Step In Metabolism Research Articles

Drug Interactions with Antihypertensives.

Hypertension is remarkably prevalent, affecting an estimated 1.13 billion people worldwide. It often requires the use of multi-drug regimens and is commonly associated with a myriad of other comorbidities which increase medication use. The pervasive use of antihypertensive medications combined with the presence of polypharmacy in many hypertensive patients results in significant risk of drug interactions. This review will summarize the relevant literature to assist clinicians in mitigating drug interaction risks when prescribing antihypertensives. Pharmacokinetic interactions affect drug disposition in the body and can occur at the steps of absorption, distribution, metabolism, or elimination of involved medications. Data has established the calcium channel blockers, namely, diltiazem and verapamil, as potent inhibitors of CYP3A4, and the majority of significant drug interactions involving antihypertensives are attributable to these two agents. Although less common, pharmacokinetic drug interactions with other antihypertensive classes have also been identified. Pharmacodynamic drug interactions with antihypertensives lead to synergy or antagonism of blood pressure lowering effects and can increase or mitigate adverse effects depending on the agents involved. Knowledge is emerging about drug-induced phenoconversion, a phenomenon whereby a drug interaction results in a drug metabolizing phenotype that is different than that predicted by an individual's genotype. Antihypertensive use in patients with comorbidities and polypharmacy increases the likelihood of encountering important drug-drug interactions. Dedicated efforts to better understand the relationship between pharmacokinetic drug interactions and pharmacogenomic information is important to advance efforts related to personalized medicine.

Abstract LT009: Stromal BCAT1 drives branched-chain ketoacid dependency in stromal-rich PDAC tumours

Abstract Branched chain amino acids (BCAAs) in cancer serve as requisite precursors for protein synthesis, maintaining metabolite pools in the tricarboxylic acid (TCA) cycle, and sustaining production of nucleotides and lipids. However, the role of stromal cancer associated fibroblasts (CAFs) in support of BCAA metabolism in tumors is still poorly understood. Since most studies in pancreatic cancers have focused on systemic or cancer cell autonomous BCAA metabolism, understanding cancer-stromal ecosystem requires insight into the intersection of cancer-associated transformations in the stroma with reprogramming of their BCAA metabolism. Deciphering the precise role of various cellular components in BCAA metabolism of tumors is complicated by conflicting evidence from past studies and the challenging nature of the intricate tumor microenvironment (TME). Neither systemic in vivo BCAA metabolism nor cancer cells’ BCAA metabolism alone is sufficient to dissect the stromal role. The difficulty in understanding BCAA metabolism in the tumor milieu is exacerbated by nutrient-scarcity, exchange reactions, and metabolite sharing between cancer and stromal cells. Both, the fibrotic environment and nutrient scarcity are difficult to mimic in aggressive murine PDAC models. The metabolic fates of the BCAAs, leucine, valine, and isoleucine, are cell- and tissue-dependent. BCAA transaminases (BCAT1/2), first deaminate BCAAs to branched chain a-ketoacids (BCKAs). The second step in BCAA metabolism involves irreversible BCKA oxidation catalyzed by the mitochondrial BCKA dehydrogenase (BCKDH) complex. Further, oxidation of BCKAs results in succinyl-CoA and acetyl-CoA that act as anaplerotic or ketogenic sources for the TCA cycle. Our recent study revealed differential BCAA metabolism in cancer and stromal compartments of PDAC tumors. We identified a strikingly higher BCAA catabolic flux in CAFs but increased BCKA oxidative flux in cancer cells. Further, CAF-secreted BCKAs were used for maintaining protein synthesis, augmenting TCA cycle metabolite pools, and increasing oxidative phosphorylation in cancer cells. To corroborate the mechanistic underpinnings discovered in our human CAF and cancer cell-line model, we employed two patient-derived models: circulating tumor cells (CTCs) and tumor slice cultures. Collectively, we elucidated an undiscovered metabolic-signaling crosstalk between PDAC and stromal cells and demonstrated that targeting BCAA metabolism in PDAC tumors could mitigate PDAC aggression. Ziwen Zhu, Abhinav Achreja, Noah Meurs, Olamide Animasahun, Sarah Owen, Anjali Mittal, Pooja Parikh, TingWen Lo, Janusz Franco-Barraza, Jiaqi Shi, Mara Sherman, Edna Cuikerman, Andrew Pickering, Anirban Maitra, Vaibhav Sahai, Meredith Morgan, Sunitha Nagrath, Thedore Lawrence, Deepak Nagrath, "Tumor Reprogrammed Stromal BCAT1 Fuels Branched Chain Ketoacid Dependency in Stromal-Rich PDAC Tumors", Nature Metabolism, 2 (8), 2020. Citation Format: Deepak Nagrath. Stromal BCAT1 drives branched-chain ketoacid dependency in stromal-rich PDAC tumours [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT009.

Chemistry, Cyclophosphamide, Cancer Chemotherapy, and Serendipity: Sixty Years On

Cambridge Dictionary: serendipity | noun | the phenomenon of finding interesting or valuable things by chance.
 The year 2019 marked the 60th anniversary of the approval of cyclophosphamide (CP) as an anticancer by the U.S. Food & Drug Administration in 1959 for the treatment of lymphoma. Between 1959 and 2019 there were ~50,000 publications listed in PubMed that have CP in the title and/or abstract, with these annual numbers showing a continual increase, and over 1,800 such articles in 2019 alone. The discovery of CP is a prime example of serendipity in science, which also applies to key elements of the metabolism and pharmacological basis for the specificity of the cytotoxicity of CP toward cancer cells. Phosphoramide mustard (PM), HO(H2N)P(O)N(CH2CH2Cl)2, the principal metabolite of CP with DNA alkylating activity, was synthesized and reported by Friedman and Seligman in 1954 prior to the discovery of CP. Interestingly, the original drug design premise for synthesizing PM, which was based on elevated phosphamidase enzyme activity in cancer cells proved to be incorrect. While this wrong premise also led to the synthesis of CP, as a six-membered ring cyclic phosphamidase-activated precursor of PM, the actual metabolic conversion of CP to PM was subsequently found to involve a surprisingly complex array of metabolites and metabolic pathways, all completely unrelated to phosphamidase. Although the molecular structure of CP has an asymmetrically substituted, i.e. chiral phosphorus center, the racemic mixture of the Rp and Sp enantiomers of CP was used throughout its initial investigations and subsequent clinical trials despite the involvement of an initial enzyme-mediated metabolic activation step, which could, in principle, be stereoselective for only one of the enantiomers of CP. Stereochemical investigations along those lines were eventually carried out, but the results did not warrant replacement of racemic CP with either enantiomer in the clinic. Amazingly, there are now ~4,000 structural congeners of PM listed Chemical Abstracts, but none have led to an anticancer drug superior to CP. This account provides a synopsis of the key chemistry and stereochemistry investigations that comprise this story of CP, as a remarkable instance of serendipity in science, and my chance involvement in the unfolding of this fascinating story.

Methanol dehydrogenase, a key enzyme of one-carbon metabolism: a review

One-carbon compounds such as methanol and methane are cheap and readily available feedstocks for biomanufacturing. Oxidation of methanol to formaldehyde catalyzed by methanol dehydrogenase (MDH) is a key step of microbial one-carbon metabolism. A variety of MDHs that depend on different co-factors and possess different enzymatic properties have been discovered from native methylotrophs. Nicotinamide adenine dinucleotide (NAD)-dependent MDHs are widely used in constructing synthetic methylotrophs, whereas this type of MDH usually suffers from low methanol oxidation activity and low affinity to methanol. Consequently, methanol oxidation is considered as a rate-limiting step of methanol metabolism in synthetic methylotrophs. To accelerate methanol oxidation, thereby improving the methanol utilization efficiency of synthetic methylotrophs, massive researches have focused on discovery and engineering of MDHs. In this review, we summarize the ongoing efforts to discover, characterize, and engineer various types of MDHs as well as the applications of MDHs in synthetic methylotrophs. Directed evolution of MDH and construction of multi-enzyme complexes are described in detail. In the future prospective part, we discuss the potential strategies of growth-coupled protein evolution and rational protein design for acquisition of superior MDHs.

Bioanalysis of drugs and their metabolites by chiral electromigration techniques (2010-2020).

The further development and application of capillary electromigration techniques for the enantioselective determination of drugs and their metabolites in body fluids, tissues, and in vitro preparations during the 2010 to 2020 time period continued to proof their usefulness and attractiveness in bioanalysis. This review discusses the principles and important aspects of capillary electrophoresis- based chiral drug bioassays, provides a survey of the assays reported during the past 10 years and presents an overview of the key achievements encountered in that time period. For systems with charged chiral selectors, special attention is paid on assays that feature field-amplified sample injection to enable the determination of ppb levels of analytes and optimized online incubation procedures for the rapid assessment of a metabolic pathway. Applications discussed encompass the pharmacokinetics of drug enantiomers in vivo and in vitro, the impact of inhibitors on metabolic steps, the elucidation of the stereoselectivity of drug metabolism in vivo and in vitro, and drug enantiomers in toxicological, forensic, and doping analysis.

Abstract PD15-09: Sox4 and smarca4 upregulate glycolysis-driven tumor proliferation through hexokinase 2 in tnbc

Abstract Background Triple-negative breast cancer (TNBC) accounts for nearly 1-in-4 breast cancer related deaths in the United States each year despite representing 10-12% of newly diagnosed cases. This aggressive disease, which is largely synonymous with the basal-like molecular subtype, predominantly affects younger women and women of African American decent. Despite a number of recent advances, overall survival has not drastically improved, highlighting the need to better understand mechanisms regulating tumor growth and progression as a means to identify novel therapeutic opportunities in these patients. Methods In order to identify the functional impact of overexpression of the oncogenic transcription factor SOX4 and its co-factor SMARCA4 in TNBC, RNAseq and ChIPseq analyses were used to identify SMARCA4-dependent and -independent SOX4 gene expression programs. Analyses of more than 3,000 tumor samples from the METABRIC and TCGA cohorts was used to investigate the functional implications and clinical association of these gene expression programs in human tumors. Mass spectrometry (MS)-based metabolomic profiling in conjunction with genetic and pharmacological-based in vitro studies were used to demonstrate the impact of these programs on tumor cell metabolism, cell viability and proliferation. Results We, and others, have previously demonstrated that the oncogenic transcription factor SOX4 is overexpressed in basal-like tumors. In order to identify mechanisms by which this protein mediates breast cancer tumorigenesis, we first identified SOX4 co-factors. Immunoprecipitation follow by MS was used to identify the SWI/SNF ATPase SMARCA4 as a prominent SOX4 cofactor that is concurrently overexpressed in basal-like tumors. Mechanistic studies showed that this complex is required to maintain an active open chromatin conformation at regulatory regions upstream of SOX4-regulated genes. ChIPseq and RNAseq-based analyses were next used to investigate the role of this complex in TNBC signaling and tumorigenesis. Our analyses identified SMARCA4-dependent and -independent SOX4 gene expression programs and demonstrated that SMARCA4-dependent SOX4 signaling is associated with poor clinical outcome. Further in silico analyses of human tumors indicate that each program mediates distinct down-stream signaling; SMARCA4-dependent signaling is associated with altered tumor metabolism and increased proliferative signaling. Consistent with this premise, in vitro studies showed that SOX4 and SMARCA4 are essential for breast tumor cell proliferation, survival and colony formation. Metabolomic profiling of breast cancer cell lines demonstrated that SOX4 overexpression resulted in increased glycolysis. The effect of SOX4 and SMARCA4 on cell metabolism was validated by demonstrating that this complex can mediate intracellular glucose consumption. Finally, we determined that SOX4 and SMARCA4 cooperatively regulate mRNA expression of hexokinase 2 (HK2), which catalyzes for the first step in glucose metabolism, and demonstrated that HK2 activity is essential for SOX4/SMARCA4-regulation of glycolysis. Importantly, in vitro genetic and pharmacological studies identified a link between HK2 activity, glycolysis and SOX4/SMARCA4-mediated cell proliferation and viability in TNBC. Conclusions Our studies have identified SOX4 and its transcriptional cofactor SMARCA4 as being uniformly overexpressed in basal-like or TNBC tumors. These analyses demonstrate that SOX4 and SMARCA4 cooperatively promote tumorigenesis in TNBC cells, in part, by modulating glycolysis and cell proliferation through transcriptional regulation of HK2. Given the drug-ability of the glycolysis pathway, these results have potential clinical implications for treatment of TNBC patients. Citation Format: Pooja Khanna, Gaurav Mehta, Michael L Gatza. Sox4 and smarca4 upregulate glycolysis-driven tumor proliferation through hexokinase 2 in tnbc [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-09.

Plasma periostin as a biomarker of osteoporosis in postmenopausal women with type 2 diabetes.

Periostin, as an emerging biomarker, is involved in multiple steps in bone metabolism. This study aimed to investigate the correlation between periostin levels and bone mineral density as well as bone turnover markers in postmenopausal women with type 2 diabetes (T2DM). This study was a cross-sectional study that included 164 postmenopausal women with T2DM as study subjects and 32 age-matched nondiabetic postmenopausal women with normal bone mineral density (BMD) as healthy control subjects. A total of 164 subjects with T2DM were then divided into three groups according to BMD: the normal BMD group (n = 29), the osteopenia group (n = 70), and the osteoporosis group (n = 65). The clinical data of all subjects along with the relevant biochemical parameter data were collected. Plasma periostin was detected using an enzyme-linked immunosorbent assay (ELISA). Plasma periostin levels were significantly increased in T2DM patients with normal BMD compared with healthy controls (p < 0.05). In the diabetic group, plasma periostin levels were significantly elevated with decreased BMD, were positively correlated with osteocalcin levels (r = 0.162, p = 0.039) and were inversely associated with femoral neck BMD (r = - 0.308, p < 0.001) and total femur BMD (r = - 0.295, p < 0.001). In the case of chronic complications, periostin levels were slightly increased in individuals with complications of diabetic retinopathy, diabetic nephropathy and fracture (p > 0.05). The current study demonstrated that plasma periostin levels were significantly associated with BMD in patients with T2DM, and periostin might act as a novel biochemical marker of osteoporosis in postmenopausal women with type 2 diabetes.

Glucose-6-phosphate induced changed of stomatal aperture in an irradiance dependent manner

Hexokinase catalyses hexose phosphorylation, which is the key step of sucrose metabolism. In this study, stomatal apertures of Arabidopsis epidermal peel were detected with or without exogenous application of mannose, fructose, glucose, and glucose-6-phosphate (G-6P). The results here showed that G-6P, but not glucose itself, induces stomatal closure in Arabidopsis. Furthermore, detection of stomatal apertures of Arabidopsis hexokinase loss of function with exogenous application of glucose showed that glucose induced stomatal closure was not due to osmotic pressure and it triggered guard cell ROS production depend on hexokinase activity. The effect of irradiance and G-6P on regulation of Arabidopsis stomatal aperture was investigated. The data obtained here indicated that G-6P induced changes of stomatal aperture depend on irradiance.

Three ATP-dependent phosphorylating enzymes in the first committed step of dihydroxyacetone metabolism in Gluconobacter thailandicus NBRC3255.

Dihydroxyacetone (DHA), a chemical suntan agent, is produced by the regiospecific oxidation of glycerol with Gluconobacter thailandicus NBRC3255. However, this microorganism consumes DHA produced in the culture medium. Here, we attempted to understand the pathway for DHA metabolism in NBRC3255 to minimize DHA degradation. The two gene products, NBRC3255_2003 (DhaK) and NBRC3255_3084 (DerK), have been annotated as DHA kinases in the NBRC 3255 draft genome. Because the double deletion derivative for dhaK and derK showed ATP-dependent DHA kinase activity similar to that of the wild type, we attempted to purify DHA kinase from ∆dhaK ∆derK cells to identify the gene for DHA kinase. The identified gene was NBRC3255_0651, of which the product was annotated as glycerol kinase (GlpK). Mutant strains with several combinations of deletions for the dhaK, derK, and glpK genes were constructed. The single deletion strain ∆glpK showed approximately 10% of wild-type activity and grew slower on glycerol than the wild type. The double deletion strain ∆derK ∆glpK and the triple deletion strain ∆dhaK ∆derK ∆glpK showed DHA kinase activity less than a detection limit and did not grow on glycerol. In addition, although ΔderK ΔglpK consumed a small amount of DHA in the late phase of growth, ∆dhaK ΔderK ΔglpK did not show DHA consumption on glucose-glycerol medium. The transformants of the ∆dhaK ΔderK ΔglpK strain that expresses one of the genes from plasmids showed DHA kinase activity. We concluded that all three DHA kinases, DhaK, DerK, and GlpK, are involved in DHA metabolism of G. thailandicus. KEY POINTS: • Dihydroxyacetone (DHA) is produced but degraded by Gluconobacter thailandicus. • Phosphorylation rather than reduction is the first committed step in DHA metabolism. • Three kinases are involved in DHA metabolism with the different properties.

Physiological Perspectives on the Use of Triheptanoin as Anaplerotic Therapy for Long Chain Fatty Acid Oxidation Disorders.

Inborn errors of mitochondrial fatty acid oxidation (FAO) comprise the most common group of disorders identified through expanded newborn screening mandated in all 50 states in the United States, affecting 1:10,000 newborns. While some of the morbidity in FAO disorders (FAODs) can be reduced if identified through screening, a significant gap remains between the ability to diagnose these disorders and the ability to treat them. At least 25 enzymes and specific transport proteins are responsible for carrying out the steps of mitochondrial fatty acid metabolism, with at least 22 associated genetic disorders. Common symptoms in long chain FAODs (LC-FAODs) in the first week of life include cardiac arrhythmias, hypoglycemia, and sudden death. Symptoms later in infancy and early childhood may relate to the liver or cardiac or skeletal muscle dysfunction, and include fasting or stress-related hypoketotic hypoglycemia or Reye-like syndrome, conduction abnormalities, arrhythmias, dilated or hypertrophic cardiomyopathy, and muscle weakness or fasting- and exercise-induced rhabdomyolysis. In adolescent or adult-onset disease, muscular symptoms, including rhabdomyolysis, and cardiomyopathy predominate. Unfortunately, progress in developing better therapeutic strategies has been slow and incremental. Supplementation with medium chain triglyceride (MCT; most often a mixture of C8–12 fatty acids containing triglycerides) oil provides a fat source that can be utilized by patients with long chain defects, but does not eliminate symptoms. Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: oxidative phosphorylation (OXPHOS), FAO, and the tricarboxylic (TCA) cycle, also called the Krebs cycle. Cell and mouse studies have identified a deficiency in TCA cycle intermediates in LC-FAODs, thought to be due to a depletion of odd chain carbon compounds in patients treated with a predominantly MCT fat source. Triheptanoin (triheptanoyl glycerol; UX007, Ultragenyx Pharmaceuticals) is chemically composed of three heptanoate (seven carbon fatty acid) molecules linked to glycerol through ester bonds that has the potential to replete TCA cycle intermediates through production of both acetyl-CoA and propionyl-CoA through medium chain FAO. Compassionate use, retrospective, and recently completed prospective studies demonstrate significant reduction of hypoglycemic events and improved cardiac function in LC-FAOD patients, but a less dramatic effect on muscle symptoms.

Pseudouridine site assignment by high-throughput in vitro RNA pseudouridylation and sequencing.

Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNAs. High-throughput pseudouridine profiling of eukaryotic mRNAs from cells has revealed novel sites of modification across the transcriptome. Pseudouridine affects RNA structure and RNA-protein interactions with the potential to influence many steps of mRNA metabolism and thereby affect gene expression. Identifying the mechanisms by which individual pseudouridines sites are modified by pseudouridine synthases (PUS) will facilitate studies on the molecular functions of Ψ. Multiple pseudouridine synthases are expressed in all organisms and might direct pseudouridylation of diverse cellular RNAs, but the RNA targets of many enzymes and their specificity determinants remain to be defined. We developed a high-throughput in vitro pseudouridylation assay followed by sequencing that allows validation of candidate sites identified in cells, assignment of sites as direct targets of PUS and interrogation of the RNA sequence and structural features that direct modification. We also implemented an analysis pipeline to assign Ψ sites from these data, including an updated approach to peak-calling that accounts for noisy signal from low-abundance transcripts.

Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.

NAD+ is a redox cofactor essential to the proper functioning of a variety of important metabolic pathways, including key steps in mitochondrial energy metabolism. In addition, it serves as a signaling substrate for enzymes such as sirtuins and the poly-ADP ribosyl-polymerase family of enzymes. Sirtuins, which are NAD+-dependent protein deacylases, harness changes in cellular NAD+ concentrations to produce changes in protein acylation status, thereby affecting downstream functions including energy metabolism, stress resistance, and cell survival. Thus, the availability of NAD+ in cells, or in specific organelles such as the mitochondrion, regulates downstream signaling and key biological processes. This concept has driven a need for researchers to easily and precisely measure NAD+ concentrations in biological samples. We herein describe several protocols for the measurement of NAD+ and NADH concentrations in tissues, cells, or subcellular compartments such as mitochondria. These protocols include a cycling assay that can quickly measure NAD+ or NADH levels using a plate reader equipped with fluorescence measurement capabilities. This plate assay relies only upon commercially available materials in addition to the biological samples of interest. In addition, we describe a protocol employing stable isotope-labeled NAD+ as an internal standard to determine biological NAD+ content by isotope-dilution methods. This method requires mass spectrometry to ratio endogenous NAD+ with exogenous isotope-labeled NAD+ to obtain quantification using HPLC and mass spectrometry.

An in vitro Coupled Assay for PEPC with Control of Bicarbonate Concentration.

Phosphoenolpyruvate carboxylase (PEPC) catalyzes a critical step in carbon metabolism in plants and bacteria, the irreversible reaction between bicarbonate and phosphoenolpyruvate to produce the C4 compound oxaloacetate. This enzyme is particularly important in the context of C4 photosynthesis, where it is the initial carbon-fixing enzyme. Many studies have used kinetic approaches to characterize the properties of PEPCs from different species, different post-translational states, and after mutagenesis. Most of these studies have worked at a fixed saturating concentration of bicarbonate. Controlling the concentration of bicarbonate is difficult at low concentrations because of equilibration with atmospheric CO2. We describe here a simple, repeatable, and gas-tight assay system for PEPC that allows bicarbonate concentrations to be controlled above ca. 50 µM.

Linker residues regulate the activity and stability of hexokinase 2, a promising anticancer target

Hexokinase (HK) catalyzes the first step in glucose metabolism, making it an exciting target for the inhibition of tumor initiation and progression due to their elevated glucose metabolism. The upregulation of hexokinase-2 (HK2) in many cancers and its limited expression in normal tissues make it a particularly attractive target for the selective inhibition of cancer growth and the eradication of tumors with limited side effects. The design of such safe and effective anticancer therapeutics requires the development of HK2-specific inhibitors that will not interfere with other HK isozymes. As HK2 is unique among HKs in having a catalytically active N-terminal domain (NTD), we have focused our attention on this region. We previously found that NTD activity is affected by the size of the linker helix-α13 that connects the N- and C-terminal domains of HK2. Three nonactive site residues (D447, S449, and K451) at the beginning of the linker helix-α13 have been found to regulate the NTD activity of HK2. Mutation of these residues led to increased dynamics, as shown via hydrogen deuterium exchange analysis and molecular dynamic simulations. D447A contributed the most to the enhanced dynamics of the NTD, with reduced calorimetric enthalpy of HK2. Similar residues exist in the C-terminal domain (CTD) but are unnecessary for HK1 and HK2 activity. Thus, we postulate these residues serve as a regulatory site for HK2 and may provide new directions for the design of anticancer therapeutics that reduce the rate of glycolysis in cancer through specific inhibition of HK2.

Fatty Acid Metabolism as a Potential Therapeutic Target in Thyroid Carcinoma

Background: Papillary thyroid carcinoma (PTC) accounts for the majority of thyroid cancer and affects a large number of individuals. The pathogenesis of PTC has not been completely elucidated thus far. Metabolic reprogramming is a common feature in tumours. Our previous research revealed the reprogramming of lipid metabolism in PTC. Further studies on lipid metabolism reprogramming may help elucidate the pathogenesis of PTC. Methods: Clinical samples of PTC and para-tumour tissue were analysed using lipidomic, proteomic, and metabolomic approaches. A multi-omics integrative strategy was adopted to identify the important pathways in PTC. The findings were further confirmed using western blotting, tissue microarray, and bioinformatics. Findings: The differentially expressed substances included 146 lipids, 2975 proteins, and 217 metabolites in PTC samples. Multi-omics data and the results of integrated analysis revealed that the three steps of fatty acid metabolism (hydrolysis, transportation, and oxidation) were significantly enhanced in PTC. The key enzymes in the three steps, namely LPL, FATP2, and CPT1A, respectively, were overexpressed. Moreover, their expression was strongly associated with the TNM stage, lymph node metastasis, and PTC prognosis. In addition, the levels of several types of phospholipids and sphingolipids increased significantly in PTC. Interpretation: Our data suggested that enhanced fatty acid metabolism supplied additional energy and substrates for tumour growth, invasion, and metastasis in PTC. This may help elucidate the mechanism underlying PTC pathogenesis and identify the potential therapeutic targets for PTC. Funding Information: Work in the authors' laboratory is supported by grants from National Natural Science Foundation of China (No. 81702647, NO. 81972819), Taishan Scholar Foundation of Shandong Province (No. tspd20181201), Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong, Natural Science Foundation of Shandong Province (No. ZR201702190483, No. ZR2020YQ57), Key Technology Research and Development Program of Shandong (No. 2016GSF201161). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: This prospective study was approved by the institutional ethical committee of Qilu Hospital of Shandong University, Shandong Province, China. All the specimens were collected in the Qilu Hospital of Shandong University. The participants provided written informed consent for the use of thyroid tissue specimens for research.

Limb Remote Ischemic Conditioning Ameliorates Cognitive Impairment in Rats with Chronic Cerebral Hypoperfusion by Regulating Glucose Transport.

Cognitive impairment is closely associated with the slowing of glucose metabolism in the brain. Glucose transport, a rate-limiting step of glucose metabolism, plays a key role in this phenomenon. Previous studies have reported that limb remote ischemic conditioning (LRIC) improves cognitive performance in rats with chronic cerebral hypoperfusion (CCH). Here, we determined whether LRIC could ameliorate cognitive impairment in rats with CCH by regulating glucose transport. A total of 170 male Sprague-Dawley rats were used. Animals subjected to permanent double carotid artery occlusion (2VO) were assigned to the control or LRIC treatment group. LRIC was applied beginning 3 days after the 2VO surgery. We found that LRIC can improve learning and memory; decrease the ratio of ADP/ATP; increase glucose content; upregulate the expression of pAMPKα, GLUT1 and GLUT3; and increase the number of GLUT1 and GLUT3 transporters in cerebral cortical neurons. The expression of GLUT1 and GLUT3 in the cortex displayed a strong correlation with learning and memory. Pearson correlation analysis showed that the levels of GLUT1 and GLUT3 are correlated with neurological function scores. All of these beneficial effects of LRIC were ablated by application of the AMPK inhibitor, dorsomorphin. In summary, LRIC ameliorated cognitive impairment in rats with CCH by regulating glucose transport via the AMPK/GLUT signaling pathway. We conclude that AMPK-mediated glucose transport plays a key role in LRIC. These data also suggest that supplemental activation of glucose transport after CCH may provide a clinically applicable intervention for improving cognitive impairment.

The Severity of the Clinical Expression of Lysosomal Acid Lipase in Patients with Cryptogenic Cirrhosis

Lysosomal acid lipase is an enzyme that intervenes in the last steps of lipid metabolism to hydrolyze cholesteryl esters. Patients with cryptogenic cirrhosis present a clear deficiency of lysosomal acid lipase with an unknown mechanism. Design: The present study has an analytical and retrospective design, of a sample of 55 patients with cryptogenic cirrhosis. The degree of association of lysosomal acid lipase was determined with the results of the enzymes alanine aminotransferase and alkaline phosphatase, as well as with the clinical manifestations of portal hypertension and splenic volume. The sensitivity and specificity of the test were determined for the diagnosis of the manifestations of portal hypertension. Results: The most frequent complication of portal hypertension was variceal hemorrhage with 40%, followed by ascites with 32.7%, and last, by hepatic encephalopathy, with 18.2%. Association by the x² test was without statistical significance with values of 0.177, 0.299, and 0.184 for encephalopathy, variceal hemorrhage, and ascites, respectively. The association of lysosomal acid lipase and splenic volume utilizing the Student t-test had a low degree of association and p >0.05. Through ROC curves, we obtained AUROC results close to 0.5. Conclusion: It is established that there is no correlation of the levels of activity of lysosomal acid lipase with the values of alanine aminotransferase and alkaline phosphatase, as well as with the presence or absence of clinical manifestations and by ultrasound of portal hypertension. Lysosomal acid lipase is not a good test for the diagnosis of the clinical manifestations of portal hypertension.

Crystal structures of γ-glutamylmethylamide synthetase provide insight into bacterial metabolism of oceanic monomethylamine

Monomethylamine (MMA) is an important climate-active oceanic trace gas and ubiquitous in the oceans. γ-Glutamylmethylamide synthetase (GmaS) catalyzes the conversion of MMA to γ-glutamylmethylamide, the first step in MMA metabolism in many marine bacteria. The gmaS gene occurs in ∼23% of microbial genomes in the surface ocean and is a validated biomarker to detect MMA-utilizing bacteria. However, the catalytic mechanism of GmaS has not been studied because of the lack of structural information. Here, the GmaS from Rhodovulum sp. 12E13 (RhGmaS) was characterized, and the crystal structures of apo-RhGmaS and RhGmaS with different ligands in five states were solved. Based on structural and biochemical analyses, the catalytic mechanism of RhGmaS was explained. ATP is first bound in RhGmaS, leading to a conformational change of a flexible loop (Lys287-Ile305), which is essential for the subsequent binding of glutamate. During the catalysis of RhGmaS, the residue Arg312 participates in polarizing the γ-phosphate of ATP and in stabilizing the γ-glutamyl phosphate intermediate; Asp177 is responsible for the deprotonation of MMA, assisting the attack of MMA on γ-glutamyl phosphate to produce a tetrahedral intermediate; and Glu186 acts as a catalytic base to abstract a proton from the tetrahedral intermediate to finally generate glutamylmethylamide. Sequence analysis suggested that the catalytic mechanism of RhGmaS proposed in this study has universal significance in bacteria containing GmaS. Our results provide novel insights into MMA metabolism, contributing to a better understanding of MMA catabolism in global carbon and nitrogen cycles.

Накопление нейтральных липидов и β-каротина дрожжами Yarrowia lipolytica на среде с сахарозой в качестве источника углерода

Recombinant Yarrowia lipolytica yeast has been used as a host strain for the expression of the Saccharomyces cerevisiae ScSUC2 gene and Y. lipolytica YlHXK1 gene encoding invertase and hexakinase, respectively. The expression was carried out on the background of the enhanced pathway of the synthesis of neutral lipids. This allowed the yeast to efficiently utilize glucose as a single carbon source. The engineered strain accumulated neutral lipids in amount of 50.7% of biomass dry weight, when cultured in tubes in minimal medium with nitrogen limitation and high sucrose content. The next metabolic engineering step was the use of the CRISPR-Cas9 editing system to introduce a heterologous β-carotene synthesis pathway by the expression of the Mucor circinelloides genes CarRP and CarB, encoding the bifunctional enzyme phytoene synthase/licopene-β-cyclase (carRP) and phytoene dehydrogenase (carB), as well as an increase in the expression level of the Y. lipolytica YlGGS1 gene, encoding geranyl diphosphate synthase enzyme. The β-carotene production on a sucrose-containing medium was shown for the first time; it amounted for 24.0 mg/g dry biomass, or 406.9 mg/L, on the 5th day of cultivation in tubes in a rich medium with a sucrose portioned supply (50 g/L). The corresponding values for a rich medium with a higher sucrose concentration (90 g/L) were 21.9 mg/g dry biomass and 625.8 mg/L. Yarrowia lipolytica, β-carotene, sucrose, CRISPR-Cas9 The plasmids pdKu70Yl-URA3, pLTet-SP-CAT, pARS-Cre-reverse, pMW-att-Cm and pGPD1Yl were kindly provided by Ph.D. I.A. Laptev ("Kurchatov Institute"-GOSNIIGENETIKA NRC). The work was carried out using the equipment of the Multipurpose Scientific Facility of the "Russian State Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»-GOSNIIGENETIKA. The work was supported by state assignment no. 14 of 25.07.2012 AAAA-A20-120093090016-9. The quantitative analysis of lipids and β-carotene was carried out by V.A. Zolottsev within the framework of the "Long-Term Program of Fundamental Scientific Research in the Russian Federation (2021-2030)".

Case Study 12: Roadmap to Quantifying Ago2-Mediated siRNA Metabolic Activation Kinetics.

Therapeutic siRNA is a prodrug that requires Ago2-mediated site-specific hydrolysis of the sense strand before RNA interference can occur. Although this metabolic activation step was first described 15years ago, the kinetics of this reaction, and its relationship to in vivo siRNA efficacy, remains unexplored in the literature. To provide a roadmap to address these gaps, we describe a liquid chromatography-mass spectrometry method to monitor formation of the cleaved sense-strand metabolites in a reconstituted system. In the absence of metabolite standards for quantitation, we apply an ionization efficiency correction across a panel of siRNA molecules and find that it improves in vitro-in vivo correlation in a transgenic mouse model. Finally, we lay out a case for why Michaelis-Menten kinetics will likely be inadequate for describing Ago2-mediated metabolic activation kinetics, and propose several alternative models that can be solved numerically and applied to quantitated kinetic data when it becomes available.