Regulation of hypothalamic reactive oxygen species and feeding behavior by phosphorylation of the beta 2 thyroid hormone receptor isoform.

Unlike other thyroid hormone receptors (THRs), the beta 2 isoform (THRB2) has a restricted expression pattern and is uniquely and abundantly phosphorylated at a conserved serine residue S101 (S102 in humans). Using tagged and or phosphorylation-defective (S101A) THRB2 mutant mice, we show that THRB2 is present in a large subset of POMC neurons and mitigates ROS accumulation during ROS-triggering events, such as fasting/refeeding or high fat diet (HFD). Excessive ROS accumulation in mutant POMC neurons was accompanied by a skewed production of orexigenic/anorexigenic hormones, resulting in elevated food intake. The prolonged exposure to pathogenic hypothalamic ROS levels during HFD feeding lead to a significant loss of POMC neurons in mutant versus wild-type (WT) mice. In cultured cells, the presence of WT THRB2 isoform, but not other THRs, or THRB2S101A, reduced ROS accumulation upon exogenous induction of oxidative stress by tert-butyl hydroperoxide. The protective function of phospho-THRB2 (pTHRB2) did not require thyroid hormone (TH), suggesting a TH-independent role of the THRB2 isoform, and phospho-S101 in particular, in regulating oxidative stress. We propose that pTHRB2 has a fundamental role in neuronal protection against ROS cellular damage, and mitigates hypothalamic pathological changes found in diet-induced obesity.

Reduction of neutral lipid reservoirs, bioconversion and untargeted metabolomics reveal distinct roles for vitamin K isoforms on lipid metabolism.

Vitamin K isoforms are known as co-factors for the synthesis of blood-clotting proteins, but several other bioactivities were reported. In this work, we isolated a vitamin K1-analogue (OH-PhQ) from the cyanobacterium Tychonema sp. LEGE 07196 with lipid reducing activity. OH-PhQ reduced neutral lipid reservoirs with an EC50 value of 31 µM after 48 h exposure in zebrafish larvae, while other vitamin K isoforms had EC50 values of 21.1 µM (K2) and 1.2 µM (K3). No lipid reducing activity was observed for K1 up to 50 µM. The presence of vitamin K isoforms was studied in zebrafish after exposure (OH-PhQ, K1, K2 and K3), and a clear preference for bioconversion was observed to retain K1 and OH-PhQ. Untargeted metabolomics revealed different biological effects for vitamin K isoforms on the subclass and metabolite level, but similarities were present on the compound class level, particularly on the regulation of glycerophospholipids. Our data showed for the first time a lipid reducing activity of OH-PhQ and performed a comparative analysis of vitamin K isoforms, which could be important for the development of future nutraceuticals or food supplements.

Krüppel-like factor 4 in transcriptional control of the three unique isoforms of Agouti-related peptide in mice.

Agouti-related peptide (AgRP/Agrp) within the hypothalamic arcuate nucleus (ARC) contributes to the control of energy balance, and dysregulated Agrp may contribute to metabolic adaptation during prolonged obesity. In mice, three isoforms of Agrp are encoded via distinct first exons. Agrp-A (ENSMUST00000005849.11) contributed 95% of total Agrp in mouse ARC, whereas Agrp-B (ENSMUST00000194654.2) dominated in placenta (73%). Conditional deletion of Klf4 from Agrp-expressing cells (Klf4Agrp-KO mice) reduced Agrp mRNA and increased energy expenditure but had no effects on food intake or the relative abundance of Agrp isoforms in the ARC. Chronic high-fat diet feeding masked these effects of Klf4 deletion, highlighting the context-dependent contribution of KLF4 to Agrp control. In the GT1-7 mouse hypothalamic cell culture model, which expresses all three isoforms of Agrp (including Agrp-C, ENSMUST00000194091.6), inhibition of extracellular signal-regulated kinase (ERK) simultaneously increased KLF4 binding to the Agrp promoter and stimulated Agrp expression. In addition, siRNA-mediated knockdown of Klf4 reduced expression of Agrp. We conclude that the expression of individual isoforms of Agrp in the mouse is dependent upon cell type and that KLF4 directly promotes the transcription of Agrp via a mechanism that is superseded during obesity.NEW & NOTEWORTHY In mice, three distinct isoforms of Agouti-related peptide are encoded via distinct first exons. In the arcuate nucleus of the hypothalamus, Krüppel-like factor 4 stimulates transcription of the dominant isoform in lean mice, but this mechanism is altered during diet-induced obesity.

Strength in numbers: An isoform variety of homogalacturonan modifying enzymes may contribute to pollen tube fitness.

Pectin is a major component of the cell wall in land plants. It plays crucial roles in cell wall assembly, cell growth, shaping, and signaling. The relative abundance of pectin in the cell wall is particularly high in rapidly growing organ regions and cell types. Homogalacturonan (HG), a polymer of 1,4-linked α-D-galacturonic acid, is a major pectin constituent in growing and dividing plant cells. In pollen tubes, an extremely rapidly growing cell type, HG is secreted at and inserted into the apical cell wall and is subject to further modification in muro by HG modifying enzymes (HGMEs). These enzymes, including pectin esterases and depolymerases, have multiple isoforms, some of which are specifically expressed in pollen. Given the importance of pectin chemistry for the fitness of pollen tubes, it is of interest to interrogate the potentially crucial roles these isoforms play in pollen germination and elongation. It is hypothesized that different HGME isoforms, through their action on apoplastic HG, may generate differential methylation and acetylation patterns endowing HG polysaccharides with specific, spatially and temporally varying properties that lead to a fine-tuned pattern of cell wall modification. In addition, these isoforms may be differentially activated and/or inhibited depending on the local conditions that may vary at subcellular resolution. In this Update we review the different HGME isoforms identified in recent years in Arabidopsis thaliana and postulate that the multiplicity of these isoforms may allow for specialized substrate recognition and conditional activation, leading to a sophisticated regulation scheme exemplified in the process that governs the dynamic properties of the cell wall in pollen tube growth.

Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

Nitric oxide is required for lung alveolarization revealed by deficiency of argininosuccinate lyase.

Inhaled nitric oxide (NO) therapy has been reported to improve lung growth in premature newborns. However, the underlying mechanisms by which NO regulates lung development remain largely unclear. NO is enzymatically produced by three isoforms of nitric oxide synthase (NOS) enzymes. NOS knockout mice are useful tools to investigate NO function in the lung. Each single NOS knockout mouse does not show obvious lung alveolar phenotype, likely due to compensatory mechanisms. While mice lacking all three NOS isoforms display impaired lung alveolarization, implicating NO plays a pivotal role in lung alveolarization. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing L-arginine, the sole precursor for NOS-dependent NO synthesis. ASL is also required for channeling extracellular L-arginine into a NO-synthetic complex. Thus, ASL deficiency (ASLD) is a non-redundant model for cell-autonomous, NOS-dependent NO deficiency. Here, we assessed lung alveolarization in ASL-deficient mice. Hypomorphic deletion of Asl (AslNeo/Neo) results in decreased lung alveolarization, accompanied with reduced level of S-nitrosylation in the lung. Genetic ablation of one copy of Caveolin-1, which is a negative regulator of NO production, restores total S-nitrosylation as well as lung alveolarization in AslNeo/Neo mice. Importantly, NO supplementation could partially rescue lung alveolarization in AslNeo/Neo mice. Furthermore, endothelial-specific knockout mice (VE-Cadherin Cre; Aslflox/flox) exhibit impaired lung alveolarization at 12 weeks old, supporting an essential role of endothelial-derived NO in the enhancement of lung alveolarization. Thus, we propose that ASLD is a model to study NO-mediated lung alveolarization.

Effects of Creatine Supplementation on the Myostatin Pathway and Myosin Heavy Chain Isoforms in Different Skeletal Muscles of Resistance-Trained Rats.

Creatine has been used to maximize resistance training effects on skeletal muscles, including muscle hypertrophy and fiber type changes. This study aimed to evaluate the impact of creatine supplementation on the myostatin pathway and myosin heavy chain (MyHC) isoforms in the slow- and fast-twitch muscles of resistance-trained rats. Twenty-eight male Wistar rats were divided into four groups: a sedentary control (Cc), sedentary creatine supplementation (Cr), resistance training (Tc), and resistance training combined with creatine supplementation (Tcr). Cc and Tc received standard commercial chow; Cr and Tcr received a 2% creatine-supplemented diet. Tc and Tcr performed a resistance training protocol on a ladder for 12 weeks. Morphology, MyHC isoforms, myostatin, follistatin, and ActRIIB protein expressions were analyzed in soleus and white gastrocnemius portion samples. The results were analyzed using two-way ANOVA and Tukey's test. Tc and Tcr exhibited higher performance than their control counterparts. Resistance training increased the ratio between muscle and body weight, the cross-sectional area, as well as the interstitial collagen fraction. Resistance training alone increased MyHC IIx and follistatin while reducing myostatin (p < 0.001) and ActRIIB (p = 0.040) expressions in the gastrocnemius. Resistance training induced skeletal muscle hypertrophy and interstitial remodeling, which are more evident in the gastrocnemius muscle. The effects were not impacted by creatine supplementation.

The presence of ß'1-COP and ß'2-COP is required for female and male gametophyte development.

Coat protein I (COPI) and Coat protein II (COPII) coated vesicles mediate protein transport in the early secretory pathway. Although several components of COPII vesicles have been shown to have an essential role in Arabidopsis gametogenesis, the function of COPI components in gametogenesis has not been studied in detail. COPI consists of a heptameric complex made of α, ß, ß', γ, δ, ɛ, and ζ-COP subunits and most subunits have several isoforms in Arabidopsis. We have found that two isoforms of the ß'-COP subunit, ß'1-COP and ß'2-COP, are required for female and male gametophyte development. Reciprocal crosses between wild type plants and plants heterozygous for T-DNA insertions in ß'1-COP and ß'2-COP showed that ß'1ß'2-cop gametophytes are not transmitted.

Ancient medicinal plant rosemary contains a highly efficacious and isoform-selective KCNQ potassium channel opener.

Voltage-gated potassium (Kv) channels in the KCNQ subfamily serve essential roles in the nervous system, heart, muscle and epithelia. Different heteromeric KCNQ complexes likely serve distinct functions in the brain but heteromer subtype-specific small molecules for research or therapy are lacking. Rosemary (Salvia rosmarinus) is an evergreen plant used medicinally for millennia for neurological and other disorders. Here, we report that rosemary extract is a highly efficacious opener of heteromeric KCNQ3/5 channels, with weak effects on KCNQ2/3. Using functional screening we find that carnosic acid, a phenolic diterpene from rosemary, is a potent, highly efficacious, PIP2 depletion-resistant KCNQ3 opener with lesser effects on KCNQ5 and none on KCNQ1 or KCNQ2. Carnosic acid is also highly selective for KCNQ3/5 over KCNQ2/3 heteromers. Medicinal chemistry, in silico docking, and mutagenesis reveal that carboxylate-guanidinium ionic bonding with an S4-5 linker arginine underlies the KCNQ3 opening proficiency of carnosic acid, the effects of which on KCNQ3/5 suggest unique therapeutic potential and a molecular basis for ancient neurotherapeutic use of rosemary.

Selenium-analogs based on natural sources as cancer-associated carbonic anhydrase isoforms IX and XII inhibitors.

In the relentless search for new cancer treatments, organoselenium compounds, and carbonic anhydrase (CA) inhibitors have emerged as promising drug candidates. CA isoforms IX and XII are overexpressed in many types of cancer, and their inhibition is associated with potent antitumor/antimetastatic effects. Selenium-containing compounds, particularly selenols, have been shown to inhibit tumour-associated CA isoforms in the nanomolar range since the properties of the selenium atom favour binding to the active site of the enzyme. In this work, two series of selenoesters (1a-19a and 1b-19b), which gathered NSAIDs, carbo/heterocycles, and fragments from natural products, were evaluated against hCA I, II, IX, and XII. Indomethacin (17b) and flufenamic acid (19b) analogs exhibited selectivity for tumour-associated isoform IX in the low micromolar range. In summary, selenoesters that combine NSAIDs with fragments derived from natural sources have been developed as promising nonclassical inhibitors of the tumour-associated CA isoforms.

A group of novel VEGF splice variants as alternative therapeutic targets in renal cell carcinoma.

The efficacy of anti-angiogenic treatment by targeting VEGF/VEGF receptors in metastatic clear cell renal cell carcinoma (ccRCC) varies from patient to patient. Discovering the reasons behind this variability could lead to the identification of relevant therapeutic targets. Thus, we investigated the novel splice variants of VEGF that are less efficiently inhibited by anti-VEGF/VEGFR targeting than the conventional isoforms. By in silico analysis, we identified a novel splice acceptor in the last intron of the VEGF gene resulting in an insertion of 23 bp in VEGF mRNA. Such an insertion can shift the open-reading frame in previously described splice variants of VEGF (VEGFXXX ), leading to a change in the C-terminal part of the VEGF protein. Next, we analysed the expression of these alternatively spliced VEGF new isoforms (VEGFXXX/NF ) in normal tissues and in RCC cell lines by qPCR and ELISA, and we investigated the role of VEGF222/NF (equivalent to VEGF165 ) in physiological and pathological angiogenesis. Our in vitro data demonstrated that recombinant VEGF222/NF stimulated endothelial cell proliferation and vascular permeability by activating VEGFR2. In addition, VEGF222/NF overexpression enhanced proliferation and metastatic properties of RCC cells, whereas downregulation of VEGF222/NF resulted in cell death. We also generated an in vivo model of RCC by implanting RCC cells overexpressing VEGF222/NF in mice, which we treated with polyclonal anti-VEGFXXX/NF antibodies. VEGF222/NF overexpression enhanced tumour formation with aggressive properties and a fully functional vasculature, while treatment with anti-VEGFXXX/NF antibodies slowed tumour growth by inhibiting tumour cell proliferation and angiogenesis. In a patient cohort from the NCT00943839 clinical trial, we investigated the relationship between plasmatic VEGFXXX/NF levels, resistance to anti-VEGFR therapy and survival. High plasmatic VEGFXXX/NF levels correlated with shorter survival and lower efficacy of anti-angiogenic drugs. Our data confirmed the existence of new VEGF isoforms that could serve as novel therapeutic targets in patients with RCC that are resistant to anti-VEGFR therapy.

Genome-Wide Identification, Characterization and Expression Profiling of Potato (Solanum tuberosum) Frataxin (FH) Gene.

Frataxin (FH) plays a crucial role in the biogenesis of mitochondria and the regulation of iron in the cells of various organisms. However, there has been very little research on FH in plants. In this study, the potato FH gene (StFH) was identified and characterized using a genome-wide approach, and its sequence was compared to those of FH genes from Arabidopsis, rice, and maize. The FH genes were found to have a lineage-specific distribution and were more conserved in monocots than in dicots. While multiple copies of FH genes have been reported in some species, including plants, only one isoform of FH was found in potato. The expression of StFH in leaves and roots was analyzed under two different abiotic stress conditions, and the results showed that StFH was upregulated more in leaves and that its expression levels increased with the severity of the stress. This is the first study to examine the expression of an FH gene under abiotic stress conditions.

Targeting carnitine palmitoyltransferase 1 isoforms in the hypothalamus: A promising strategy to regulate energy balance.

Tackling the growing incidence and prevalence of obesity urgently requires uncovering new molecular pathways with therapeutic potential. The brain, and in particular the hypothalamus, is a major integrator of metabolic signals from peripheral tissues that regulate functions such as feeding behavior and energy expenditure. In obesity, hypothalamic capacity to sense nutritional status and regulate these functions is altered. An emerging line of research is that hypothalamic lipid metabolism plays a critical role in regulating energy balance. Here, we focus on the carnitine palmitoyltransferase 1 (CPT1) enzyme family responsible for long-chain fatty acid metabolism. The evidence suggests that two of its isoforms expressed in the brain, CPT1A and CPT1C, play a crucial role in hypothalamic lipid metabolism, and their promise as targets in food intake and bodyweight management is currently being intensively investigated. In this review we describe and discuss the metabolic actions and potential up- and downstream effectors of hypothalamic CPT1 isoforms, and posit the need to develop innovative nanomedicine platforms for selective targeting of CPT1 and related nutrient sensors in specific brain areas as potential next-generation therapy to treat obesity.

ERß1 Sensitizes and ERß2 Desensitizes ERα-Positive Breast Cancer Cells to the Inhibitory Effects of Tamoxifen, Fulvestrant and Their Combination with All-Trans Retinoic Acid.

Adjuvant endocrine therapy (AET) is the treatment of choice for early-stage estrogen receptor alpha (ERα)-positive breast cancer (BC). However, almost 40% of tamoxifen-treated cases display no response or a partial response to AET, thus increasing the need for new treatment options and strong predictors of the therapeutic response of patients at high risk of relapse. In addition to ERα, BC research has focused on ERß1 and ERß2 (isoforms of ERß), the second ER isotype. At present, the impact of ERß isoforms on ERα-positive BC prognosis and treatment remains elusive. In the present study, we established clones of MCF7 cells constitutively expressing human ERß1 or ERß2 and investigated their role in the response of MCF7 cells to antiestrogens [4-hydroxytamoxifen (OHΤ) and fulvestrant (ICI182,780)] and retinoids [all-trans retinoic acid (ATRA)]. We show that, compared to MCF7 cells, MCF7-ERß1 and MCF7-ERß2 cells were sensitized and desensitized, respectively, to the antiproliferative effect of the antiestrogens, ATRA and their combination and to the cytocidal effect of the combination of OHT and ATRA. Analysis of the global transcriptional changes upon OHT-ATRA combinatorial treatment revealed uniquely regulated genes associated with anticancer effects in MCF7-ERß1 cells and cancer-promoting effects in MCF7-ERß2 cells. Our data are favorable to ERß1 being a marker of responsiveness and ERß2 being a marker of resistance of MCF7 cells to antiestrogens alone and in combination with ATRA.

Zingiberene, a non-zinc-binding class I HDAC inhibitor: A novel strategy for the management of neuropathic pain.

BACKGROUND: Even though numerous Histone deacetylase inhibitors (HDACi) have been approved for the treatment of different types of cancer, and others are in clinical trials for the treatment of neurodegenerative diseases, the main problem related to the clinical use of available HDACi is their low isoform selectivity which causes undesirable effects and inevitably limits their therapeutic application. Previously, we demonstrated that a standardized Zingiber officinalis Roscoe rhizome extract (ZOE) reduced neuroinflammation through HDAC1 inhibition in a mice model of neuropathy, and this activity was related to terpenes fraction. HYPOTHESIS/PURPOSE: The aim of this work was to identify the ZOE constituent responsible for the activity on HDAC1 and to study its possible application in trauma-induced neuropathic pain. METHODS: The ability of ZOE and its terpenes fraction (ZTE) to inhibit HDAC and SIRT isoforms activity and protein expression was assessed in vitro. Then, a structure-based virtual screening approach was applied to predict which constituent could be responsible for the activity. In the next step, the activity of selected compound was tested in an in vitro model of neuroinflammation and in an in vivo model of peripheral neuropathy (SNI). RESULTS: ZTE resulted to be more potent than ZOE on HDAC1, 2, and 6 isoforms, while ZOE was more active on HDAC8. Zingiberene (ZNG) was found to be the most promising HDAC1 inhibitor, with an IC50 of 2.3 ± 0.1 µM. A non-zinc-binding mechanism of inhibition was proposed based on molecular docking. Moreover, the oral administration of ZNG reduced thermal hyperalgesia and mechanical allodynia in animals with neuropathy after 60 min from administration, and decreased HDAC-1 levels in the spinal cord microglia. CONCLUSION: We found a new non-zinc-dependent inhibitor of HDAC class I, with a therapeutic application in trauma-related neuropathic pain forms in which microglia-spinal overexpression of HDAC1 occurs. The non-zinc-binding mechanism has the potential to reduce off target effects, leading to a higher selectivity and better safety profile, compared to other HDAC inhibitors.

The full-length Auxin Response Factor 8 isoform ARF8.1 controls pollen cell wall formation and directly regulates TDF1, AMS and MS188 expression.

Auxin Response Factor 8 plays a key role in late stamen development: its splice variants ARF8.4 and ARF8.2 control stamen elongation and anther dehiscence. Here, we characterized the role of ARF8 isoforms in pollen fertility. By phenotypic and ultrastructural analysis of arf8-7 mutant stamens, we found defects in pollen germination and viability caused by alterations in exine structure and pollen coat deposition. Furthermore, tapetum degeneration, a prerequisite for proper pollen wall formation, is delayed in arf8-7 anthers. In agreement, the genes encoding the transcription factors TDF1, AMS, MS188 and MS1, required for exine and pollen coat formation, and tapetum development, are downregulated in arf8-7 stamens. Consistently, the sporopollenin content is decreased, and the expression of sporopollenin synthesis/transport and pollen coat protein biosynthetic genes, regulated by AMS and MS188, is reduced. Inducible expression of the full-length isoform ARF8.1 in arf8-7 inflorescences complements the pollen (and tapetum) phenotype and restores the expression of the above transcription factors. Chromatin immunoprecipitation-quantitative polymerase chain reaction assay revealed that ARF8.1 directly targets the promoters of TDF1, AMS and MS188. In conclusion, the ARF8.1 isoform controls pollen and tapetum development acting directly on the expression of TDF1, AMS and MS188, which belong to the pollen/tapetum genetic pathway.

Impact of Common Vitamin D-Binding Protein Isoforms on Supplemental Vitamin D3 and/or Calcium Effects on Colorectal Adenoma Recurrence Risk: A Secondary Analysis of a Randomized Clinical Trial.

Importance: Variants in the vitamin D-binding protein (DBP) gene (GC) encode DBP isoforms that may affect vitamin D metabolism. However, whether these isoforms modify the effects of vitamin D3 and/or calcium supplementation on colorectal adenoma recurrence is unclear. We hypothesized that supplementation effects may be stronger among those with the DBP2 isoform (encoded by the rs4588*A allele), which is associated with vitamin D deficiency and modified the associations of circulating vitamin D with risk for colorectal neoplasms in observational studies. Objective: To estimate supplemental vitamin D3 and/or calcium effects on colorectal adenoma recurrence according to 3 common DBP isoforms (DBP1s, DBP1f, DBP2) encoded by 2 missense variants: rs7041 (NG_012837.3:g.57904T>G NP_001191235.1:p.Asp432Glu) and rs4588 (NG_012837.3:g.57915C>A NP_001191235.1:p.Thr436Lys). Design, Setting, and Participants: Secondary analysis of a randomized, double-blind, placebo-controlled clinical trial of 2259 participants with a recently diagnosed adenoma and no remaining polyps after complete colonoscopy in the US from July 1, 2004, to August 31, 2013. The current analyses were performed from August 12, 2019, to July 16, 2022. Interventions: Daily vitamin D3 (1000 IU), calcium (1200 mg), both, or placebo. Main Outcomes and Measures: One or more adenomas diagnosed during 3 to 5 years of follow-up. Treatment effects were estimated according to DBP isoform as risk ratios (RRs) and 95% CIs using Poisson regression analysis. Results: Of the 2259 participants randomized (mean [SD] age, 58 [6.8] years; 1033 [64%] men), 1604 non-Hispanic White participants (chosen to avoid population stratification bias) were included in the analysis. Among those with the DBP2 isoform (rs4588*AC or AA), the RRs (95% CI) for adenoma recurrence were 0.84 (0.72-1.00) with vitamin D3 relative to no vitamin D3, 0.83 (95% CI, 0.70-0.99) with calcium relative to no calcium, and 0.76 (95% CI, 0.59-0.98) with both agents relative to neither agent. Conversely, among those without DBP2 (rs4588*CC), the corresponding values were 1.08 (95% CI, 0.93-1.26; P = .03 for interaction) with vitamin D3 relative to no vitamin D3, 0.98 (95% CI, 0.84-1.14; P = .37 for interaction) with calcium relative to no calcium, and 1.09 (0.88-1.36; P = .03 for interaction) with both agents relative to neither agent. Among DBP2 homozygotes (rs4588*AA), the RR for adenoma recurrence was 0.57 (95% CI, 0.31-1.08) with both agents relative to neither agent. Conclusions and Relevance: The findings of this secondary analysis of a randomized clinical trial suggest that individuals with the DBP2 isoform-encoding rs4588*A allele may particularly benefit from vitamin D3 and/or calcium supplementation for colorectal adenoma prevention. Trial Registration: ClinicalTrials.gov Identifier: NCT00153816.

Identification of a second 16-hydroxytabersonine-O-methyltransferase suggests an evolutionary relationship between alkaloid and flavonoid metabolisms in Catharanthus roseus.

The medicinal plant Catharanthus roseus biosynthesizes many important drugs for human health, including the anticancer monoterpene indole alkaloids (MIAs) vinblastine and vincristine. Over the past decades, the continuous increase in pharmaceutical demand has prompted several research groups to characterize MIA biosynthetic pathways for considering future metabolic engineering processes of supply. In line with previous work suggesting that diversification can potentially occur at various steps along the vindoline branch, we were here interested in investigating the involvement of distinct isoforms of tabersonine-16-O-methyltransferase (16OMT) which plays a pivotal role in the MIA biosynthetic pathway. By combining homology searches based on the previously characterized 16OMT1, phylogenetic analyses, functional assays in yeast, and biochemical and in planta characterizations, we identified a second isoform of 16OMT, referred to as 16OMT2. 16OMT2 appears to be a multifunctional enzyme working on both MIA and flavonoid substrates, suggesting that a constrained evolution of the enzyme for accommodating the MIA substrate has probably occurred to favor the apparition of 16OMT2 from an ancestral specific flavonoid-O-methyltransferase. Since 16OMT1 and 16OMT2 displays a high sequence identity and similar kinetic parameters for 16-hydroxytabersonine, we postulate that 16OMT1 may result from a later 16OMT2 gene duplication accompanied by a continuous neofunctionalization leading to an almost complete loss of flavonoid O-methyltransferase activity. Overall, these results participate in increasing our knowledge on the evolutionary processes that have likely led to enzyme co-optation for MIA synthesis.

Molecular isolation and expression analysis of hemocyanin isoform 2 of Macrobrachium rosenbergii.

OBJECTIVE: Hemocyanin is a copper-bearing protein in the hemolymph of many arthropods and mollusks and functions as an oxygen transport and important nonspecific immune protein. METHODS: In this study, complementary DNA of hemocyanin isoform 2 of the prawn Macrobrachium rosenbergii (MrHc2) was isolated by rapid amplification of cDNA ends and mRNA expression was characterized to elucidate molecular basis of its function. RESULT: With a molecular mass of 77.3 kDa, MrHc2 contained three domains: hemocyanin-all-alpha, hemocyanin-copper-containing, and hemocyanin-immunoglobulin-like domains. Molecular phylogenetic analysis revealed that MrHc2 belongs to the γ-type subunit and is closely related to hemocyanin subunit 1 of the palaemonid shrimp Macrobrachium nipponense. In addition, MrHc2 resided in a different clade relative to hemocyanin (MrHc) of M. rosenbergii (α-type subunit) and in a different subclade relative to the hemocyanin proteins of penaeid shrimp. The messenger RNA transcript of MrHc2 was highly expressed in the hepatopancreas and weakly expressed in the gills, intestine, stomach, muscle, and hemocytes. Upon challenge with M. rosenbergii nodavirus (MrNV), the expression of MrHc2 was 1.96-, 2.93-, and 1.96-fold on days 3, 4, and 5, respectively, and then gradually declined to basal levels on day 7. CONCLUSION: This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV.

Influence of Vitamins A and D on the Expression of MicroRNA27-3p Isoforms and GATA3 in Experimental Autoimmune Encephalomyelitis.

Vitamins A, D, and microRNAs contribute to T cell differentiation into TH2 phenotypes. We investigated the molecular mechanisms and effects of vitamin A and D on the expression of GATA3 and miR-27-3p isoforms in experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein, mixed with Complete Freund's Adjuvant, together with injection of pertussis toxin. Treatments began one day before immunization with (200 µg and 100 ng of vitamin A and vitamin D per mouse, respectively, and vitamin A+D (100 µg+50 ng) per mouse. Expression levels of GATA3 and miR­27­3p isoforms were measured in the CNS and splenocytes by real-time RT-PCR. The expression level of GATA3 in the mice spinal cords and splenocytes was increased in the vitamin A and A+D-treated EAE mice at 24 h and 48 h after restimulation by 10 µg and 40 µg of myelin oligodendrocyte glycoprotein. Vitamins A and D and their combination upregulated the miR-27-3p isoforms compared with EAE mice with no treatments. We also demonstrated that miR-273p isoform expression was altered in splenocytes of vitamin-treated EAE mice. The results showed a positive correlation between splenocyte GATA3 levels and miR-27-3p isoform expression. The protective impacts of vitamins A and D in EAE mice may be mediated by the upregulation of GATA3. However, it is not specified whether suppression of GATA3-targeting miRNAs of the miR-27-3p family is involved in this effect. These results do not rule out the possibility that miR-27-3p isoforms might have beneficial effects by targeting other transcripts, such as GluA2 and NR2B.