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Non-ribosomal peptide synthetases are mega-enzyme assembly lines that synthesize many clinically useful compounds. As a gatekeeper, they have an adenylation (A)-domain that controls substrate specificity and plays an important role in product structural diversity. This review summarizes the natural distribution, catalytic mechanism, substrate prediction methods, and in vitro biochemical analysis of the A-domain. Taking genome mining of polyamino acid synthetases as an example, we introduce research on mining non-ribosomal peptides based on A-domains. We discuss how non-ribosomal peptide synthetases can be engineered based on the A-domain to obtain novel non-ribosomal peptides. This work provides guidance for screening non-ribosomal peptide-producing strains, offers a method to discover and identify A-domain functions, and will accelerate the engineering and genome mining of non-ribosomal peptide synthetases.Key points• Introducing adenylation domain structure, substrate prediction, and biochemical analysis methods• Advances in mining homo polyamino acids based on adenylation domain analysis• Creating new non-ribosomal peptides by engineering adenylation domains

Amoxicillin is one of the most widely used antibacterial drugs. It is known to be the first reason of adverse effects caused by medications in human and animals. Antibacterial drugs, combined with a delivery agent, could provide desirable therapeutic effects with decreased toxicity and reduce the emergence of antimicrobial resistant bacteria. The aim of this study was to compare the toxicity of amoxicillin, polyphosphate ester amoxicillin complex and phosphate ester on mice. Synthesis of the polyphosphate ester (P4) was performed via polycondensation technique, using PEG-400. Animal studies were performed in accordance with the European Convention for the Protection of Vertebrate Animals (Strasbourg, 1986). BALB/c mice were treated by intramuscular injection with saline 0.2 mL (control), amoxicillin 0.2 mL (15 mg/kg); polyphosphate ester complex with amoxicillin 0.2 mL (with amoxicillin content 15 mg/kg) and polyphosphate ester 0.2 mL. Blood biochemical analysis and histology of liver, spleen and kidney were used to assess toxicity. Blood biochemical analysis indicates that P4 did not induce changes in liver and kidneys. Specifically, blood biochemical indicis that represent functional state and cell structure of these organs were within normal physiological values: ALT (56 ± 15.96 U/L), AST (265 ± 37.50U/L), urea (4.4 ± 1.45 mmol/L), creatinine 62.8 ± 5.17 mmol/L, cholesterol 3.5 ± 0.56 mmol/L, total protein 55.9 ± 4.60 g/L, glucose 8.1 ± 0.55 mmol/L. However, the analysis of organ to body weight ratio showed decreased liver ratio (p ≤ 0.05) in mice injected with polyphosphate ester (P4). Histological examination of the liver didn’t show severe pathological changes. There were single places with mild portal vein inflammation in liver of mice receiving amoxicillin and amoxicillin complexed with polyphosphate ester. P4 separately in some places caused cell cytoplasm granulation in liver. No spleen alterations were observed. Overall, the results of this study showed that P4 polyphosphate ester alone and in complex with amoxicillin does not cause renal, hepatic and splenic toxicity in mice. Thus, polyphosphate ester P4 can serve as a safe drug carrier for antimicrobial drugs. It is planned to carry out more extensive studies on other animal species to study its biocompatibility and effectiveness of antimicrobial activity in a complex with antimicrobials.

Hydrophilic interaction chromatography (HILIC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) were optimised for the direct determination of gadolinium-based contrast agents in tap water. In comparison to our previous work, a new developed zwitterionic HILIC column (ZIC- c HILIC) was used for speciation of Gd-containing contrast agents. The limit of quantification (LOQ) for the five contrast agents Gd-BOPTA, Gd-DPTA-BMA, Gd-BT-DO3A, Gd-DOTA and Gd-DTPA are in the range of 5–12 ng Gd per litre. Additionally, a new internal standard, Pr-DOTA, was investigated to correct intensity drifts, minor and major changes in the sample volumes and possible matrix effects. With the speciation method described, tap water samples from the area of Berlin were analysed and for the first time, three Gd species, Gd-BT-DO3A, Gd-DOTA and Gd-BOPTA, were found in tap water samples at concentrations of about 10–20 ng Gd per litre. These are the same Gd species which have been previously detected predominantly in surface waters of the Berlin area.

The use of cold plasma in the treatment of seeds before sowing presents a promising technique for sustainable agriculture. The objective of this study is to evaluate the effect of cold plasma treatment on the morphology of wheat seeds (Triticum aestivum L. ‘Dacic’ and ‘Otilia’), their germination, biochemical composition, and the nutritional quality of wheat grass. Wheat seeds were morphologically and elementally characterized by atomic force microscopy (AFM), scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDX), X-ray computer tomography (CT), and particle-induced X-ray emission (PIXE). Helium was used as a working gas for plasma generation and the analysis of the species produced showed the presence of NOγ, OH, N2 and N2+ and O. Evaluation of germination and plant growth for 10 days (wheat grass stage) highlighted a specific trend for each cultivar. The biochemical analysis of wheat grass highlighted an increase in the chlorophyll content in the plasma-treated variants, an increase in the flavonoid and polyphenol content in ‘Dacic’-treated variant, while the soluble protein content, antioxidant activity, and color were not affected. The analysis of the nutritional quality of wheat grass by the FT-NIR analytical technique highlighted an increase in the ash content in the plasma-treated wheat cultivars, while the humidity, proteins, neutral detergent fiber (NDF), acid detergent fiber (ADF), and energy values were not affected.

This study assessed the genetic diversity between durum wheat landraces (L1-L12) and bread wheat landraces (L1-L12) using a comparative biochemical (SDS-PAGE) and bioinformatics analysis of their major gluten proteins: LMW-GSs, HMW-GSs, and gliadin fractions. Biochemical analysis revealed significant polymorphism in both species, with approximately 24-26% of the assessed loci showing variability. Cluster analysis based on the overall protein profiles successfully grouped all genotypes into three distinct clusters. Specific focus on the HMW-GSs and omega-gliadins revealed molecular markers related to end-use quality. Significant allelic polymorphism was detected at the Glu-1 loci: the Glu-A1 subunit 1 was fixed in durum wheat but rare in bread wheat, while Glu-A1 subunit 2 showed the inverse distribution. Diverse alleles (7, 8, 17, 18) were found at Glu-B1. Furthermore, bread wheat generally had five omega-gliadin subunits, compared to only four in durum wheat. Crucially, hierarchical clustering using the combined HMW-GS and omega-gliadin data provided a robust molecular signature, cleanly separating all genotypes into two clusters (Durum and Bread). This demonstrates the effectiveness of these two protein groups in distinguishing the wheat types, reflecting quality-related genetic differences. The subsequent bioinformatics analysis of 3D structures showed that most fractions (LMW-GSs, gamma-gliadin, and alpha/beta-gliadin) are highly conserved, with only minor variations in the N-terminal regions. In contrast, HMW-GSs and omega-gliadin exhibited significant structural divergence in their central repetitive domains. HMW-GS variations in beta-turn and beta-spiral distribution directly correlated with dough viscoelastic properties. Notably, durum omega-gliadin was found to be longer and contained more rigid polyproline type II helices, highlighting key structural differences influencing wheat quality.

Bovine coronavirus (BCoV) is a major viral pathogen linked to respiratory and enteric problems in newborn calves. The goal of this study is to look into the molecular occurrence, haemato-biochemical changes, and risk factors associated with the occurrence of BCoV infection in cattle calves at different dairy farms and small households in the district of Jhelum, Pakistan. From July 2020 to June 2021,200 faecal samples were collected from newborn cattle calves exhibiting symptoms of diarrhoea and dysentery. SandC Biotech Bovine Coronavirus Antigen Rapid Test Kits were used to screen samples, which were then subjected to RT-PCR for molecular characterization. For the haemato-biochemical analysis, blood was taken from calves infected with BCoV. A questionnaire was used to collect information about the risk factors associated with the occurrence of BCoV infection. BCoV infection was found in 3.5% (7/200) of calves using diagnostic screening kits and 3.0% (6/200) of calves using RT-PCR. Breed, age, sex, vomiting, preious history of diarrhoea, body conditions, food type, colostrum feeding, deworming history, living environment, seasonal variation, and cohabitation with other animals were all assumed risk factors for BCoV infection (p<0.05). There was a significant (p<0.001) decrease in MCV (fl) and a significant (p<0.006) increase in TLC (1x103/cm3) on haematological analysis in BCoV-infected calves. TEC (1x106/cm3), WBC (1x106/cm3), RBC (1x106/cm3), and Hct% were significantly (p<0.05) higher. Similarly, infected calves had a non-significant (p<0.090) increase in K (mEq/L) and a significant (p<0.000) decrease in Na (mEq/L), Ca (mmol/L), Cu (mol/L), and Fe (mol/L) on biochemical analysis. It was concluded that the occurrence of BCoV infection was predisposed by assumed risk factors, and haemato-biochemical alterations were observed in BCoV infected calves.

Background: Exercise-induced muscle damage (EIMD) is the most common health risk in training. So far, EIMD diagnosis predominantly relies on blood biochemical analysis or medical imaging. EIMD prediction by using saliva shows great prospects in public fitness. Methods: A total of 18 participants performed high-intensity rowing training. Blood biochemical indicator and pain analyses indicated EIMD occurrence. Pseudo-targeted metabolomics techniques were utilized to analyze changes in salivary metabolites after exercise. Results: A total of 43 salivary metabolites significantly increased while 31 salivary metabolites significantly decreased after exercise. The upregulated metabolites were related to hormone secretion, antioxidation, and muscle repair. A partial least squares discriminant analysis model was established, and three potential salivary biomarkers for EIMD prediction were screened. The sensitivity and specificity of single biomarkers achieved more than 88.9% and 94.4% in classification of EIMD occurrence, respectively. The accuracy of classification increased to ~100% with multiple metabolites. Conclusion: Salivary metabolites significantly changed after high-intensity rowing training and EIMD occurrence. Some salivary metabolites exhibited similar trends with blood biochemical indicators. Salivary biomarkers have great prospects in EIMD prediction, and better performance was achieved with multiple salivary metabolites.

Fluorescent lights are typically used as light source in indoor horticultural production, including micropropagation. However, light emitting diodes (LEDs) have been recently used for plant growth under controlled environment. Major advantage of LEDs is wavelength specificity, that allows to adequately adjust the spectra according to plant needs. The possibility of using LED as primary light source for the micropropagation of Pyrus communis L. has been investigated in this work. It was proceeded to the optimization of a protocol of micropropagation using a LED lamp as the primary light source inside of the growth chamber with specific wavelengths, to reduce energy consumption and increase the quality of the micro propagated plants. Explants were maintained in a growth chamber and exposed to three different continuous spectrum LED lamps (AP67, NS1, G2) as a primary light source and fluorescent lamps (control) for 4 weeks. At the end of four weeks period, it was proceeded to the morphometric and biochemical analysis. Shoot and leaf growths were more influenced by LED lamps as compared to fluorescent lamps (control) in both cultivars. The results showed that the shoots of William and San Giovanni cultivars showed significant differences in morphological and physiological traits, as well as in chlorophyll, carotenoid, and MDA contents. Highest number of neo-formed shoots and nodes were observed in the plantlets of cv William under AP67 LED followed by NS1 and G2 LED lights respectively as compared to the white light (control), whereas same trend was observed in cv San Giovanni under AP67 LED, but it showed higher shoots and node numbers under control LED lamps as compared to both NS1 and G2. The photosynthetic pigments were significantly decreased in leaves of both cultivars when grown under LEDs as compared to the control fluorescent lamps. Moreover, the AP67 LED light had also significant effects on the protein and MDA contents in the leaves of both cultivars in comparison to all other treatments. This work underlines the importance of the modulation of light sources in relation with different species and varieties, allowing optimizing the proliferation phase and the efficiency of Pyrus communis L. micropropagation protocol. Moreover, this protocol can be improved with further studies to examine the response of the plantlets to the ex-vitro acclimatization because the possibility of using LED light for the micropropagation of pear can be considered as a valuable alternative for its ecologically sustainable production.

Various endogenous and exogenous agents drive the un-directed formation of covalent bonds between proteins and DNA. These complex molecules are of great biological relevance, as can derive in mutations, but are difficult to study because of their heterogeneous chemical properties. New analytical approaches with sufficient detection capabilities to detect and quantify these compounds can help to standardize study models based on synthesized standards. The use of atomic spectrometry can provide quantitative information on the DNA-protein cross-link reaction yield along with basic stoichiometry of the products, based on internal elemental tags, sulfur from Cys and Met amino acids, and phosphorus from the DNA. A new instrumental approach to remove isobaric and polyatomic interferences from 31 P + and 32 S + was developed recently, with state-of-the-art for interference removal that captures 31 P + in Q1; it reacts with O 2 in an octopole collision-reaction cell generating 47 PO + , therefore allowing detection in Q3 without 31 NOH + / 48 Ca/ 47 Ti interferences. Similarly, 32 S + is reacted to 48 SO + , eliminating the polyatomic interferences at m / z  = 32. In conjunction with the high resolving power of high-performance liquid chromatography (HPLC), this newer technology was applied by to the product purification of a DNA-protein cross link model and some preliminary structural studies.