A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna.

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6ß-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer.

Enhancing Human Treg Cell Induction through Engineered Dendritic Cells and Zinc Supplementation.

Regulatory T (Treg) cells hold promise for the ultimate cure of immune-mediated diseases. However, how to effectively restore Treg function in patients remains unknown. Previous reports suggest that activated dendritic cells (DCs) de novo synthesize locally high concentrations of 1,25-dihydroxy vitamin D, i.e., the active vitamin D or 1,25(OH)2D by upregulating the expression of 25-hydroxy vitamin D 1α-hydroxylase. Although 1,25(OH)2D has been shown to induce Treg cells, DC-derived 1,25(OH)2D only serves as a checkpoint to ensure well-balanced immune responses. Our animal studies have shown that 1,25(OH)2D requires high concentrations to generate Treg cells, which can cause severe side effects. In addition, our animal studies have also demonstrated that dendritic cells (DCs) overexpressing the 1α-hydroxylase de novo synthesize the effective Treg-inducing 1,25(OH)2D concentrations without causing the primary side effect of hypercalcemia (i.e., high blood calcium levels). This study furthers our previous animal studies and explores the efficacy of the la-hydroxylase-overexpressing DCs in inducing human CD4+FOXP3+regulatory T (Treg) cells. We discovered that the effective Treg-inducing doses of 1,25(OH)2D were within a range. Additionally, our data corroborated that the 1α-hydroxylase-overexpressing DCs synthesized 1,25(OH)2D within this concentration range in vivo, thus facilitating effective Treg cell induction. Moreover, this study demonstrated that 1α-hydroxylase expression levels were pivotal for DCs to induce Treg cells because physiological 25(OH)D levels were sufficient for the engineered but not parental DCs to enhance Treg cell induction. Interestingly, adding non-toxic zinc concentrations significantly augmented the Treg-inducing capacity of the engineered DCs. Our new findings offer a novel therapeutic avenue for immune-mediated human diseases, such as inflammatory bowel disease, type 1 diabetes, and multiple sclerosis, by integrating zinc with the 1α-hydroxylase-overexpressing DCs.

The impact of physical activity on promoter-specific methylation of genes involved in the redox-status and disease progression: A longitudinal study on post-surgery female breast cancer patients undergoing medical treatment.

Most anticancer treatments act on oxidative-stress pathways by producing reactive oxygen species (ROS) to kill cancer cells, commonly resulting in consequential drug-induced systemic cytotoxicity. Physical activity (PA) has arisen as an integrative cancer therapy, having positive health effects, including in redox-homeostasis. Here, we investigated the impact of an online supervised PA program on promoter-specific DNA methylation, and corresponding gene expression/activity, in 3 antioxidants- (SOD1, SOD2, and CAT) and 3 breast cancer (BC)-related genes (BRCA1, L3MBTL1 and RASSF1A) in a population-based sample of women diagnosed with primary BC, undergoing medical treatment. We further examined mechanisms involved in methylating and demethylating pathways, predicted biological pathways and interactions of exercise-modulated molecules, and the functional relevance of modulated antioxidant markers on parameters related to aerobic capacity/endurance, physical fatigue and quality of life (QoL). PA maintained levels of SOD activity in blood plasma, and at the cellular level significantly increased SOD2 mRNA (≈+77 %), contrary to their depletion due to medical treatment. This change was inversely correlated with DNA methylation in SOD2 promoter (≈-20 %). Similarly, we found a significant effect of PA only on L3MBTL1 promoter methylation (≈-25 %), which was inversely correlated with its mRNA (≈+43 %). Finally, PA increased TET1 mRNA levels (≈+15 %) and decreased expression of DNMT3B mRNA (≈-28 %). Our results suggest that PA-modulated DNA methylation affects several signalling pathways/biological activities involved in the cellular oxidative stress response, chromatin organization/regulation, antioxidant activity and DNA/protein binding. These changes may positively impact clinical outcomes and improve the response to cancer treatment in post-surgery BC patients.

Functional characterization of a cold related flavanone 3-hydroxylase from Tetrastigma hemsleyanum: an in vitro, in silico and in vivo study.

Tetrastigma hemsleyanum Diels et Gilg, a traditional Chinese medicine, frequently suffers from cold damage in the winter, leading to lower yields. There is a pressing need to improve cold resistance; however, the mechanisms underlying T. hemsleyanum responses to cold stress are still not clearly understood. Here, we explored the function of the flavanone 3-hydroxylase gene (ThF3H) in T. hemsleyanum under cold treatment. The open reading frame of ThF3H is 1092 bp and encodes 363 amino acid residues. In vitro, the ThF3H enzyme was expressed in E. coli and successfully catalyzed naringenin and eriodictyol into dihydrokaempferol and dihydroquercetin, respectively. ThF3H exhibited a higher affinity for naringenin than for eriodictyol, which was in accordance with an in silico molecular docking analysis. The optimal pH and temperature for ThF3H activity were 7.0 and 30 °C, respectively. In vivo, overexpression of the ThF3H gene enhanced the cold tolerance of transgenic Arabidopsis lines, which was likely due to the increase in flavonoids. Collectively, the function of a cold-related ThF3H in the flavonoid biosynthesis pathway may be helpful for improving the cold tolerance of T. hemsleyanum through molecular breeding techniques.

In vitro regulation of fibroblast growth factor 23 by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D synthesized by osteocyte-like MC3T3-E1 cells.

Fibroblast growth factor 23 (FGF23) is produced and secreted by osteocytes and is essential for maintaining phosphate homeostasis. One of the main regulators of FGF23, 1,25-dihydroxyvitamin D (1,25(OH)2D3), is primarily synthesized in the kidney from 25-hydroxyvitamin D (25(OH)D) by 1α-hydroxylase (encoded by CYP27B1). Hitherto, it is unclear whether osteocytes can convert 25(OH)D and thereby allow for 1,25(OH)2D3 to induce FGF23 production and secretion locally. Here, we differentiated MC3T3-E1 cells toward osteocyte-like cells expressing and secreting FGF23. Treatment with 10-6 M 25(OH)D resulted in conversion of 25(OH)D to 150 pmol/L 1,25(OH)2D3 and increased FGF23 expression and secretion, but the converted amount of 1,25(OH)2D3 was insufficient to trigger an FGF23 response, so the effect on FGF23 was most likely directly caused by 25(OH)D. Interestingly, combining phosphate with 25(OH)D resulted in a synergistic increase in FGF23 expression and secretion, likely due to activation of additional signaling pathways by phosphate. Blockage of the vitamin D receptor (VDR) only partially abolished the effects of 25(OH)D or 25(OH)D combined with phosphate on Fgf23, while completely inhibiting the upregulation of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1), encoding for 24-hydroxylase. RNA sequencing and in silico analyses showed that this could potentially be mediated by the nuclear receptors Retinoic Acid Receptor ß (RARB) and Estrogen Receptor 2 (ESR2). Taken together, we demonstrate that osteocytes are able to convert 25(OH)D to 1,25(OH)2D3, but this is insufficient for FGF23 activation, implicating a direct effect of 25(OH)D in the regulation of FGF23, which occurs at least partially independent from its cognate VDR. Moreover, phosphate and 25(OH)D synergistically increase expression and secretion of FGF23, which warrants investigating consequences in patients receiving a combination of vitamin D analogues and phosphate supplements. These observations help us to further understand the complex relations between phosphate, vitamin D, and FGF23.

Exploring the bioremediation capability of petroleum-contaminated soils for enhanced environmental sustainability and minimization of ecotoxicological concerns.

The bioremediation of soils contaminated with petroleum hydrocarbons (PHCs) has emerged as a promising approach, with its effectiveness contingent upon various types of PHCs, i.e., crude oil, diesel, gasoline, and other petroleum products. Strategies like genetically modified microorganisms, nanotechnology, and bioaugmentation hold potential for enhancing remediation of polycyclic aromatic hydrocarbon (PAH) contamination. The effectiveness of bioremediation relies on factors such as metabolite toxicity, microbial competition, and environmental conditions. Aerobic degradation involves enzymatic oxidative reactions, while bacterial anaerobic degradation employs reductive reactions with alternative electron acceptors. Algae employ monooxygenase and dioxygenase enzymes, breaking down PAHs through biodegradation and bioaccumulation, yielding hydroxylated and dihydroxylated intermediates. Fungi contribute via mycoremediation, using co-metabolism and monooxygenase enzymes to produce CO2 and oxidized products. Ligninolytic fungi transform PAHs into water-soluble compounds, while non-ligninolytic fungi oxidize PAHs into arene oxides and phenols. Certain fungi produce biosurfactants enhancing degradation of less soluble, high molecular-weight PAHs. Successful bioremediation offers sustainable solutions to mitigate petroleum spills and environmental impacts. Monitoring and assessing strategy effectiveness are vital for optimizing biodegradation in petroleum-contaminated soils. This review presents insights and challenges in bioremediation, focusing on arable land safety and ecotoxicological concerns.

Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes.

Introduction: The potentially unlimited number of cardiomyocyte (CMs) derived from human induced pluripotent stem cells (hiPSCs) in vitro facilitates high throughput applications like cell transplantation for myocardial repair, disease modelling, and cardiotoxicity testing during drug development. Despite promising progress in these areas, a major disadvantage that limits the use of hiPSC derived CMs (hiPSC-CMs) is their immaturity. Methods: Three hiPSC lines (PCBC-hiPSC, DP3-hiPSCs, and MLC2v-mEGFP hiPSC) were differentiated into CMs (PCBC-CMs, DP3-CMs, and MLC2v-CMs, respectively) with or without retinoic acid (RA). hiPSC-CMs were either maintained up to day 30 of contraction (D30C), or D60C, or purified using lactate acid and used for experiments. Purified hiPSC-CMs were cultured in basal maturation medium (BMM) or BMM supplemented with ascorbic acid (AA) for 14 days. The AA treated and non-treated hiPSC-CMs were characterized for sarcomeric proteins (MLC2v, TNNI3, and MYH7), ion channel proteins (Kir2.1, Nav1.5, Cav1.2, SERCA2a, and RyR), mitochondrial membrane potential, metabolomics, and action potential. Bobcat339, a selective and potent inhibitor of DNA demethylation, was used to determine whether AA promoted hiPSC-CM maturation through modulating DNA demethylation. Results: AA significantly increased MLC2v expression in PCBC-CMs, DP3-CMs, MLC2v-CMs, and RA induced atrial-like PCBC-CMs. AA treatment significantly increased mitochondrial mass, membrane potential, and amino acid and fatty acid metabolism in PCBC-CMs. Patch clamp studies showed that AA treatment induced PCBC-CMs and DP3-CMs adaptation to a ventricular-like phenotype. Bobcat339 inhibited MLC2v protein expression in AA treated PCBC-CMs and DP3-CMs. DNA demethylation inhibition was also associated with reduced TET1 and TET2 protein expressions and reduced accumulation of the oxidative product, 5 hmC, in both PCBC-CMs and DP3-CMs, in the presence of AA. Conclusions: Ascorbic acid induced MLC2v protein expression and promoted ventricular-like CM subtype in hiPSC-CMs. The effect of AA on hiPSC-CM was attenuated with inhibition of TET1/TET2 mediated DNA demethylation.

Biosynthesis of kratom opioids.

Mitragynine, an analgesic alkaloid from the plant Mitragyna speciosa (kratom), offers a safer alternative to clinical opioids such as morphine, owing to its more favorable side effect profile. Although kratom has been traditionally used for stimulation and pain management in Southeast Asia, the mitragynine biosynthesis pathway has remained elusive. We embarked on a search for mitragynine biosynthetic genes from the transcriptomes of kratom and other members of the Rubiaceae family. We studied their functions in vitro and in vivo. Our investigations led to the identification of several reductases and an enol methyltransferase that forms a new clade within the SABATH methyltransferase family. Furthermore, we discovered a methyltransferase from Hamelia patens (firebush), which catalyzes the final step. With the tryptamine 4-hydroxylase from the psychedelic mushroom Psilocybe cubensis, we accomplished the four-step biosynthesis for mitragynine and its stereoisomer, speciogynine in both yeast and Escherichia coli when supplied with tryptamine and secologanin. Although we have yet to pinpoint the authentic hydroxylase and methyltransferase in kratom, our discovery completes the mitragynine biosynthesis. Through these breakthroughs, we achieved the microbial biosynthesis of kratom opioids for the first time. The remarkable enzyme promiscuity suggests the possibility of generating derivatives and analogs of kratom opioids in heterologous systems.

Effects of Scutellaria strigillosa Hemsl. extract on HepG2 cell proliferation and apoptosis through binding to aspartate ß-hydroxylase.

This study aims to examine the impacts of Scutellaria strigillosa Hemsl. (SSH) on the proliferation, apoptosis of human hepatoma cell HepG2 and screen the bioactive components. We found that SSH extract inhibited HepG2 proliferation, arrested cell division prior to S phase. Additionally, SSH extract exposure induced apoptosis, and increased the proportions of late apoptotic cells. Specifically, we focus on the inhibitory effect of SSH extract on aspartate ß-hydroxylase, a key therapeutic target of hepatocellular carcinoma closely related with the proliferation and apoptosis of HepG2. We found SSH extract with notable inhibitory activity against aspartate ß-hydroxylase, elucidated the main bioactive constituents by HPLC-Q-TOF/MS and Molecular docking analysis. In conclusion, these results provided the antiproliferative and proapoptotic effects of SSH on HepG2 cell, elucidated the main bioactive constituents based on aspartate ß-hydroxylase inhibition. These data revealed the potential value of SSH and its bioactive components for the prevention and treatment of liver cancer for the first time.

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification.

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.

Structures of L-proline trans-hydroxylase reveal the catalytic specificity and provide deeper insight into AKG-dependent hydroxylation.

L-Proline hydroxylase is a member of the non-heme Fe2+/α-ketoglutarate (AKG)-dependent hydroxylase family that catalyzes the reaction from L-proline to hydroxy-L-proline, which is widely used in drug synthesis, biochemistry, food supplementation and cosmetic industries. Here, the first crystal structure of L-proline trans-hydroxylase and its complexes with substrate and product are reported, which reveal the structural basis of trans-cis proline hydroxylation selectivity. Structure comparison with other AKG-dependent hydroxylases identifies conserved amino acid residues, which may serve as signatures of in-line or off-line AKG binding modes in the AKG-dependent enzyme family.

Selenite as a dual apoptotic and ferroptotic agent synergizes with EGFR and KRAS inhibitors with epigenetic interference.

BACKGROUND: Selenium, an essential trace element, has previously been investigated as a pro-apoptotic and DNA demethylation agent. It sensitizes the response to chemotherapy in patients who were refractory to cytotoxic agents. Meanwhile, ferroptosis is a novel approach to cancer treatment by triggering cell death and reversing drug resistance. The role of selenium in treating cancer cells harboring druggable oncogenic alterations and its underlying mechanism are largely unknown. RESULTS: We treated lung adenocarcinoma cell lines-EGFR-mutant H1975 (H1975 EGFR p.L858R and p.T790M) and KRAS-mutant H358 (H358 KRAS p.G12C), with sodium selenite to examine its effect on cell apoptosis, ferroptosis, and DNA methylation, as well as its interaction with existing targeted therapy, osimertinib, and adagrasib. We observed selenite to be a dual apoptotic and ferroptotic agent on lung cancer cells, associated with the activation of p38-ATF4-DDIT3 axis in the unfolded protein response. Ferroptosis induction was more remarkable in H1975 than H358. Selenite also altered cellular DNA methylation machinery through downregulating DNMT1 and upregulating TET1, though not as a major mechanism of its activity. Low-dose selenite synergized with osimertinib in EGFR-mutant H1975, and with adagrasib in KRAS-mutant H358, with stronger synergism observed in H1975. CONCLUSION: These results suggest that selenite is a potential apoptotic and ferroptotic drug candidate for the treatment of especially EGFR- and potentially KRAS-mutant lung cancer.

Molecular characterization of a feruloyl-CoA 6'-hydroxylase involved in coumarin biosynthesis in Clematis terniflora DC.

Coumarin is an important secondary metabolite that affects plant physiology. It is a lactone of cis-o-hydroxycinnamic acid and widely exists in medicinal plants. Clematis terniflora DC. is a plant belonging to Ranunculaceae and is rich in variety of coumarins. Feruloyl-CoA 6'-hydroxylase has been reported as a key enzyme in the formation of coumarin basic skeleton only in some common plants, however, its evidence in other species is still lacking especially for the biosynthesis of coumarins in C. terniflora. In the present study, we identified a feruloyl-CoA 6'-hydroxylase CtF6'H in C. terniflora, and functional characterization indicated that CtF6'H could hydroxylate feruloyl-CoA to 6-hydroxyferuloyl-CoA. Furthermore, the expression level of CtF6'H was differed among different tissues in C. terniflora, while under UV-B radiation, the level of CtF6'H was increased in the leaves. Biochemical characteristics and subcellular location showed that CtF6'H was mainly present in the cytosol. The crystal structure of CtF6'H was simulated by homology modeling to predict the potential residues affecting enzyme activity. This study provides the additional evidence of feruloyl-CoA 6'-hydroxylase in different plant species and enriches our understanding of biosynthetic mechanism of coumarin in C. terniflora.

Inborn errors of bile acid metabolism in Japan.

Bile acids are a category of steroids biosynthesized from cholesterol in the liver. Inborn errors of their metabolism are inherited in an autosomal recessive manner, resulting in enzyme deficiencies affecting the bile acid biosynthetic pathway. These defects in the pathway cause accumulation of unusual bile acids or bile alcohols. Unusual bile acids are highly cytotoxic, causing injury to the liver. These unusual bile acids damage hepatocytes, resulting in cholestatic liver injury beginning in infancy. Except for cerebrotendinous xanthomatosis and some secondary defects, various inborn errors of bile acid metabolism (IEBAM) have been reported from Japan, affecting eight patients including three with 3ß-hydroxy-Δ5 -C27 -steroid dehydrogenase/isomerase deficiency, three with Δ4 -3-oxosteroid 5ß-reductase deficiency, one with oxysterol 7α-hydroxylase deficiency, and one with bile acid-CoA: amino acid N-acyltransferase deficiency. Distinctive laboratory findings in patients with 3ß-hydroxy-Δ5 -C27 -steroid dehydrogenase/isomerase deficiency, Δ4 -3-oxosteroid 5ß-reductase deficiency, and oxysterol 7α-hydroxylase deficiency include normal serum γ-glutamyltransferase and total bile acids concentrations despite presence of cholestasis (elevated serum direct bilirubin) from infancy. Pediatricians and pediatric surgeons who suspect a case of IEBAM should obtain urinary and serum bile acid analyses using gas or liquid chromatography-mass spectrometry as well as genetic analyses. Available treatments include oral cholic acid, chenodeoxycholic acid, glycocholic acid, and ursodeoxycholic acid; fat-soluble vitamin supplementation; and liver transplantation. Early diagnosis and treatment can offer a good outcome.

Molecular Characterization of a Stereoselective and Promiscuous Flavanone 3-Hydroxylase from Carthamus tinctorius L.

Flavanone 3-hydroxylases (F3Hs) belong to the 2-oxoglutarate-dependent dioxygenase family and play an important role in plant flavonoid biosynthesis. However, the stereoselective catalytic mechanism and substrate promiscuity of this type of enzyme are not well understood. In this study, we identified and biochemically characterized CtF3H1, an F3H from Carthamus tinctorius, a plant used in traditional Chinese medicine that exhibits high stereoselectivity and substrate promiscuity toward structurally diverse (2S)-flavanones. Isothermal titration calorimetry revealed that CtF3H1 exhibits distinctly different binding behaviors with (2S)-flavanone (2S-naringenin) and (2R)-flavanone (2R-naringenin), and these differences govern its stereoselectivity. An investigation of the structure-activity relationships between the enzyme and its substrates demonstrated that 7-OH and/or 4'-OH are necessary for regio- and stereoselective 3-hydroxylation of (2S)-flavanones. Homology modeling and molecular docking combined with site-directed mutagenesis identified the amino acid residues necessary for hydroxylation. These findings demonstrate the potential versatility of CtF3H1 in regio- and stereohydroxylation and provide molecular insights into the catalytic mechanism of F3H for further enzyme engineering.

Hypercalcemia worsened after vitamin D supplementation in a sarcoidosis patient: A case report.

RATIONALE: There are many causes of hypercalcemia, with hyperparathyroidism and malignancy accounting for 90% of cases. Sarcoidosis and the intake of vitamin D supplements may also cause hypercalcemia, although the occurrence rate is low if only one is involved. We herein report a sarcoidosis patient who developed hypercalcemia after taking cholecalciferol (vitamin D supplement) for a year. PATIENT CONCERN: A 62-year-old Japanese man presented with hypercalcemia and acute kidney injury along with symptoms of fatigue and appetite loss while being followed up for sarcoidosis. DIAGNOSES: We determined that a combination of cholecalciferol supplementation and sarcoidosis had led to hypercalcemia for several reasons. First, hypercalcemia had not been noted when this patient had first been admitted due to sarcoidosis-related respiratory failure several years earlier, which we presumed that was the highest sarcoidosis disease activity. Second, low serum 25-OH Vit.D3 and high 1,25-(OH)2 Vit.D3 levels were noted despite cholecalciferol supplementation for a year, suggesting that 1-α-hydroxylase overexpression caused by sarcoidosis accelerated the conversion from 25-OH Vit.D3 to 1,25-(OH)2 Vit.D3. INTERVENTIONS: Although initially resistant to preservative management, the hypercalcemia promptly improved after starting corticosteroid treatment. OUTCOMES: Hypercalcemia and acute kidney injury were normalized after corticosteroid treatment. LESSONS: We should be aware of patients' medications, especially in patients with granulomatosis disease. The concomitant measurement of 25-OH Vit.D3 and 1,25-(OH)2 Vit.D3 levels is useful for determining the cause of hypercalcemia.

Manganese-driven CoQ deficiency.

Overexposure to manganese disrupts cellular energy metabolism across species, but the molecular mechanism underlying manganese toxicity remains enigmatic. Here, we report that excess cellular manganese selectively disrupts coenzyme Q (CoQ) biosynthesis, resulting in failure of mitochondrial bioenergetics. While respiratory chain complexes remain intact, the lack of CoQ as lipophilic electron carrier precludes oxidative phosphorylation and leads to premature cell and organismal death. At a molecular level, manganese overload causes mismetallation and proteolytic degradation of Coq7, a diiron hydroxylase that catalyzes the penultimate step in CoQ biosynthesis. Coq7 overexpression or supplementation with a CoQ headgroup analog that bypasses Coq7 function fully corrects electron transport, thus restoring respiration and viability. We uncover a unique sensitivity of a diiron enzyme to mismetallation and define the molecular mechanism for manganese-induced bioenergetic failure that is conserved across species.

Integrated Metabolite and Transcriptome Profiling-Mediated Gene Mining of Sida cordifolia Reveals Medicinally Important Genes.

Sida cordifolia is a medicinal shrub that is conventionally used in the Indian system of medicine;however, the genes contributing to its medicinal properties have been minimally explored, thus limiting its application. High-throughputsequencing and Liquid Chromatography with tandem mass spectrometry(LC-MS/MS) technologies were applied to unravel the medicinally important bioactive compounds. As a result, transcriptomic sequencing generated more than 12 GB of clean data, and 187,215 transcripts were obtained by de novoassembly. These transcripts were broadly classified into 20 classes, based on the gene ontology classification, and 6551 unigenes were annotated using Kyoto Encyclopedia of Genes and Genomes (KEGG) database with more than 142 unigenes involved in the biosynthesis of secondary metabolites. LC-MS/MS analysis of three tissues of Sida cordifolia revealed that acacetin and procyanidin are some important metabolites identified thatcontribute to its medicinal value. Several key enzymes witha crucial role in phenylpropanoid and flavonoid biosynthetic pathways were identified, especially phenylalanine ammonia lyase, which might be an important rate-limiting enzyme. Real-Time Quantitative Reverse Transcription Polymerase chain reaction (qRT-PCR) analysis revealed enzymes, such as Phenylalanine ammonia lyase (PAL), Cinnamyl alcohol dehydrogenase 1 (CAD), Cinnamoyl-CoA reductase 1 (CF1) and Trans cinnamate 4-monooxygenase(TCM), which were predominantly expressed in root compared to leaf and stem tissue. The study provides a speculative insight for the screening of active metabolites and metabolic engineering in Sida cordifolia.

Discovery of New Phenylacetone Monooxygenase Variants for the Development of Substituted Indigoids through Biocatalysis.

Indigoids are natural pigments obtained from plants by ancient cultures. Romans used them mainly as dyes, whereas Asian cultures applied these compounds as treatment agents for several diseases. In the modern era, the chemical industry has made it possible to identify and develop synthetic routes to obtain them from petroleum derivatives. However, these processes require high temperatures and pressures and large amounts of solvents, acids, and alkali agents. Thus, enzyme engineering and the development of bacteria as whole-cell biocatalysts emerges as a promising green alternative to avoid the use of these hazardous materials and consequently prevent toxic waste generation. In this research, we obtained two novel variants of phenylacetone monooxygenase (PAMO) by iterative saturation mutagenesis. Heterologous expression of these two enzymes, called PAMOHPCD and PAMOHPED, in E. coli was serendipitously found to produce indigoids. These interesting results encourage us to characterize the thermal stability and enzyme kinetics of these new variants and to evaluate indigo and indirubin production in a whole-cell system by HPLC. The highest yields were obtained with PAMOHPCD supplemented with L-tryptophan, producing ~3000 mg/L indigo and ~130.0 mg/L indirubin. Additionally, both enzymes could oxidize and produce several indigo derivatives from substituted indoles, with PAMOHPCD being able to produce the well-known Tyrian purple. Our results indicate that the PAMO variants described herein have potential application in the textile, pharmaceutics, and semiconductors industries, prompting the use of environmentally friendly strategies to obtain a diverse variety of indigoids.

[The albino mechanism of a new theanine-rich tea cultivar 'Fuhuang 2'].

To explore the mechanism of tea albino variation and high theanine formation, 'Fuyun 6' and a new theanine-rich tea cultivar 'Fuhuang 2' were as materials in this study, pigment content, metabolome and transcriptome of the two cultivars were analyzed by ultramicroelectron microscopy, widely targeted metabolomics, targeted metabolomics and transcriptomics. The results showed that five catechins, theobromine, caffeine, and 20 free amino acids, including theanine, glutamine, arginine, etc., were identified by targeted metabolomics. The amino acid content of 'Fuhuang 2' was significantly higher than that of 'Fuyun 6', and the theanine content was as high as 57.37 mg/g in 'Fuhuang 2'. The ultrastructure of leaves showed that the chloroplast cell structure of 'Fuhuang 2' was fuzzy, most of the grana lamellae were arranged in disorder, with large gaps, and the thylakoids were filiform. The determination of pigments showed that compared with 'Fuyun 6', the contents of chlorophyll A and B, carotenoids, flavonoids and other pigments of 'Fuhuang 2' decreased significantly, some important pigment-related-genes, such as chlorophyllase (CLH), 9-cis-epoxycarotenoid dioxygenase (NCED), flavonoid 3ß-hydroxylase (F3H) and flavonoid 3', 5'-hydroxylase (F3'5'H) were significantly changed. Compared with 'Fuyun 6', 'Fuhuang 2' identified 138 significantly changed metabolites (SCMs) and 658 differentially expressed genes (DEGs). KEGG enrichment analysis showed that SCMs and DEGs were significantly enriched in amino acid biosynthesis, glutathione metabolism and TCA cycle. In general, the albino phenotype of 'Fuhuang 2' may be caused by a deficiency in photosynthetic proteins, chlorophyll metabolism genes and chlorophyll content. The accumulation of high theanine in 'Fuhuang 2' may be due to the low nitrogen consumption in yellowed leaves and the lack of carbon skeleton, amino and nitrogen resources are stored more effectively, resulting in the up regulation of metabolites and related gene expression in the amino acid synthesis pathway, theanine has become a significant accumulation of nitrogen-containing compounds in yellowed leaves.