Mechanistic safety assessment via multi-omic characterisation of systemic pathway perturbations following in vivo MAT2A inhibition.

The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.

[PET/CT with 11C-methionine in assessment of brain glioma metabolism]. / Pozitronnaya emissionnaya tomografiya v sochetanii s komp'yuternoi tomografiei i 11S-metioninom v otsenke metabolizma gliom golovnogo mozga.

OBJECTIVE: To study 11C-methionine (MET) metabolism in gliomas using CNS tumor biobank imaging data. MATERIAL AND METHODS: MRI and 11C-MET PET/CT were performed in 225 patients (49±14 years, M/F=84/101) according to standard protocols with analysis of 11C-MET accumulation index and volumetric parameters (V_FLAIR, V_PET and V_PET/FLAIR). These results were compared with molecular genetic testing and 2-year overall survival. RESULTS: We examined 225 patients with gliomas (97 glioblastomas, 70 astrocytomas, 58 oligodendrogliomas). Accumulation index and volume of 11C-MET in glioblastomas were significantly higher in the general group (AI=2.90, Se 69%, Sp 76%, AUC 0.76; V_PET=24.3 cm3, Se 67%, Sp 60%, AUC 0.65; V_PET/FLAIR 0.46, Se 60%, Sp 69%, AUC 0.67) and within the group of astrocytomas (AI=2.93, Se 68%, Sp 89%, AUC 0.84; V_PET=8.06 cm3, Se 91%, Sp 35%, AUC 0.66; V_PET/FLAIR 0.27, Se 77%, Sp 60%, AUC 0.71). The median 2-year overall survival in patients with glioblastomas was 13 months that was significantly lower compared to IDH «+¼ gliomas (p<0.0001). There was a relationship between high accumulation index of 11C-MET and shorter overall survival in patients with glioblastomas. Significantly higher AI >3.59 (Se 89%, Sp 67%, AUC 0.79) was additionally obtained in subgroup of patients with glioblastomas >50 years (n=34) for EGFR «+¼ tumors. CONCLUSION: We found variable 11C-MET metabolism in WHO 2021 gliomas and confirmed significant difference in metabolic activity and volume of 11C-MET accumulation in glioblastomas compared to IDH «+¼ gliomas. Moreover, we revealed the relationship between high accumulation index and shorter survival. Analysis of 11C-MET metabolism in patients over 50 years old revealed higher accumulation index in the EGFR «+¼ group. Further comparison of these imaging methods and assessment of other significant mutations are necessary to identify the anatomical and metabolic patterns of IDH «+¼ gliomas.

Elemental sulfur concentration can be used as a rapid, reliable, and cost-effective predictor of sulfur amino acid content of soybean seeds.

In this study, we have examined the feasibility of using elemental sulfur content of soybean seeds as a proxy for the overall sulfur amino acid content of soybean seeds. Earlier, we have identified by high throughput ionomic phenotyping several high and low sulfur containing soybean lines from the USDA Soybean Germplasm Collection. Here, we measured the cysteine and methionine content of select soybean lines by high-performance liquid chromatography. Our results demonstrate that those soybean lines which had high elemental sulfur content also had a higher cysteine and methionine content when compared to soybean lines with low elemental sulfur. SDS-PAGE and immunoblot analysis revealed that the accumulation of Bowman Birk protease inhibitor and lunasin in soybean seeds may only be marginally correlated with the elemental sulfur levels. However, we found a positive correlation between the levels of trypsin and chymotrypsin inhibitor activities and elemental sulfur and sulfur amino acid content of the seeds. Thus, elemental sulfur content and/or protease inhibitor activity measurement can be utilized as a rapid and cost-effective method to predict the overall sulfur amino acid content of soybean seeds. Our findings will benefit breeders in their endeavors to develop soybean cultivars with enhanced sulfur amino acid content.

Assessment of methionine plus cystine requirement of tambaqui (Colossoma macropomum) based on zootechnical performance, body composition, erythrogram, and plasmatic and hepatic metabolites.

Tambaqui (Colossoma macropomum) is a species of great cultural and economic importance in aquaculture in the Amazon region. Methionine is considered the first limiting sulfur amino acid in practical fish diets, which encourages investigating its use in diets for tambaqui. This study aimed to verify the digestible methionine plus cystine (Met + Cys) requirement in diets for tambaqui (89.52 ± 0.53 g) for 60 days. The treatments investigated were: 6.50, 7.80, 9.10, 10.40, 11.70, and 13.00 g Met + Cys kg diet-1. The estimated requirement based on final weight, weight gain, feed conversion ratio, and specific growth rate was 9.04, 8.92, 8.91, and 8.58 g Met + Cys kg diet-1, respectively, while on body protein deposition, body fat deposition, body ash deposition, and nitrogen retention efficiency was 9.29, 9.20, 9.19, and 8.72 g Met + Cys kg diet-1, respectively. Linear regression demonstrated that increased digestible Met + Cys in the diet decreased plasma total protein, globulin, and liver total protein levels. Quadratic regression showed that the highest value for liver glycogen was found with a 10.40 g Met + Cys kg diet-1. Another quadratic regression demonstrated a lower hepatic aspartate aminotransferase (AST) enzymatic activity in fish fed between 7.80 and 11.70 g Met + Cys kg diet-1. The different treatments did not influence the erythrogram. In conclusion, when considering an integrative view of the results for growth performance, whole-body deposition, and liver parameters without harming the physiological and metabolic status, we recommended choosing a diet with digestible Met + Cys between 8.58 and 9.29 g kg- 1 for tambaqui.

[Assessment of the role of nutritional and genetic determinants in the formation of the risk of diseases associated with folate cycle disorders in the population of the Omsk region].

A number of essential nutrients are involved in the folate cycle, and its effectiveness depends on the sufficient intake of them. In addition, polymorphic variants of the methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) and methionine synthase (MTR) genes affect a wide range of biochemical reactions of the folate cycle and should also be considered as a risk factor for the development of a number of diseases. The purpose of this research was to study the prevalence of these risk factors. Material and methods. The prevalence of polymorphisms of the folate cycle genes: C677T polymorphism of the MTHFR gene and A66G polymorphism of the MTRR gene in a random stratified (by sex and age) sample of the adult population of the Omsk region [n=139, 51 men, 88 women, aged 18 to 75 years, median age 37 (26; 48) years] was studied. The identification of polymorphisms was carried out by the method of allele-specific polymerase chain reaction with an electrophoretic detection scheme. Using the food intake frequency questionnaire, the dietary intake of nutrients involved in the folate cycle was determined: B vitamins (B6, B2, B9, B12), methionine, choline, in a representative stratified sample of residents of the Omsk region [n=421, 177 men, 244 women, aged 18 to 83 years, median age 37 (23; 57) years]. Results. MTHFR genotypes (A222V С677T C>T) were distributed as follows: CC-type - 51.3%, CT - 41.0%, TT - 7.7%; MTRR genotypes (I22M A>G): AA type - 57.9%, AG - 30.3%, GG - 11.8%. The analysis of actual nutrition showed consumption below the recommended dietary intake of folates in 88.2% persons, vitamin B2 and choline - in 40.5%, vitamin B6 - in 29.2%, methionine - in 22.0%. Vitamin B12 intake was within the recommended range. Conclusion. The totality of the data presented indicates the combined influence and wide distribution of factors that determine the low efficiency of the folate cycle, and, as a result, a high risk of developing a characteristic pathology for the adult population of the region, which determines the need and priorities for prevention measures, including healthy nutrition.

Does the addition of choline and/or betaine to diets reduce the methionine requirements of laying quails? Assessment of performance and egg antioxidant capacity.

The purpose of the present study was to assess the performance, quality of eggs internally and externally, and antioxidant capacity of yolks in laying quails with the administration of choline and betaine to diets containing reduced methionine levels. A total of 150 Japanese laying quails (Coturnix coturnix japonica) at the 10-wk age were randomly assigned to 6 experimental groups, each consisting of 5 replicates and 5 birds for 10 wk. The treatment diets were designed by adding the following substances: 0.45% methionine (C), 0.30% methionine (LM), 0.30% methionine + 0.15% choline (LMC), 0.30% methionine + 0.20% betaine (LMB), 0.30% methionine + 0.075% choline + 0.10% betaine (LMCB1), 0.30% methionine + 0.15% choline + 0.20% betaine (LMCB2). The treatments did not affect performance, egg production, or egg internal quality (P > 0.05). No significant effect was determined on the damaged egg rate (P > 0.05), but the egg-breaking strength, eggshell thickness, and eggshell relative weight decreased in the LMCB2 group (P < 0.05). Regarding lipid peroxidation, treatments did not affect the yolk 2,2 diphenyl-1-picrylhydrazyl value (P > 0.05), although the lowest thiobarbituric acid reactive substances value was observed in the LMB compared to the control group (P < 0.05). It may be summarized that methionine can be decreased to levels of 0.30% for laying quail diets with no negative effect on performance, egg production, or egg internal quality, whereas the combination of methionine (0.30%) and betaine (0.2%) could improve antioxidant stability of eggs over the 10-wk experimental period. These findings provide useful information to the traditional recommendations on the requirements of laying quail. However, further studies are needed to test whether these effects persist throughout extended study periods.

The Assessment of Glioblastoma Metabolic Activity via 11C-Methionine PET and Radiomics.

Nowadays, the quantitative analysis of PET/CT data in patients with glioblastoma is not strictly standardized in the clinic and does not exclude the human factor. This study aimed to evaluate the relationship between the radiomic features of glioblastoma 11C-methionine PET images and the tumor-to-normal brain (T/N) ratio determined by radiologists in clinical routine. PET/CT data were obtained for 40 patients (mean age 55 ± 12 years; 77.5% men) with a histologically confirmed diagnosis of glioblastoma. Radiomic features were calculated for the whole brain and tumor-containing regions of interest using the RIA package for R. We redesigned the original RIA functions for GLCM and GLRLM calculation to reduce computation time significantly. Machine learning over radiomic features was applied to predict T/N with the best median correlation between the true and predicted values of 0.73 (p = 0.01). The present study showed a reproducible linear relationship between 11C-methionine PET radiomic features and a T/N indicator routinely assessed in brain tumors. Radiomics enabled utilizing texture properties of PET/CT neuroimaging that may reflect the biological activity of glioblastoma and can potentially augment the radiological assessment.

A novel method for in silico assessment of Methionine oxidation risk in monoclonal antibodies: Improvement over the 2-shell model.

Over the past decade, therapeutic monoclonal antibodies (mAbs) have established their role as valuable agents in the treatment of various diseases ranging from cancers to infectious, cardiovascular and autoimmune diseases. Reactive groups of the amino acids within these proteins make them susceptible to many kinds of chemical modifications during manufacturing, storage and in vivo circulation. Among these reactions, the oxidation of methionine residues to their sulfoxide form is a commonly observed chemical modification in mAbs. When the oxidized methionine is in the complementarity-determining region (CDR), this modification can affect antigen binding and thus abrogate biological activity. For these reasons, it is essential to identify oxidation liabilities during the antibody discovery and development phases. Here, we present an in silico method, based on protein modeling and molecular dynamics simulations, to predict the oxidation-liable residues in the variable region of therapeutic antibodies. Previous studies have used the 2-shell water coordination number descriptor (WCN) to identify methionine residues susceptible to oxidation. Although the WCN descriptor successfully predicted oxidation liabilities when the residue was solvent exposed, the method was much less accurate for partially buried methionine residues. Consequently, we introduce a new descriptor, WCN-OH, that improves the accuracy of prediction of methionine oxidation susceptibility by extending the theoretical framework of the water coordination number to incorporate the effects of polar amino acids side chains in close proximity to the methionine of interest.

[Variations in fecal microbiota of first episode schizophrenia associated with clinical assessment and serum metabolomics].

OBJECTIVE: To explore the role of the microbiota in drug naïve first-onset schizophrenia patients and to seek evidence from multidimensional longitudinal analyses of the intestinal microbiome and clinical phenotype with antipsychotic drugs (APDs) therapy. METHODS: In this study, 28 drug naïve first onset schizophrenia patients and age-, gender- and education-matched 29 healthy controls were included, and the patients were treated with APDs. We collected fecal and serum samples at baseline and after 6 weeks of treatment to identify the different microbiota strains and analyse their correlation with clinical symptoms and serum metabolites. The 16S rRNA genes of the gut microbiota were sequenced, and the diversity and relative abundance at the phylum and genus levels were analyzsed in detail. The PANSS score, BMI changed value, and serum metabolome were included in the data analyses. RESULTS: A multiomics study found a potential connection among the clinical phenotype, microbiota and metabolome. The species diversity analyses revealed that the alpha diversity index (chao1, ACE, and goods_coverage) in the schizophrenia APDs group was significantly lower than that in the control group, and the schizophrenia group had clear demarcation from the control group. The microbiota composition analysis results showed that the relative abundance of the genera of Bacteroides, Streptococcus, Romboutsia, and Eubacterium ruminantium group significantly changed after APDs treatment in the schizophrenia patients. These strains could reflect the APDs treatment effect. More genera had differences between the patient and control groups. The LEfSe analysis showed that Prevotella_9 and Bacteroides were enriched in schizophrenia, while Blautia, Dialister, and Roseburia were enriched in the control group. The correlation analysis between microbiota and clinical symptoms showed that Bifidobacterium in schizophrenia was positively correlated with the PANSS reduction rate of the general psychopathology scale. The BMI changed value was positively correlated with the alteration of Clostridium_sensu_stricto_1 during treatment and the baseline abundance of Bacteroides. Moreover, metabolomic data analysis revealed a significant correlation between specific genera and metabolites, such as L-methionine, L-proline, homovanillic acid, N-acetylserotonin, and vitamin B6. CONCLUSION: Our study found some microbiota features in schizophrenia patients and healthy controls, and several strains were correlated with APDs effects. Furthermore, the multiomics analysis implies the intermediate role of microbiota between antipsychotic effects and serum metabolites and provides new evidence to interpret the difference from multiple levels in the pathogenesis and pharmacological mechanism of schizophrenia.

Development and assessment of diabetic nephropathy prediction model using hub genes identified by weighted correlation network analysis.

Diabetic nephropathy (DN) is one microvascular complication of diabetes. About 30% of diabetic patients can develop DN, which is closely related to the high incidence and mortality of heart diseases, and then develop end-stage renal diseases. Therefore, early detection and screening of high-risk patients with DN is important. Herein, we explored the differences of serum transcriptomics between DN and non-DN in type II diabetes mellitus (T2DM) patients. We obtained 110 target genes using weighted correlation network analysis. Gene Ontology enrichment analysis indicates these target genes are mainly related to membrane adhesion, alpha-amino acid biosynthesis, metabolism, and binding, terminus, inhibitory synapse, clathrinid-sculpted vesicle, kinase activity, hormone binding, receptor activity, and transporter activity. Kyoto Encyclopedia of Genes and Genomes analysis indicates the process of DN in diabetic patients can involve synaptic vesicle cycle, cysteine and methionine metabolism, N-Glycan biosynthesis, osteoclast differentiation, and cAMP signaling pathway. Next, we detected the expression levels of hub genes in a retrospective cohort. Then, we developed a risk score tool included in the prediction model for early DN in T2DM patients. The prediction model was well applied into clinical practice, as confirmed by internal validation and several other methods. A novel DN risk model with relatively high prediction accuracy was established based on clinical characteristics and hub genes of serum detection. The estimated risk score can help clinicians develop individualized intervention programs for DN in T2DM. External validation data are required before individualized intervention measures.

Emission of volatile sulphur compounds during swine manure composting: Source identification, odour mitigation and assessment.

This study aimed to identify the sources of volatile sulphur compounds (VSCs) and evaluate their mitigation by ferric oxide (Fe2O3) during swine manure composting. Four chemicals, including l-cysteine, l-methionine, sodium sulphite, and sodium sulphate, were further added to simulate organic and inorganic sulphur-containing substances in swine manure to track VSC sources during composting. Results show that sulphur simulants induced the emission of six common VSCs, including methyl sulphide (Me2S), dimethyl sulphide (Me2SS), carbonyl sulphide (COS), carbon disulphide (CS2), methyl mercaptan (MeSH), and ethyl mercaptan (EtSH), during swine manure composting. Of them, COS, CS2, MeSH and Me2SS were predominantly contributed by the biodegradation of methionine and cysteine, while Me2S and EtSH were dominated by the reduction of sulphite and sulphate. Further Fe2O3 addition at 1.5 % of total wet weight of composting materials immobilized elemental sulphur and inhibited sulphate reduction to reduce the emission of VSCs by 46.7-80.9 %. Furthermore, odour assessment indicated that adding Fe2O3 into composting piles significantly reduced the odour intensity level to below 4, the odour value of VSCs by 47.1-81.3 %, and thus the non-carcinogenic risk by 68.4 %.

11C-Methionine PET/CT in Assessment of Multiple Myeloma Patients: Comparison to 18F-FDG PET/CT and Prognostic Value.

Multiple myeloma (MM) is the second most common haematological malignancy and remains incurable despite therapeutic advances. 18F-FDG (FDG) PET/CT is a relevant tool MM for staging and it is the reference imaging technique for treatment evaluation. However, it has limitations, and investigation of other PET tracers is required. Preliminary results with L-methyl-[11C]- methionine (MET), suggest higher sensitivity than 18F-FDG. This study aimed to compare the diagnostic accuracy and prognostic value of 1FDG and MET in MM patients. We prospectively compared FDG and MET PET/CT for assessment of bone disease and extramedullary disease (EMD) in a series of 52 consecutive patients (8 smoldering MM, 18 newly diagnosed MM and 26 relapsed MM patients). Bone marrow (BM) uptake patterns and the detection of focal lesions (FLs) and EMD were compared. Furthermore, FDG PET parameters with known MM prognostic value were explored for both tracers, as well as total lesion MET uptake (TLMU). Median patient age was 61 years (range, 37-83 years), 54% were male, 13% of them were in stage ISS (International Staging System) III, and 31% had high-risk cytogenetics. FDG PET/CT did not detect active disease in 6 patients, while they were shown to be positive by MET PET/CT. Additionally, MET PET/CT identified a higher number of FLs than FDG in more than half of the patients (63%). For prognostication we focussed on the relapsed cohort, due to the low number of progressions in the two other cohorts. Upon using FDG PET/CT in relapsed patients, the presence of more than 3 FLs (HR 4.61, p = 0.056), more than 10 FLs (HR 5.65, p = 0.013), total metabolic tumor volume (TMTV) p50 (HR 4.91, p = 0.049) or TMTV p75 (HR 5.32, p = 0.016) were associated with adverse prognosis. In MET PET/CT analysis, TMTV p50 (HR 4.71, p = 0.056), TMTV p75 (HR 6.27, p = 0.007), TLMU p50 (HR 8.8, p = 0.04) and TLMU p75 (HR 6.3, p = 0.007) adversely affected PFS. This study confirmed the diagnostic and prognostic value of FDG in MM. In addition, it highlights that MET has higher sensitivity than FDG PET/CT for detection of myeloma lesions, including FLs. Moreover, we show, for the first time, the prognostic value of TMTV and TLMU MET PET/CT in the imaging evaluation of MM patients.

Mechanistic assessment of tolerance to iron deficiency mediated by Trichoderma harzianum in soybean roots.

AIMS: Iron (Fe) deficiency in soil is a continuing problem for soybean (Glycine max L.) production, partly as a result of continuing climate change. This study elucidates how Trichoderma harzianum strain T22 (TH) mitigates growth retardation associated with Fe-deficiency in a highly sensitive soybean cultivar. METHODS AND RESULTS: Soil TH supplementation led to mycelial colonization and the presence of UAOX1 gene in roots that caused substantial improvement in chlorophyll score, photosynthetic efficiency and morphological parameters, indicating a positive influence on soybean health. Although rhizosphere acidification was found to be a common feature of Fe-deficient soybean, the upregulation of Fe-reductase activity (GmFRO2) and total phenol secretion were two of the mechanisms that substantially increased the Fe availability by TH. Heat-killed TH applied to soil caused no improvement in photosynthetic attributes and Fe-reductase activity, confirming the active role of TH in mitigating Fe-deficiency. Consistent increases in tissue Fe content and increased Fe-transporter (GmIRT1, GmNRAMP2a, GmNRAMP2b and GmNRAMP7) mRNA levels in roots following TH supplementation were observed only under Fe-deprivation. Root cell death, electrolyte leakage, superoxide (O2 •- ) and hydrogen peroxide (H2 O2 ) substantially declined due to TH in Fe-deprived plants. Further, the elevation of citrate and malate concentration along with the expression of citrate synthase (GmCs) and malate synthase (GmMs) caused by TH suggest improved chelation of Fe in Fe-deficient plants. Results also suggest that TH has a role in triggering antioxidant defence by increasing the activity of glutathione reductase (GR) along with elevated S-metabolites (glutathione and methionine) to stabilize redox status under Fe-deficiency. CONCLUSIONS: TH increases the availability and mobilization of Fe by inducing Fe-uptake pathways, which appears to help provide resistance to oxidative stress associated with Fe-shortage in soybean. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate that while Fe deficiency does not affect the rate or degree of TH hyphal association in soybean roots, the beneficial effects of TH alone may be Fe deficiency-dependent.

A Comprehensive Assessment of All-Oleate Polysorbate 80: Free Fatty Acid Particle Formation, Interfacial Protection and Oxidative Degradation.

PURPOSE: Enzymatic polysorbate (PS) degradation and resulting free fatty acid (FFA) particles are detrimental to biopharmaceutical drug product (DP) stability. Different types and grades of polysorbate have varying propensity to form FFA particles. This work evaluates the homogenous all-oleate (AO) PS80 alongside heterogeneous PS20 and PS80 grades in terms its propensity to form FFA particles and other important attributes like interfacial protection and oxidation susceptibility. METHODS: FFA particle formation rates were compared by degrading PS using non-immobilized hydrolases and fast degrading DP formulations. Interfacial protection of monoclonal antibodies (mAbs) was assessed by agitation studies in saline using non-degraded and degraded PS. Several antioxidants were assessed for their ability to mitigate AO PS80 oxidation and subsequent mAb oxidation by a 40°C placebo stability study and a 2, 2'-Azobis (2-amidinopropane) dihydrochloride stress model, respectively. RESULTS: Visible and subvisible particles were significantly delayed in AO PS80 formulations compared with heterogeneous PS20 and PS80 formulations. Non-degraded AO PS80 was less protective of mAbs against the air-water interface compared with heterogeneous PS20. Interfacial protection by AO PS80 improved upon degradation owing to high surface activity of FFAs. Diethylenetriaminepentaacetic acid (DTPA) completely mitigated AO PS80 oxidation unlike L-methionine and N-Acetyl-DL-Tryptophan. However, DTPA did not mitigate radical mediated mAb oxidation. CONCLUSION: AO PS80 is a promising alternative to reduce FFA particle formation compared with other PS types and grades. However, limitations observed here---such as lower protection against interfacial stresses and higher propensity for oxidation---need to be considered in assessing the risk/benefit ratio in using AO PS80.

Multiparametric ultrasound imaging for the assessment of normal versus steatotic livers.

Liver disease is increasing in prevalence across the globe. We present here a multiparametric ultrasound (mpUS) imaging approach for assessing nonalcoholic fatty liver disease (NALFD). This study was performed using rats (N = 21) that were fed either a control or methionine and choline deficient (MCD) diet. A mpUS imaging approach that includes H-scan ultrasound (US), shear wave elastography, and contrast-enhanced US measurements were then performed at 0 (baseline), 2, and 6 weeks. Thereafter, animals were euthanized and livers excised for histological processing. A support vector machine (SVM) was used to find a decision plane that classifies normal and fatty liver conditions. In vivo mpUS results from control and MCD diet fed animals reveal that all mpUS measures were different at week 6 (P < 0.05). Principal component analysis (PCA) showed that the H-scan US data contributed the highest percentage to the classification among the mpUS measurements. The SVM resulted in 100% accuracy for classification of normal and high fat livers and 92% accuracy for classification of normal, low fat, and high fat livers. Histology findings found considerable steatosis in the MCD diet fed animals. This study suggests that mpUS examinations have the potential to provide a comprehensive estimation of the main components of early stage NAFLD.

Multi-criteria assessment of pea protein quality in rats: a comparison between casein, gluten and pea protein alone or supplemented with methionine.

The objective of this study was to assess the nutritional quality of pea protein isolate in rats and to evaluate the impact of methionine (Met) supplementation. Several protein diets were studied: pea protein, casein, gluten, pea protein-gluten combination and pea protein supplemented with Met. Study 1: Young male Wistar rats (n 8/group) were fed the test diets ad libitum for 28 d. The protein efficiency ratio (PER) was measured. Study 2: Adult male Wistar rats (n 9/group) were fed the test diets for 10 d. A protein-free diet group was used to determine endogenous losses of N. The rats were placed in metabolism cages for 3 d to assess N balance, true faecal N digestibility and to calculate the Protein Digestible-Corrected Amino Acid Score (PDCAAS). They were then given a calibrated meal and euthanised 6 h later for collection of digestive contents. The true caecal amino acid (AA) digestibility was determined, and the Digestible Indispensable Amino Acid Score (DIAAS) was calculated. Met supplementation increased the PER of pea protein (2·52 v. 1·14, P < 0·001) up to the PER of casein (2·55). Mean true caecal AA digestibility was 94 % for pea protein. The DIAAS was 0·88 for pea protein and 1·10 with Met supplementation, 1·29 for casein and 0·25 for gluten. Pea protein was highly digestible in rats under our experimental conditions, and Met supplementation enabled generation of a mixture that had a protein quality that was not different from that of casein.

Understanding Oxidation Propensity in GCSF and Assessment of its Safety and Efficacy.

PURPOSE: To understand the impact of methionine oxidation in GCSF on efficacy (neutrophil production/activation) and safety (biochemical and histopathological changes). METHODS: Nine GCSF biosimilars were analyzed for the levels of residual iron and copper content. Oxidation in GCSF was induced by H2O2 treatment and four samples were prepared: wtGCSF (no oxidation), MetO (1138), MetO (1,138,127) and MetO (1138,127,122). These samples were used to evaluate binding affinity with the GCSF receptor (GCSFR) using biolayer interferometry, thermal stability using circular dichroism and in vitro potency using a relevant cell-based assay. In vivo pharmacodynamics examined changes in neutrophil production upon GCSF methionine oxidation, with the outcome correlated with the differential expression of genes implicated in the GCSF mediated neutrophil activation/ maturation. Pre-clinical safety studies including biochemical and histopathological changes were also performed. RESULTS: Met 122 and Met 127 have the most deleterious effect on the potency. Lower binding affinity with GCSFR was identified as the underlying cause for lower efficacy and potency. Role of Asp 110 in GCSF as the critical residue having adverse impact on efficacy in context of methionine oxidation has been elucidated. Impairment of in vitro binding affinity with GCSF manifests as in vivo pharmacodynamic differences via differential expression of downstream genes required for neutrophil maturation. CONCLUSION: The data from the present study suggests that methionine oxidation in GCSF is a critical quality attribute that needs careful monitoring and control during commercial manufacturing and subsequent supply chain stages.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.

[C-methionine PET in assessment of brain lesions in patients with glial tumors after combined treatment]. / PÉT s S-metioninom v otsenke porazhenii golovnogo mozga u bol'nykh s glial'nymi opukholiami posle kombinirovannogo lecheniia.

Positron emission tomography (PET) with amino acid-based radiopharmaceuticals is considered as an effective method to diagnose continued growth of cerebral gliomas, but the variability of 11C-methionine uptake by brain lesions of different genesis after combined treatment still remains poorly understood. The aim of this study was to explore the information value of 11C-methionine PET in delimitating progression of cerebral gliomas and stable disease and to assess the risk of tumor recurrence at different values of the 11C-methionine uptake index. MATERIAL AND METHODS: We performed a retrospective analysis of the results of 11C-methionine PET or PET/CT in 324 patients suspected for continued growth of cerebral tumor based on magnetic resonance imaging (MRI) findings. A quantitative analysis of the results included calculation of the 11C-methionine uptake index (UI). RESULTS: A ROC analysis revealed that the specificity of PET in the diagnosis of continued tumor growth (CTG) was 98%, and the sensitivity was 71% for a UI of more than 1.9. We found that 98% of lesions with a negative level of RP uptake were related to radiation brain lesions (RBLs) or residual tumors combined with radiation pathomorphims. The UI in a range of 1.2-1.6 in 75% of lesions characterized a stable disease, but 25.5% of the lesions represented continued glioma growth. The proportion of recurrences increased to 40% in a UI range of 1.6-1.9, and 95.5% of brain lesions with a UI of more than 1.9 were tumor recurrences. Therefore, high 11C-methionine uptake with the UI above 1.9 in brain lesions characterized by radiological signs of disease progression is a highly specific indicator of CTG; however, the UI may significantly vary during tumor growth, and a substantial fraction of recurrent gliomas may have lower radiopharmaceutical uptake. In the case of borderline UI values, early dynamic control or complementary additional MRI or CT techniques should be used.