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Capture of a photon by an opsin visual pigment isomerizes its 11-cis-retinaldehyde (11cRAL) chromophore to all-trans-retinaldehyde (atRAL), which subsequently dissociates. To restore light sensitivity, the unliganded apo-opsin combines with another 11cRAL to make a new visual pigment. Two enzyme pathways supply chromophore to photoreceptors. The canonical visual cycle in retinal pigment epithelial cells supplies 11cRAL at low rates. The photic visual cycle in Müller cells supplies cones with 11-cis-retinol (11cROL) chromophore precursor at high rates. Although rods can only use 11cRAL to regenerate rhodopsin, cones can use 11cRAL or 11cROL to regenerate cone visual pigments. We performed a screen in zebrafish retinas and identified ZCRDH as a candidate for the enzyme that converts 11cROL to 11cRAL in cone inner segments. Retinoid analysis of eyes from Zcrdh-mutant zebrafish showed reduced 11cRAL and increased 11cROL levels, suggesting impaired conversion of 11cROL to 11cRAL. By microspectrophotometry, isolated Zcrdh-mutant cones lost the capacity to regenerate visual pigments from 11cROL. ZCRDH therefore possesses all predicted properties of the cone 11cROL dehydrogenase. The human protein most similar to ZCRDH is RDH12. By immunocytochemistry, ZCRDH was abundantly present in cone inner segments, similar to the reported distribution of RDH12. Finally, RDH12 was the only mammalian candidate protein to exhibit 11cROL-oxidase catalytic activity. These observations suggest that RDH12 in mammals is the functional ortholog of ZCRDH, which allows cones, but not rods, to regenerate visual pigments from 11cROL provided by Müller cells. This capacity permits cones to escape competition from rods for visual chromophore in daylight-exposed retinas.

Pathogenic variants in retinol dehydrogenase 5 (RDH5) attenuate supply of 11-cis-retinal to photoreceptors leading to a range of clinical phenotypes including night blindness because of markedly slowed rod dark adaptation and in some patients, macular atrophy. Current animal models (such as Rdh5-/- mice) fail to recapitulate the functional or degenerative phenotype. Addressing this need for a relevant animal model we present a new domestic cat model with a loss-of-function missense mutation in RDH5 (c.542G > T; p.Gly181Val). As with patients, affected cats have a marked delay in recovery of dark adaptation. In addition, the cats develop a degeneration of the area centralis (equivalent to the human macula). This recapitulates the development of macular atrophy that is reported in a subset of patients with RDH5 mutations and is shown in this paper in seven patients with biallelic RDH5 mutations. There is notable variability in the age at onset of the area centralis changes in the cat, with most developing changes as juveniles but some not showing changes over the first few years of age. There is similar variability in development of macular atrophy in patients and while age is a risk factor, it is hypothesized that genetic modifying loci influence disease severity, and we suspect the same is true in the cat model. This novel cat model provides opportunities to improve molecular understanding of macular atrophy and test therapeutic interventions for RDH5-associated retinopathies.

Psoriasis is a chronic immune-mediated disease characterized by excessive proliferation of epidermal keratinocytes and increased immune cell infiltration to the skin. Although it is well-known that psoriasis pathogenesis is driven by aberrant production of proinflammatory cytokines, the mechanisms underlying the imbalance between proinflammatory and anti-inflammatory cytokine expression are incompletely understood. In this study, we report that the transcriptional coregulators CtBP1 and 2 can transactivate a common set of proinflammatory genes both in the skin of imiquimod-induced mouse psoriasis model and in human keratinocytes and macrophages stimulated by imiquimod. We find that mice overexpressing CtBP1 in epidermal keratinocytes display severe skin inflammation phenotypes with increased expression of T helper type 1 and T helper type 17 cytokines. We also find that the expression of CtBPs and CtBP-target genes is elevated both in human psoriatic lesions and in the mouse imiquimod psoriasis model. Moreover, we were able to show that topical treatment with a peptidic inhibitor of CtBP effectively suppresses the CtBP-regulated proinflammatory gene expression and thus attenuates psoriatic inflammation in the imiquimod mouse model. Together, our findings suggest to our knowledge previously unreported strategies for therapeutic modulation of the immune response in inflammatory skin diseases.

Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.

The retinol dehydrogenase Rdh10 catalyzes the rate-limiting reaction that converts retinol into retinoic acid (RA), an autacoid that regulates energy balance and reduces adiposity. Skeletal muscle contributes to preventing adiposity, by consuming nearly half the energy of a typical human. We report sexually dimorphic differences in energy metabolism and muscle function in Rdh10+/- mice. Relative to wild-type (WT) controls, Rdh10+/- males fed a high-fat diet decrease reliance on fatty-acid oxidation and experience glucose intolerance and insulin resistance. Running endurance decreases 40%. Rdh10+/- females fed this diet increase fatty acid oxidation and experience neither glucose intolerance nor insulin resistance. Running endurance increases 220%. We therefore assessed RA function in the mixed-fiber type gastrocnemius muscles (GM), which contribute to running, rather than standing, and are similar to human GM. RA levels in Rdh10+/- male GM decrease 38% relative to WT. Rdh10+/- male GM increase expression of Myog and reduce Eif6 mRNAs, which reduce and enhance running endurance, respectively. Cox5A, complex IV activity, and ATP decrease. Increased centralized nuclei reveal existence of muscle malady and/or repair in GM fibers. Comparatively, RA in Rdh10+/- female GM decreases by less than half the male decrease, from a more modest decrease in Rdh10 and an increase in the estrogen-induced retinol dehydrogenase Dhrs9. Myog mRNA decreases. Cox5A, complex IV activity, and ATP increase. Centralized GM nuclei do not increase. We conclude that Rdh10/RA affects whole body energy use and insulin resistance partially through sexual dimorphic effects on skeletal muscle gene expression, structure, and mitochondria activity.

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each samples copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.

Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH2) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH2 with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH2 derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4-/-Rdh8-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH2 or other tested amino acid derivatives of Ret-NH2. Ret-NH2-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.

The process of obtaining ascorbic acid (AA) via intestinal absorption and blood circulation is carrier-mediated utilizing the AA transporters SVCT1 and SVCT2, which are expressed in the intestine and brain (SVCT2 in abundance). AA concentration is decreased in Alzheimer's disease (AD), but information regarding the status of intestinal AA uptake in the AD is still lacking. We aimed here to understand how AA homeostasis is modulated in a transgenic mouse model (5xFAD) of AD. AA levels in serum from 5xFAD mice were markedly lower than controls. Expression of oxidative stress response genes (glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1)) were significantly increased in AD mice jejunum, and this increase was mitigated by AA supplementation. Uptake of AA in the jejunum was upregulated. This increased AA transport was caused by a marked increase in SVCT1 and SVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression. A significant increase in the expression of HNF1α and specific protein 1 (Sp1), which drive SLC23A1 and SLC23A2 promoter activity, respectively, was observed. Expression of hSVCT interacting proteins GRHPR and CLSTN3 were also increased. SVCT2 protein and mRNA expression in the hippocampus of 5xFAD mice was not altered. Together, these investigations reveal adaptive up-regulation of intestinal AA uptake in the 5xFAD mouse model.

Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.

To realize the promise of CRISPR-Cas9-based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We addressed this challenge by profiling transcriptional, translational, and posttranslational reporters using CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach allowed us to connect an entire library of guides to their individual phenotypic consequences using pooled sequencing. CiBER-seq profiling fully recapitulated the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged transfer RNA (tRNA) accumulation activated ISR reporter transcription. Notably, tRNA insufficiency also activated the reporter, independent of the uncharged tRNA sensor. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.

BackgroundTwenty-five germline variants are associated with adult diffuse glioma, and some of these variants have been shown to be associated with particular subtypes of glioma. We hypothesized that additional germline variants could be identified if a genome-wide association study (GWAS) were performed by molecular subtype.MethodsA total of 1320 glioma cases and 1889 controls were used in the discovery set and 799 glioma cases and 808 controls in the validation set. Glioma cases were classified into molecular subtypes based on combinations of isocitrate dehydrogenase (IDH) mutation, telomerase reverse transcriptase (TERT) promoter mutation, and 1p/19q codeletion. Logistic regression was applied to the discovery and validation sets to test for associations of variants with each of the subtypes. A meta-analysis was subsequently performed using a genome-wide P-value threshold of 5 × 10-8.ResultsNine variants in or near D-2-hydroxyglutarate dehydrogenase (D2HGDH) on chromosome 2 were genome-wide significant in IDH-mutated glioma (most significant was rs5839764, meta P = 2.82 × 10-10). Further stratifying by 1p/19q codeletion status, one variant in D2HGDH was genome-wide significant in IDH-mutated non-codeleted glioma (rs1106639, meta P = 4.96 × 10-8). Further stratifying by TERT mutation, one variant near FAM20C (family with sequence similarity 20, member C) on chromosome 7 was genome-wide significant in gliomas that have IDH mutation, TERT mutation, and 1p/19q codeletion (rs111976262, meta P = 9.56 × 10-9). Thirty-six variants in or near GMEB2 on chromosome 20 near regulator of telomere elongation helicase 1 (RTEL1) were genome-wide significant in IDH wild-type glioma (most significant was rs4809313, meta P = 2.60 × 10-10).ConclusionsPerforming a GWAS by molecular subtype identified 2 new regions and a candidate independent region near RTEL1, which were associated with specific glioma molecular subtypes.

Polyketides are a large class of structurally and functionally diverse natural products with important bioactivities. Many polyketides are synthesized by reducing type II polyketide synthases (PKSs), containing transiently interacting standalone enzymes. During synthesis, ketoreductase (KR) catalyzes regiospecific carbonyl to hydroxyl reduction, determining the product outcome, yet little is known about what drives specific KR-substrate interactions. In this study, computational approaches were used to explore KR-substrate interactions based on previously solved apo and mimic cocrystal structures. We found five key factors guiding KR-substrate binding. First, two major substrate binding motifs were identified. Second, substrate length is the key determinant of substrate binding position. Third, two key residues in chain length specificity were confirmed. Fourth, phosphorylation of substrates is critical for binding. Finally, packing/hydrophobic effects primarily determine the binding stability. The molecular bases revealed here will help further engineering of type II PKSs and directed biosynthesis of new polyketides.

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L-1 h-1, a titer of 275 g L-1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.

BACKGROUND:The outer peels of pomegranate (Punica granatum L.) possess two groups of polyphenols that have health beneficial properties: anthocyanins (ATs, which also affect peel color); and hydrolysable tannins (HTs). Their biosynthesis intersects at 3-dehydroshikimate (3-DHS) in the shikimate pathway by the activity of shikimate dehydrogenase (SDH), which converts 3-DHS to shikimate (providing the precursor for AT biosynthesis) or to gallic acid (the precursor for HTs biosynthesis) using NADPH or NADP+ as a cofactor. The aim of this study is to gain more knowledge about the factors that regulate the levels of HTs and ATs, and the role of SDH. RESULTS:The results have shown that the levels of ATs and HTs are negatively correlated in the outer fruit peels of 33 pomegranate accessions, in the outer peels of two fruits exposed to sunlight, and in those covered by paper bags. When calli obtained from the outer fruit peel were subjected to light/dark treatment and osmotic stresses (imposed by different sucrose concentrations), it was shown that light with high sucrose promotes the synthesis of ATs, while dark at the same sucrose concentration promotes the synthesis of HTs. To verify the role of SDH, six PgSDHs (PgSDH1, PgSDH3-1,2, PgSDH3a-1,2 and PgSDH4) were identified in pomegranate. The expression of PgSDH1, which presumably contributes to shikimate biosynthesis, was relatively constant at different sucrose concentrations. However, the transcript levels of PgSDH3s and PgSDH4 increased with the accumulation of gallic acid and HTs under osmotic stress, which apparently accumulates to protect the cells from the stress. CONCLUSIONS:The results strongly suggest that the biosynthesis of HTs and ATs competes for the same substrate, 3-DHS, and that SDH activity is regulated not only by the NADPH/NADP+ ratio, but also by the expression of the PgSDHs. Since the outer peel affects the customer's decision regarding fruit consumption, such knowledge could be utilized for the development of new genetic markers for breeding pomegranates having higher levels of both ATs and HTs.

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallography, were exploited to reach these conclusions and provide additional insights.

Despite the enormous potential shown by recent biorefineries, the current bioeconomy still encounters multifaceted challenges. To develop a sustainable biorefinery in the future, multidisciplinary research will be essential to tackle technical difficulties. Herein, we leveraged a known plant genetic engineering approach that results in aldehyde-rich lignin via down-regulation of cinnamyl alcohol dehydrogenase (CAD) and disruption of monolignol biosynthesis. We also report on renewable deep eutectic solvents (DESs) synthesized from phenolic aldehydes that can be obtained from CAD mutant biomass. The transgenic Arabidopsis thaliana CAD mutant was pretreated with the DESs and showed a twofold increase in the yield of fermentable sugars compared with wild type (WT) upon enzymatic saccharification. Integrated use of low-recalcitrance engineered biomass, characterized by its aldehyde-type lignin subunits, in combination with a DES-based pretreatment, was found to be an effective approach for producing a high yield of sugars typically used for cellulosic biofuels and biobased chemicals. This study demonstrates that integration of renewable DES with plant genetic engineering is a promising strategy in developing a closed-loop process.

While rods in the mammalian retina regenerate rhodopsin through a well-characterized pathway in cells of the retinal pigment epithelium (RPE), cone visual pigments are thought to regenerate in part through an additional pathway in Müller cells of the neural retina. The proteins comprising this intrinsic retinal visual cycle are unknown. Here, we show that RGR opsin and retinol dehydrogenase-10 (Rdh10) convert all-trans-retinol to 11-cis-retinol during exposure to visible light. Isolated retinas from Rgr+/+ and Rgr-/- mice were exposed to continuous light, and cone photoresponses were recorded. Cones in Rgr-/- retinas lost sensitivity at a faster rate than cones in Rgr+/+ retinas. A similar effect was seen in Rgr+/+ retinas following treatment with the glial cell toxin, α-aminoadipic acid. These results show that RGR opsin is a critical component of the Müller cell visual cycle and that regeneration of cone visual pigment can be driven by light.

The retinoid cycle is a metabolic process in the vertebrate retina that continuously regenerates 11-cis-retinal (11-cisRAL) from the all-trans-retinal (atRAL) isomer. atRAL accumulation can cause photoreceptor degeneration and irreversible visual dysfunction associated with incurable blinding retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and atrophic age-related macular degeneration (AMD). The underlying cellular mechanisms leading to retinal degeneration remain uncertain, although previous studies have shown that atRAL promotes calcium influx associated with cell apoptosis. To identify compounds that mitigate the effects of atRAL toxicity, here we developed an unbiased and robust image-based assay that can detect changes in intracellular calcium levels in U2OS cells. Using our assay in a high-throughput screen of 2,400 compounds, we noted that selective estrogen receptor modulators (SERMs) potently stabilize intracellular calcium and thereby counteract atRAL-induced toxicity. In a light-induced retinal degeneration mouse model (Abca4 -/- Rdh8 -/-), raloxifene (a benzothiophene-type scaffold SERM) prevented the onset of photoreceptor apoptosis and thus protected the retina from degeneration. The minor structural differences between raloxifene and one of its derivatives (Y 134) had a major impact on calcium homeostasis after atRAL exposure in vitro, and we verified this differential impact in vivo In summary, the SERM raloxifene has structural and functional neuroprotective effects in the retina. We propose that the highly sensitive image-based assay developed here could be applied for the discovery of additional drug candidates preventing photoreceptor degeneration.

Epithelial integrity is maintained by the cytoskeleton and through cell adhesion. However, it is not yet known how a deregulated cytoskeleton is associated with cancer. We identified cancer-related regulator of actin dynamics (CRAD) as frequently mutated or transcriptionally downregulated in colorectal cancer. We found that CRAD stabilizes the cadherin-catenin-actin complex via capping protein inhibition. The loss of CRAD inhibits F-actin polymerization and subsequently disrupts the cadherin-catenin-actin complex, which leads to β-catenin release and Wnt signalling hyperactivation. In mice, CRAD knockout induces epithelial cell integrity loss and Wnt signalling activation, resulting in the development of intestinal mucinous adenoma. With APC mutation, CRAD knockout initiates and accelerates mucinous and invasive adenoma development in the colorectum. These results define CRAD as a tumour suppressor, the inactivation of which deregulates the cytoskeleton and hyperactivates Wnt signalling thus initiating mucinous colorectal cancer. Our study reveals the unexpected roles of an actin cytoskeletal regulator in maintaining epithelial cell integrity and suppressing tumorigenesis.

High performance microprobes for combined sensing of glucose and choline were fabricated using microcontact printing (μCP) to transfer choline oxidase (ChOx) and glucose oxidase (GOx) onto targeted sites on microelectrode arrays (MEAs). Most electroenzymatic sensing sites on MEAs for neuroscience applications are created by manual enzyme deposition, which becomes problematic when the array feature size is less than or equal to ∼100 μm. The μCP process used here relies on use of soft lithography to create features on a polydimethylsiloxane (PDMS) microstamp that correspond to the dimensions and array locations of targeted, microscale sites on a MEA. Precise alignment of the stamp with the MEA is also required to transfer enzyme only onto the specified microelectrode(s). The dual sensor fabrication process began with polyphenylenediamine (PPD) electrodeposition on all Pt microelectrodes to block common interferents (e.g., ascorbic acid and dopamine) found in brain extracellular fluid. Next, a chitosan film was electrodeposited to serve as an adhesive layer. The two enzymes, ChOx and GOx, were transferred onto different microelectrodes of 2 × 2 arrays using two different PDMS stamps and a microscope for stamp alignment. Using constant potential amperometry, the combined sensing microprobe was confirmed to have high sensitivity for choline and glucose (286 and 117 μA mM cm-2, respectively) accompanied by low detection limits (1 and 3 μM, respectively) and rapid response times (≤2 s). This work demonstrates the use of μCP for facile creation of multianalyte sensing microprobes by targeted deposition of enzymes onto preselected sites of a microelectrode array.

Continuous regeneration of the 11-cis-retinal visual chromophore from all-trans-retinal is critical for vision. Insufficiency of 11-cis-retinal arising from the dysfunction of key proteins involved in its regeneration can impair retinal health, ultimately leading to loss of human sight. Delayed recovery of visual sensitivity and night blindness caused by inadequate regeneration of the visual pigment rhodopsin are typical early signs of this condition. Excessive concentrations of unliganded, constitutively active opsin and increased levels of all-trans-retinal and its byproducts in photoreceptors also accelerate retinal degeneration after light exposure. Exogenous 9-cis-retinal iso-chromophore can reduce the toxicity of ligand-free opsin but fails to prevent the buildup of retinoid photoproducts when their clearance is defective in human retinopathies, such as Stargardt disease or age-related macular degeneration. Here we evaluated the effect of a locked chromophore analog, 11-cis-6-membered ring-retinal against bright light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Using in vivo imaging techniques, optical coherence tomography, scanning laser ophthalmoscopy, and two-photon microscopy, along with in vitro histologic analysis of retinal morphology, we found that treatment with 11-cis-6-membered ring-retinal before light stimulation prevented rod and cone photoreceptor degradation and preserved functional acuity in these mice. Moreover, additive accumulation of 11-cis-6-membered ring-retinal measured in the eyes of these mice by quantitative liquid chromatography-mass spectrometry indicated stable binding of this retinoid to opsin. Together, these results suggest that eliminating excess of unliganded opsin can prevent light-induced retinal degeneration in Abca4-/-Rdh8-/- mice.

Noninvasive imaging of visual system components in vivo is critical for understanding the causal mechanisms of retinal diseases and for developing therapies for their treatment. However, ultraviolet light needed to excite endogenous fluorophores that participate in metabolic processes of the retina is highly attenuated by the anterior segment of the human eye. In contrast, 2-photon excitation fluorescence imaging with pulsed infrared light overcomes this obstacle. Reducing retinal exposure to laser radiation remains a major barrier in advancing this technology to studies in humans. To increase fluorescence intensity and reduce the requisite laser power, we modulated ultrashort laser pulses with high-order dispersion compensation and applied sensorless adaptive optics and custom image recovery software and observed an over 300% increase in fluorescence of endogenous retinal fluorophores when laser pulses were shortened from 75 fs to 20 fs. No functional or structural changes to the retina were detected after exposure to 2-photon excitation imaging light with 20-fs pulses. Moreover, wide bandwidth associated with short pulses enables excitation of multiple fluorophores with different absorption spectra and thus can provide information about their relative changes and intracellular distribution. These data constitute a substantial advancement for safe 2-photon fluorescence imaging of the human eye.

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4 -/- Rdh8 -/- mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential.

Pharmacological dosing of all-trans-retinoic acid (atRA) controls adiposity in rodents by inhibiting adipogenesis and inducing fatty acid oxidation. Retinol dehydrogenases (Rdh) catalyze the first reaction that activates retinol into atRA. This study examined postnatal contributions of Rdh10 to atRA biosynthesis and physiological functions of endogenous atRA. Embryonic fibroblasts from Rdh10 heterozygote hypomorphs or with a total Rdh10 knockout exhibit decreased atRA biosynthesis and escalated adipogenesis. atRA or a retinoic acid receptor (RAR) pan-agonist reversed the phenotype. Eliminating one Rdh10 copy in vivo (Rdh10+/- ) yielded a modest decrease (≤25%) in the atRA concentration of liver and adipose but increased adiposity in male and female mice fed a high-fat diet (HFD); increased liver steatosis, glucose intolerance, and insulin resistance in males fed an HFD; and activated bone marrow adipocyte formation in females, regardless of dietary fat. Chronic dosing with low-dose atRA corrected the metabolic defects. These data resolve physiological actions of endogenous atRA, reveal sex-specific effects of atRA in vivo, and establish the importance of Rdh10 to metabolic control by atRA. The consequences of a modest decrease in tissue atRA suggest that impaired retinol activation may contribute to diabesity, and low-dose atRA therapy may ameliorate adiposity and its sequelae of glucose intolerance and insulin resistance.

Regeneration following tissue damage often necessitates a mechanism for cellular re-programming, so that surviving cells can give rise to all cell types originally found in the damaged tissue. This process, if unchecked, can also generate cell types that are inappropriate for a given location. We conducted a screen for genes that negatively regulate the frequency of notum-to-wing transformations following genetic ablation and regeneration of the wing pouch, from which we identified mutations in the transcriptional co-repressor C-terminal Binding Protein (CtBP). When CtBP function is reduced, ablation of the pouch can activate the JNK/AP-1 and JAK/STAT pathways in the notum to destabilize cell fates. Ectopic expression of Wingless and Dilp8 precede the formation of the ectopic pouch, which is subsequently generated by recruitment of both anterior and posterior cells near the compartment boundary. Thus, CtBP stabilizes cell fates following damage by opposing the destabilizing effects of the JNK/AP-1 and JAK/STAT pathways.

Regeneration following tissue damage often necessitates a mechanism for cellular re-programming, so that surviving cells can give rise to all cell types originally found in the damaged tissue. This process, if unchecked, can also generate cell types that are inappropriate for a given location. We conducted a screen for genes that negatively regulate the frequency of notum-to-wing transformations following genetic ablation and regeneration of the wing pouch, from which we identified mutations in the transcriptional co-repressor C-terminal Binding Protein (CtBP). When CtBP function is reduced, ablation of the pouch can activate the JNK/AP-1 and JAK/STAT pathways in the notum to destabilize cell fates. Ectopic expression of Wingless and Dilp8 precede the formation of the ectopic pouch, which is subsequently generated by recruitment of both anterior and posterior cells near the compartment boundary. Thus, CtBP stabilizes cell fates following damage by opposing the destabilizing effects of the JNK/AP-1 and JAK/STAT pathways.

Obesity is associated with poorer outcome for many cancers. Previously, we observed that adipocytes protect acute lymphoblastic leukemia (ALL) cells from the anthracycline, daunorubicin. In this study, it is determined whether adipocytes clear daunorubicin from the tumor microenvironment (TME). Intracellular daunorubicin concentrations were evaluated using fluorescence. Daunorubicin and its largely inactive metabolite, daunorubicinol, were analytically measured in media, cells, and tissues using liquid chromatography/mass spectrometry (LC/MS). Expression of daunorubicin-metabolizing enzymes, aldo-keto reductases (AKR1A1, AKR1B1, AKR1C1, AKR1C2, AKR1C3, and AKR7A2) and carbonyl reductases (CBR1, CBR3), in human adipose tissue, were queried using public databases and directly measured by quantitative PCR (qPCR) and immunoblot. Adipose tissue AKR activity was measured by colorimetric assay. Adipocytes absorbed and efficiently metabolized daunorubicin to daunorubicinol, reducing its antileukemia effect in the local microenvironment. Murine studies confirmed adipose tissue conversion of daunorubicin to daunorubicinol in vivo Adipocytes expressed high levels of AKR and CBR isoenzymes that deactivate anthracyclines. Indeed, adipocyte protein levels of AKR1C1, AKR1C2, and AKR1C3 are higher than all other human noncancerous cell types. To our knowledge, this is the first demonstration that adipocytes metabolize and inactivate a therapeutic drug. Adipocyte-mediated daunorubicin metabolism reduces active drug concentration in the TME. These results could be clinically important for adipocyte-rich cancer microenvironments such as omentum, breast, and marrow. As AKR and CBR enzymes metabolize several drugs, and can be expressed at higher levels in obese individuals, this proof-of-principle finding has important implications across many diseases.Implications: Adipocyte absorption and metabolism of chemotherapies can reduce cytotoxicity in cancer microenvironments, potentially contributing to poorer survival outcomes. Mol Cancer Res; 15(12); 1704-13. ©2017 AACR.

The N-terminal half of adenovirus e1a assembles multimeric complexes with host proteins that repress innate immune responses and force host cells into S-phase. In contrast, the functions of e1a's C-terminal interactions with FOXK, DCAF7, and CtBP are unknown. We found that these interactions modulate RAS signaling, and that a single e1a molecule must bind all three of these host proteins to suppress activation of a subset of IFN-stimulated genes (ISGs). These ISGs were otherwise induced in primary respiratory epithelial cells at 12 hr p.i. This delayed activation of ISGs required IRF3 and coincided with an ∼10-fold increase in IRF3 from protein stabilization. The induced IRF3 bound to chromatin and localized to the promoters of activated ISGs. While IRF3, STAT1/2, and IRF9 all greatly increased in concentration, there were no corresponding mRNA increases, suggesting that e1a regulates the stabilities of these key activators of innate immune responses, as shown directly for IRF3.

Small secreted proteins (SSPs), along with lignocellulose degrading enzymes, are integral components of the secretome of Pleurotus ostreatus, a white rot fungus. In this study, we identified 3 genes (ssp1, 2 and 3) encoding proteins that are annotated as SSPs and that exhibited of ~4,500- fold expression, 24 hr following exposure to the toxic compound 5-hydroxymethylfurfural (HMF). Homologues to genes encoding these SSPs are present in the genomes of other basidiomycete fungi, however the role of SSPs is not yet understood. SSPs, aryl-alcohol oxidases (AAO) and the intracellular aryl-alcohol dehydrogenases (AAD) were also produced after exposure to other aryl-alcohols, known substrates and inducers of AAOs, and during idiophase (after the onset of secondary metabolism). A knockdown strain of ssp1 exhibited reduced production of AAO-and AAD-encoding genes after HMF exposure. Conversely, a strain overexpressing ssp1 exhibited elevated expression of genes encoding AAOs and ADD, resulting in a 3-fold increase in enzymatic activity of AAOs, as well as increased expression and protein abundance of versatile peroxidase 1, which directly degrades lignin. We propose that in addition to symbionts and pathogens, SSPs also have roles in saprophytes and function in P. ostreatus as components of the ligninolytic system.

Decaprenylphosphoryl-β-d-ribose-2'-epimerase (DprE1) is a druggable target which is being exploited for the development of new anti-TB agents. In the present work, we report developing a pharmacophore model and performing virtual screening of Asinex database using the developed pharmacophore model to get eight hits as potential DprE1 inhibitors. The hits were used as leads to design new 3-phenylpyrazolo[1,5- a ]pyrimidine-2,7(1 H ,4 H )-dione based potential anti-TB agents. On the basis of the identified lead molecules, a total of 18 compounds were synthesized and evaluated for their anti-TB activity by using MABA. ADMET predictions for all the compounds revealed that these compounds have drug-like and lead-like properties. One of the final compounds was found to exhibit potent anti-TB activity against Mycobacterium bovis .Communicated by Ramaswamy H. Sarma.

Benzothiazole-bearing compounds have emerged as potential noncovalent DprE1 (decaprenylphosphoryl-β-d-ribose-2'-epimerase) inhibitors active against Mycobacterium tuberculosis . Based on structure-based virtual screening (PDB ID: 4KW5), a focused library of thirty-one skeletally diverse benzothiazole amides was prepared, and the compounds were assessed for their antitubercular activity against M.tb H37Ra. Most potent compounds 3b and 3n were further evaluated against the M.tb H37Rv strain by the microdilution assay method. Among the compounds evaluated, bis - benzothiazole amide 3n emerged as a hit molecule and demonstrated promising antitubercular activity with minimum inhibitory concentration (MIC) values of 0.45 μg/mL and 8.0 μg/mL against H 37 Ra and H 37 Rv, respectively. Based on the preliminary hit molecule ( 3n) , a focused library of 12 more bis-benzothiazole amide derivatives was further prepared by varying the substituents on either side to obtain new leads and generate a structure-activity relationship (SAR). Among these compounds, 6a, 6c , and 6d demonstrated remarkable antitubercular activity with MIC values of 0.5 μg/mL against H37Ra and 1.0, 2.0, and 8.0 μg/mL against H 37 Rv, respectively. The most active compound, 6a , also displayed significant efficacy against four drug-resistant tuberculosis strains. Compound 6a was assessed for in vitro cytotoxicity against the HepG2 cell line, and it displayed insignificant cytotoxicity. Furthermore, time-kill kinetic studies demonstrated time- and dose-dependent bactericidal activity of this compound. The GFP release assay revealed that compound 6a targets the inhibition of a cell wall component. SNPs in dprE-1 gene assessment revealed that compound 6a binds to tyrosine at position 314 of DprE1 and replaces it with histidine, causing resistance similar to that of standard TCA1. In silico docking studies further suggest that the strong noncovalent interactions of these compounds may lead to the development of potent noncovalent DprE1 inhibitors.

The Mycobacterium cell wall is a capsule-like structure comprising of various layers of biomolecules such as mycolic acid, peptidoglycans, and arabinogalactans, which provide the Mycobacteria a sort of cellular shield. Drugs like isoniazid, ethambutol, cycloserine, delamanid, and pretomanid inhibit cell wall synthesis by inhibiting one or the other enzymes involved in cell wall synthesis. Many enzymes present across these layers serve as potential targets for the design and development of newer anti-TB drugs. Some of these targets are currently being exploited as the most druggable targets like DprE1, InhA, and MmpL3. Many of the anti-TB agents present in clinical trials inhibit cell wall synthesis. The present article covers a systematic perspective of developing cell wall inhibitors targeting various enzymes involved in cell wall biosynthesis as potential drug candidates for treating Mtb infection., (© 2024 John Wiley & Sons Ltd.)

The genetic control underlying natural variation in lignin content and composition in trees is not fully understood. We performed a systems genetic analysis to uncover the genetic regulation of lignin biosynthesis in a natural 'SwAsp' population of aspen (Populus tremula) trees. We analyzed gene expression by RNA sequencing (RNA-seq) in differentiating xylem tissues, and lignin content and composition using Pyrolysis-GC-MS in mature wood of 268 trees from 99 genotypes. Abundant variation was observed for lignin content and composition, and genome-wide association study identified proteins in the pentose phosphate pathway and arabinogalactan protein glycosylation among the top-ranked genes that are associated with these traits. Variation in gene expression and the associated genetic polymorphism was revealed through the identification of 312 705 local and 292 003 distant expression quantitative trait loci (eQTL). A co-expression network analysis suggested modularization of lignin biosynthesis and novel functions for the lignin-biosynthetic CINNAMYL ALCOHOL DEHYDROGENASE 2 and CAFFEOYL-CoA O-METHYLTRANSFERASE 3. PHENYLALANINE AMMONIA LYASE 3 was co-expressed with HOMEOBOX PROTEIN 5 (HB5), and the role of HB5 in stimulating lignification was demonstrated in transgenic trees. The systems genetic approach allowed linking natural variation in lignin biosynthesis to trees´ responses to external cues such as mechanical stimulus and nutrient availability., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Tuberculosis remains the second deadliest infectious disease in humans and a public health threat due to the emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains. Therefore, it is urgent to identify new anti-tuberculosis treatments and novel therapeutic targets to prevent the emergence of resistance. In recent years, the study of anti-tuberculosis properties of nitroaromatic compounds has led to the identification of two novel biological targets, the deazaflavin (F 420 )-dependent nitroreductase Ddn and the decaprenylphosphoryl-β-d-ribose 2'-epimerase DprE1. This review aims to show why Ddn and DprE1 are promising therapeutic targets and highlight nitroaromatic compounds interest in developing new anti-tuberculosis treatments active against MDR-TB and XDR-TB. Despite renewed interest in the development of new anti-tuberculosis nitroaromatic compounds, pharmaceutical companies often exclude nitro-containing molecules from their drug discovery programs because of their toxic and mutagenic potential. This exclusion results in missed opportunities to identify new nitroaromatic compounds and promising therapeutic targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Emerging threats of multi-drug resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR) tuberculosis led to the discovery of a novel target which was entitled Decaprenylphosphoryl-β-D-ribose 2'-epimerase (DprE1) enzyme. DprE1 is composed of two isoforms, decaprenylphosphoryl-β-D-ribose oxidase (DprE1) and decaprenylphosphoryl-D-2-keto erythro pentose reductase (DprE2). The enzymes, DprE1 and DprE2, regulate the two-step epimerization process to form DPA (Decaprenylphosphoryl arabinose) from DPX (Decaprenylphosphoryl-D-ribose), which is the sole precursor in the cell wall synthesis of arabinogalactan (AG) and lipoarabinomannan (LAM). Target-based and whole-cell-based screening played an imperative role in the identification of the druggable target, DprE1, whereas the druggability of the DprE2 enzyme is not proved yet. To date, diverse scaffolds of heterocyclic and aromatic ring systems have been reported as DprE1 inhibitors based on their interaction mode, i.e. covalent, and non-covalent inhibitors. This review describes the structure-activity relationship (SAR) of reported covalent and non-covalent inhibitors to enlighten about the crucial pharmacophoric features required for DprE1 inhibition, along with in-silico studies which characterize the amino acid residues responsible for covalent and non-covalent interactions.Communicated by Ramaswamy H. Sarma.