Epigenetic scars of CD8+ T cell exhaustion persist after cure of chronic infection in humans.

T cell exhaustion is an induced state of dysfunction that arises in response to chronic infection and cancer. Exhausted CD8+ T cells acquire a distinct epigenetic state, but it is not known whether that chromatin landscape is fixed or plastic following the resolution of a chronic infection. Here we show that the epigenetic state of exhaustion is largely irreversible, even after curative therapy. Analysis of chromatin accessibility in HCV- and HIV-specific responses identifies a core epigenetic program of exhaustion in CD8+ T cells, which undergoes only limited remodeling before and after resolution of infection. Moreover, canonical features of exhaustion, including super-enhancers near the genes TOX and HIF1A, remain 'epigenetically scarred.' T cell exhaustion is therefore a conserved epigenetic state that becomes fixed and persists independent of chronic antigen stimulation and inflammation. Therapeutic efforts to reverse T cell exhaustion may require new approaches that increase the epigenetic plasticity of exhausted T cells.

Complete integration of carbene-transfer chemistry into biosynthesis.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Unbiased insights into the multiplicity of the CYP46A1 brain effects in 5XFAD mice treated with low dose-efavirenz.

Cytochrome P450 46A1 (CYP46A1) is the CNS-specific cholesterol 24-hydroxylase that controls cholesterol elimination and turnover in the brain. In mouse models, pharmacologic CYP46A1 activation with low-dose efavirenz or by gene therapy mitigates the manifestations of various brain disorders, neurologic, and nonneurologic, by affecting numerous, apparently unlinked biological processes. Accordingly, CYP46A1 is emerging as a promising therapeutic target; however, the mechanisms underlying the multiplicity of the brain CYP46A1 activity effects are currently not understood. We proposed the chain reaction hypothesis, according to which CYP46A1 is important for the three primary (unifying) processes in the brain (sterol flux through the plasma membranes, acetyl-CoA, and isoprenoid production), which in turn affect a variety of secondary processes. We already identified several processes secondary to changes in sterol flux and herein undertook a multiomics approach to compare the brain proteome, acetylproteome, and metabolome of 5XFAD mice (an Alzheimer's disease model), control and treated with low-dose efavirenz. We found that the latter had increased production of phospholipids from the corresponding lysophospholipids and a globally increased protein acetylation (including histone acetylation). Apparently, these effects were secondary to increased acetyl-CoA production. Signaling of small GTPases due to their altered abundance or abundance of their regulators could be affected as well, potentially via isoprenoid biosynthesis. In addition, the omics data related differentially abundant molecules to other biological processes either reported previously or new. Thus, we obtained unbiased mechanistic insights and identified potential players mediating the multiplicity of the CYP46A1 brain effects and further detailed our chain reaction hypothesis.

Synthesis, in vitro Anti-HIV-1RT evaluation, molecular modeling, DFT and acute oral toxicity studies of some benzotriazole derivatives.

This study synthesized and evaluated a series of benzotriazole derivatives denoted 3(a-j) and 6(a-j) for their anti-HIV-1 RT activities compared to the standard drug efavirenz. Notably, compound 3 h, followed closely by 6 h, exhibited significant anti-HIV-1 RT efficacy relative to the standard drug. In vivo oral toxicity studies were conducted for the most active compound 3 h, confirming its nontoxic nature to ascertain the safety profile. By employing molecular docking techniques, we explored the potential interactions between the synthesized compounds (ligands) and a target biomolecule (protein)(PDB ID 1RT2) at the molecular level. We undertook the molecular dynamics study of 3 h, the most active compound, within the active binding pocket of the cocrystallized structure of HIV-1 RT (PDB ID 1RT2). We aimed to learn more about how biomolecular systems behave, interact, and change at the atomic or molecular level over time. Finally, the DFT-derived HOMO and LUMO orbitals, as well as analysis of the molecular electrostatic potential map, aid in discerning the reactivity characteristics of our molecule.

Iron Heme Enzyme-Catalyzed Cyclopropanations with Diazirines as Carbene Precursors: Computational Explorations of Diazirine Activation and Cyclopropanation Mechanism.

The mechanism of cyclopropanations with diazirines as air-stable and user-friendly alternatives to commonly employed diazo compounds within iron heme enzyme-catalyzed carbene transfer reactions has been studied by means of density functional theory (DFT) calculations of model systems, quantum mechanics/molecular mechanics (QM/MM) calculations, and molecular dynamics (MD) simulations of the iron carbene and the cyclopropanation transition state in the enzyme active site. The reaction is initiated by a direct diazirine-diazo isomerization occurring in the active site of the enzyme. In contrast, an isomerization mechanism proceeding via the formation of a free carbene intermediate in lieu of a direct, one-step isomerization process was observed for model systems. Subsequent reaction with benzyl acrylate takes place through stepwise C-C bond formation via a diradical intermediate, delivering the cyclopropane product. The origin of the observed diastereo- and enantioselectivity in the enzyme was investigated through MD simulations, which indicate a preferred formation of the cis-cyclopropane by steric control.

Efficacy and effect on lipid profiles of switching to ainuovirine-based regimen versus continuing efavirenz-based regimen in people with HIV-1: 24-week results from a real-world, retrospective, multi-center cohort study.

Ainuovirine (ANV), a novel non-nucleoside reverse-transcriptase inhibitor (NNRTI), was approved in China in 2021. In a previous randomized phase 3 trial, ANV demonstrated non-inferior efficacy relative to efavirenz (EFV) and was associated with lower rates of dyslipidemia. In this study, we aimed to explore lipid changes in treatment-experienced people with human immunodeficiency virus (HIV)-1 (PWH) switching to ANV from EFV in real world. At week 24, 96.65% of patients in the ANV group and 93.25% in the EFV group had HIV-1 RNA levels below the limit of quantification (LOQ). Median changes from baseline in CD4 +T cell counts (37.0 vs 36.0 cells/µL, P = 0.886) and CD4+/CD8 +ratio (0.03 vs 0.10, P = 0.360) were similar between the two groups. The ANV group was superior to the EFV group in mean changes in total cholesterol (TC, -0.06 vs 0.26 mmol/L, P = 0.006), triglyceride (TG, -0.6 vs 0.14 mmol/L, P < 0.001), high-density lipoprotein cholesterol (HDL-C, 0.09 vs 0.08 mmol/L, P = 0.006), and low-density lipoprotein cholesterol (LDL-C, -0.18 vs 0.29 mmol/L, P < 0.001) at week 24. We also observed that a higher proportion of patients demonstrated improved TC (13.55% vs 4.45%, P = 0.015) or LDL-C (12.93% vs 6.89%, P = 0.017), and a lower proportion of patients showed worsened LDL-C (5.57% vs 13.52%, P = 0.017) with ANV than with EFV at week 24. In conclusion, we observed good efficacy and favorable changes in lipids in switching to ANV from EFV in treatment-experienced PWH in real world, indicating a promising switching option for PWH who may be more prone to metabolic or cardiovascular diseases.

Population pharmacokinetics of ravidasvir in adults with chronic hepatitis C virus infection and impact of antiretroviral treatment.

Ravidasvir (RDV) is a novel NS5A inhibitor that exhibits potent pan-genotypic inhibition of hepatitis C virus (HCV) replication. Sofosbuvir (SOF) plus RDV was demonstrated to be efficacious and safe in adults with active HCV infection, including those living with HIV (LWHIV), in the STORM-C-1 trial. We assessed the population pharmacokinetics (PK) of RDV in a sub-study nested within STORM-C-1 conducted in Thailand and Malaysia. SOF (400 mg) plus RDV (200 mg) was administered orally once daily for 12 weeks to adults with chronic HCV infection, but without cirrhosis and for 24 weeks to those with compensated cirrhosis. Intensive and sparse PK samples were collected at 4, 8, and 12 weeks after treatment initiation. Population PK parameters of RDV and the impact of covariates were evaluated using nonlinear mixed-effects modeling. Five hundred ninety-four participants were included, 235 (40%) had compensated cirrhosis, and 189 (32%) were LWHIV. RDV plasma concentrations were best described by a two-compartment model with first-order elimination. Oral clearance (CL/F) and volume of distribution (Vd/F) parameters were allometrically scaled on fat-free mass. Concomitant antiretroviral treatment (ART) increased RDV CL/F by 30%-60%, with efavirenz-based ART having the largest impact. Females had 16% lower RDV CL/F than males, and higher albumin levels reduced RDV central volume of distribution. While several covariates impact RDV CL/F and Vd/F, the effect on RDV exposures was not clinically relevant based on the efficacy data reported in this diverse Asian adult population. There were no meaningful drug-drug interactions in adults LWHIV on ART.

Influenza C virus susceptibility to antivirals with different mechanisms of action.

Antiviral susceptibility of influenza viruses was assessed using a high-content imaging-based neutralization test. Cap-dependent endonuclease inhibitors, baloxavir and AV5116, were superior to AV5115 against type A viruses, and AV5116 was most effective against PA mutants tested. However, these three inhibitors displayed comparable activity (EC50 8-22 nM) against type C viruses from six lineages. Banana lectin and a monoclonal antibody, YA3, targeting the hemagglutinin-esterase protein effectively neutralized some, but not all, type C viruses.

An update on topical therapies for psoriasis.

PURPOSE OF REVIEW: Topical therapies are a mainstay of treatment for mild psoriasis and may be a useful adjunct in treatment of moderate-to-severe psoriasis. This review summarizes recent advances in topical therapies for psoriasis and currently available treatments. RECENT FINDINGS: Topical aryl hydrocarbon receptor modulators (tapinarof) and topical phosphodiesterase-4 inhibitors (roflumilast) have been proven effective in randomized controlled trials for psoriasis. Although topical JAK inhibitors have also been studied, none are currently licensed for treatment of psoriasis. Topical corticosteroids and vitamin D analogues remain the most commonly used and widely available topical treatments for psoriasis. Cost may limit use of novel topical agents. SUMMARY: Although the novel topical agents tapinarof and roflumilast are licensed for treatment of psoriasis by the FDA in the United States, they have not yet been licensed in Europe, and it remains to be seen whether they will be limited by cost.

Dolutegravir as First- or Second-Line Treatment for HIV-1 Infection in Children.

BACKGROUND: Children with human immunodeficiency virus type 1 (HIV-1) infection have limited options for effective antiretroviral treatment (ART). METHODS: We conducted an open-label, randomized, noninferiority trial comparing three-drug ART based on the HIV integrase inhibitor dolutegravir with standard care (non-dolutegravir-based ART) in children and adolescents starting first- or second-line ART. The primary end point was the proportion of participants with virologic or clinical treatment failure by 96 weeks, as estimated by the Kaplan-Meier method. Safety was assessed. RESULTS: From September 2016 through June 2018, a total of 707 children and adolescents who weighed at least 14 kg were randomly assigned to receive dolutegravir-based ART (350 participants) or standard care (357). The median age was 12.2 years (range, 2.9 to 18.0), the median weight was 30.7 kg (range, 14.0 to 85.0), and 49% of the participants were girls. By design, 311 participants (44%) started first-line ART (with 92% of those in the standard-care group receiving efavirenz-based ART), and 396 (56%) started second-line ART (with 98% of those in the standard-care group receiving boosted protease inhibitor-based ART). The median follow-up was 142 weeks. By 96 weeks, 47 participants in the dolutegravir group and 75 in the standard-care group had treatment failure (estimated probability, 0.14 vs. 0.22; difference, -0.08; 95% confidence interval, -0.14 to -0.03; P = 0.004). Treatment effects were similar with first- and second-line therapies (P = 0.16 for heterogeneity). A total of 35 participants in the dolutegravir group and 40 in the standard-care group had at least one serious adverse event (P = 0.53), and 73 and 86, respectively, had at least one adverse event of grade 3 or higher (P = 0.24). At least one ART-modifying adverse event occurred in 5 participants in the dolutegravir group and in 17 in the standard-care group (P = 0.01). CONCLUSIONS: In this trial involving children and adolescents with HIV-1 infection who were starting first- or second-line treatment, dolutegravir-based ART was superior to standard care. (Funded by ViiV Healthcare; ODYSSEY ClinicalTrials.gov number, NCT02259127; EUDRACT number, 2014-002632-14; and ISRCTN number, ISRCTN91737921.).

Roflumilast ameliorates GAN diet-induced non-alcoholic fatty liver disease by reducing hepatic steatosis and fibrosis in ob/ob mice.

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent progressive liver disease. Currently, there is only one drug for NAFLD treatment, and the options are limited. Phosphodiesterase-4 (PDE-4) inhibitors have potential in treating NAFLD. Therefore, this study aims to investigate the effect of roflumilast on NAFLD. Here, we fed ob/ob mice to induce the NAFLD model by GAN diet. Roflumilast (1 mg/kg) was administered orally once daily. Semaglutide (20 nmol/kg), used as a positive control, was injected subcutaneously once daily. Our findings showed that roflumilast has beneficial effects on NAFLD. Roflumilast prevented body weight gain and improved lipid metabolism in ob/ob-GAN NAFLD mice. In addition, roflumilast decreased hepatic steatosis by down-regulating the expression of hepatic fatty acid synthesis genes (SREBP1c, FASN, and CD36) and improving oxidative stress. Roflumilast not only reduced liver injury by decreasing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, but also ameliorated hepatic inflammation by reducing the gene expression of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6). Roflumilast lessened liver fibrosis by inhibiting the expression of fibrosis mRNA (TGFß1, α-SMA, COL1a1, and TIMP-1). Collectively, roflumilast could ameliorate NAFLD, especially in reducing hepatic steatosis and fibrosis. Our findings suggested a PDE-4 inhibitor roflumilast could be a potential drug for NAFLD.

Genome-wide identification of MAPK family in papaya (Carica papaya) and their involvement in fruit postharvest ripening.

BACKGROUND: Papaya (Carica papaya) is an economically important fruit cultivated in the tropical and subtropical regions of China. However, the rapid softening rate after postharvest leads to a short shelf-life and considerable economic losses. Accordingly, understanding the mechanisms underlying fruit postharvest softening will be a reasonable way to maintain fruit quality and extend its shelf-life. RESULTS: Mitogen-activated protein kinases (MAPKs) are conserved and play essential roles in response to biotic and abiotic stresses. However, the MAPK family remain poorly studied in papaya. Here, a total of nine putative CpMAPK members were identified within papaya genome, and a comprehensive genome-wide characterization of the CpMAPKs was performed, including evolutionary relationships, conserved domains, gene structures, chromosomal locations, cis-regulatory elements and expression profiles in response to phytohormone and antioxidant organic compound treatments during fruit postharvest ripening. Our findings showed that nearly all CpMAPKs harbored the conserved P-loop, C-loop and activation loop domains. Phylogenetic analysis showed that CpMAPK members could be categorized into four groups (A-D), with the members within the same groups displaying high similarity in protein domains and intron-exon organizations. Moreover, a number of cis-acting elements related to hormone signaling, circadian rhythm, or low-temperature stresses were identified in the promoters of CpMAPKs. Notably, gene expression profiles demonstrated that CpMAPKs exhibited various responses to 2-chloroethylphosphonic acid (ethephon), 1-methylcyclopropene (1-MCP) and the combined ascorbic acid (AsA) and chitosan (CTS) treatments during papaya postharvest ripening. Among them, both CpMAPK9 and CpMAPK20 displayed significant induction in papaya flesh by ethephon treatment, and were pronounced inhibition after AsA and CTS treatments at 16 d compared to those of natural ripening control, suggesting that they potentially involve in fruit postharvest ripening through ethylene signaling pathway or modulating cell wall metabolism. CONCLUSION: This study will provide some valuable insights into future functional characterization of CpMAPKs, and hold great potential for further understanding the molecular mechanisms underlying papaya fruit postharvest ripening.

Influence of efavirenz and 8-hydroxy-efavirenz plasma levels on cognition and central nervous system side effects.

OBJECTIVES: To investigate whether efavirenz (EFV) or 8-hydroxy-EFV (8-OH-EFV) plasma levels are associated with neurocognitive impairment and central nervous system (CNS) side effects. METHODS: We conducted a cross-sectional analysis to explore the potential links between EFV/8-OH-EFV levels and cognitive performance or CNS-related side effects in patients screened within a randomized trial involving a switch from EFV to rilpivirine. The Mann-Whitney test was employed to compare drug levels in patients with or without cognitive impairment, depression, anxiety, sleep disorder or CNS symptoms. Additionally, Spearman's test was used to assess correlations between drug levels and test scores. RESULTS: Among 104 patients, neither EFV nor 8-OH-EFV levels were linked to cognitive impairment, although trends towards higher EFV levels were observed in those with impaired executive function (p = 0.055) and language performances (p = 0.021). On the other hand, elevated 8-OH-EFV levels, but not EFV levels, were associated with more CNS side effects (222 vs. 151 ng/mL, p = 0.027), depressive symptoms (247 vs. 164 ng/mL, p = 0.067) and sleep impairment (247 vs. 164 ng/mL, p = 0.078). Consistently, a trend towards a correlation between EFV levels and lower z-scores in executive function and motor function was observed, while 8-OH-EFV levels, but not EFV levels, were directly correlated with symptom scores. CONCLUSIONS: Higher levels of 8-OH-EFV were associated with CNS side effects, while EFV levels were only marginally associated with cognitive performance, thus suggesting that EFV and its metabolite may act differently in determining detrimental neurological effects.

Assessment of weight gain in adult patients living with HIV receiving first-line dolutegravir-based or efavirenz-based ART regimens in routine care clinics in Tshwane district, South Africa: An observational study.

INTRODUCTION: Although dolutegravir (DTG) is deemed stable, safe, cost-effective, and clinically beneficial, it also carries the risk of side effects, including observed weight gain among patients on DTG-based antiretroviral therapy (ART) regimens. We compared weight changes among adults (≥18 years) initiating tenofovir disoproxil fumarate, lamivudine, and dolutegravir (TLD) or tenofovir disoproxil fumarate, emtricitabine, and efavirenz (TEE) regimens and those switching from TEE to TLD (TEE-to-TLD switchers) in three large primary care facilities in South Africa METHODS: We conducted a retrospective longitudinal record review using patient medical records, extracting relevant demographic and clinical data from October 2018 to June 2021 from randomly selected adults who initiated TLD or TEE (initiators) and adult TEE-to-TLD switchers. We assessed weight, body mass index (BMI), and percentage weight changes for both groups and fitted linear regression and generalized linear models to determine factors associated with weight and BMI change and percentage weight change ≥10%, respectively, among treatment initiators. We fitted linear mixed-effect models among TEE-to-TLD switchers to consider repeated measures. RESULTS: Of 860 initiators, 450 (52.3%) initiated on TEE and 410 (47.7%) on TLD, with median follow-up of 1.4 years and 1.0 year, respectively. At initiation, 43.3% on TEE and 40.8% on TLD were overweight or obese. TLD initiators had an adjusted higher mean weight gain of 1.6 kg (p < 0.001) and mean BMI gain of 0.51 kg/m2 (p < 0.001) than TEE initiators. Independent risk factors for higher mean weight and BMI included age ≥50 years, male, on ART for >12 months, initial BMI of <18.5 kg/m2, and CD4 counts <200 cells/µL. Of 298 TEE-to-TLD switchers, 36.6% were overweight or obese at TEE initiation. Comparing before and after TLD switch, TEE-to-TLD switchers had an adjusted mean weight of 1.2 kg less while on TLD (p = 0.026). Being overweight and CD4 counts >350 cells/µL were independent risk factors for lower weight gain after TLD switch. CONCLUSIONS: We report more weight gain among TLD than among TEE initiators, although to a lesser extent than previously reported. TEE-to-TLD switchers experienced less weight gain after TLD switch; return to health before receiving TLD may be a contributory factor. The current findings are reassuring for those switching to a DTG-based regimen.

ß2-Adrenoreceptor Agonists, Montelukast, and Parkinson Disease Risk.

OBJECTIVE: This study was undertaken to examine the association between montelukast use, ß2-adrenoreceptor (ß2AR) agonist use, and later Parkinson disease (PD). METHODS: We ascertained use of ß2AR agonists (430,885 individuals) and montelukast (23,315 individuals) from July 1, 2005 to June 30, 2007, and followed 5,186,886 PD-free individuals from July 1, 2007 to December 31, 2013 for incident PD diagnosis. We estimated hazard ratios and 95% confidence intervals using Cox regressions. RESULTS: We observed 16,383 PD cases during on average 6.1 years of follow-up. Overall, use of ß2AR agonists and montelukast were not related to PD incidence. A 38% lower PD incidence was noted among high-dose montelukast users when restricted to PD registered as the primary diagnosis. INTERPRETATION: Overall, our data do not support inverse associations between ß2AR agonists, montelukast, and PD. The prospect of lower PD incidence with high-dose montelukast exposure warrants further investigation, especially with adjustment for high-quality data on smoking. ANN NEUROL 2023;93:1023-1028.

The Cytochrome P450 2C8*3 Variant (rs11572080) Is Associated with Improved Asthma Symptom Control in Children and Altered Lipid Mediator Production and Inflammatory Response in Human Bronchial Epithelial Cells.

This study investigated an association between the cytochrome P450 (CYP) 2C8*3 polymorphism with asthma symptom control in children and changes in lipid metabolism and pro-inflammatory signaling by human bronchial epithelial cells (HBECs) treated with cigarette smoke condensate (CSC). CYP genes are inherently variable in sequence, and while such variations are known to produce clinically relevant effects on drug pharmacokinetics and pharmacodynamics, the effects on endogenous substrate metabolism and associated physiologic processes are less understood. In this study, CYP2C8*3 was associated with improved asthma symptom control among children: Mean asthma control scores were 3.68 (n = 207) for patients with one or more copies of the CYP2C8*3 allele versus 4.42 (n = 965) for CYP2C8*1/*1 (P = 0.0133). In vitro, CYP2C8*3 was associated with an increase in montelukast 36-hydroxylation and a decrease in linoleic acid metabolism despite lower mRNA and protein expression. Additionally, CYP2C8*3 was associated with reduced mRNA expression of interleukin-6 (IL-6) and C-X-C motif chemokine ligand 8 (CXCL-8) by HBECs in response to CSC, which was replicated using the soluble epoxide hydrolase inhibitor, 12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid. Interestingly, 9(10)- and 12(13)- dihydroxyoctadecenoic acid, the hydrolyzed metabolites of 9(10)- and 12(13)- epoxyoctadecenoic acid, increased the expression of IL-6 and CXCL-8 mRNA by HBECs. This study reveals previously undocumented effects of the CYP2C8*3 variant on the response of HBECs to exogenous stimuli. SIGNIFICANCE STATEMENT: These findings suggest a role for CYP2C8 in regulating the epoxyoctadecenoic acid:dihydroxyoctadecenoic acid ratio leading to a change in cellular inflammatory responses elicited by environmental stimuli that exacerbate asthma.

Ethylene-regulated asymmetric growth of the petal base promotes flower opening in rose (Rosa hybrida).

Flowers are the core reproductive structures and key distinguishing features of angiosperms. Flower opening to expose stamens and gynoecia is important in cases where pollinators much be attracted to promote cross-pollination, which can enhance reproductive success and species preservation. The floral opening process is accompanied by the coordinated movement of various floral organs, particularly petals. However, the mechanisms underlying petal movement and flower opening are not well understood. Here, we integrated anatomical, physiological, and molecular approaches to determine the petal movement regulatory network using rose (Rosa hybrida) as a model. We found that PETAL MOVEMENT-RELATED PROTEIN1 (RhPMP1), a homeodomain transcription factor (TF) gene, is a direct target of ETHYLENE INSENSITIVE3, a TF that functions downstream of ethylene signaling. RhPMP1 expression was upregulated by ethylene and specifically activated endoreduplication of parenchyma cells on the adaxial side of the petal (ADSP) base by inducing the expression of RhAPC3b, a gene encoding the core subunit of the Anaphase-Promoting Complex. Cell expansion of the parenchyma on the ADSP base was subsequently enhanced, thus resulting in asymmetric growth of the petal base, leading to the typical epinastic movement of petals and flower opening. These findings provide insights into the pathway regulating petal movement and associated flower-opening mechanisms.�.

Synthesis of Trifluoromethylated Monoterpenes by an Engineered Cytochrome P450.

Protein engineering of cytochrome P450s has enabled these biocatalysts to promote a variety of abiotic reactions beyond nature's repertoire. Integrating such non-natural transformations with microbial biosynthetic pathways could allow sustainable enzymatic production of modified natural product derivatives. In particular, trifluoromethylation is a highly desirable modification in pharmaceutical research due to the positive effects of the trifluoromethyl group on drug potency, bioavailability, and metabolic stability. This study demonstrates the biosynthesis of non-natural trifluoromethyl-substituted cyclopropane derivatives of natural monoterpene scaffolds using an engineered cytochrome P450 variant, P411-PFA. P411-PFA successfully catalyzed the transfer of a trifluoromethyl carbene from 2-diazo-1,1,1-trifluoroethane to the terminal alkenes of several monoterpenes, including L-carveol, carvone, perilla alcohol, and perillartine, to generate the corresponding trifluoromethylated cyclopropane products. Furthermore, integration of this abiotic cyclopropanation reaction with a reconstructed metabolic pathway for L-carveol production in Escherichia coli enabled one-step biosynthesis of a trifluoromethylated L-carveol derivative from limonene precursor. Overall, amalgamating synthetic enzymatic chemistry with established metabolic pathways represents a promising approach to sustainably produce bioactive natural product analogs.

The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.

Effect of Roflumilast, a Selective PDE4 Inhibitor, on Bone Phenotypes in ADO2 Mice.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We previously created mouse models of ADO2 (p.G213R) with one of the most common mutations (G215R) as found in humans and demonstrated that this mutation in mice phenocopies the human disease of ADO2. Previous studies have shown that roflumilast (RF), a selective phosphodiesterase 4 (PDE4) inhibitor that regulates the cAMP pathway, can increase osteoclast activity. We also observed that RF increased bone resorption in both wild-type and ADO2 heterozygous osteoclasts in vitro, suggesting it might rescue bone phenotypes in ADO2 mice. To test this hypothesis, we administered RF-treated diets (0, 20 and 100 mg/kg) to 8-week-old ADO2 mice for 6 months. We evaluated bone mineral density and bone micro-architecture using longitudinal in-vivo DXA and micro-CT at baseline, and 6-, 12-, 18-, and 24-week post-baseline time points. Additionally, we analyzed serum bone biomarkers (CTX, TRAP, and P1NP) at baseline, 12-, and 24-week post-baseline. Our findings revealed that RF treatment did not improve aBMD (whole body, femur, and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group treated with a normal diet. Furthermore, we did not observe any significant changes in serum levels of bone biomarkers due to RF treatment in these mice. Overall, our results indicate that RF does not rescue the osteopetrotic bone phenotypes in ADO2 heterozygous mice.