A fundamental study on postmortem submersion interval estimation by metabolomics analyzing of gastrocnemius muscle from submersed rat models in freshwater.

In forensic practice, determining the postmortem submersion interval (PMSI) and cause-of-death of cadavers in aquatic ecosystems has always been challenging task. Traditional approaches are not yet able to address these issues effectively and adequately. Our previous study proposed novel models to predict the PMSI and cause-of-death based on metabolites of blood from rats immersed in freshwater. However, with the advance of putrefaction, it is hardly to obtain blood samples beyond 3 days postmortem. To further assess the feasibility of PMSI estimation and drowning diagnosis in the later postmortem phase, gastrocnemius, the more degradation-resistant tissue, was collected from drowned rats and postmortem submersion model in freshwater immediately after death, and at 1 day, 3 days, 5 days, 7 days, and 10 days postmortem respectively. Then the samples were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the dynamic changes of the metabolites. A total of 924 metabolites were identified. Similar chronological changes of gastrocnemius metabolites were observed in the drowning and postmortem submersion groups. The difference in metabolic profiles between drowning and postmortem submersion groups was only evident in the initial 1 day postmortem, which was faded as the PMSI extension. Nineteen metabolites representing temporally-dynamic patterns were selected as biomarkers for PMSI estimation. A regression model was built based on these biomarkers with random forest algorithm, which yielded a mean absolute error (± SE) of 5.856 (± 1.296) h on validation samples from an independent experiment. These findings added to our knowledge of chronological changes in muscle metabolites from submerged vertebrate remains during decomposition, which provided a new perspective for PMSI estimation.

Nano-biocatalysts for food applications; immobilized enzymes within different nanostructures.

The rapid progress in modern technologies and paying more attention to food safety has prompted new green technologies superior than chemical methods in the food industry. In this regard, enzymes can decrease the usage of chemical reactions but they are sensitive to environmental effects (pH and temperature). In addition, enzymes are scarcely possible to be reused. Consequently, their application as natural catalysts is restricted. Using nanotechnology and the possibility of enzyme immobilization on nanomaterials has led to nanobiocatalysts, resulting from the integration of nanotechnology and biotechnology. Nanocarriers have individual features like nanoscale size, excellent surface/volume ratio, and diversity in construction to improve the activity, efficiency, stability, and storage stability of enzymes. Nanobiocatolysts have a wide range of applications in purification, extraction, clarification, production, and packaging of various products in the food industry. Furthermore, the application of nanobiocatalysts to identify specific components of food contaminants such as microorganisms or their metabolites, heavy metals, antibiotics, and residual pesticides has been successful due to the high accuracy of detection. This review investigates the integration of nanotechnology and food enzymes, the nanomaterials used to create nanobiocatalysts and their application, along with the possible risks and legal aspects of nanomaterials in food bioprocesses.

Oleo-foams and emulsion-foams as lipid-based foam systems: a review of their formulation, characterization, and applications.

Lipid-based foam systems (LBFs) have grown in popularity recently because of their effectiveness and potential uses. As a result, in order to stabilize them, considerable work has been put into developing more biodegradable and environmentally friendly materials. However, the use of natural stabilizing agents has been constrained due to a lack of thorough knowledge of them. This review offers insightful data that will encourage more studies into the development and use of LBFs. Emulsifiers or gelling agents, as well as new preparation and characterization methods, can be used to increase or prolong the functional performance of LBFs. Special emphasis has been given on the connections between their structures and properties and expanding the range of industries in which they can be applied. In conclusion, it is crucial to gain a deeper understanding of the preparation mechanisms and influencing factors in order to improve the quality of foam products and create novel LBFs.

A preliminary study on early postmortem submersion interval (PMSI) estimation and cause-of-death discrimination based on nontargeted metabolomics and machine learning algorithms.

Postmortem submersion interval (PMSI) estimation and cause-of-death discrimination of corpses in water have long been challenges in forensic practice. Recently, many studies have linked postmortem metabolic changes with PMI extension, providing a potential strategy for estimating PMSI using the metabolome. Additionally, there is a lack of potential indicators with high sensitivity and specificity for drowning identification. In the present study, we profiled the untargeted metabolome of blood samples from drowning and postmortem submersion rats at different PMSIs within 24 h by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 601 metabolites were detected. Four different machine learning algorithms, including random forest (RF), partial least squares (PLS), support vector machine (SVM), and neural network (NN), were used to compare the efficiency of the machine learning methods. Nineteen metabolites with obvious temporal regularity were selected as candidate biomarkers according to "IncNodePurity." Robust models were built with these biomarkers, which yielded a mean absolute error of 1.067 h. Additionally, 36 other metabolites were identified to build the classifier model for discriminating drowning and postmortem submersion (AUC = 1, accuracy = 95%). Our results demonstrated the potential application of metabolomics combined with machine learning in PMSI estimation and cause-of-death discrimination.

Candidate biomarkers in brown adipose tissue for post-mortem diagnosis of fatal hypothermia.

Post-mortem diagnosis of fatal hypothermia (FHT) is challenging in forensic practice because traditional morphological and biochemical methods lack specificity. Recent studies have reported that brown adipose tissue (BAT) is activated during cold-induced non-shivering thermogenesis in mammals, but BAT has not been used to diagnose FHT. The aim of this study was to identify novel biomarkers in BAT for FHT based on morphological changes and differential protein expression. Two FHT animal models were created by exposing mice to 4 or -20 °C at 50% humidity. Morphologically, the unilocular lipid droplet content was significantly increased in BAT of FHT model mice compared with that of control mice. Proteomics analysis revealed a total of 283 and 266 differentially expressed proteins (DEPs) between the 4 or -20 °C FHT subgroups and control group, respectively. In addition, 140 proteins were shared between the FHT subgroups. GO and KEGG analyses revealed that the shared DEPs were mainly enriched in pathways associated with metabolism, oxidative phosphorylation, and thermogenesis. Further screening (|log2FC| > 1.6, q-value (FDR) < 0.05) identified GMFB, KDM1A, DDX6, RAB1B, SHMT-1, CLPTM1, and LMF1 as candidate biomarkers of FHT. Subsequent validation experiments were performed in FHT model mice using classic immunohistochemistry and western blotting. RAB1B and GMFB expression was further verified in BAT specimens from human cases of FHT. The results demonstrate that BAT can be used as a target organ for FHT diagnosis employing RAB1B and GMFB as biological markers, thus providing a new strategy for the post-mortem diagnosis of FHT in forensic practice.

Inferring Postmortem Submersion Interval in Rats Found in Water Based on Vitreous Humor Metabolites.

OBJECTIVES: The metabolomics technique of LC-MS/MS combined with data analysis was used to detect changes and differences in metabolic profiles in the vitreous humor of early rat carcasses found in water, and to explore the feasibility of its use for early postmortem submersion interval (PMSI) estimation and the cause of death determination. METHODS: The experimental model was established in natural lake water with 100 SD rats were randomly divided into a drowning group (n=50) and a postmortem (CO2 suffocation) immediately submersion group (n=50). Vitreous humor was extracted from 10 rats in each group at 0, 6, 12, 18 and 24 h postmortem for metabolomics analyses, of which 8 were used as the training set to build the model, and 2 were used as test set. PCA and PLS multivariate statistical analysis were performed to explore the differences in metabolic profiles among PMSI and causes of death in the training set samples. Then random forest (RF) algorithm was used to screen several biomarkers to establish a model. RESULTS: PCA and PLS analysis showed that the metabolic profiles had time regularity, but no differences were found among different causes of death. Thirteen small molecule biomarkers with good temporal correlation were selected by RF algorithm. A simple PMSI estimation model was constructed based on this indicator set, and the data of the test samples showed the mean absolute error (MAE) of the model was 0.847 h. CONCLUSIONS: The 13 metabolic markers screened in the vitreous humor of rat corpses in water had good correlations with the early PMSI. The simplified PMSI estimation model constructed by RF can be used to estimate the PMSI. Additionally, the metabolic profiles of vitreous humor cannot be used for early identification of cause of death in water carcasses.

Electrochemiluminescence sensor based on cyclic peptides-recognition and Au nanoparticles assisted graphitic carbon nitride for glucose determination.

A glucose (Glu) sensor was designed by introducing synthetic cyclic peptides (CPs) as recognition receptors and Au nanoparticles assisted graphitic carbon nitride (AuNPs/g-C3N4) for electrochemiluminescence (ECL) enhancement. The synthetic CP receptor (cyclo-[-CNDNHCRDNDC-]) with natural active center of Glu binding protein can mimic the interactions between Glu and Glu binding protein to specifically capture Glu. The AuNPs were reduced on g-C3N4 and formed a new nanohybrid that can be applied as an ECL emitter. The AuNPs/g-C3N4 effectively ameliorated the ECL response of bare g-C3N4. The ECL enhancement mechanism was theoretically speculated through computer simulation. Glu quantification was conducted by recording ECL shifts induced by the binding of Glu to CPs. The linear detection range of the fabricated CPs-based ECL sensor was 1 to 100 mmol L-1, and the detection limit (LOD) was 0.57 nmol L-1 (S / N = 3). The CP-based ECL sensor also showed good specificity, repeatability, stability, and favorable recoveries in sample analysis. This work offer a promising analytical method for Glu assay in clinical diagnostics and bioprocess monitoring.

Development of a Broad-Specific Competitive ELISA for First-Generation Cephalosporin Antibiotics in Animal-Derived Foods Samples.

The abuse of antibiotics, such as the cephalosporins in livestock and aquaculture productions, usually causes the widespread antibiotic resistance due to their growth-promoting effects. In this study, cephalexin was chosen as the hapten molecule to prepare a broad-spectrum rabbit polyclonal antibody for cephalosporin antibiotics. The obtained antibody exhibited broad cross-reactivity ranging from 0.05% to 100% with 10 cephalosporins. Based on this antibody, we developed a broad-specific indirect competitive ELISA (ic-ELISA) for cefalexin, cefradine, cefadroxil and cefazolin with the half maximal inhibitory concentration (IC50) ranging from 0.72 to 2.99 ng/mL in working buffer. For animal-derived food samples with spiked cephalosporins, the ic-ELISA exhibited an excellent recovery ranging from 72.3% to 95.6%. To verify the accuracy of this proposed ic-ELISA, its detection performance was evaluated utilizing the high-performance liquid chromatography with satisfactory results. This study confirmed that: firstly, the prepared antibody can be used as a class-specific recognition element to develop immunoassays for cephalosporin antibiotics; and secondly, the developed ic-ELISA provided a new tool for broad-spectrum detection of first-generation cephalosporins in animal-derived foods.

A novel approach for the forensic diagnosis of drowning by microbiological analysis with next-generation sequencing and unweighted UniFrac-based PCoA.

The diagnosis of drowning is one of the major challenges in forensic practice, especially when the corpse is in a state of decomposition. Novel indicators of drowning are desired in the field of forensic medicine. In the past decade, aquatic bacteria have attracted great attention from forensic experts because they can easily enter the blood circulation with drowning medium, and some of them can proliferate in the corpse. Recently, the advent of next-generation sequencing (NGS) has created new opportunities to efficiently analyze whole microbial communities and has catalyzed the development of forensic microbiology. We presumed that NGS could be a potential method for diagnosing drowning. In the present study, we verified this hypothesis by fundamental experiments in drowned and postmortem-submersed rat models. Our study revealed that detecting the bacterial communities with NGS and processing the data in a transparent way with unweighted UniFrac-based principal coordinates analysis (PCoA) could clearly discriminate the skin, lung, blood, and liver specimens of the drowning group and postmortem submersion group. Furthermore, the acquired information could be used to identify new cases. Taken together, these results suggest that we could build a microbial database of drowned and postmortem-submersed victims by NGS and subsequently use a bioinformatic method to diagnose drowning in future forensic practice.

Intermolecular Radical Addition to Ketoacids Enabled by Boron Activation.

The intermolecular radical addition to the carbonyl group is difficult due to the facile fragmentation of the resulting alkoxyl radical. To date, the intermolecular radical addition to ketones, a valuable approach to construct quaternary carbon centers, remains a formidable synthetic challenge. Here, we report the first visible-light-induced intermolecular alkyl boronic acid addition to α-ketoacids enabled by the Lewis acid activation. The in situ boron complex formation is confirmed by various spectroscopic measurements and mechanistic probing experiments, which facilitates various alkyl boronic acid addition to the carbonyl group and prevents the cleavage of the newly formed C-C bond. Diversely substituted lactates can be synthesized from readily available alkyl boronic acids and ketoacids at room temperature merely under visible light irradiation, without any additional reagent. This boron activation approach can be extended to alkyl dihydropyridines as radical precursors with external boron reagents for primary, secondary, and tertiary alkyl radical additions. The pharmaceutically useful anticholinergic precursors are easily scaled up in multigrams under metal-free conditions in flow reactors.

PAQR4 has a tumorigenic effect in human breast cancers in association with reduced CDK4 degradation.

Progestin and adipoQ receptor 4 (PAQR4) is a member of the PAQR family, and the members within this family are involved in the regulation of a number of biological processes including metabolism and cancer development. The potential function of PAQR4 in human cancers is unknown. Analysis of ONCOMINE database reveals that PAQR4 is highly expressed in human breast cancers. We confirmed this finding by analyzing 82 human breast cancers samples. PAQR4 mRNA level was significantly upregulated in human breast cancer samples compared with their corresponding para-cancerous histological normal tissues (P < 0.0001). The mRNA level of PAQR4 was negatively correlated with disease-free survival (P < 0.0001) and overall survival of the patients (P = 0.001). Knockdown of PAQR4 in human breast cancer cells SUM159 and MCF7 suppressed cell proliferation. In contrast, overexpression of PAQR4 in SUM159 cells enhanced cell proliferation and colony formation. In a tumor xenograft model, overexpression of PAQR4 promoted tumor growth of SUM159 cells in vivo, while PAQR4 knockdown suppressed the tumor growth. PAQR4 was able to negatively regulate cyclin-dependent kinases 4 (CDK4) protein level in the breast cancer cells. Knockdown of PAQR4 accelerated degradation of CDK4 together with upregulation of CDK4 polyubiquitination. On the other hand, overexpression of PAQR4 slowed down CDK4 protein degradation and reduced CDK4 polyubiquitination. Collectively, these data at the cellular, animal and human levels indicate that PAQR4 has a tumorigenic effect on human breast cancers, and such effect is associated with a modulatory activity of PAQR4 on protein degradation of CDK4.

Design of Cyclic Peptide Based Glucose Receptors and Their Application in Glucose Sensing.

Glucose assay is of great scientific significance in clinical diagnostics and bioprocess monitoring, and to design a new glucose receptor is necessary for the development of more sensitive, selective, and robust glucose detection techniques. Herein, a series of cyclic peptide (CP) glucose receptors were designed to mimic the binding sites of glucose binding protein (GBP), and CPs' sequence contained amino acid sites Asp, Asn, His, Asp, and Arg, which constituted the first layer interactions of GBP. The properties of these CPs used as a glucose receptor or substitute for the GBP were studied by using a quartz crystal microbalance (QCM) technique. It was found that CPs can form a self-assembled monolayer at the Au quartz electrode surface, and the monolayer's properties were characterized by using cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. The CPs' binding affinity to saccharide (i.e., galactose, fructose, lactose, sucrose, and maltose) was investigated, and the CPs' sensitivity and selectivity toward glucose were found to be dependent upon the configuration,i.e., the amino acids sequence of the CPs. The cyclic unit with a cyclo[-CNDNHCRDNDC-] sequence gave the highest selectivity and sensitivity for glucose sensing. This work suggests that a synthetic peptide bearing a particular functional sequence could be applied for developing a new generation of glucose receptors and would find huge application in biological, life science, and clinical diagnostics fields.

Visible-Light-Induced Alkoxyl Radical Generation Enables Selective C(sp(3))-C(sp(3)) Bond Cleavage and Functionalizations.

The alkoxyl radical is an important reactive intermediate in mechanistic studies and organic synthesis; however, its current generation from alcohol oxidation heavily relies on transition metal activation under strong oxidative conditions. Here we report the first visible-light-induced alcohol oxidation to generate alkoxyl radicals by cyclic iodine(III) reagent catalysis under mild reaction conditions. The ß-fragmentation of alkoxyl radicals enables selective C(sp(3))-C(sp(3)) bond cleavage and alkynylation/alkenylation reactions with various strained cycloalkanols, and for the first time with linear alcohols.

Generation of Alkoxyl Radicals by Photoredox Catalysis Enables Selective C(sp(3))-H Functionalization under Mild Reaction Conditions.

Reported herein is the first visible-light-induced formation of alkoxyl radicals from N-alkoxyphthalimides, and the Hantzsch ester as the reductant is crucial for the reaction. The selective hydrogen atom abstraction by the alkoxyl radical enables C(sp(3))-H allylation and alkenylation reactions under mild reaction conditions at room temperature. Broad substrate variations, including a structurally complexed steroid, undergo the C(sp(3))-H functionalization reaction effectively with high regio- and chemoselectivity.

Validation and application of modeling algorithms for the design of molecularly imprinted polymers.

In the study, four different semiempirical algorithms, modified neglect of diatomic overlap, a reparameterization of Austin Model 1, complete neglect of differential overlap and typed neglect of differential overlap, have been applied for the energy optimization of template, monomer, and template-monomer complexes of imprinted polymers. For phosmet-, estrone-, and metolcarb-imprinted polymers, the binding energies of template-monomer complexes were calculated and the docking configures were assessed in different molar ratio of template/monomer. It was found that two algorithms were not suitable for calculating the binding energy in template-monomers complex system. For the other algorithms, the obtained optimum molar ratio of template and monomers were consistent with the experimental results. Therefore, two algorithms have been selected and applied for the preparation of enrofloxacin-imprinted polymers. Meanwhile using a different molar ratio of template and monomer, we prepared imprinted polymers and nonimprinted polymers, and evaluated the adsorption to template. It was verified that the experimental results were in good agreement with the modeling results. As a result, the semiempirical algorithm had certain feasibility in designing the preparation of imprinted polymers.

Encapsulation of iron within whey protein-pectin nanocomplexes: Fabrication, characterization, and optimization.

Iron is an important micronutrient that cannot be added directly into food products due to potential reactions with the food matrix, impact on color, and taste. Complexed biopolymeric nanocarriers can overcome these challenges particularly for oral delivery of iron, but selecting appropriate biopolymers, their ratio and pH of complexation is very important. In this study, whey protein concentrate (WPC)-pectin nanocomplexes were prepared at different concentrations (WPC 4, 6 and 8%; pectin 0.5, 0.75 and 1%), and pH (3, 6 and 9) to encapsulate iron. The smallest carriers were observed at pH 3; higher pH led to higher zeta potential (zero to -32.5 mV). Encapsulation efficiency of iron in nanocarriers formulated at pH = 3, 6 and 9 were 87.83, 75.92 and 20%, respectively. Scanning electron microscopy revealed the spherical particles at pH 3. To conclude, a WPC to pectin ratio of 4: 1 at pH 3 was the best conditions for loading iron.

Natural oleogelators for the formulation of oleogels by considering their rheological and textural perspective; a review.

A well-known method for reducing or swapping out undesirable and controversial fats in food is oleogelation. To quantify the effects of droplets-particle inclusion on the textural aspects of gelled systems, a thorough understanding of rheological behavior of oleogels (OGs) is necessary. Otherwise stated, a rational grasp of rheological characterization is essential for food development, optimization, and processing (when touching or putting food into the mouth, rheological flow qualities influence our perception). This narrative review primarily intends to investigate rheological and textural characteristics of various oleogelator-based OGs, such as operative connection between hardness, distortion, stresses, and rheological parameters like viscosity, elasticity, and viscoelasticity, as well as flow behavior and recovery. Expanding oleogelators concentration and synergistic interactions between them increase robustness and moduli values, as compared to single oleogelators. However, given the lack of information on the connection between the OGs' macroscopic rheological characteristics and their microstructural characteristics, this review presents state-of-the-art overview of various oleogelator-based OGs, highlighting the importance of structure-rheology relationships of OGs to provide advanced knowledge on the development of innovative OGs.

A sensitive lateral flow immunoassay relying on time-resolved fluorescent microspheres immune probe for determination of ceftiofur and its metabolite.

Ceftiofur (CEF) is an antimicrobial agent with high efficiency and low toxicity, desfuroylceftiofur is its main metabolite, but they are also have potential harm to human health. In this study, ceftiofur was combined with carrier proteins to get artificial antigens. A specific antibody (pAb) against CEF and desfuroylceftiofur was prepared. A sensitive and rapid paper-based sensor relying on time-resolved fluorescent microspheres (TRFMs) immune probes was developed, which were time-resolved fluorescent immunochromatographic strips (TRFMs-LFIA). The concentrations of T line and C line, activated pH, antibody volume and probe volume were optimized. Quantitative limits of detection (qLODs) of TRFMs-LFIA for CEF and desfuroylceftiofur were 0.97 ng/mL and 0.41 ng/mL, respectively. And 50 % inhibiting concentrations (IC50) were 12.92 ng/mL and 12.58 ng/mL, respectively. Pretreatment procedures of real samples were simple and rapid. Detection time of TRFMs-LFIA strip was 15 min. Qualitative analysis of CEF and desfuroylceftiofur was achieved under a UV light, quantitative analysis was implemented with a fluorescent immunoassay analyzer. The average recovery rates ranged from 91.4 % to 107.7 % and corresponding coefficients of variation (CV) was 1.5%-9.7 %. Concentration levels of artificially-spiked samples were measured by TRFMs-LFIA and compared with detection results of High performance liquid chromatography (HPLC), which showed a good accordance. These results indicated that the proposed assay can provide an effective strategy for on-site detection of CEF and desfuroylceftiofur simultaneously.

FGCD: a database of fungal gene clusters related to secondary metabolism.

Fungal secondary metabolites are not necessary for growth, but they are important for fungal metabolism and ecology because they provide selective advantages for competition, survival and interactions with the environment. These various metabolites are widely used as medicinal precursors and insecticides. Secondary metabolism genes are commonly arranged in clusters along chromosomes, which allow for the coordinate control of complete pathways. In this study, we created the Fungal Gene Cluster Database to store, retrieve, and visualize secondary metabolite gene cluster information across fungal species. The database was created by merging data from RNA sequencing, Basic Local Alignment Search Tool, genome browser, enrichment analysis and the R Shiny web framework to visualize and query putative gene clusters. This database facilitated the rapid and thorough examination of significant gene clusters across fungal species by detecting, defining and graphically displaying the architecture, organization and expression patterns of secondary metabolite gene clusters. In general, this genomic resource makes use of the tremendous chemical variety of the products of these ecologically and biotechnologically significant gene clusters to our further understanding of fungal secondary metabolism. Database URL: https://www.hebaubioinformatics.cn/FungalGeneCluster/.

Microfluidic-oriented green synthesis of pepsin-doped gold nanoparticles for colorimetric and photothermal dual-readout detection of food hazards.

Gold nanoparticles (AuNPs)-based immunochromatographic assay has gained popularity as a rapid detection method for food hazards. Synthesizing highly stable AuNPs in a rapid, simple and environmentally friendly manner is a key focus in this field. Here, we present a green microfluidic strategy for the rapid, automated, and size-controllable synthesis of pepsin-doped AuNPs (AuNPs@Pep) by employing glucose-pepsin as a versatile reducing agent and stabilizer. Through combining the colorimetric and photothermal (PoT) properties of AuNPs@Pep, both "signal-off" and "signal-on" formats of microfluidic paper analytical devices (PADs) were developed for detection of a small molecule antibiotic, florfenicol, and an egg allergen, ovalbumin. Compared to the colorimetric mode, a 4-fold and 3-fold improvement in limit of detection was observed in the "signal-off" detection of florfenicol and the "signal-on" detection of ovalbumin, respectively. The results demonstrated the practicality of AuNPs@Pep as a colorimetric/PoT dual-readout probe for immunochromatographic detection of food hazards at different molecular scales.