Characteristic Amino Acid Research Articles

Azurin a potent anticancer and antimicrobial agent isolated from a novel Pseudomonas aeruginosa strain

Azurin, a bacterial blue-copper protein, has garnered significant attention as a potential anticancer drug in recent years. Among twenty Pseudomonas aeruginosa isolates, we identified one isolate that demonstrated potent and remarkable azurin synthesis using the VITEK 2 system and 16S rRNA sequencing. The presence of the azurin gene was confirmed in the genomic DNA using specific oligonucleotide primers, and azurin expression was also detected in the synthesized cDNA, which revealed that the azurin expression is active. Furthermore, crude azurin protein was extracted, precipitated using 70% ammonium sulfate, dialyzed, and subjected to purification using carboxymethyl-Sephadex in affinity chromatography as a cheap method for purification. The partially purified azurin protein was characterized using polyacrylamide gel electrophoresis, energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Notably, qualitative elemental analysis by EDX showed the presence of copper and sulfur, corresponding to the copper-core and disulfide-bridge, respectively, in the purified azurin fraction. Moreover, FTIR spectroscopy revealed characteristic amide I and II absorption peaks (1500–1700 cm− 1), revealing the possible secondary structure of azurin. The results of NMR revealed the presence of characteristic amino acids such as methionine and cysteine, which confirmed the EDX results for sulfur-containing amino acids. Purified azurin exhibited antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Klebsiella pneumoniae. Additionally, its anticancer properties were determined using the MTT assay and cell cycle analysis, revealing a preference for inhibiting the MCF7 breast cancer cell line where breast cancer is most common in Egypt. Overall, the research findings suggest that the local isolate, P. aeruginosa strain 105, could be a potential source of azurin protein for incorporation into cancer treatment strategies.

Recombination and Pathogenicity Analysis of NADC30-like and QYYZ-like PRRSV Strains in South China.

Since 2010, the Lineage 1 (NADC30-like) and Lineage 3 (QYYZ-like) strains of PRRSV-2 have become widespread in China, undergoing recombination and resulting in varying virulent and immune-evading mutants that have severely impacted the pig farming industry. In this study, we isolated one NADC30-like strain (GXHX20211106) and one QYYZ-like strain (GXGG202007) from pig serum samples collected in southern China. Comparative genomic analysis revealed that GXHX20211106 shares 90% identity with NADC30, while GXGG202007 shows 91.4% similarity to QYYZ. Both strains exhibit characteristic amino acids deletions or insertions in the Nsp2 region. Phylogenetic analyses further classified GXHX20211106 within Lineage 1.8 (NADC30-like) and GXGG202007 in Lineage 3 (QYYZ-like). Recombination analysis demonstrated that GXHX20211106 resulted from recombination between NADC30-like and HP-PRRSV-like strains, while GXGG202007 originated from recombination events involving QYYZ-like, VR-2332-like, and HP-PRRSV-like strains. Pathogenicity studies in piglets indicated that both isolates caused moderate clinical signs, with GXHX20211106 showing higher virulence compared to GXGG202007. No deaths were recorded in the infected piglets. These findings highlight the critical role of recombination in shaping PRRSV virulence and underscore the need for ongoing surveillance and control measures to mitigate the impact of recombinant PRRSV strains in southern China.

Recombinant Anti-PF4 Antibodies Derived from Patients with Vaccine-Induced Immune Thrombocytopenia and Thrombosis (VITT) Facilitate Research and Laboratory Diagnosis of VITT.

Adenoviral vector-based vaccines against COVID-19 rarely cause vaccine-induced immune thrombocytopenia and thrombosis (VITT), a severe adverse reaction caused by IgG antibodies against platelet factor 4 (PF4). To study VITT, patient samples are crucial but have become a scarce resource. Recombinant antibodies (rAbs) derived from VITT patient characteristic amino acid sequences of anti-PF4 IgG are an alternative to study VITT pathophysiology. Amino acid sequences of the variable region of immunoglobulin light and heavy chain of anti-PF4 IgG derived from VITT patients were obtained by mass spectrometry sequencing and rAbs were synthetized by reverse-engineering. Six different rAbs were produced: CR23003, CR23004, and CR23005 (from a patient vaccinated with Jcovden, Johnson & Johnson-Janssen (Beerse, Belgium)), CR22046, and CR22050 and CR22066 (from two different patients vaccinated with Vaxzevria, AstraZeneca (Cambridge, UK)). These rAbs were further characterized using anti-PF4 and anti-PF4/heparin IgG ELISAs, rapid anti-PF4 and anti-PF4/polyanion chemiluminescence assays, and PF4-induced platelet activation assay (PIPA) and their capacity to induce procoagulant platelets. rAbs bound to PF4 alone, but not to PF4/polyanion complexes in rapid chemiluminescence assays. Chemiluminescence assays and both anti-PF4 IgG and anti-PF4 IgG/heparin ELISA showed concentration-dependent PF4 binding of all six rAbs, however, with different reactivities among them. PIPA showed a similar, concentration-dependent platelet activation pattern. rAbs varied in their reactivity and the majority of the tested rAbs were able to induce procoagulant platelets. The six rAbs derived from VITT patients reflect VITT-typical binding capacities and the ability to activate platelets. Therefore, these rAbs offer an attractive new option to study VITT pathophysiology.

Cloning and application in synthetic biology of chalcone synthase gene from Lithocarpus litseifolius

Lithocarpus litseifolius is rich in the chalcones phloridzin and trilobatin, the biosynthesis pathways of which have not been fully demonstrated. Chalcone synthase(CHS) is the first key rate-limiting enzyme in the biosynthesis of flavonoids in plants. To explore the functions of CHS gene family in chalcone synthesis of L. litseifolius, this study screened out two CHS genes(LlCHS1 and LlCHS2) from the transcriptome data of this plant, and then bioinformatics analysis and functional characterization were performed for the two genes. The bioinformatics analysis showed that LlCHS1 and LlCHS2 were acidic hydrophilic stable proteins with no transmembrane domain, composed of 395 and 390 amino acid residues, respectively. Both of them contained the characteristic amino acid sequence "WGVLFGFGPGL" and highly conserved active sites(Cys-164, Phe-215, His-303, and Asn-336) of the CHS family. The phylogenetic tree showed that LlCHS1 shared the same clade with similar genes in Aquilaria sinensis, and LlCHS2 was closely related to similar genes in Malus domestica. Under exogenous addition of phloretic acid, co-expression of LlCHS1 or LlCHS2 with Aa4CL from Aromatoleum aromaticum in Escherichia coli catalyzed the production of phloretin from phloretic acid. This study laid a theoretical foundation for revealing the functions of CHS in plants and provided new enzymatic modules for producing phloretin by synthetic biology.

Chondrogenic Cancer Grading by Combining Machine and Deep Learning with Raman Spectra of Histopathological Tissues

Raman spectroscopy (RS) is a promising tool for cancer diagnosis. In particular, in the last years several studies have demonstrated how the diagnostic performances of RS can be significantly improved by employing machine learning (ML) algorithms for the interpretation of Raman-based data. Recently, it has been demonstrated that RS can perform an accurate classification of chondrosarcoma tissues. Chondrosarcoma is a cancer of bones, that can occur in the soft tissues near the bones. It is normally characterized by three different malignant degrees and a benign counterpart, knows as enchondroma. In line with these findings, in this paper, we exploited ML algorithms to distinguish, as well as possible, between the three grades of chondrosarcoma and to distinguish between chondrosarcoma and enchondroma. We obtained a high level of accuracy of classification by analyzing a dataset composed of a relatively small number of Raman spectra, collected in a previous study by one of the authors of this paper. Such spectra were acquired from micrometric tissue sections with a confocal Raman microscope. We tested the classification performances of a support vector machine (SVM) and a random forest classifier (RFC), as representatives of ML algorithms, and two versions of the multi-layer perceptron (MLPC) as representatives of deep learning (DL). These models, especially RFC and MLPC, showed excellent classification performances, with accuracy reaching 99.7%. This outcome makes the aforementioned models a promising route for future improvements of diagnostic devices focused on detecting cancerous bone tissues. Alongside the diagnostic purpose, the aforementioned approach allowed us to identify characteristic molecules, i.e., amino acids, nucleic acids, and bioapatites, relevant for obtaining the final diagnostic response, through the use of a tool named by us Raman Band Identification (RBI). The method to evaluate RBI is the most important contribution of this paper, because RBI could represent a relevant parameter for the identification of biochemical processes on the basis of the tumor progression and associated malignant degree. In turn, the spectral bands highlighted by RBI could provide precious indicators in an attempt to restrict the spectral acquisition to specific Raman bands. This last objective could help to reduce the amount of experimental data needed to obtain an accurate final grading outcome, with a consequent reduction in the computational cost.

Metabolomics revealed pharmacodynamic effects of aspirin and indobufen in patients after percutaneous transluminal angioplasty surgery.

Aspirin and indobufen are commonly used therapeutic drugs for the prevention of vascular restenosis (VR) after percutaneous transluminal angioplasty surgery. They both exhibited antiplatelet effects but molecular mechanisms underlying metabolic changes induced by them remain unclear. In this study, we collected plasma samples from patients on aspirin medication (n = 5), patients on indobufen medication, patients with no medication after PTA, and healthy controls (CKs) (n = 5). Our investigation aimed to reveal the metabolic processes in patients during vascular restenosis and its amelioration through drug therapy using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our data showed significant alterations in amino acid and choline metabolism in patients without medication after PTA. Aspirin and indobufen were able to regulate these metabolic pathways to alleviate VR symptoms. We identified several characteristic amino acids, including pro-leu, L-citrulline, his-glu, and L-glutamate, as important biomarkers for VR assessment in patients without medication after PTA. A total of 17 and 4 metabolites involved in arginine and phenylalanine metabolism were specifically induced by aspirin and indobufen, respectively. Their expression levels were significantly regulated by aspirin or indobufen, nearly reaching normal levels. Taken together, our identification of metabolites involved in metabolic changes affected by aspirin and indobufen medication enhances the understanding of VR pathology after PTA. This may help identify early diagnostic biomarkers and therapeutic targets.

HulaCCR1, a pump-like cation channelrhodopsin discovered in a lake microbiome

Channelrhodopsins are light-gated ion channels consisting of seven transmembrane helices and a retinal chromophore, which are used as popular optogenetic tools for modulating neuronal activity. Cation channelrhodopsins (CCRs), first recognized as the photoreceptors in the chlorophyte Chlamydomonas reinhardtii, have since been identified in diverse species of green algae, as well in other unicellular eukaryotes. The CCRs from non-chlorophyte species are commonly referred to as bacteriorhodopsin-like cation channelrhodopsins, or BCCRs, as most of them feature the three characteristic amino acid residues of the “DTD motif” in the third transmembrane helix (TM3 or helix C) matching the canonical DTD motif of the well-studied archaeal light-driven proton pump bacteriorhodopsin. Here, we report characterization of HulaCCR1, a novel BCCR identified through metatranscriptomic analysis of a unicellular eukaryotic community in Lake Hula, Israel. Interestingly, HulaCCR1 has an ETD motif in which the first residue of the canonical motif is substituted for glutamate. Electrophysiological measurements of the wild-type and a mutant with a DTD motif of HulaCCR1 suggest the critical role of the first glutamate in spectral tuning and channel gating. Additionally, HulaCCR1 exhibits long extensions at the N- and C-termini. Photocurrents recorded from a truncated variant without the signal peptide predicted at the N-terminus were diminished, and membrane localization of the truncated variant significantly decreased, indicating that the signal peptide is important for membrane trafficking of HulaCCR1. These characteristics of HulaCCR1 would be related to a new biological significance in the original unidentified species, distinct from those known for other BCCRs.

Comparison of gel and functional properties of gelatin derived from two jellyfish Stomolophus meleagris and Rhopilema esculentum kishinouye

The gel and functional properties of jellyfish gelatin from Stomolophus meleagris (SG) and Rhopilema esculentum kishinouye (RG) species were investigated, and commercial fish gelatin (FG) was used as a control. In comparison to FG, SG and RG had similar X-ray diffraction (XRD) and fourier transform infrared (FTIR) pattern with shorter amide A band position, representing the formation of triple helices and hydrogen bonds due to polar groups in gelatin, and indicating a more ordered structure. Moreover, FG and RG presented elastic dominance while SG exhibited viscous dominance in a dose-dependent behavior from 10% to 20%, with G′ increasing approximately 86–27038 times when comparing SG and RG at 1 Hz due to their different characteristic amino acid composition. RG showed the least water mobility and most abundant hydrogen proton density, thus supporting greater gel strength than SG. Furthermore, SG showed a sparse and loose network with large voids, while FG and RG showed dense and homogeneous gel networks with smaller holes, which was conductive to the resistance of external forces. In addition, SG showed superior foam stability (20%–200% increase) and emulsifying stability index (ESI) (12%–86% increase) than those of FG and RG in a dose-dependent manner due to larger pores and more hydrophobic amino acid contents. The comparable gelation behaviors of RG to FG and remarkable interfacial properties of SG to FG supported the possible application of SG and RG as marine-based gelling, foaming and emulsifying agents in the food sector.

Characterization of flavor profile of Steamed beef with rice flour using gas chromatography-ion mobility spectrometry combined with intelligent sensory (Electronic nose and tongue).

The intelligent senses (Electronic nose and tongue), were combined with headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) and free amino acid were used in combination to determine the aroma and taste components during the processing of Chinese traditional dish Steamed beef with rice flour (SBD). The findings revealed that E-nose and E-tongue, could clearly distinguish and identify the aroma and taste of SBD. A total of 66 volatile substances and 19 free amino acids were identified by HS-GC-IMS and amino acid analyzer, respectively. The highest contribution to aroma in the production of SBD was alcohols, esters and aldehydes. Further analysis of relative odor activity showed that 3-Methylbutanol-D, 3-Methylbutanol-M and 3-Methylthio propanal is the marinating stage (T2) main aroma components. Ethyl 3-methylbutanoate-M and Ethyl 3-methylbutanoate-D were the main aroma components in the seasoning stage (T3). Additionally, the calculation of the taste activity value showed that Glutamic contributed significantly to the umami of SBD. Alanine was a representative taste component in the marinating stage (T2), while Proline, Aspartic, Lysine, Glutamic, Valine, Arginine, and Histidine were characteristic amino acids of the seasoning stage (T3). Consequently, this study offers valuable insights into the industrial-scale production and flavor regulation of SBD products.

Genetic characterization and phylogenetic analysis of the S genes of porcine epidemic diarrhea virus isolates from China from 2020 to 2023.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that has been the main cause of diarrhea in piglets since 2010 in China. The aim of this study was to investigate sequence variation and recombination events in the spike (S) gene of PEDV isolates from China. Thirty complete S gene sequences were obtained from PEDV-positive samples collected in six provinces in China from 2020 to 2023. Phylogenetic analysis showed that 10% (3/30) belonged to subtype GII-a, 6.67% (2/30) were categorized as subtype GII-b, 66.67% (20/30) were categorized as subtype GII-c, and 16.66% (5/30) were clustered with the S-INDEL strains. Amino acid sequence alignments showed that, when compared to strains of other subtypes, the GII-c strains had two characteristic amino acid substitutions (N139D and I289M). Five S-INDEL subtype strains had a single amino acid deletion (139N) and four amino acid substitutions (N118G, T137S, A138S, and D141G). Recombination analysis allowed six putative recombination events to be identified, one involving recombination between GII-c strains, two involving GII-c and GII-b strains, two involving GII-c and GI-a strains, and one involving GII-a and GI-b strains. These results suggest that recombination between PEDV strains has been common and complex in recent years and is one of the main reasons for the continuous variation of PEDV strains.

Fusion of spectra and texture features of hyperspectral imaging for quantification and visualization of characteristic amino acid contents in beef

Characteristic amino acids is an important indicator for evaluating the nutritional value and flavor parameter of beef. To accurately quantify and visualize of arginine and alanine content in beef, the feasibility of visible near-infrared hyperspectral multivariate calibration analysis and data fusion was explored. Three methods were used to select the optimal feature wavelength and fusion multi-level texture information, and to develop the prediction performance of linear, non-linear and neural network models in different signals. Compared to all models, the linear model demonstrated superior performance in terms of characteristic spectral and fused spectral. Among, the more effective prediction performances emerged from CARS-ASM-ENT-PLSR model with RP2 = 0.9211, RMSEP = 0.1252 mg/100 g and RPDp = 3.49 for alanine. The UVE-ASM-ENT-HOM-COR-PLSR model for arginine prediction achieved a good performance of RP2 = 0.8596, RMSEP = 0.8596 mg/100 g and RPDp = 2.62. Finally, visualization plots of arginine and alanine content distribution were generated. This study shows that the data fusion method provides a new approach for rapid evaluation of CAA content.

S-1-Propenylcysteine Enhances Endurance Capacity of Mice by Stimulating Fatty Acid Metabolism via Muscle Isoform of Carnitine Acyltransferase-1

BackgroundEndurance is an important capacity to sustain healthy lifestyles. Aged garlic extract (AGE) has been reported to exert an endurance-enhancing effect in clinical and animal studies, although little is known about its active ingredients and mechanism of action. ObjectivesThis study investigated the potential effect of S-1-propenylcysteine (S1PC), a characteristic sulfur amino acid in AGE, on the swimming endurance of mice, and examined its mechanism of action by a metabolomics-based approach. MethodsMale Institute of Cancer Research (ICR) mice (6 wk old) were orally administered either water (control) or S1PC (6.5 mg/kg/d) for 2 wk. The swimming duration to exhaustion was measured at 24 h after the final administration. Nontargeted metabolomic analysis was conducted on the plasma samples obtained from mice after 40-min submaximal swimming bouts. Subsequently, the enzyme activity of carnitine acyltransferase-1 (CPT-1) and the content of malonyl-coenzyme A (CoA), acetyl-CoA, and adenosine triphosphate (ATP) were quantified in heart, skeletal muscles, and liver of mice. ResultsThe duration time of swimming was substantially increased in the S1PC-treated mice as compared with the control group. Metabolomic analysis revealed significant alterations in the plasma concentration of the metabolites involved in fatty acid metabolism, in particular medium- or long-chain acylcarnitines in the mice treated with S1PC. Moreover, the administration of S1PC significantly enhanced the CPT-1 activity with the concomitant decrease in the malonyl-CoA content in the heart and skeletal muscles. These effects of S1PC were accompanied by the elevation of the acetyl-CoA and ATP levels to enhance the energy production in those tissues. ConclusionsS1PC is a key constituent responsible for the endurance-enhancing effect of AGE. This study suggests that S1PC helps provide energy during endurance exercise by increasing fatty acid metabolism via CPT-1 activation in the heart and skeletal muscles.

Effects of various substances on the binding of keratin monomers to S. maltophilia DHHJ cells for the induction of keratinase production.

Biodegradation effectiveness of S. maltophilia DHHJ is determined by its ability to attach to the hydrolyzed feather keratin monomers. This binding capacity can be influenced by many components in the culture medium. Keratin monomers from feathers or those produced by gene overexpression can induce keratinase production in S. maltophilia DHHJ, and several proteases lack the ability to degrade keratin fragments and cysteines. In this study, we co-incubated FITC-labelled keratin monomers with S. maltophilia DHHJ cells in the presence of BSA, DNA, ATP, and several metal ions, and measured fluorescence values and keratinase activity. BSA was found to compete with keratins for cell binding sites, resulting in less keratinase production. DNA did not interfere with cellular binding to keratins revealing unchanged keratinase level. ATP, along with metal ions, enhanced the cellular binding capacity to keratins and increased the production of keratinase by S. maltophilia DHHJ. Fragments of keratin monomers degraded by proteases reduced the ability of cells to bind to keratin and affected enzyme production. Cysteine, a characteristic amino acid of feather keratin, did not have an effect on cellular binding to keratin monomer or on keratinase production. This study will facilitate the tweaking of catalytic parameters to improve feather biodegradation by S. maltophilia DHHJ.

Orthotospovirus iridimaculaflavi (iris yellow spot virus): An emerging threat to onion cultivation and its transmission by Thrips tabaci in India

The yellow spot disease caused by the virus species Orthotospovirus iridimaculaflavi (Iris yellow spot virus-IYSV), belonging to the genus Orthotospovirus, the family Tospoviridae, order Bunyavirales and transmitted by Thrips tabaci Lindeman. At present, emerging as a major threat in onion (Allium cepa) in Tamil Nadu, India. The yellow spot disease incidence was found to be 53–73 % in six districts out of eight major onion-growing districts surveyed in Tamil Nadu during 2021–2023. Among the onion cultivars surveyed, the cultivar CO 5 was the most susceptible to IYSV. The population of thrips was nearly 5–9/plant during vegetative and flowering stages. The thrips infestation was 34–60 %. The tospovirus involved was confirmed as IYSV through DAS-ELISA, followed by molecular confirmation through RT-PCR using the nucleocapsid (N) gene. The predominant thrips species present in onion crops throughout the growing seasons was confirmed as Thrips tabaci based on the nucleotide sequence of the MtCOI gene. The mechanical inoculation of IYSV in different hosts viz., Vigna unguiculata, Gomphrena globosa, Chenopodium amaranticolor, Chenopodium quinoa and Nicotiana benthamiana resulted in chlorotic and necrotic lesion symptoms. The electron microscopic studies with partially purified sap from onion lesions revealed the presence of spherical to pleomorphic particles measuring 100–230 nm diameter. The transmission of IYSV was successful with viruliferous adult Thrips tabaci in cowpea (Cv. CO7), which matured from 1st instar larva fed on infected cowpea leaves (24 h AAP). Small brown necrotic symptoms were produced on inoculated plants after an interval of four weeks. The settling preference of non-viruliferous and viruliferous T. tabaci towards healthy and infected onion leaves resulted in the increased preference of non-viruliferous thrips towards infected (onion-61.33 % and viruliferous thrips towards healthy onion leaves (75.33 %). The study isolates shared 99–100 % identity at a nucleotide and amino acid level with Indian isolates of IYSV in the N gene. The multiple alignment of the amino acid sequence of the N gene of IYSV isolates collected from different locations and IYSV isolates from the database revealed amino acid substitution in the isolate ITPR4. All the IYSV isolates from India exhibited characteristic amino acid substitution of serine at the 6th position in the place of threonine in the isolates from Australia, Japan and USA. The phylogenetic analysis revealed the monophyletic origin of the IYSV isolates in India.

Correlation of glutamate dehydrogenase gene polymorphism and mRNA expression level with free glutamate content in the clam Meretrix petechialis

Free glutamate is a characteristic amino acid with a fresh taste. A high free glutamate content can increase the flavour of clams and better meet consumer demands. GDH is involved in the production of glutamate from a-ketoglutarate and determines the speed and direction of reaction in the glutamate metabolic pathway. In this study, we cloned a full-length cDNA of MpGDH homologues in the clam Meretrix petechialis and further investigated the relationship among MpGDH gene polymorphisms, gene expression levels, enzyme activity and glutamate content traits. A significant positive correlation of MpGDH gene expression with free glutamate content was observed (r = 0.62, p < 0.05). The relationship of variation in MpGDH gene expression with its SNP genotype and SNP haplotype was assessed. Five SNPs were found to be significantly associated with MpGDH gene expression (p < 0.05), from which we constructed two main haplotypes due to linkage disequilibrium. The results showed that haplotype H2 may be an effective haplotype associated with high MpGDH gene expression and high free glutamate content. The genetic effect of different haplotypes was validated by different family materials, which showed that MpGDH gene expression level of the H1H2 heterozygous genotype were significantly higher than those of the homozygous genotype H1H1 (p < 0.05). In summary, our findings revealed the potential influence of the MpGDH polymorphism on the free glutamate content and provided molecular biological information for the selective breeding of good-quality traits of M. petechialis.

Structural and Computational Insights into Dynamics and Intermediate States of Orexin 2 Receptor Signaling.

Orexin 2 receptor (OX2R) is a G protein-coupled receptor (GPCR) whose activation is crucial to regulation of the sleep-wake cycle. Recently, inactive and active state structures were determined from X-ray crystallography and cryo-electron microscopy single particle analysis, and the activation mechanisms have been discussed based on these static data. GPCRs have multiscale intermediate states during activation, and insights into these dynamics and intermediate states may aid the precise control of intracellular signaling by ligands in drug discovery. Molecular dynamics (MD) simulations are used to investigate dynamics induced in response to thermal perturbations, such as structural fluctuations of main and side chains. In this study, we proposed collective motions of the TM domain during activation by performing 30 independent microsecond-scale MD simulations for various OX2R systems and applying relaxation mode analysis. The analysis results suggested that TM3 had a vertical structural movement relative to the membrane surface during activation. In addition, we extracted three characteristic amino acid residues on TM3, i.e., Q1343.32, V1423.40, and R1523.50, which exhibited large conformational fluctuations. We quantitatively evaluated the changes in their equilibrium during activation in relation to the movement of TM3. We also discuss the regulation of ligand binding recognition and intracellular signal selectivity by changes in the equilibrium of Q1343.32 and R1523.50, respectively, according to MD simulations and GPCR database. Additionally, the OX2R-Gi signaling complex is stabilized in the conformation resembling a non-canonical (NC) state, which was previously proposed as an intermediate state during activation of neurotensin 1 receptor. Insights into the dynamics and intermediate states during activation gained from this study may be useful for developing biased agonists for OX2R.

Structure and biological activity in vitro of Flagellin and its mutants from Escherichia coli Nissle 1917.

Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliCEcN) and constructed two mutants with varying degrees of deletion in its highly variable regions (HVRs). We found that there was a difference in immune stimulation levels between the two mutants, with the mutant lacking the D2-D3 domain pair of FliCEcN having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliCEcN and its two mutants and measured their levels of Caco-2 cell stimulation to explore the impact of different domains in the HVRs of FliCEcN on its structure and immune efficacy. This study utilized AlphaFold2, SERS (Surface-enhanced Raman spectroscopy), and CD (circular dichroism) techniques to analyze the structural characteristics of FliCEcN and its mutants, FliCΔ174-506 and FliCΔ274-406, and tested their immune effects by stimulating Caco-2 cells in vitro. The resultsindicate that the D2andD3 domainsof FliCEcNhave more complex interactionscomparedtothe D1-D2 domainpair., and thesedomains alsoplay a role in molecular docking with TLR5 (Toll-like receptor 5). Furthermore, FliCΔ274-406 hasmore missing side chain and characteristic amino acid peaks than FliCΔ174-506. The FliCEcN group was found to stimulatehigherlevelsofIL-10 (interleukin 10) secretion, while the FliCΔ174-506 and FliCΔ274-406 groups had higher levels of IL-6 (interleukin 6) and TNF-α (tumor necrosis factor-α) secretion. In summary, the deletion of different domains in the HVRs of FliCEcN affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.

Isolation and molecular characterization of lumpy skin disease virus from Tamil Nadu, India during the outbreaks from 2020 to 2022.

Lumpy skin disease (LSD) caused by LSD virus is a WOAH notifiable, high-impact, transboundary poxviral disease of bovines. The first official report of LSDV in India is from Odisha state during August 2019. Since then, cases have been reported from many states including Tamil Nadu, a Southern state of India. The present study deals with isolation and molecular characterization of LSDV from Tamil Nadu during the period August 2020 to July 2022. LSDV was isolated in embryonated chicken eggs (ECE) and BHK 21 cells and was characterized based on P32, RPO30, and GPCR genes. The phylogenetic analysis revealed that Tamil Nadu isolates from India are closely related to other Indian strains, Kenyan strains and strains from neighboring countries such as Bangladesh, Nepal, and Myanmar confirming the common exotic source for the transboundary spread across borders. The presence of unique signature of amino acid (aa) at specific positions (A11, T12, T34, S99, and P199) in the GPCR sequence confirmed the identity of LSDV. A twelve nucleotide (nt94-105) insertion and corresponding aa (TILS) at 30-33 position was found in GPCR sequence and characteristic amino acid proline at 98 position (P98) in the RPO30 gene sequence of our isolates was similar to strains from Bangladesh, Nepal, and Myanmar. Further, dissimilarity of our isolates from Neethling like vaccine strains confirms the circulation of virulent filed strains responsible for the outbreaks.

Identification and analysis of gemstone binding materials on imperial rank belts excavated from the tomb of Murongzhi

AbstractThis paper analyzes and identifies the binders used in the jewel settings of the Baodian and Diexie belts excavated from the tomb of Murongzhi in Wuwei, Gansu, China. Fourier transform infrared spectroscopy (FTIR) indicated the presence of protein in the binder samples. The result of gas chromatography–mass spectrometry (GC‐MS) shows that samples from both contained hydroxyproline, a characteristic amino acid for animal glue. Principal component analysis was performed on one of the samples, and it was concluded that the binder was a mixture of hide glue and egg glue. Further analysis using scanning electron microscopy with energy‐dispersive X‐ray (SEM‐EDX) and laser Raman spectroscopy (Raman) found that a red particulate matter visually identified in the binder was cinnabar, which was apparently added to binder in order to modify the color of the jewels. This study provides a basis for the conservation and restoration of future finds of elite jewelry. It also shows how imperial jewelry was made during the Tang Dynasty. Finally, the analysis of these heraldic belts provides a key insight the life and role of an exiled prince in the Tang court.

Evaluating the capability of soybean peptides as calcium ion carriers: a study through sequence analysis and molecular dynamics simulations.

Calcium homeostasis imbalance in the body can lead to a variety of chronic diseases. Supplement efficiency is essential. Peptide calcium chelate, a fourth-generation calcium supplement, offers easy absorption and minimal side effects. Its effectiveness relies on peptide's calcium binding capacity. However, research on amino acid sequences in peptides with high calcium binding capacity (HCBC) is limited, affecting the efficient identification of such peptides. This study used soybean peptides (SP), separated and purified by gel chromatography, to obtain HCBC peptide (137.45 μg mg-1) and normal peptide (≤95.78 μg mg-1). Mass spectrometry identified the sequences of these peptides, and an analysis of the positional distribution of characteristic amino acids followed. Two HCBC peptides with sequences GGDLVS (271.55 μg mg-1) and YEGVIL (272.54 μg mg-1) were discovered. Molecular dynamics showed that when either aspartic acid is located near the N-terminal's middle, or glutamic acid is near the end, or in cases of continuous Asp or Glu, the binding speed, probability, and strength between the peptide and calcium ions are superior compared to those at other locations. The study's goal was to clarify how the positions of characteristic amino acids in peptides affect calcium binding, aiding in developing peptide calcium chelates as a novel calcium supplement.