Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells.

BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models.  CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Patient-derived castration-resistant prostate cancer model revealed CTBP2 upregulation mediated by OCT1 and androgen receptor.

BACKGROUND: Prostate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation. METHODS: In this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database. RESULTS: Genome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC. CONCLUSIONS: Our findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management.

The Integration of Transcriptome and Metabolome Analyses Provides Insights into the Determinants of the Wood Properties in Toona ciliata.

Toona ciliata, also known as Chinese mahogany, is a high-quality and fast-growing wood species with a high economic value. The wood properties of T. ciliata of different provenances vary significantly. In this study, we conducted comprehensive transcriptome and metabolome analyses of red and non-red T. ciliata wood cores of different provenances to compare their wood properties and explore the differential metabolites and genes that govern the variation in their wood properties. Through combined analyses, three differential genes and two metabolites were identified that are possibly related to lignin synthesis. The lignin content in wood cores from T. ciliata of different provenances shows significant variation following systematic measurement and comparisons. The gene Tci09G002190, one of the three differential genes, was identified as a member of the CAD (Cinnamyl alcohol dehydrogenase) gene family of T. ciliata, which is associated with lignin synthesis. Our data provide insights into the determinants of the wood properties in T. ciliata, providing a solid foundation for research into the subsequent mechanisms of the formation of T. ciliata wood.

Reconstructing dynamics correlation network to simultaneously improve activity and stability of 2,3-butanediol dehydrogenase by design of distal interchain disulfide bonds.

The complete enzyme catalytic cycle includes substrate binding, chemical reaction and product release, in which different dynamic conformations are adopted. Due to the complex relationship among enzyme activity, stability and dynamics, the directed evolution of enzymes for improved activity or stability commonly leads to a trade-off in stability or activity. It hence remains a challenge to engineer an enzyme to have both enhanced activity and stability. Here, we have attempted to reconstruct the dynamics correlation network involved with active center to improve both activity and stability of a 2,3-butanediol dehydrogenase (2,3-BDH) by introducing inter-chain disulfide bonds. A computational strategy was first applied to evaluate the effect of introducing inter-chain disulfide bond on activity and stability of three 2,3-BDHs, and the N258C mutation of 2,3-BDH from Corynebacterium glutamicum (CgBDH) was proved to be effective in improving both activity and stability. In the results, CgBDH-N258C showed a different unfolding curve from the wild type, with two melting temperatures (Tm) of 68.3 °C and 50.8 °C, 19.7 °C and 2 °C higher than 48.6 °C of the wild type. Its half-life was also improved by 14.8-fold compared to the wild type. Catalytic efficiency (kcat/Km) of the mutant was increased by 7.9-fold toward native substrate diacetyl and 8.8-fold toward non-native substrate 2,5-hexanedione compared to the wild type. Molecular dynamics simulations revealed that an interaction network formed by Cys258, Arg162, Ala144 and the catalytic residues was reconstructed in the mutant and the dynamics change caused by the disulfide bond could be propagated through the interactions network. This improved the enzyme stability and activity by decreasing the flexibility and locking more "reactive" pose, respectively. Further construction of mutations including A144G showing a 44-fold improvement in catalytic efficiency toward meso-2,3-BD confirmed the role of modifying dynamics correlation network in tunning enzyme activity and selectivity. This study provided important insights into the relationship among dynamics, enzyme catalysis and stability, and will be useful in the designing new enzymes with co-evolution of stability, activity and selectivity.

Laser capture microdissection transcriptome (LCM RNA-seq) reveals BcDFR is a key gene in anthocyanin synthesis of non-heading Chinese cabbage.

BACKGROUND: Purple non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis] has become popular because of its richness in anthocyanin. However, anthocyanin only accumulates in the upper epidermis of leaves. Further studies are needed to investigate the molecular mechanisms underlying the specific accumulation of it. RESULTS: In this study, we used the laser capture frozen section method (LCM) to divide purple (ZBC) and green (LBC) non-heading Chinese cabbage leaves into upper and lower epidermis parts (Pup represents the purple upper epidermis, Plow represents the purple lower epidermis, Gup represents the green upper epidermis, Glow represents the green lower epidermis). Through transcriptome sequencing, we found that the DIHYDROFLAVONOL 4-REDUCTASE-encoding gene BcDFR, is strongly expressed in Pup but hardly in others (Plow, Gup, Glow). Further, a deletion and insertion in the promoter of BcDFR in LBC were found, which may interfere with BcDFR expression. Subsequent analysis of gene structure and conserved structural domains showed that BcDFR is highly conserved in Brassica species. The predicted protein-protein interaction network of BcDFR suggests that it interacts with almost all functional proteins in the anthocyanin biosynthesis pathway. Finally, the results of the tobacco transient expression also demonstrated that BcDFR promotes the synthesis and accumulation of anthocyanin. CONCLUSIONS: BcDFR is specifically highly expressed on the upper epidermis of purple non-heading Chinese cabbage leaves and regulates anthocyanin biosynthesis and accumulation. Our study provides new insights into the functional analysis and transcriptional regulatory network of anthocyanin-related genes in purple non-heading Chinese cabbage.

LncRNA CTBP1-AS inhibits TP63-mediated activation of S100A14 during prostate cancer progression.

Long noncoding RNAs (lncRNAs) have emerged as important molecules and potential new targets for human cancers. This study investigates the function of lncRNA CTBP1 antisense RNA (CTBP1-AS) in prostate cancer (PCa) and explores the entailed molecular mechanism. Aberrantly expressed genes potentially correlated with PCa progression were probed using integrated bioinformatics analyses. A cohort of 68 patients with PCa was included, and their tumor and para-cancerous tissues were collected. CTBP1-AS was highly expressed in PCa tissues and cells and associated with poor patient prognosis. By contrast, tumor protein p63 (TP63) and S100 calcium binding protein A14 (S100A14) were poorly expressed in the PCa tissues and cells. CTBP1-AS did not affect TP63 expression; however it blocked the TP63-mediated transcriptional activation of S100A14, thereby reducing its expression. CTBP1-AS silencing suppressed proliferation, apoptosis resistance, migration, invasion, and tumorigenicity of PCa cell lines, while its overexpression led to inverse results. The malignant phenotype of cells was further weakened by TP63 overexpression but restored following artificial S100A14 silencing. In conclusion, this study demonstrates that CTBP1-AS plays an oncogenic role in PCa by blocking TP63-mediated transcriptional activation of S100A14. This may provide insight into the management of PCa.

Adaptive Optics Retinal Imaging in RDH12-Associated Early Onset Severe Retinal Dystrophy.

Purpose: RDH12 is among the most common genes found in individuals with early-onset severe retinal (EOSRD). Adaptive optics scanning light ophthalmoscopy (AOSLO) enables resolution of individual rod and cone photoreceptors in the retina. This study presents the first AOSLO imaging of individuals with RDH12-associated EOSRD. Methods: Case series of patients who attended Moorfields Eye Hospital (London, UK). Spectral-domain optical coherence tomography, near-infrared reflectance (NIR), and blue autofluorescence imaging were analyzed. En face image sequences of photoreceptors were recorded using either of two AOSLO modalities. Cross-sectional analysis was undertaken for seven patients and longitudinal analysis for one patient. Results: Nine eyes from eight patients are presented in this case series. The mean age at the time of the assessment was 11.2 ± 6.5 years of age (range 7-29). A subfoveal continuous ellipsoid zone (EZ) line was present in eight eyes. Posterior pole AOSLO revealed patches of cone mosaics. Average cone densities at regions of interest 0.5° to the fovea ranged from 12,620 to 23,660 cells/mm2, whereas intercell spacing ranged from 7.0 to 9.7 µm. Conclusions: This study demonstrates that AOSLO can provide useful high-quality images in patients with EOSRD, even during childhood, with nystagmus, and early macular atrophy. Cones at the posterior pole can appear as scattered islands or, possibly later in life, as a single subfoveal conglomerate. Detailed image analysis suggests that retinal pigment epithelial stress and dysfunction may be the initial step toward degeneration, with NIR being a useful tool to assess retinal well-being in RDH12-associated EOSRD.

Retinol dehydrogenase 12 (RDH12) knock out may cause hyperuricemia phenotype in mice.

Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorder. And several gene loci and transporter proteins that associated with uric acid transport functions have been identified. Retinol Dehydrogenase 12 (RDH12), recognized for its role in safeguarding photoreceptors, and our study investigated the potential impact of Rdh12 mutations on other organs and diseases, particularly hyperuricemia. We assessed Rdh12 mRNA expression levels in various tissues and conducted serum biochemical analyses in Rdh12-/- mice. Compared with the wild type, significant alterations in serum uric acid levels and kidney-related biochemical indicators have been revealed. Then further analysis, including quantitative RT-PCR of gene expression in the liver and kidney, highlighted variations in the expression levels of specific genes linked to hyperuricemia. And renal histology assessment exposed mild pathological lesions in the kidneys of Rdh12-/- mice. In summary, our study suggests that Rdh12 mutations impact not only retinal function but also contribute to hyperuricemia and renal disease phenotypes in mice. Our finding implies that individuals with Rdh12 mutations may be prone to hyperuricemia and gout, emphasizing the significance of preventive measures and regular examinations in daily life.

Identify CTBP1-DT as an immunological biomarker that promotes lipid synthesis and apoptosis resistance in KIRC.

Recently, mounting evidence has highlighted the essential function of the C-terminal binding protein-1 divergent transcript (CTBP1-DT) in malignancies. However, its role in kidney renal clear cell carcinoma (KIRC) remains largely unknown. Our study aimed to identify the potential function of CTBP1-DT in KIRC. RT-qPCR, Kaplan-Meier survival analysis, Cox regression analysis, and nomogram analysis were utilized to determine the expression and effects of CTBP1-DT on survival. The subcellular localization of CTBP1-DT was determined using RNA fluorescence in situ hybridization (FISH). To investigate the functions of CTBP1-DT in regulating KIRC cell proliferation, migration, invasion, lipid synthesis, and apoptosis, we conducted CCK8, EdU, Transwell, and Oil Red O staining and cell apoptosis staining assays. The relationships between CTBP1-DT and the tumor microenvironment were investigated with multiple bioinformatics analysis algorithms and databases, including CYBERSORT, TIMER2, Spearman correlation test, tumor mutation burden (TMB), microsatellite instability (MSI), and immunophenoscore (IPS). According to our results, CTBP1-DT is a lncRNA located in the nucleus that is significantly upregulated in KIRC and is correlated with better clinical outcomes. Downregulating CTBP1-DT inhibited cell viability, migration, invasion, and lipid synthesis but triggered cell apoptosis. Additionally, we explored the potential effect of CTBP1-DT in regulating immune cell infiltration in KIRC and other malignancies. Furthermore, CTBP1-DT could be used to predict the effectiveness of targeted drugs and immune checkpoint inhibitors. In conclusion, we identified CTBP1-DT as a potential immunological biomarker and discovered the potential role of CTBP1-DT in regulating lipid synthesis and apoptosis resistance.

Alcohol dehydrogenase system acts as the sole pathway for methanol oxidation in Desulfofundulus kuznetsovii strain TPOSR.

Desulfofundulus kuznetsovii is a thermophilic, spore-forming sulphate-reducing bacterium in the family Peptococcaceae. In this study, we describe a newly isolated strain of D. kuznetsovii, strain TPOSR, and compare its metabolism to the type strain D. kuznetsovii 17T. Both strains grow on a large variety of alcohols, such as methanol, ethanol and propane-diols, coupled to the reduction of sulphate. Strain 17T metabolizes methanol via two routes, one involving a cobalt-dependent methyl transferase and the other using a cobalt-independent alcohol dehydrogenase. However, strain TPOSR, which shares 97% average nucleotide identity with D. kuznetsovii strain 17T, lacks several genes from the methyl transferase operon found in strain 17T. The gene encoding the catalytically active methyl transferase subunit B is missing, indicating that strain TPOSR utilizes the alcohol dehydrogenase pathway exclusively. Both strains grew with methanol during cobalt starvation, but growth was impaired. Strain 17T was more sensitive to cobalt deficiency, due to the repression of its methyl transferase system. Our findings shed light on the metabolic diversity of D. kuznetsovii and their metabolic differences of encoding one or two routes for the conversion of methanol.

L-2 hydroxyglutaric aciduria: report of a Mexican-Mayan patient with the mutation c.569C>T and response to vitamin supplements and levocarnitine.

Background: L-2-hydroxyglutaric aciduria (L2HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by pathogenic variants in the L2HGDH gene which encodes mitochondrial 2-hydroxyglutarate dehydrogenase. Here, we report a case of L2HGA in a Mexican-Mayan patient with a homozygous mutation at L2HGDH gene and clinical response to vitamin supplements and levocarnitine. Case report: A 17-year-old, right-handed female patient with long-term history of seizures, developmental delay and ataxia was referred to a movement disorders specialist for the evaluation of tremor. Her brain MRI showed typical findings of L2HGA. The diagnosis was corroborated with elevated levels of 2-hydroxyglutaric acid in urine and genetic test which revealed a homozygous genetic known variant c.569C>T in exon 5 of L2HGDH gene. She was treated with levocarnitine and vitamin supplements, showing improvement in tremor and gait. Discussion: To our knowledge this is the first report of a Mexican patient with L2HGA. This case adds information about a rare condition in a different ethnic group and supports the findings of other authors which encountered symptomatic improvement with the use of flavin adenine dinucleotide (and its precursor riboflavin), and levocarnitine. Highlights: We report the first case of Mexican-Mayan patient with L2HGA showing a missense homozygous mutation in L2HGDH gene, and improvement of symptoms with vitamin supplements and levocarnitine.

WRKY33 negatively regulates anthocyanin biosynthesis and cooperates with PHR1 to mediate acclimation to phosphate starvation.

Anthocyanin accumulation is acknowledged as a phenotypic indicator of phosphate (Pi) starvation. However, negative regulators of this process and their molecular mechanisms remain largely unexplored. In this study, we demonstrate that WRKY33 acts as a negative regulator of phosphorus-status-dependent anthocyanin biosynthesis. WRKY33 regulates the expression of the gene encoding dihydroflavonol 4-reductase (DFR), a rate-limiting enzyme in anthocyanin production, both directly and indirectly. WRKY33 binds directly to the DFR promoter to repress its expression and also interferes with the MBW complex through interacting with PAP1 to indirectly influence DFR transcriptional activation. Under -Pi conditions, PHR1 interacts with WRKY33, and the protein level of WRKY33 decreases; the repression of DFR expression by WRKY33 is thus attenuated, leading to anthocyanin accumulation in Arabidopsis. Further genetic and biochemical assays suggest that PHR1 is also involved in regulating factors that affect WRKY33 protein turnover. Taken together, our findings reveal that Pi starvation represses WRKY33, a repressor of anthocyanin biosynthesis, to finely tune anthocyanin biosynthesis. This "double-negative logic" regulation of phosphorus-status-dependent anthocyanin biosynthesis is required for the maintenance of plant metabolic homeostasis during acclimation to Pi starvation.

Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor.

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4-V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.

Clinical and Genetic Characterization of RDH12-Retinal Dystrophy in a South American Cohort.

PURPOSE: To characterize the largest cohort of individuals with retinol dehydrogenase 12 (RDH12)-retinal dystrophy to date, and the first one from South America. DESIGN: Retrospective multicenter international study. SUBJECTS: Seventy-eight patients (66 families) with an inherited retinal dystrophy and biallelic variants in RDH12. METHODS: Review of clinical notes, ophthalmic images, and molecular diagnosis. MAIN OUTCOME MEASURES: Visual function, retinal imaging, and characteristics were evaluated and correlated. RESULTS: Thirty-seven individuals self-identified as Latino (51%) and 34 as White (47%). Sixty-nine individuals (88%) had Leber congenital amaurosis (LCA)/early-onset severe retinal dystrophy. Macular and midperipheral atrophy were seen in all patients from 3 years of age. A novel retinal finding was a hyperautofluorescent ring in 2 young children with LCA. Thirty-nine patients (50%) had subsequent visits, with mean follow-up of 6.8 ± 7.3 (range, 0-29) years. Eight variants (21%) were previously unreported, and the most frequent variant was c.295C>A, p.Leu99Ile, present in 52 alleles of 32 probands. Individuals with LCA homozygous for p.Leu99Ile (31%) had a later age of onset, a slower rate of best-corrected visual acuity decrease, the largest percentage of patients with mild visual impairment, and were predicted to reach legal blindness at an older age than the rest of the cohort. CONCLUSIONS: By describing the largest molecularly confirmed cohort to date, improved understanding of disease progression was possible. Our detailed characterization aims to support research and the development of novel therapies that may have the potential to reduce or prevent vision loss in individuals with RDH12-associated retinal dystrophy. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures.

A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor.

The C-terminal binding protein (CtBP) is a transcriptional corepressor that plays critical roles in development, tumorigenesis, and cell fate. CtBP proteins are structurally similar to alpha hydroxyacid dehydrogenases and feature a prominent intrinsically disordered region in the C terminus. In the mammalian system, CtBP proteins lacking the C-terminal domain (CTD) are able to function as transcriptional regulators and oligomerize, putting into question the significance of this unstructured domain for gene regulation. Yet, the presence of an unstructured CTD of ∼100 residues, including some short motifs, is conserved across Bilateria, indicating the importance of maintaining this domain over evolutionary time. To uncover the significance of the CtBP CTD, we functionally tested naturally occurring Drosophila isoforms of CtBP that possess or lack the CTD, namely CtBP(L) and CtBP(S). We used the CRISPRi system to recruit dCas9-CtBP(L) and dCas9-CtBP(S) to endogenous promoters to directly compare their transcriptional impacts in vivo. Interestingly, CtBP(S) was able to significantly repress transcription of the Mpp6 promoter, while CtBP(L) was much weaker, suggesting that the long CTD may modulate CtBP's repression activity. In contrast, in cell culture, the isoforms behaved similarly on a transfected Mpp6 reporter gene. The context-specific differences in activity of these two developmentally regulated isoforms suggests that the CTD may help provide a spectrum of repression activity suitable for developmental programs.

Metabolic engineering of Escherichia coli for enhanced production of 1,3-butanediol from glucose.

1,3-Butanediol (1,3-BDO) finds versatile applications in the cosmetic, chemical, and food industries. This study focuses on the metabolic engineering of Escherichia coli K12 to achieve efficient production of 1,3-BDO from glucose via acetoacetyl-CoA, 3-hydroxybutyryl-CoA, and 3-hydroxybutyraldehyde. The accumulation of an intermediary metabolite (pyruvate) and a byproduct (3-hydroxybutyric acid) was reduced by disruption of the negative transcription factor (PdhR) for pyruvate dehydrogenase complex (PDHc) and employing an efficient alcohol dehydrogenase (YjgB), respectively. Additionally, to improve NADPH availability, carbon flux was redirected from the Embden-Meyerhof-Parnas (EMP) pathway to the Entner-Doudoroff (ED) pathway. One resulting strain achieved a record-high titer of 790 mM (∼71.1 g/L) with a yield of 0.65 mol/mol for optically pure (R)-1,3-BDO, with an enantiomeric excess (e.e.) value of 98.5 %. These findings are useful in the commercial production of 1,3-BDO and provide valuable insights into the development of an efficient cell factory for other acetyl-CoA derivatives.

Identification and functional characterization of a novel aldo-keto reductase from Aloe vera.

MAIN CONCLUSION: The present investigation profoundly asserted the catalytic potential of plant-based aldo-ketoreductase, postulating its role in polyketide biosynthesis and providing new insights for tailored biosynthesis of vital plant polyketides for therapeutics. Plants hold great potential as a future source of innovative biocatalysts, expanding the possibilities within chemical reactions and generating a variety of benefits. The aldo-keto reductase (AKR) superfamily includes a huge collection of NAD(P)H-dependent oxidoreductases that carry out a variety of redox reactions essential for biosynthesis, detoxification, and intermediary metabolism. The present study involved the isolation, cloning, and purification of a novel aldo-ketoreductase (AvAKR) from the leaves of Aloe vera (Aloe barbadensis Miller) by heterologous gene expression in Escherichia coli based on the unigene sequences of putative ketoreductase and cDNA library screening by oligonucleotide hybridization. The in-silico structural analysis, phylogenetic relationship, and molecular modeling were outranged to approach the novelty of the sequence. Additionally, agroinfiltration of the candidate gene tagged with a green fluorescent protein (GFP) was employed for transient expression in the Nicotiana benthamiana to evaluate the sub-cellular localization of the candidate gene. The AvAKR preferred cytoplasmic localization and shared similarities with the known plant AKRs, keeping the majority of the conserved active-site residues in the AKR superfamily enzymes. The enzyme facilitated the NADPH-dependent reduction of various carbonyl substrates, including benzaldehyde and sugars, proclaiming a broad spectrum range. Our study successfully isolated and characterized a novel aldo-ketoreductase (AvAKR) from Aloe vera, highlighting its versatile NADPH-dependent carbonyl reduction proficiency therewith showcasing its potential as a versatile biocatalyst in diverse redox reactions.

Preparation of polyhydroxyalkanoate-based magnetic microspheres for carbonyl reductase purification and immobilization.

A polyhydroxyalkanoate (PHA) magnetic microsphere was designed for one-step purification and immobilization of a novel carbonyl reductase (RLSR5) from recombinant Escherichia coli lysate. The hydrophobic core of this microsphere was composed of a highly biocompatible polymer, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), in which magnetic Fe3O4 particles were embedded during solvent evaporation. The hydrophilic shell of the fusion protein formed by PHA particle-binding protein (PhaP) and RLSR5 (PR) was expressed in recombinant E. coli. The magnetic core of Fe3O4@PHBHHx directly purified the hydrophilic shell from the E. coli lysate, and the two self-assembled to form Fe3O4@PHBHHx-PR through hydrophobic and hydrophilic interactions, eliminating the separation of the fusion protein. The microstructure, magnetic properties, morphology, size, and dispersion of Fe3O4@PHBHHx-PR were investigated by XRD, VSM, SEM, TEM, elemental mapping and DLS. It was found that Fe3O4@PHBHHx-PR correctly assembled, with a well dispersed spherical structure at the nanoscale and superparamagnetism properties. The amount of RLSR5 immobilized on PHA microspheres reached 121.9 mg/g. The Fe3O4@PHBHHx-PR was employed to synthesize (R)-tolvaptan with 99 % enantiomeric excess and 97 % bioconversion efficiency, and the catalyst maintained 78.6 % activity after 10 recovery cycles. These PHA magnetic microspheres are versatile carriers for enzyme immobilization and demonstrate improved stability and reusability of the free enzyme.

Conditional Dependency of LP-184 on Prostaglandin Reductase 1 is Synthetic Lethal in Pancreatic Cancers with DNA Damage Repair Deficiencies.

The greater efficacy of DNA-damaging drugs for pancreatic adenocarcinoma (PDAC) relies on targeting cancer-specific vulnerabilities while sparing normal organs and tissues due to their inherent toxicities. We tested LP-184, a novel acylfulvene analog, for its activity in preclinical models of PDAC carrying mutations in the DNA damage repair (DDR) pathways. Cytotoxicity of LP-184 is solely dependent on prostaglandin reductase 1 (PTGR1), so that PTGR1 expression robustly correlates with LP-184 cytotoxicity in vitro and in vivo. Low-passage patient-derived PDAC xenografts with DDR deficiencies treated ex vivo are more sensitive to LP-184 compared with DDR-proficient tumors. Additional in vivo testing of PDAC xenografts for their sensitivity to LP-184 demonstrates marked tumor growth inhibition in models harboring pathogenic mutations in ATR, BRCA1, and BRCA2. Depletion of PTGR1, however, completely abrogates the antitumor effect of LP-184. Testing combinatorial strategies for LP-184 aimed at deregulation of nucleotide excision repair proteins ERCC3 and ERCC4 established synergy. Our results provide valuable biomarkers for clinical testing of LP-184 in a large subset of genetically defined characterized refractory carcinomas. High PTGR1 expression and deleterious DDR mutations are present in approximately one third of PDAC making these patients ideal candidates for clinical trials of LP-184.