A novel AST2 mutation generated upon whole-genome transformation of Saccharomyces cerevisiae confers high tolerance to 5-Hydroxymethylfurfural (HMF) and other inhibitors.

Development of cell factories for conversion of lignocellulosic biomass hydrolysates into biofuels or bio-based chemicals faces major challenges, including the presence of inhibitory chemicals derived from biomass hydrolysis or pretreatment. Extensive screening of 2526 Saccharomyces cerevisiae strains and 17 non-conventional yeast species identified a Candida glabrata strain as the most 5-hydroxymethylfurfural (HMF) tolerant. Whole-genome (WG) transformation of the second-generation industrial S. cerevisiae strain MD4 with genomic DNA from C. glabrata, but not from non-tolerant strains, allowed selection of stable transformants in the presence of HMF. Transformant GVM0 showed the highest HMF tolerance for growth on plates and in small-scale fermentations. Comparison of the WG sequence of MD4 and GVM1, a diploid segregant of GVM0 with similarly high HMF tolerance, surprisingly revealed only nine non-synonymous SNPs, of which none were present in the C. glabrata genome. Reciprocal hemizygosity analysis in diploid strain GVM1 revealed AST2N406I as the only causative mutation. This novel SNP improved tolerance to HMF, furfural and other inhibitors, when introduced in different yeast genetic backgrounds and both in synthetic media and lignocellulose hydrolysates. It stimulated disappearance of HMF and furfural from the medium and enhanced in vitro furfural NADH-dependent reducing activity. The corresponding mutation present in AST1 (i.e. AST1D405I) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2N406I and in presence of high inhibitor concentrations. Our work provides a powerful genetic tool to improve yeast inhibitor tolerance in lignocellulosic biomass hydrolysates and other inhibitor-rich industrial media, and it has revealed for the first time a clear function for Ast2 and Ast1 in inhibitor tolerance.

Progression from remodeling to hibernation of ribosomes in zinc-starved mycobacteria.

Zinc starvation in mycobacteria leads to remodeling of ribosomes, in which multiple ribosomal (r-) proteins containing the zinc-binding CXXC motif are replaced by their motif-free paralogues, collectively called C- r-proteins. We previously reported that the 70S C- ribosome is exclusively targeted for hibernation by mycobacterial-specific protein Y (Mpy), which binds to the decoding center and stabilizes the ribosome in an inactive and drug-resistant state. In this study, we delineate the conditions for ribosome remodeling and hibernation and provide further insight into how zinc depletion induces Mpy recruitment to C- ribosomes. Specifically, we show that ribosome hibernation in a batch culture is induced at an approximately two-fold lower cellular zinc concentration than remodeling. We further identify a growth phase in which the C- ribosome remains active, while its hibernation is inhibited by the caseinolytic protease (Clp) system in a zinc-dependent manner. The Clp protease system destabilizes a zinc-bound form of Mpy recruitment factor (Mrf), which is stabilized upon further depletion of zinc, presumably in a zinc-free form. Stabilized Mrf binds to the 30S subunit and recruits Mpy to the ribosome. Replenishment of zinc to cells harboring hibernating ribosomes restores Mrf instability and dissociates Mpy from the ribosome. Finally, we demonstrate zinc-responsive binding of Mpy to ribosomes in Mycobacterium tuberculosis (Mtb) and show Mpy-dependent antibiotic tolerance of Mtb in mouse lungs. Together, we propose that ribosome hibernation is a specific and conserved response to zinc depletion in both environmental and pathogenic mycobacteria.

Elucidating aromatic acid tolerance at low pH in Saccharomyces cerevisiae using adaptive laboratory evolution.

Toxicity from the external presence or internal production of compounds can reduce the growth and viability of microbial cell factories and compromise productivity. Aromatic compounds are generally toxic for microorganisms, which makes their production in microbial hosts challenging. Here we use adaptive laboratory evolution to generate Saccharomyces cerevisiae mutants tolerant to two aromatic acids, coumaric acid and ferulic acid. The evolution experiments were performed at low pH (3.5) to reproduce conditions typical of industrial processes. Mutant strains tolerant to levels of aromatic acids near the solubility limit were then analyzed by whole genome sequencing, which revealed prevalent point mutations in a transcriptional activator (Aro80) that is responsible for regulating the use of aromatic amino acids as the nitrogen source. Among the genes regulated by Aro80, ESBP6 was found to be responsible for increasing tolerance to aromatic acids by exporting them out of the cell. Further examination of the native function of Esbp6 revealed that this transporter can excrete fusel acids (byproducts of aromatic amino acid catabolism) and this role is shared with at least one additional transporter native to S. cerevisiae (Pdr12). Besides conferring tolerance to aromatic acids, ESBP6 overexpression was also shown to significantly improve the secretion in coumaric acid production strains. Overall, we showed that regulating the activity of transporters is a major mechanism to improve tolerance to aromatic acids. These findings can be used to modulate the intracellular concentration of aromatic compounds to optimize the excretion of such products while keeping precursor molecules inside the cell.

Remdesivir targets a structurally analogous region of the Ebola virus and SARS-CoV-2 polymerases.

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.

Severe delayed hypersensitivity reactions to IL-1 and IL-6 inhibitors link to common HLA-DRB1*15 alleles.

OBJECTIVES: Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, delayed hypersensitivity reaction (DHR). We observed DRESS to inhibitors of interleukin 1 (IL-1) or IL-6 in a small group of patients with Still's disease with atypical lung disease. We sought to characterise features of patients with Still's disease with DRESS compared with drug-tolerant Still's controls. We analysed human leucocyte antigen (HLA) alleles for association to inhibitor-related DHR, including in a small Kawasaki disease (KD) cohort. METHODS: In a case/control study, we collected a multicentre series of patients with Still's disease with features of inhibitor-related DRESS (n=66) and drug-tolerant Still's controls (n=65). We retrospectively analysed clinical data from all Still's subjects and typed 94/131 for HLA. European Still's-DRESS cases were ancestry matched to International Childhood Arthritis Genetics Consortium paediatric Still's cases (n=550) and compared for HLA allele frequencies. HLA association also was analysed using Still's-DRESS cases (n=64) compared with drug-tolerant Still's controls (n=30). KD subjects (n=19) were similarly studied. RESULTS: Still's-DRESS features included eosinophilia (89%), AST-ALT elevation (75%) and non-evanescent rash (95%; 88% involving face). Macrophage activation syndrome during treatment was frequent in Still's-DRESS (64%) versus drug-tolerant Still's (3%; p=1.2×10-14). We found striking enrichment for HLA-DRB1*15 haplotypes in Still's-DRESS cases versus INCHARGE Still's controls (p=7.5×10-13) and versus self-identified, ancestry-matched Still's controls (p=6.3×10-10). In the KD cohort, DRB1*15:01 was present only in those with suspected anakinra reactions. CONCLUSIONS: DRESS-type reactions occur among patients treated with IL-1/IL-6 inhibitors and strongly associate with common HLA-DRB1*15 haplotypes. Consideration of preprescription HLA typing and vigilance for serious reactions to these drugs are warranted.

Microbial phenotypic heterogeneity in response to a metabolic toxin: Continuous, dynamically shifting distribution of formaldehyde tolerance in Methylobacterium extorquens populations.

While microbiologists often make the simplifying assumption that genotype determines phenotype in a given environment, it is becoming increasingly apparent that phenotypic heterogeneity (in which one genotype generates multiple phenotypes simultaneously even in a uniform environment) is common in many microbial populations. The importance of phenotypic heterogeneity has been demonstrated in a number of model systems involving binary phenotypic states (e.g., growth/non-growth); however, less is known about systems involving phenotype distributions that are continuous across an environmental gradient, and how those distributions change when the environment changes. Here, we describe a novel instance of phenotypic diversity in tolerance to a metabolic toxin within wild-type populations of Methylobacterium extorquens, a ubiquitous phyllosphere methylotroph capable of growing on the methanol periodically released from plant leaves. The first intermediate in methanol metabolism is formaldehyde, a potent cellular toxin that is lethal in high concentrations. We have found that at moderate concentrations, formaldehyde tolerance in M. extorquens is heterogeneous, with a cell's minimum tolerance level ranging between 0 mM and 8 mM. Tolerant cells have a distinct gene expression profile from non-tolerant cells. This form of heterogeneity is continuous in terms of threshold (the formaldehyde concentration where growth ceases), yet binary in outcome (at a given formaldehyde concentration, cells either grow normally or die, with no intermediate phenotype), and it is not associated with any detectable genetic mutations. Moreover, tolerance distributions within the population are dynamic, changing over time in response to growth conditions. We characterized this phenomenon using bulk liquid culture experiments, colony growth tracking, flow cytometry, single-cell time-lapse microscopy, transcriptomics, and genome resequencing. Finally, we used mathematical modeling to better understand the processes by which cells change phenotype, and found evidence for both stochastic, bidirectional phenotypic diversification and responsive, directed phenotypic shifts, depending on the growth substrate and the presence of toxin.

Phase variation in Mycobacterium tuberculosis glpK produces transiently heritable drug tolerance.

The length and complexity of tuberculosis (TB) therapy, as well as the propensity of Mycobacterium tuberculosis to develop drug resistance, are major barriers to global TB control efforts. M. tuberculosis is known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a mechanism of genetically encoded but rapidly reversible drug tolerance in M. tuberculosis caused by transient frameshift mutations in a homopolymeric tract (HT) of 7 cytosines (7C) in the glpK gene. Inactivating frameshift mutations associated with the 7C HT in glpK produce small colonies that exhibit heritable multidrug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the same glpK HT region. These reversible frameshift mutations in the 7C HT of M. tuberculosis glpK occur in clinical isolates, accumulate in M. tuberculosis-infected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction of dosR and sigH and repression of kstR regulons, similar to that observed in other in vitro models of M. tuberculosis tolerance. These results suggest that GlpK phase variation may contribute to drug tolerance, treatment failure, and relapse in human TB. Drugs effective against phase-variant M. tuberculosis may hasten TB treatment and improve cure rates.

Epistasis between antibiotic tolerance, persistence, and resistance mutations.

Understanding the evolution of microorganisms under antibiotic treatments is a burning issue. Typically, several resistance mutations can accumulate under antibiotic treatment, and the way in which resistance mutations interact, i.e., epistasis, has been extensively studied. We recently showed that the evolution of antibiotic resistance in Escherichia coli is facilitated by the early appearance of tolerance mutations. In contrast to resistance, which reduces the effectiveness of the drug concentration, tolerance increases resilience to antibiotic treatment duration in a nonspecific way, for example when bacteria transiently arrest their growth. Both result in increased survival under antibiotics, but the interaction between resistance and tolerance mutations has not been studied. Here, we extend our analysis to include the evolution of a different type of tolerance and a different antibiotic class and measure experimentally the epistasis between tolerance and resistance mutations. We derive the expected model for the effect of tolerance and resistance mutations on the dynamics of survival under antibiotic treatment. We find that the interaction between resistance and tolerance mutations is synergistic in strains evolved under intermittent antibiotic treatment. We extend our analysis to mutations that result in antibiotic persistence, i.e., to tolerance that is conferred only on a subpopulation of cells. We show that even when this population heterogeneity is included in our analysis, a synergistic interaction between antibiotic persistence and resistance mutations remains. We expect our general framework for the epistasis in killing conditions to be relevant for other systems as well, such as bacteria exposed to phages or cancer cells under treatment.

The Impact of P-Glycoprotein on Opioid Analgesics: What's the Real Meaning in Pain Management and Palliative Care?

Opioids are widely used in cancer and non-cancer pain management. However, many transporters at the blood-brain barrier (BBB), such as P-glycoprotein (P-gp, ABCB1/MDR1), may impair their delivery to the brain, thus leading to opioid tolerance. Nonetheless, opioids may regulate P-gp expression, thus altering the transport of other compounds, namely chemotherapeutic agents, resulting in pharmacoresistance. Other kinds of painkillers (e.g., acetaminophen, dexamethasone) and adjuvant drugs used for neuropathic pain may act as P-gp substrates and modulate its expression, thus making pain management challenging. Inflammatory conditions are also believed to upregulate P-gp. The role of P-gp in drug-drug interactions is currently under investigation, since many P-gp substrates may also act as substrates for the cytochrome P450 enzymes, which metabolize a wide range of xenobiotics and endobiotics. Genetic variability of the ABCB1/MDR1 gene may be accountable for inter-individual variation in opioid-induced analgesia. P-gp also plays a role in the management of opioid-induced adverse effects, such as constipation. Peripherally acting mu-opioid receptors antagonists (PAMORAs), such as naloxegol and naldemedine, are substrates of P-gp, which prevent their penetration in the central nervous system. In our review, we explore the interactions between P-gp and opioidergic drugs, with their implications in clinical practice.

Activation of spinal PDGFRß in microglia promotes neuronal autophagy via p38 MAPK pathway in morphine-tolerant rats.

The adverse side effects of opioids, especially antinociceptive tolerance, limit their clinical application. A recent study reported that platelet-derived growth factor receptor ß (PDGFRß) blockage selectively inhibited morphine tolerance. Autophagy has been reported to contribute to the cellular and behavioral responses to morphine. However, little is known about the relationship between PDGFRß and autophagy in the mechanisms of morphine tolerance. In this study, rats were intrathecally administered with morphine twice daily for 7 days to induce antinociceptive tolerance, which was evaluated using a tail-flick latency test. By administration autophagy inhibitor 3-Methyladenine, PDGFRß inhibitor imatinib, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 hydrochloride and minocycline hydrochloride, western blot, immunofluorescence, and transmission electron microscopy techniques were used to elucidate the roles of PDGFRß, autophagy, and related signaling pathways in morphine tolerance. This study demonstrated for the first time that spinal PDGFRß in microglia promotes autophagy in gamma-aminobutyric acid (GABA) interneurons through activating p38 MAPK pathway during the development of morphine tolerance, which suggest a potential strategy for preventing the development of morphine tolerance clinically, thereby improving the use of opioids in pain management.

Deubiquitinating enzyme inhibitor alleviates cyclin A1-mediated proteasome inhibitor tolerance in mixed-lineage leukemia.

Drug resistance is a significant obstacle to effective cancer treatment. Drug resistance develops from initially reversible drug-tolerant cancer cells, which offer therapeutic opportunities to impede cancer relapse. The mechanisms of resistance to proteasome inhibitor (PI) therapy have been investigated intensively, however the ways by which drug-tolerant cancer cells orchestrate their adaptive responses to drug challenges remain largely unknown. Here, we demonstrated that cyclin A1 suppression elicited the development of transient PI tolerance in mixed-lineage leukemia (MLL) cells. This adaptive process involved reversible downregulation of cyclin A1, which promoted PI resistance through cell-cycle arrest. PI-tolerant MLL cells acquired cyclin A1 dependency, regulated directly by MLL protein. Loss of cyclin A1 function resulted in the emergence of drug tolerance, which was associated with patient relapse and reduced survival. Combination treatment with PI and deubiquitinating enzyme (DUB) inhibitors overcame this drug resistance by restoring cyclin A1 expression through chromatin crosstalk between histone H2B monoubiquitination and MLL-mediated histone H3 lysine 4 methylation. These results reveal the importance of cyclin A1-engaged cell-cycle regulation in PI resistance in MLL cells, and suggest that cell-cycle re-entry by DUB inhibitors may represent a promising epigenetic therapeutic strategy to prevent acquired drug resistance.

Heterogeneity in populations of enterohaemorrhagic Escherichia coli undergoing D-serine adaptation.

Phenotypic and genetic heterogeneities are conserved features of prokaryotic populations. During periods of stress, this programmed diversity increases the likelihood that variants within the population will survive the adverse conditions, allowing for proliferation. Phenotypic heterogeneity can have a mutational or indeed a non-mutational basis as observed in bet-hedging strategies adopted by antibiotic-tolerant persister cells. Genetic variants can arise by phase variation (slip-strand mispairing, promoter inversions etc.), nucleotide polymorphisms resulting from replication errors or larger rearrangements such as deletions and insertions. In the face of selective pressures, these alterations may be neutral, beneficial or deleterious.We recently described the genetic basis of tolerance to a normally toxic metabolite, D-serine (D-ser) in enterohaemorrhagic E. coli (EHEC). Here we summarize our work in the context of population dynamics, provide further discussion on the distinction between these tolerance mechanisms and the importance of heterogeneity for maximising adaptive potential.

Adaptive Laboratory Evolution Restores Solvent Tolerance in Plasmid-Cured Pseudomonas putida S12: a Molecular Analysis.

Pseudomonas putida S12 is inherently solvent tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of P. putida S12 carries several gene clusters involved in solvent tolerance, and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent tolerance of this strain. Whole-genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% [vol/vol]). We identified mutations in an RND efflux pump regulator, arpR, that resulted in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit ß', a global two-component regulatory system (GacA/GacS), and a putative AraC family transcriptional regulator, Afr. Transcriptomic analysis further revealed a constitutive downregulation of energy-consuming activities in ALE-derived strains, such as flagellar assembly, FoF1 ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of the solvent tolerance trait in P. putida S12 lacking its megaplasmid.IMPORTANCE Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of Pseudomonas putida; however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured P. putida S12 strain to regain its solvent tolerance. Through these adapted strains, we began to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in P. putida This work sheds light on the possible genetic engineering targets to enhance solvent tolerance in Pseudomonas putida as well as other bacteria.

Local Adaptation of Legionella pneumophila within a Hospital Hot Water System Increases Tolerance to Copper.

In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental isolates from hot water system biofilm and water and from cooling tower water. After a 1-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/liter) for over 672 h. Complete loss of culturability was observed for three isolates following copper exposure to 5 mg/liter for 672 h. Two sequence type 1427 (ST1427)-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, maintained higher culturability. The expression of the copper resistance gene copA evaluated by reverse transcription-quantitative PCR (RT-qPCR) was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole-genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a health care facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone.IMPORTANCELegionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In health care facilities, copper levels in water can vary, depending on water quality, plumbing materials, and age. This study evaluated the impact of the isolation site (water versus biofilm, hot water system versus cooling tower) within building water systems. Closely related strains isolated from a health care facility hot water system exhibited variable tolerance to copper stress, shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressors such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.

Repeated ethanol intoxications of Drosophila melanogaster adults increases the resistance to ethanol of their progeny.

BACKGROUND: For decades, Drosophila melanogaster has been used as a model organism to understand the genetics and neurobiology of ethanol intoxication and tolerance. Previous research has shown that acute and chronic pre-exposures to ethanol can trigger the development of functional ethanol tolerance in flies and has unveiled some of the genetic pathways involved in the process. To our knowledge, however, no previous work has systematically explored whether repeated intoxications of adult flies can affect the ethanol tolerance of their progeny. METHODS: Adult flies were intoxicated several times (once daily, over several days), and their F1 and F2 progeny were subjected to a functional tolerance test in which flies are exposed to ethanol and video recorded twice within 5 hr. Their behavior was subsequently analyzed to determine how long it took them to become sedated during the first and second exposures. One- and 2-way ANOVAs were used to determine whether parental treatment had an effect on their progeny's baseline resistance and/or acquired functional tolerance to ethanol. RESULTS: Parental flies that were intoxicated several times produced F1 and F2 progeny with a significantly higher resistance to ethanol than progeny from unexposed controls. Further, parental intoxications inconsistently increased the progeny's capacity to develop rapid functional tolerance upon re-exposure to ethanol. The transmission of increased ethanol resistance to progeny lasted several days after the last parental intoxication. CONCLUSION: To our knowledge, this is the first demonstration that repeated parental daily intoxications affect the progeny's response to ethanol in fruit flies. Our findings support the use of D. melanogaster to explore conserved pathways underlying the transmission of ethanol tolerance and can help in the identificaton of novel strategies for managing alcohol use disorder.

HipBA-promoter structures reveal the basis of heritable multidrug tolerance.

Multidrug tolerance is largely responsible for chronic infections and caused by a small population of dormant cells called persisters. Selection for survival in the presence of antibiotics produced the first genetic link to multidrug tolerance: a mutant in the Escherichia coli hipA locus. HipA encodes a serine-protein kinase, the multidrug tolerance activity of which is neutralized by binding to the transcriptional regulator HipB and hipBA promoter. The physiological role of HipA in multidrug tolerance, however, has been unclear. Here we show that wild-type HipA contributes to persister formation and that high-persister hipA mutants cause multidrug tolerance in urinary tract infections. Perplexingly, high-persister mutations map to the N-subdomain-1 of HipA far from its active site. Structures of higher-order HipA-HipB-promoter complexes reveal HipA forms dimers in these assemblies via N-subdomain-1 interactions that occlude their active sites. High-persistence mutations, therefore, diminish HipA-HipA dimerization, thereby unleashing HipA to effect multidrug tolerance. Thus, our studies reveal the mechanistic basis of heritable, clinically relevant antibiotic tolerance.

Deletion of Tlr3 reduces acute tolerance to alcohol and alcohol consumption in the intermittent access procedure in male mice.

Pharmacological studies implicate toll-like receptor 3 (TLR3) signaling in alcohol drinking. We examined the role of TLR3 in behavioral responses to alcohol and GABAergic drugs by studying Tlr3 -/- mice. Because of opposing signaling between TLR3 and MyD88 pathways, we also evaluated Myd88 -/- mice. Ethanol consumption and preference decreased in male but not in female Tlr3 -/- mice during two-bottle choice every-other-day (2BC-EOD) drinking. There were no genotype differences in either sex during continuous or limited-access drinking. Null mutations in Tlr3 or Myd88 did not alter conditioned taste aversion to alcohol and had small or no effects on conditioned place preference. The Tlr3 null mutation did not alter acute alcohol withdrawal. Male, but not female, Tlr3 -/- mice took longer than wild-type littermates to recover from ataxia by ethanol or diazepam and longer to recover from sedative-hypnotic effects of ethanol or gaboxadol, indicating regulation of GABAergic signaling by TLR3. Acute functional tolerance (AFT) to alcohol-induced ataxia was decreased in Tlr3 -/- mice but was increased in Myd88 -/- mice. Thus, MyD88 and TLR3 pathways coordinately regulate alcohol consumption and tolerance to intoxicating doses of alcohol and GABAergic drugs. Despite similar alcohol metabolism and similar amounts of total alcohol consumed during 2BC and 2BC-EOD procedures in C57BL/6J mice, only 2BC-EOD drinking induced tolerance to alcohol-induced ataxia. Ataxia recovery was inversely correlated with level of drinking in wild-type and Tlr3 -/- littermates. Thus, deleting Tlr3 reduces alcohol consumption by reducing AFT to alcohol and not by altering tolerance induced by 2BC-EOD drinking.

Genome-wide association across Saccharomyces cerevisiae strains reveals substantial variation in underlying gene requirements for toxin tolerance.

Cellulosic plant biomass is a promising sustainable resource for generating alternative biofuels and biochemicals with microbial factories. But a remaining bottleneck is engineering microbes that are tolerant of toxins generated during biomass processing, because mechanisms of toxin defense are only beginning to emerge. Here, we exploited natural diversity in 165 Saccharomyces cerevisiae strains isolated from diverse geographical and ecological niches, to identify mechanisms of hydrolysate-toxin tolerance. We performed genome-wide association (GWA) analysis to identify genetic variants underlying toxin tolerance, and gene knockouts and allele-swap experiments to validate the involvement of implicated genes. In the process of this work, we uncovered a surprising difference in genetic architecture depending on strain background: in all but one case, knockout of implicated genes had a significant effect on toxin tolerance in one strain, but no significant effect in another strain. In fact, whether or not the gene was involved in tolerance in each strain background had a bigger contribution to strain-specific variation than allelic differences. Our results suggest a major difference in the underlying network of causal genes in different strains, suggesting that mechanisms of hydrolysate tolerance are very dependent on the genetic background. These results could have significant implications for interpreting GWA results and raise important considerations for engineering strategies for industrial strain improvement.

Repression of transcription factor AtWRKY47 confers tolerance to boron toxicity in Arabidopsis thaliana.

Boron (B) excess gives rise to a serious agricultural problem. In this study, we identified a B toxicity responsive transcription factor AtWRKY47 in Arabidopsis thaliana. The T-DNA insertion mutants Atwrky47 showed enhanced tolerance to B toxicity with better growth parameters under high B conditions compared to wild-type Col-0 plants. Quantitative analysis of AtWRKY47 mRNA abundance indicated that it was down-regulated under B toxicity conditions. Fluorescently labeled AtWRKY47 protein was localized in nucleus. In contrast to the phenotype of Atwrky47 mutants, overexpression of AtWRKY47 in Col-0 background resulted in lower biomass, less chlorophyll content, and increased sensitivity to B toxicity. More importantly, the B concentration in shoots was higher in the overexpression lines and lower in the Atwrky47 mutants than in Col-0 plants, respectively. These results demonstrate that AtWRKY47 gene plays a key role in regulating plant tolerance to B toxicity.

Disulfiram Abrogates Morphine Tolerance-A Possible Role of µ-Opioid Receptor-Related G-Protein Activation in the Striatum.

One of the key strategies for effective pain management involves delaying analgesic tolerance. Early clinical reports indicate an extraordinary effectiveness of off-label disulfiram-an agent designed for alcohol use disorder-in potentiating opioid analgesia and abrogation of tolerance. Our study aimed to determine whether sustained µ-opioid signaling upon disulfiram exposure contributes to these phenomena. Wistar rats were exposed to acute and chronic disulfiram and morphine cotreatment. Nociceptive thresholds were assessed with the mechanical Randal-Selitto and thermal tail-flick tests. µ-opioid receptor activation in brain structures important for pain processing was carried out with the [35S]GTPγS assay. The results suggest that disulfiram (12.5-50 mg/kg i.g.) augmented morphine antinociception and diminished morphine (25 mg/kg, i.g.) tolerance in a supraspinal, opioid-dependent manner. Disulfiram (25 mg/kg, i.g.) induced a transient enhancement of µ-opioid receptor activation in the periaqueductal gray matter (PAG), rostral ventromedial medulla (RVM), hypothalamus, prefrontal cortex and the dorsal striatum at day 1 of morphine treatment. Disulfiram rescued µ-opioid receptor signaling in the nucleus accumbens and caudate-putamen 14 days following morphine and disulfiram cotreatment. The results of this study suggest that striatal µ-opioid receptors may contribute to the abolition of morphine tolerance following concomitant treatment with disulfiram.