Saturation Mutagenesis of Burkholderia cepacia R34 2,4-Dinitrotoluene Dioxygenase at DntAc Valine 350 for Synthesizing Nitrohydroquinone, Methylhydroquinone, and Methoxyhydroquinone

Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc α-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 ± 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 ± 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 ± 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 ± 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 ± 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 ± 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 ± 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 ± 0.1 nmol/min/mg of protein from m-cresol. V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol. The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 ± 2.6 nmol/min/mg of protein), forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene. Hence, mutagenesis of wild-type DDO through active-site engineering generated variants with relatively high rates toward a previously uncharacterized class of substituted phenols for the nitroarene dioxygenases; seven previously uncharacterized substrates were evaluated for wild-type DDO, and four novel monooxygenase-like products were found for the DDO variants V350F and V350M (methoxyhydroquinone, methylhydroquinone, 2-hydroxybenzyl alcohol, and 3-nitrocatechol)

Protein Engineering of the Archetypal Nitroarene Dioxygenase of Ralstonia sp. Strain U2 for Activity on Aminonitrotoluenes and Dinitrotoluenes through Alpha-Subunit Residues Leucine 225, Phenylalanine 350, and Glycine 407

Naphthalene dioxygenase (NDO) from Ralstonia sp. strain U2 has not been reported to oxidize nitroaromatic compounds. Here, saturation mutagenesis of NDO at position F350 of the α-subunit (NagAc) created variant F350T that produced 3-methyl-4-nitrocatechol from 2,6-dinitrotoluene (26DNT), that released nitrite from 23DNT sixfold faster than wild-type NDO, and that produced 3-amino-4-methyl-5-nitrocatechol and 2-amino-4,6-dinitrobenzyl alcohol from 2-amino-4,6-dinitrotoluene (2A46DNT) (wild-type NDO has no detectable activity on 26DNT and 2A46DNT). DNA shuffling identified the beneficial NagAc mutation G407S, which when combined with the F350T substitution, increased the rate of NDO oxidation of 26DNT, 23DNT, and 2A46DNT threefold relative to variant F350T. DNA shuffling of NDO nagAcAd also generated the NagAc variant G50S/L225R/A269T with an increased rate of 4-amino-2-nitrotoluene (4A2NT; reduction product of 2,4-dinitrotoluene) oxidation; from 4A2NT, this variant produced both the previously uncharacterized oxidation product 4-amino-2-nitrocresol (enhanced 11-fold relative to wild-type NDO) as well as 4-amino-2-nitrobenzyl alcohol (4A2NBA; wild-type NDO does not generate this product). G50S/L225R/A269T also had increased nitrite release from 23DNT (14-fold relative to wild-type NDO) and generated 2,3-dinitrobenzyl alcohol (23DNBA) fourfold relative to wild-type NDO. The importance of position L225 for catalysis was confirmed through saturation mutagenesis; relative to wild-type NDO, NDO variant L225R had 12-fold faster generation of 4-amino-2-nitrocresol and production of 4A2NBA from 4A2NT as well as 24-fold faster generation of nitrite and 15-fold faster generation of 23DNBA from 23DNT. Hence, random mutagenesis discovered two new residues, G407 and L225, that influence the regiospecificity of Rieske non-heme-iron dioxygenases

Obesity does not modify the effect of continuous positive airway pressure on insulin resistance in adults with obstructive sleep apnoea

Funding Information: Support statement: This study was supported by grant P01-HL094307 from the National Heart, Lung, and Blood Institute (principal investigator: A.I. Pack). Funding information for this article has been deposited with the Crossref Funder Registry.This study found no evidence that obesity significantly modifies the effect of 4 months of CPAP treatment on HOMA-IR. Longer duration of CPAP treatment may be needed in order to reduce insulin resistance and determine whether obesity modifies the effect. https://bit.ly/3CtX7jZ.Non peer reviewe

Changes in sleepiness and 24-h blood pressure following 4 months of CPAP treatment are not mediated by ICAM-1.

To access publisher's full text version of this article click on the hyperlink belowObjective: Continuous positive airway pressure (CPAP) therapy reduces circulating intercellular adhesion molecule 1 (ICAM-1) in adults with obstructive sleep apnea (OSA). ICAM-1 levels may affect the daytime sleepiness and elevated blood pressure associated with OSA. We evaluated the association of changes from baseline in ICAM-1 with changes of objective and subjective measures of sleepiness, as well as 24-h ambulatory blood pressure monitoring (ABPM) measures, following 4 months of CPAP treatment. Methods: The study sample included adults with newly diagnosed OSA. Plasma ICAM-1, 24-h ABPM, Epworth Sleepiness Scale (ESS), and psychomotor vigilance task (PVT) were obtained at baseline and following adequate CPAP treatment. The associations between changes in natural log ICAM-1 and changes in the number of lapses on PVT, ESS score, and 24-h mean arterial blood pressure (MAP) were assessed using multivariate regression models, controlling for a priori baseline covariates of age, sex, BMI, race, site, smoking status, physical activity, anti-hypertensive medications, AHI, and daily hours of CPAP use. Results: Among 140 adults (83% men), mean (± SD) body mass index (BMI) was 31.5 ± 4.2 kg/m2, and apnea-hyopnea index (AHI) was 36.8 ± 15.3 events/h. Sleepiness measures, although not ICAM-1 or ABPM measures, improved significantly following CPAP treatment. We observed no statistically significant associations between the change in ICAM-1 and changes in sleepiness, MAP, or other ABPM measures. Conclusion: Changes in ICAM-1 levels were not related to changes in sleepiness or ABPM following CPAP treatment of adults with OSA. Future work should explore whether or not other biomarkers may have a role in mediating these treatment outcomes in adults with OSA. Keywords: Ambulatory blood pressure monitoring; Continuous positive airway pressure; Intercellular adhesion molecule-1; Obstructive sleep apnea; Sleepiness.United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Heart Lung & Blood Institute (NHLBI

Leveraging immune resistance archetypes in solid cancer to inform next-generation anticancer therapies.

Anticancer immunotherapies, such as immune checkpoint inhibitors, bispecific antibodies, and chimeric antigen receptor T cells, have improved outcomes for patients with a variety of malignancies. However, most patients either do not initially respond or do not exhibit durable responses due to primary or adaptive/acquired immune resistance mechanisms of the tumor microenvironment. These suppressive programs are myriad, different between patients with ostensibly the same cancer type, and can harness multiple cell types to reinforce their stability. Consequently, the overall benefit of monotherapies remains limited. Cutting-edge technologies now allow for extensive tumor profiling, which can be used to define tumor cell intrinsic and extrinsic pathways of primary and/or acquired immune resistance, herein referred to as features or feature sets of immune resistance to current therapies. We propose that cancers can be characterized by immune resistance archetypes, comprised of five feature sets encompassing known immune resistance mechanisms. Archetypes of resistance may inform new therapeutic strategies that concurrently address multiple cell axes and/or suppressive mechanisms, and clinicians may consequently be able to prioritize targeted therapy combinations for individual patients to improve overall efficacy and outcomes

Leveraging immune resistance archetypes in solid cancer to inform next-generation anticancer therapies

Anticancer immunotherapies, such as immune checkpoint inhibitors, bispecific antibodies, and chimeric antigen receptor T cells, have improved outcomes for patients with a variety of malignancies. However, most patients either do not initially respond or do not exhibit durable responses due to primary or adaptive/acquired immune resistance mechanisms of the tumor microenvironment. These suppressive programs are myriad, different between patients with ostensibly the same cancer type, and can harness multiple cell types to reinforce their stability. Consequently, the overall benefit of monotherapies remains limited. Cutting-edge technologies now allow for extensive tumor profiling, which can be used to define tumor cell intrinsic and extrinsic pathways of primary and/or acquired immune resistance, herein referred to as features or feature sets of immune resistance to current therapies. We propose that cancers can be characterized by immune resistance archetypes, comprised of five feature sets encompassing known immune resistance mechanisms. Archetypes of resistance may inform new therapeutic strategies that concurrently address multiple cell axes and/or suppressive mechanisms, and clinicians may consequently be able to prioritize targeted therapy combinations for individual patients to improve overall efficacy and outcomes