Uridine diphosphate glucuronosyltransferase 1A1 prevents the progression of liver injury.

BACKGROUND: Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) plays a crucial role in metabolizing and detoxifying endogenous and exogenous substances. However, its contribution to the progression of liver damage remains unclear. AIM: To determine the role and mechanism of UGT1A1 in liver damage progression. METHODS: We investigated the relationship between UGT1A1 expression and liver injury through clinical research. Additionally, the impact and mechanism of UGT1A1 on the progression of liver injury was analyzed through a mouse model study. RESULTS: Patients with UGT1A1 gene mutations showed varying degrees of liver damage, while patients with acute-on-chronic liver failure (ACLF) exhibited relatively reduced levels of UGT1A1 protein in the liver as compared to patients with chronic hepatitis. This suggests that low UGT1A1 levels may be associated with the progression of liver damage. In mouse models of liver injury induced by carbon tetrachloride (CCl4) and concanavalin A (ConA), the hepatic levels of UGT1A1 protein were found to be increased. In mice with lipopolysaccharide or liver steatosis-mediated liver-injury progression, the hepatic protein levels of UGT1A1 were decreased, which is consistent with the observations in patients with ACLF. UGT1A1 knockout exacerbated CCl4- and ConA-induced liver injury, hepatocyte apoptosis and necroptosis in mice, intensified hepatocyte endoplasmic reticulum (ER) stress and oxidative stress, and disrupted lipid metabolism. CONCLUSION: UGT1A1 is upregulated as a compensatory response during liver injury, and interference with this upregulation process may worsen liver injury. UGT1A1 reduces ER stress, oxidative stress, and lipid metabolism disorder, thereby mitigating hepatocyte apoptosis and necroptosis.

Mechanistic studies to understand peak tailing due to sulfinic acid- and carboxylic acid-silanophilic interactions in reversed-phase liquid chromatography.

Silanophilic interactions are a primary contributor to peak tailing of acidic pharmaceutical compounds, thus a thorough understanding is especially important for reversed-phase liquid chromatography (RPLC) method development. Herein, a sulfinic acid compound that exhibited severe peak tailing in RPLC with acidic mobile phases was carefully studied. Results indicated that the neutral protonated form of the sulfinic acid is involved in the strong interaction that leads to peak tailing, but that tailing can be mitigated with a blocking effect achieved through use of acetic acid modifier in the mobile phase. Peak tailing was also observed with other structurally-similar sulfinic acids and carboxylic acids but was, in general, less severe with the latter. The Hydrophobic Subtraction Model (HSM) was applied to six commercial C18 columns that exhibited different tailing behaviors for the sulfinic acid compound in attempts to identify key sites of interaction within the stationary phase. A combination of heated acid column wash experiments and density functional theory (DFT) calculations indicate that the differential interactions of the acids with vicinal silanol pairs in the stationary phase play a major role in peak tailing.

Self-adhesive, freeze-tolerant, and strong hydrogel electrolyte containing xanthan gum enables the high-performance of zinc-ion hybrid supercapacitors.

Hydrogel electrolyte is an ideal candidate material for flexible energy storage devices due to its excellent softness and conductivity properties. However, challenges such as the inherent mechanical weakness, the susceptibility to be frozen in low-temperature environments, and the insufficiency of hydrogel-electrode contact persist. Herein, a "Multi in One" strategy is employed to effectively conquer these difficulties by endowing hydrogels with high strength, freeze-resistance, and self-adhesive ability. Multiple hydrogen bond networks and ion crosslinking networks are constructed within the hydrogel electrolyte (PVA/PAAc/XG) containing polyvinyl alcohol (PVA), acrylic acid (AAc), and xanthan gum (XG), promoting the enhanced mechanical property, and the adhesion to electrode materials is also improved through abundant active groups. The introduction of zinc ions provides the material with superior frost resistance while also promoting electrical conductivity. Leveraging its multifunction of superior mechanical strength, anti-freeze property, and self-adhesive characteristic, the PVA/PAAc/XG hydrogel electrolyte is employed to fabricate zinc ion hybrid supercapacitors (ZHS). Remarkably, ZHS exhibits outstanding electrochemical performance and cycle stability. A remarkable capacity retention rate of 83.86 % after 10,000 charge-discharge cycles can be achieved at high current densities, even when the operational temperature decreases to -60 °C, showing great potential in the field of flexible energy storage devices.

Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability.

Multi-segmented viruses often multimerize their genomic segments to ensure efficient and stoichiometric packaging of the correct genetic cargo. In the bipartite Nodaviridae family, genome heterodimerization is also observed and conserved among different species. However, the nucleotide composition and biological function for this heterodimer remain unclear. Using Flock House virus as a model system, we developed a next-generation sequencing approach ("XL-ClickSeq") to probe heterodimer site sequences. We identified an intermolecular base-pairing site which contributed to heterodimerization in both wild-type and defective virus particles. Mutagenic disruption of this heterodimer site exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNAs. Furthermore, the disruption of this intermolecular interaction directly impacts the thermostability of the mature virions. These results demonstrate that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus have an important role on virus particle integrity and thus may impact its transmission to a new host.IMPORTANCEFlock House virus is a member of Nodaviridae family of viruses, which provides a well-studied model virus for non-enveloped RNA virus assembly, cell entry, and replication. The Flock House virus genome consists of two separate RNA molecules, which can form a heterodimer upon heating of virus particles. Although similar RNA dimerization is utilized by other viruses (such as retroviruses) as a packaging mechanism and is conserved among Nodaviruses, the role of heterodimerization in the Nodavirus replication cycle is unclear. In this research, we identified the RNA sequences contributing to Flock House virus genome heterodimerization and discovered that such RNA-RNA interaction plays an essential role in virus packaging efficiency and particle integrity. This provides significant insight into how the interaction of packaged viral RNA may have a broader impact on the structural and functional properties of virus particles.

Strong and tough poly(vinyl alcohol)/xanthan gum-based ionic conducting hydrogel enabled through the synergistic effect of ion cross-linking and salting out.

The mechanical properties of ionic conductive hydrogels (ICHs) are generally inadequate, leading to their susceptibility to breakage under external forces and consequently resulting in the failure of flexible electronic devices. In this work, a simple and convenient strategy was proposed based on the synergistic effect of ion cross-linking and salting out, in which the hydrogels consisting of polyvinyl alcohol (PVA) and xanthan gum (XG) were immersed in zinc sulfate (ZnSO4) solution to obtain ICHs with exceptional mechanical properties. The salt-out effects between PVA chains and SO42- ions along with the cross-linked network of XG chains and Zn2+ ions contribute to the desirable mechanical properties of ICHs. Notably, the mechanical properties of ICHs can be adjusted by changing the concentration of ZnSO4 solution. Consequently, the optimum fracture stress and the fracture energy can reach 3.38 MPa and 12.13 KJ m-2, respectively. Moreover, the ICHs demonstrated a favorable sensitivity (up to 2.05) when utilized as a strain sensor, exhibiting an accurate detection of human body movements across various amplitudes.

Associations of concurrent early-life famine exposure and adulthood obesity with type 2 diabetes mellitus in middle-aged Chinese.

BACKGROUND: Evidence has shown that early-life famine exposure and obesity in adulthood are independently associated with the risk of type 2 diabetes mellitus (T2DM). However, few studies had revealed the combined effect of these risk factors. METHODS: Two sets of groups from the China Health and Retirement Longitudinal Study (CHARLS) were selected. The fetal-exposure group born in 1959-1961 from 2011 wave (N = 958) and nonexposure group born in 1963-1965 from 2015 wave (N = 1540) were selected as Comparison 1. The early childhood-exposure group born in 1955-1957 from 2011 wave (N = 1510) and fetal-exposure group born in 1959-1961 from 2015 wave (N = 943) were Comparison 2. Logistic regressions were applied to examine the associations of different famine exposure periods and obesity patterns with T2DM risk. RESULTS: Compared with nonexposed participants without central overweight/obesity in adulthood, central overweight/obesity in adulthood together with nonexposure (odds ratio [OR]: 1.89, 95% confidence interval [CI]: 1.19-3.00) or fetal-exposure (OR: 1.99, 95% CI: 1.23-3.23) was associated with higher risks of T2DM. Compared with the early childhood-exposure group, the fetal-exposed participants showed higher risks of T2DM (OR: 1.30, 95% CI: 1.02-1.66). The coexistence of fetal famine exposure and central overweight/obesity in adulthood was associated with higher risks of T2DM (OR: 1.82, 95% CI: 1.19-2.79). Consistent associations were observed among males and participants from less severely affected areas. CONCLUSIONS: In conclusion, central overweight/obesity in adulthood is associated with the increased risk of T2DM, but the effect of early-life famine exposure is not very clear.

RpoS activates Salmonella Typhi biofilms and drives persistence in a Zebrafish model.

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway as established in S. Typhimurium. We adopted a genome-wide Tn5 mutation screen in S. Typhi in gallstone-mimicking conditions and generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components as Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. Overall, our work established a novel RpoS-driven paradigm for the formation of cholesterol-attached Typhi biofilms and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections.

Metabolomics Study Revealing Purines as Potential Diagnostic Biomarkers of Acute Respiratory Distress Syndrome in Patients with Community─Acquired Pneumonia.

Community-acquired pneumonia (CAP) is a significant threat to human health and the leading cause of acute respiratory distress syndrome (ARDS). We aimed to reveal the metabolic profiling whether can be used for assessing CAP with or without ARDS (nARDS) and therapeutic effects on CAP patients after treatment. Urine samples were collected at the onset and recovery periods, and metabolomics was employed to identify robust biomarkers. 19 metabolites were significantly changed in the ARDS relative to nARDS, mainly involving purines and fatty acids. After treatment, 7 metabolites in the nARDS and 14 in the ARDS were found to be significantly dysregulated, including fatty acids and amino acids. In the validation cohort, we observed that the biomarker panel consisted of N2,N2-dimethylguanosine, 1-methyladenosine, 3-methylguanine, 1-methyladenosine, and uric acid exhibited better AUCs of 0.900 than pneumonia severity index and acute physiology and chronic health evaluation II (APACHE II) scores between the ARDS and nARDS. Combining L-phenylalanine, phytosphingosine, and N-acetylaspartylglutamate as biomarkers for discriminating the nARDS and ARDS patients after treatment exhibited good AUCs of 0.811 and 0.821, respectively. The metabolic pathway and defined biomarkers may serve as crucial indicators for predicting the development of ARDS in CAP patients and for assessing therapeutic effects.

Integrated analysis of copy number variation-associated lncRNAs identifies candidates contributing to the etiologies of congenital kidney anomalies.

Congenital anomalies of the kidney and urinary tract (CAKUT) are disorders resulting from defects in the development of the kidneys and their outflow tract. Copy number variations (CNVs) have been identified as important genetic variations leading to CAKUT, whereas most CAKUT-associated CNVs cannot be attributed to a specific pathogenic gene. Here we construct coexpression networks involving long noncoding RNAs (lncRNAs) within these CNVs (CNV-lncRNAs) using human kidney developmental transcriptomic data. The results show that CNV-lncRNAs encompassed in recurrent CAKUT associated CNVs have highly correlated expression with CAKUT genes in the developing kidneys. The regulatory effects of two hub CNV-lncRNAs (HSALNG0134318 in 22q11.2 and HSALNG0115943 in 17q12) in the module most significantly enriched in known CAKUT genes (CAKUT_sig1, P = 1.150 × 10-6) are validated experimentally. Our results indicate that the reduction of CNV-lncRNAs can downregulate CAKUT genes as predicted by our computational analyses. Furthermore, knockdown of HSALNG0134318 would downregulate HSALNG0115943 and affect kidney development related pathways. The results also indicate that the CAKUT_sig1 module has function significance involving multi-organ development. Overall, our findings suggest that CNV-lncRNAs play roles in regulating CAKUT genes, and the etiologies of CAKUT-associated CNVs should take account of effects on the noncoding genome.

Pediatric adenovirus pneumonia: clinical practice and current treatment.

Adenovirus pneumonia is common in pediatric upper respiratory tract infection, which is comparatively easy to develop into severe cases and has a high mortality rate with many influential sequelae. As for pathogenesis, adenoviruses can directly damage target cells and activate the immune response to varying degrees. Early clinical recognition depends on patients' symptoms and laboratory tests, including those under 2 years old, dyspnea with systemic toxic symptoms, atelectasis or emphysema in CT image, decreased leukocytes, and significantly increased C-reaction protein (CRP) and procalcitonin (PCT), indicating the possibility of severe cases. Until now, there is no specific drug for adenovirus pneumonia, so in clinical practice, current treatment comprises antiviral drugs, respiratory support and bronchoscopy, immunomodulatory therapy, and blood purification. Additionally, post-infectious bronchiolitis obliterans (PIBO), hemophagocytic syndrome, and death should be carefully noted. Independent risk factors associated with the development of PIBO are invasive mechanical ventilation, intravenous steroid use, duration of fever, and male gender. Meanwhile, hypoxemia, hypercapnia, invasive mechanical ventilation, and low serum albumin levels are related to death. Among these, viral load and serological identification are not only "gold standard" for adenovirus pneumonia, but are also related to the severity and prognosis. Here, we discuss the progress of pathogenesis, early recognition, therapy, and risk factors for poor outcomes regarding severe pediatric adenovirus pneumonia.

Loss-of-function mutation in Omicron variants reduces spike protein expression and attenuates SARS-CoV-2 infection.

SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel amino acid mutations in the spike protein alone. While most studies focus on receptor binding domain changes, mutations in the C-terminus of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. In this study, we examined three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we found that the mutant increased spike processing, consistent with prior reports for H655Y and P681H individually. Next, we generated a single N679K mutant, finding reduced viral replication in vitro and less disease in vivo. Mechanistically, the N679K mutant had reduced spike protein in purified virions compared to wild-type; spike protein decreases were further exacerbated in infected cell lysates. Importantly, exogenous spike expression also revealed that N679K reduced overall spike protein yield independent of infection. Although a loss-of-function mutation, transmission competition demonstrated that N679K had a replication advantage in the upper airway over wild-type SARS-CoV-2 in hamsters, potentially impacting transmissibility. Together, the data show that N679K reduces overall spike protein levels during Omicron infection, which has important implications for infection, immunity, and transmission.

Trapping mode two-dimensional liquid chromatography for quantitative low-level impurity enrichment in pharmaceutical development.

Trapping mode two-dimensional liquid chromatography (2D-LC) has recently found applications in pharmaceutical analysis to clean, refocus, and enrich analytes. Given its enrichment capability, 2D-LC with multiple trappings is appealing for low-level impurity monitoring that cannot be solved by single dimensional LC (1D-LC) or unenriched 2D-LC analysis. However, the quantitative features of multi-trapping 2D-LC remain largely unknown at impurity levels from parts-per-million (ppm) to 0.15% (w/w). We present a simple heart-cutting trapping mode 2D-LC workflow using only common components and software found in typical off-the-shelf 1D-LC instruments. This robust, turn-key system's quantitative capabilities were evaluated using a variety of standard markers, demonstrating linear enrichment for up to 20 trapping cycles and achieving a recovery of over 97.0%. Next, the trapping system was applied to several real-world low-level impurity pharmaceutical case studies including (1) the identification of two unknown impurities at sub-ppm levels resulting in material discoloration, (2) the discovery of a new impurity at 0.05% (w/w) co-eluted with a known impurity, making the undesired summation above the target specification, and (3) the quantification of a potential mutagenic impurity at 10-ppm level in a poorly soluble substrate. The recovery in all studies was better than 97.0% with RSD lower than 3.0%, demonstrating accuracy and precision of the 2D-LC trapping workflow. As no specialized equipment or software is required, we envision that the system could be used to develop low-impurity monitoring methods suitable for validation and potential execution in quality-control laboratories.

A multidisciplinary approach to the identification of the protein-RNA connectome in double-stranded RNA virus capsids.

How multi-segmented double-stranded RNA (dsRNA) viruses correctly incorporate their genomes into their capsids remains unclear for many viruses, including Bluetongue virus (BTV), a Reoviridae member, with a genome of 10 segments. To address this, we used an RNA-cross-linking and peptide-fingerprinting assay (RCAP) to identify RNA binding sites of the inner capsid protein VP3, the viral polymerase VP1 and the capping enzyme VP4. Using a combination of mutagenesis, reverse genetics, recombinant proteins and in vitro assembly, we validated the importance of these regions in virus infectivity. Further, to identify which RNA segments and sequences interact with these proteins, we used viral photo-activatable ribonucleoside crosslinking (vPAR-CL) which revealed that the larger RNA segments (S1-S4) and the smallest segment (S10) have more interactions with viral proteins than the other smaller segments. Additionally, using a sequence enrichment analysis we identified an RNA motif of nine bases that is shared by the larger segments. The importance of this motif for virus replication was confirmed by mutagenesis followed by virus recovery. We further demonstrated that these approaches could be applied to a related Reoviridae member, rotavirus (RV), which has human epidemic impact, offering the possibility of novel intervention strategies for a human pathogen.

ViReMa: a virus recombination mapper of next-generation sequencing data characterizes diverse recombinant viral nucleic acids.

BACKGROUND: Genetic recombination is a tremendous source of intrahost diversity in viruses and is critical for their ability to rapidly adapt to new environments or fitness challenges. While viruses are routinely characterized using high-throughput sequencing techniques, characterizing the genetic products of recombination in next-generation sequencing data remains a challenge. Viral recombination events can be highly diverse and variable in nature, including simple duplications and deletions, or more complex events such as copy/snap-back recombination, intervirus or intersegment recombination, and insertions of host nucleic acids. Due to the variable mechanisms driving virus recombination and the different selection pressures acting on the progeny, recombination junctions rarely adhere to simple canonical sites or sequences. Furthermore, numerous different events may be present simultaneously in a viral population, yielding a complex mutational landscape. FINDINGS: We have previously developed an algorithm called ViReMa (Virus Recombination Mapper) that bootstraps the bowtie short-read aligner to capture and annotate a wide range of recombinant species found within virus populations. Here, we have updated ViReMa to provide an "error density" function designed to accurately detect recombination events in the longer reads now routinely generated by the Illumina platforms and provide output reports for multiple types of recombinant species using standardized formats. We demonstrate the utility and flexibility of ViReMa in different settings to report deletion events in simulated data from Flock House virus, copy-back RNA species in Sendai viruses, short duplication events in HIV, and virus-to-host recombination in an archaeal DNA virus.

Development of tandem-column liquid chromatographic methods for pharmaceutical compounds using simulations based on hydrophobic subtraction model parameters.

The liquid chromatography (LC) analysis of small molecule pharmaceutical compounds and related impurities is crucial in the development of new drug substances, but developing these separations is usually challenging due to analyte structural similarities. Tandem-column LC (TC-LC) has emerged as a powerful approach to achieve alternative separation selectivity compared to conventional single column separations. However, one of the bottlenecks associated with use of tandem column approaches is time-consuming column pair screening and selection. Herein, we compared critical resolution (Rc) in single column vs. TC-LC separations for a given set of small molecule pharmaceutical compounds and developed a column selection workflow that uses separation simulations based on parameters from the hydrophobic subtraction model (HSM) of reversed-phase selectivity. In this study, HSM solute parameters were experimentally determined for a small molecule pharmaceutical (Linrodostat) and ten of its related impurities using multiple linear regression of their retentions on 16 selected RPLC columns against in-house determined HSM column parameters. Rc values were calculated based on HSM database column parameters for a pool of about 200 available stationary phases in both single-phase column (2.1 mm i.d. × 100 mm) or tandem column paired (two 2.1 mm i.d. × 50 mm) formats. Four column configurations (two single and two tandem) were predicted to achieve successful separations under isocratic HSM separation conditions, with a fifth tandem pair predicted to have a single co-elution. Of these five potential candidates, one tandem pair yielded compete baseline resolution of the 11-component mixture in an experimental separation. In this specific case, the tandem column pairs outperformed single-phase columns, with better predicted and experimental Rc values for the Linrodostat mixture under the HSM separation conditions. The results reported in this study demonstrated the enormous selectivity potential of TC-LC in pharmaceutical compound separations and are consistent with our previous study that examined the potential of tandem column approaches using purely computational means, though there is room for substantial improvement in the prediction accuracy. The proposed workflow can be used to prioritize a small number of column combinations by computational means before any experiments are conducted. This is highly attractive from the point of view of time and resource savings considering over 200,000 different tandem column pairings are possible using columns for which there are data in the HSM database.

Dual-network polyvinyl alcohol/polyacrylamide/xanthan gum ionic conductive hydrogels for flexible electronic devices.

Ionic conductive hydrogels (ICHs) have received widespread attention as an ideal candidate for flexible electronic devices. However, conventional ICHs failed in widespread applications due to their inability to simultaneously possess high toughness, high ionic conductivity, and anti-freezing properties. Here, polyvinyl alcohol (PVA) and polyacrylamide (PAAm) were first dissolved in the zinc chloride solution, in which zinc ions (Zn2+) act as ionic cross-linkers and conducting ions, followed by the introduction of xanthan gum (XG) with a unique structure of trisaccharide side chains into the PVA/PAAm semi-interpenetrating network to prepare a dual-network ICHs (refers as PPXZ). Enabled by the synergistic effect of intermolecular chemical covalent cross-linking and physical cross-linking, PPXZ hydrogels exhibit significantly improved mechanical properties without sacrificing electrical conductivity. Furthermore, PPXZ hydrogels are successfully applied to flexible electronic devices, such as strain sensors and zinc ion hybrid supercapacitors, exhibiting satisfactory sensing sensitivity and cycling stability at a wide temperature range, respectively. Even at a high current density (10 A g-1), the capacity of the supercapacitor retains 88.24 % after 10,000 cycles. This strategy provides new insight for ICHs in wide temperature-applied flexible electronic devices.

Optimized integration of metabolomics and lipidomics reveals brain region-specific changes of oxidative stress and neuroinflammation in type 1 diabetic mice with cognitive decline.

INTRODUCTION: Type 1 diabetes (T1D) causes cognitive decline and has been associated with brain metabolic disorders, but its potential molecular mechanisms remain unclear. OBJECTIVES: The purpose of this study was to explore the molecular mechanisms underlying T1D-induced cognitive impairment using metabolomics and lipidomics. METHODS: We developed an optimized integration approach of metabolomics and lipidomics for brain tissue based on UPLC-Q-TOF-MS and analyzed a comprehensive characterization of metabolite and lipid profiles in the hippocampus and frontal cortex of T1D male mice with cognitive decline (T1DCD) and age-matched control (CONT) mice. RESULTS: The results show that T1DCD mice had brain metabolic disorders in a region-specific manner relative to CONT mice, and the frontal cortex exhibited a higher lipid peroxidation than the hippocampus in T1DCD mice. Based on metabolic changes, we found that microglia was activated under diabetic condition and thereby promoted oxidative stress and neuroinflammation, leading to neuronal injury, and this event was more pronounced in the frontal cortex than the hippocampus. CONCLUSION: Our results suggest that brain region-specific shifts in oxidative stress and neuroinflammation may contribute to diabetic cognitive decline, and the frontal cortex could be the more vulnerable brain region than the hippocampus.

Functional Analysis of a Methyltransferase Involved in Anthocyanin Biosynthesis from Blueberries (Vaccinium corymbosum).

Anthocyanins are natural water-soluble pigments that widely exist in plants, with various biological activities, including antioxidant, anti-obesity, and anti-diabetic activities. Currently, monomeric anthocyanins are mainly obtained through natural sources, which limits their availability. In the biosynthesis of anthocyanins, anthocyanin methyltransferases are recognized to play important roles in the water solubility and structural stability of anthocyanins. Blueberries are a rich source of anthocyanins with more than 30 chemical structures. However, the enzymes that were responsible for the methylation of anthocyanidin cores in blueberries had not been reported. Here, blueberries (Vaccinium corymbosum) have been selected as the candidate for characterization of the key enzyme. Phylogenic analysis, enzymatic activity assay, homology modeling, molecular simulation, protein expression and purification assay, site-directed mutation, isothermal titration calorimetry assay, and enzyme kinetic assay were used to identify the enzymatic function and molecular mechanism of VcOMT, which was responsible for the methylation of anthocyanidin cores. VcOMT could use delphinidin as a substrate but not cyanidin, petunidin, anthocyanins, flavonols, and flavonol glycosides. Ile191 and Glu198 were both identified as important amino acid residues for the binding interactions of anthocyanidins with VcOMT.

Intracellular alpha-fetoprotein mitigates hepatocyte apoptosis and necroptosis by inhibiting endoplasmic reticulum stress.

BACKGROUND: Endoplasmic reticulum (ER) stress contributes to the pathogenesis of chronic liver diseases, but how hepatocytes respond to ER stress has not been clarified. Alpha-fetoprotein (AFP) is secreted by hepatoma cells and elevated levels of serum AFP are associated with development of liver malignancies. AIM: To investigate whether and how AFP could regulate ER stress and hepatocyte injury. METHODS: The distribution of AFP and the degrees of ER stress in liver tissues and liver injury were characterized by histology, immunohistochemistry, and Western blot in biopsied human liver specimens, two mouse models of liver injury and a cellular model. The levels of AFP in sera and the supernatants of cultured cells were quantified by chemiluminescence. RESULTS: High levels of intracellular AFP were detected in liver tissues, particularly in the necrotic areas, from patients with chronic liver diseases and mice after carbon tetrachloride (CCl4) administration or induction of ER stress, but not from the controls. The induced intracellular AFP was accompanied by elevated activating transcription factor-6 (ATF6) expression and protein kinase R-like ER kinase (PERK) phosphorylation in mouse livers. ER stress induced AFP expression in LO2 cells and decreased their viability. ATF6, but not PERK, silencing mitigated the ER-stress-induced AFP expression in LO2 cells. Conversely, AFP silencing deteriorated the ER stress-mediated LO2 cell injury and CCl4 administration-induced liver damages by increasing levels of cleaved caspase-3, the C/enhancer binding protein homologous protein expression, mixed lineage kinase domain-like pseudokinase and PERK phosphorylation, but decreasing ATF6 expression. CONCLUSION: ER stress upregulated intra-hepatocyte AFP expression by activating ATF6 during the process of liver injury and intracellular AFP attenuated hepatocyte apoptosis and necroptosis by alleviating ER stress.