that S-MGBs do not show cross-resistance with the current diamidine drugs and are not internalized via the transporters used by diamidines. This study demonstrates that S-MGBs have significant potential as novel therapeutic agents for AAT.

Selective in vitro anti-cancer activity of non-alkylating minor groove binders.

Traditional cytotoxic agents which act through a DNA-alkylating mechanism are relatively non-specific, resulting in a small therapeutic window and thus limiting their effectiveness. In this study, we evaluate a panel of 24 non-alkylating Strathclyde Minor Groove Binders (S-MGBs), including 14 novel compounds, for in vitro anti-cancer activity against a human colon carcinoma cell line, a cisplatin-sensitive ovarian cancer cell line and a cisplatin-resistant ovarian cancer cell line. A human non-cancerous retinal epithelial cell line was used to measure selectivity of any response. We have identified several S-MGBs with activities comparable to cis-platin and carboplatin, but with better in vitro selectivity indices, particularly S-MGB-4, S-MGB-74 and S-MGB-317. Moreover, a comparison of the cis-platin resistant and cis-platin sensitive ovarian cancer cell lines reveals that our S-MGBs do not show cross resistance with cisplatin or carboplatin and that they likely have a different mechanism of action. Finally, we present an initial investigation into the mechanism of action of one compound from this class, S-MGB-4, demonstrating that neither DNA double strand breaks nor the DNA damage stress sensor protein p53 are induced. This indicates that our S-MGBs are unlikely to act through an alkylating or DNA damage response mechanism.

Changes in insulin, glucagon and ER stress precede immune activation in type 1 diabetes.

It is unknown whether there is a gene signature in pancreas which is associated with type 1 diabetes (T1D). We performed partial pancreatectomies on 30-day preinsulitic, diabetes-prone BioBreeding (BBdp) rats to prospectively identify factors involved in early prediabetes. Microarrays of the biopsies revealed downregulation of endoplasmic reticulum (ER) stress, metabolism and apoptosis. Based on these results, additional investigations compared gene expression in control (BBc) and BBdp rats age ~8, 30 and 60 days using RT-qPCR. Neonates had increased ER stress gene expression in pancreas. This was associated with decreased insulin, cleaved caspase-3 and Ins1 whereas Gcg and Pcsk2 were increased. The increase in ER stress was not sustained at 30 days and decreased by 60 days. In parallel, the liver gene profile showed a similar signature in neonates but with an early decrease of the unfolded protein response (UPR) at 30 days. This suggested that changes in the liver precede those in the pancreas. Tnf and Il1b expression was increased in BBdp pancreas in association with increased caspase-1, cleaved caspase-3 and decreased proinsulin area. Glucagon area was increased in both 30-day and 60-day BBdp rats. Increased colocalization of BIP and proinsulin was observed at 60 days in the pancreas, suggesting insulin-related ER dysfunction. We propose that dysregulated metabolism leads to ER stress in neonatal rats long before insulitis, creating a microenvironment in both pancreas and liver that promotes autoimmunity.