Systematically amino acid analysis: Advancements in extinction coefficient determination for therapeutic proteins.

Biologicals developers often face challenges in accurately determining the extinction coefficient (EC) measurement. We have successfully improved the precision and robustness of the widely recognized amino acid analysis method for EC determination, through a stepwise optimization process. Extensive analyses based on 114 observations, covering eight proteins over three years were performed, with a maximum relative standard deviation of 1.5% among multiple analysts, and a maximum deviation of 2.8% from the theoretical EC across the eight given proteins examined.

In-Depth Characterization of mAb Charge Variants by On-Line Multidimensional Liquid Chromatography-Mass Spectrometry.

In-depth characterization of charge heterogeneity is a pivotal step desired in the therapeutics antibody development. To this end, a novel on-line multidimensional liquid chromatography-mass spectrometry (MDLC-MS) method for charge variant characterization was developed to dig out potential risks on safety and efficacy. This method implemented 96-well plate fractionation and on-column preconcentration by multi-injection, thereby facilitating detection of charged species at low abundance. Eleven charge variants of mAb-A were preliminarily characterized by 2DLC(CEX × RP-C4)-MS. TRVHS and RVHS signal peptide variants of mAb-A were found in basic peaks of the CEX profile. The results supported process development in a timely manner, and the signal peptide-containing variants with potential immunogenicity were successfully removed by an optimized purification process. The retained seven charge variants of mAb-A were further characterized by 4DLC(CEX × RP-C4 × Trypsin×RP-C18)-MS. Post-translational modifications including deamidation, cyclization of N-terminal glutamine, C-terminal lysine truncation as well as proline amidation, and methionine oxidation were identified, and their potential risks were evaluated. Biological activity of the seven charge variants was evaluated by 2DLC (CEX × FcγRIIIa). Increased FcγRIIIa receptor binding affinity was observed in the acidic variants. The MDLC-MS detection can be completed in 72 h with 1.25 mg of mAb, demonstrating to be sample-economic, time-effective, and labor-saving. It provided a powerful and timely tool for charge variant characterization and met the aggressive timeline desired for antibody development.

Kynurenic acid ameliorates lipopolysaccharide-induced endometritis by regulating the GRP35/NF-κB signaling pathway.

Endometritis is a serious reproductive disease in mammals that commonly results in reproductive loss and even permanent infertility. Kynurenic acid (KYNA) is the main bioactive metabolite of tryptophan degradation and exhibits neuroprotective and anticonvulsant properties. However, little is known about the role of KYNA in achieving endometritis remission. This study investigated the protective effects and mechanisms of KYNA using a mouse model of against lipopolysaccharide (LPS)-induced endometritis. The endometritis model was induced by an intrauterine injection of LPS, and KYNA was intraperitoneally injected before and two hours after LPS treatment. Twenty-four hours after LPS administration, pathological changes in uterine tissues were observed by hematoxylin- and eosin (H&E) staining. The levels of the inflammatory factors, TNF-α and IL-1ß, were measured by ELISA. The myeloperoxidase (MPO) activity in uterine tissues was detected using MPO kits and immunohistochemistry. Furthermore, the expression of signaling pathway proteins and tight junction proteins occludin and ZO-1 in uterine tissues was detected by western blot. KYNA prominently inhibited uterine pathological injury and neutrophil infiltration and restricted the secretion of TNF-α and IL-1ß in the uteri of subjects with endometritis. Furthermore, KYNA upregulated the levels of the tight junction proteins (TJPs)occludin and ZO-1 in the uterus. In vitro, KYNA inhibited LPS-induced TNF-α and IL-1ß production, and NF-κB activation in mouse endometrial epithelial cells (mEECs). In addition, KYNA increased the expression of G protein-coupled receptor 35 (GPR35) and inhibition of GPR35 reversed the anti-inflammatory effects of KYNA. In conclusion, KYNA protected against LPS-induced endometritis by maintaining epithelial barrier permeability and suppressing proinflammatory responses via the GRP35/NF-κB signaling pathway.

Cadmium induces liver dysfunction and ferroptosis through the endoplasmic stress-ferritinophagy axis.

Cadmium (Cd) is a type of high-risk heavy metal that can damage organs such as the liver, but its mechanism is not yet clear. Ferroptosis is a newly discovered mode of regulatory cell death. We explored whether ferroptosis is involved in Cd-induced liver damage and the underlying mechanism. Our research showed that Cd induced liver damage by inducing ferroptosis, and the use of ferroptosis inhibitors reduced the degree of liver damage. Moreover, the occurrence of ferroptosis was accompanied by the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway, and inhibiting endoplasmic reticulum (ER) stress reduced ferroptosis demonstrating that ferroptosis induced by Cd is dependent on ER stress. In addition, chloroquine, a common autophagy inhibitor, mitigated ferroptosis caused by Cd exposure. Then, the iron chelator deferoxamine reduced Cd-induced lipid peroxidation and cell death, demonstrating that the iron regulation disorder caused by ferritin phagocytosis contributes to the Cd-induced ferroptosis. In conclusion, our results show that Cd-induced liver toxicity is accompanied by ferroptosis, which contributes to Cd inducing oxidative stress to trigger autophagy and ER stress to promote the process of ferroptosis.

Molecular modeling studies of atorvastatin analogues as HMGR inhibitors using 3D-QSAR, molecular docking and molecular dynamics simulations.

3-Hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) is generally regarded as targets for the treatment of hypercholesterolemia. HMGR inhibitors (more commonly known as statins) are discovered as plasma cholesterol lowering molecules. In this work, 120 atorvastatin analogues were studied using a combination of molecular modeling techniques including three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking and molecular dynamics (MD) simulation. The results show that the best CoMFA (comparative molecular field analysis) model has q(2)=0.558 and r(2)=0.977, and the best CoMSIA (comparative molecular similarity indices analysis) model has q(2)=0.582 and r(2)=0.919. Molecular docking and MD simulation explored the binding relationship of the ligand and the receptor protein. The calculation results indicated that the hydrophobic and electrostatic fields play key roles in QSAR model. After MD simulation, we found four vital residues (Lys735, Arg590, Asp690 and Asn686) and three hydrophobic regions in HMGR binding site. The calculation results show that atorvastatin analogues obtained by introduction of F atoms or gem-difluoro groups could obviously improve the inhibitory activity. The new HMGR inhibitor analogues design in this Letter had been submitted which is being currently synthesized by our laboratories.

A patient with a pulmonary hernia and chance fracture of the thoracic vertebra following a fall: A case report and literature review.

INTRODUCTION AND IMPORTANCE: Pulmonary hernia is a rare disease caused mostly by chest trauma. Patients often present with non-specific signs and symptoms. Currently, there is no unified approach to treating it. For asymptomatic pulmonary hernias, conservative treatment has been chosen in the past. However, the increasing number of cases has shown that surgery has a more positive effect on some patients with asymptomatic pulmonary hernias. CASE PRESENTATION: A 63-year-old female patient who accidentally fell from an agricultural vehicle while doing farm work presented with back pain and lower limb paralysis. Her vital signs were stable. A chest computed tomography (CT) scan showed the patient had a pulmonary hernia, thoracic vertebra Chance fracture, rib fractures, and right hydropneumothorax. The patient received an open reduction internal fixation (ORIF) of the thoracic vertebra at the trauma emergency center. The postoperative chest CT scan showed that the pulmonary hernia had reset to the chest cavity. Subsequently, the patient got a pulmonary hernia repair at the thoracic surgery department. The patient was discharged on the 19th day after the injury. Long-term follow-up showed good recovery from the thoracic trauma. CLINICAL DISCUSSION: The patient had a pulmonary hernia combined with thoracic Chance fracture and other injuries. Surgical repair achieved satisfactory results. CONCLUSION: We lack guidelines on whether to manage pulmonary hernias surgically. Patients with asymptomatic pulmonary hernias particularly should have their long-term prognoses fully evaluated. Surgery is needed if the patient has high-risk factors and severe intercostal muscle defects.

Identification and in vitro enzymatic activity analysis of the AOP2 gene family associated with glucosinolate biosynthesis in Tumorous stem mustard (Brassica juncea var. tumida).

The major enzyme encoded by the glucosinolate biosynthetic gene AOP2 is involved in catalyzing the conversion of glucoiberin (GIB) into sinigrin (SIN) in Brassicaceae crops. The AOP2 proteins have previously been identified in several Brassicaceae species, but not in Tumorous stem mustard. As per this research, the five identified members of the AOP2 family from the whole genome of Brassica juncea named BjuAOP2.1-BjuAOP2.5 were found to be evenly distributed on five chromosomes. The subcellular localization results implied that BjuAOP2 proteins were mainly concentrated in the cytoplasm. Phylogenetic analysis of the AOP2 proteins from the sequenced Brassicaceae species in BRAD showed that BjuAOP2 genes were more closely linked to Brassica carinata and Brassica rapa than Arabidopsis. In comparison with other Brassicaceae plants, the BjuAOP2 members were conserved in terms of gene structures, protein sequences, and motifs. The light response and hormone response elements were included in the BjuAOP2 genes' cis-regulatory elements. The expression pattern of BjuAOP2 genes was influenced by the different stages of development and the type of tissue being examined. The BjuAOP2 proteins were used to perform the heterologous expression experiment. The results showed that all the five BjuAOP2 proteins can catalyze the conversion of GIB to SIN with different catalytic activity. These results provide the basis for further investigation of the functional study of BjuAOP2 in Tumorous stem mustard glucosinolate biosynthesis.

A Novel Method for Monitoring N-Nitrosamines Impurities Using NH2-MIL-101(Fe) Mediated Dispersive Micro-Solid Phase Extraction Coupled with LC-MS/MS in Biopharmaceuticals.

A highly efficient and convenient method for the simultaneous determination of 12 N-nitrosamines (NAs) has been developed using an amine-functionalized metal-organic framework (NH2-MIL-101(Fe)) as sorbent for dispersive micro-solid phase extraction (D-µSPE) coupled with LC-MS/MS in biopharmaceuticals. The experimental variables involved in the extraction process (i.e., amount of the sorbent, extraction time, desorption time, ionic strength, desorption solvent and volume) were optimized to achieve the best extraction efficiency of the target analytes. Under the optimum conditions, the method was successfully validated, showing good linearity in the range of 0.5-3.0 µg/L with determination coefficients (R2) higher than 0.990, repeatability (RSD ≤ 10.0%, spiked level at 2.0 µg/L) and precision (RSD ≤ 8.2%). The limit of detection (LOD) and limit of quantitation (LOQ) were in the range of 0.005-0.025 µg/L and 0.010-0.250 µg/L, respectively. Satisfactory recoveries ranging from 82.4 to 116.8% were obtained by spiking standards at three different concentrations (0.5 µg/L, 2.0 µg/L and 3.0 µg/L). Other validation parameters, including specificity, stability, and robustness, met the validation criteria. More importantly, the plausible adsorption mechanism on NH2-MIL-101(Fe) was proposed by Fourier-transform infrared (FTIR) spectra technique. Finally, this method was successfully applied to detect trace nitrosamines in biopharmaceuticals.

Enterogenic Stenotrophomonas maltophilia migrates to the mammary gland to induce mastitis by activating the calcium-ROS-AMPK-mTOR-autophagy pathway.

BACKGROUND: Mastitis is an inflammatory disease of the mammary gland that has serious economic impacts on the dairy industry and endangers food safety. Our previous study found that the body has a gut/rumen-mammary gland axis and that disturbance of the gut/rumen microbiota could result in 'gastroenterogenic mastitis'. However, the mechanism has not been fully clarified. Recently, we found that long-term feeding of a high-concentrate diet induced mastitis in dairy cows, and the abundance of Stenotrophomonas maltophilia (S. maltophilia) was significantly increased in both the rumen and milk microbiota. Accordingly, we hypothesized that 'gastroenterogenic mastitis' can be induced by the migration of endogenous gut bacteria to the mammary gland. Therefore, this study investigated the mechanism by which enterogenic S. maltophilia induces mastitis. RESULTS: First, S. maltophilia was labelled with superfolder GFP and administered to mice via gavage. The results showed that treatment with S. maltophilia promoted the occurrence of mastitis and increased the permeability of the blood-milk barrier, leading to intestinal inflammation and intestinal leakage. Furthermore, tracking of ingested S. maltophilia revealed that S. maltophilia could migrate from the gut to the mammary gland and induce mastitis. Subsequently, mammary gland transcriptome analysis showed that the calcium and AMPK signalling pathways were significantly upregulated in mice treated with S. maltophilia. Then, using mouse mammary epithelial cells (MMECs), we verified that S. maltophilia induces mastitis through activation of the calcium-ROS-AMPK-mTOR-autophagy pathway. CONCLUSIONS: In conclusion, the results showed that enterogenic S. maltophilia could migrate from the gut to the mammary gland via the gut-mammary axis and activate the calcium-ROS-AMPK-mTOR-autophagy pathway to induce mastitis. Targeting the gut-mammary gland axis may also be an effective method to treat mastitis.

Formononetin Protects LPS-Induced Mastitis Through Suppressing Inflammation and Enhancing Blood-Milk Barrier Integrity via AhR-Induced Src Inactivation.

Formononetin (FOR), a natural flavonoid derived from Radix Astragali, has been reported to have anti-inflammatory and anti-oxidative effects. However, its protective mechanism against mastitis is still unknown. Nuclear factor kappa-B (NF-κB) signaling pathway plays an important role in inflammation, especially mastitis. Aryl hydrocarbon receptor (AhR) is involved in inflammatory regulation and defense against diseases. We investigated the protective effect of FOR on LPS-induced mastitis in mice and the effect of Ahr and NF-κB signaling pathways on the development of mastitis. In this study, mastitis model was induced by LPS injection through the nipple duct. Protective effect of FOR on LPS-induced mastitis was assessed by FOR pretreatment. The protective mechanism of FOR against mastitis was further investigated using LPS stimulation on mouse mammary epithelial cells EpH4-Ev. The results showed that LPS-induced mammary histological injury was inhibited by FOR. FOR significantly inhibited LPS-induced MPO activity. FOR administration enhanced the integrity of blood-milk barrier. In vitro and in vivo experiments showed that FOR inhibited LPS-induced NF-κB signaling pathway activation and the production of inflammatory factors TNF-α and IL-1ß. Moreover, FOR increased the expression of tight junction protein and enhanced blood-milk barrier integrity. LPS activated AhR and Src expression. But FOR induced significant increase in AhR inhibited Src phosphorylation to exert anti-inflammatory effects. In addition, AhR antagonist CH223191 reversed the inhibition of FOR on Src expression. And the inhibition of FOR on NF-κB activation and inflammatory cytokine production were reversed by AhR antagonist CH223191. In conclusion, FOR had protective effects against LPS-induced mastitis via suppressing inflammation and enhancing blood-milk barrier integrity via AhR-induced Src inactivation.

Dietary Tryptophan-Mediated Aryl Hydrocarbon Receptor Activation by the Gut Microbiota Alleviates Escherichia coli-Induced Endometritis in Mice.

Intestinal microbiota-mediated aryl hydrocarbon receptor (AhR) activation plays an important role in host-microbiota interactions and disease development. However, whether AhR activation mediates infection-induced inflammation in remote organs is not clear. The purpose of this study is to assess the effects and underlying mechanism of AhR activation and gut microbiota-mediated dietary tryptophan (Trp) metabolism on infection-induced inflammation using an Escherichia coli (E. coli)-induced endometritis model in mice. We found that AhR activation by 6-formylindolo (3,2-b) carbazole (Ficz), which is an AhR agonist derived from the photooxidation of Trp, alleviated E. coli-induced endometritis by repairing barrier function and inhibiting inflammatory responses, while inhibition of AhR by CH223191, which is a synthetic AhR antagonist, aggravated E. coli-induced endometritis. Gut dysbiosis damaged AhR activation and exacerbated E. coli-induced endometritis in mice, which responded to the reduced abundance of AhR ligand producers, such as Lactobacillus spp. Supplementation with dietary Trp ameliorated E. coli-induced endometritis in a microbiota-dependent manner, which was associated with the production of AhR ligands. Administration of AhR ligands, including indole and indole aldehyde, but not indole-3-propionic acid, rescued the protective effect of Trp on E. coli-induced endometritis in dysbiotic mice. Moreover, consumption of Lactobacillus reuteri (L. reuteri) containing AhR ligand-producing capability also alleviated E. coli-induced endometritis in mice in an AhR-dependent manner. Our results demonstrate that microbiota-mediated AhR activation is a key factor in fighting pathogen-caused inflammation, which leads to a potential strategy to regulate the gut microbiota and metabolism by dietary Trp or probiotics for the intervention of infectious diseases and reproductive health. IMPORTANCE Infection-induced endometritis is a common and frequently occurring disease in humans and animals. Accumulating evidence suggests an important role of the gut microbiota in the development of infection-induced inflammation. Whether and how gut microbiota-mediated AhR activation regulates the pathogenesis of pathogen-induced endometritis remains unknown. The current study found that AhR activation ameliorated E. coli-induced endometritis, and inhibition of AhR produced negative results. Gut dysbiosis reduced the abundance of AhR ligand producers including Lactobacillus spp., damaged AhR activation, and exacerbated E. coli-induced endometritis. Supplementation with dietary Trp, AhR ligands, and L. reuteri containing AhR ligand-producing capability alleviated E. coli-induced endometritis in mice. Our results suggest an important role of microbiota-mediated AhR activation in the pathogenesis of endometritis and provide potential strategies for the intervention of infectious diseases and reproductive health by regulating the gut microbiota and metabolism.

Synthesis and Characterization of Anatase TiO2 Microspheres Self-Assembled by Ultrathin Nanosheets.

In this paper, we report a novel anCd simple method for synthesizing the microspheres self-assembled from ultrathin anatase TiO2 nanosheets with a high percentage of (001) facets via the hydrolysis process of the single-reagent (potassium fluorotitanate). We then used optical microscopy, scanning electron microscopy, and high-resolution confocal laser Raman spectroscopy to characterize the microspheres generated under different conditions. The study found that the size of the anatase TiO2 microspheres synthesized was 0.5-3 µm. As the synthesis time increased, the corroded surface of the microspheres gradually increased, resulting in the gradual disappearance of the edges and corners of the anatase nanosheets. The exposure percentage of the (001) facets of ultrathin anatase nanosheets synthesized for 2 h at 180-200 °C are close to 100%. The microsphere whose surface is completely covered by these anatase nanosheets also has nearly 100% exposed (001) facets. This new anatase nanosheet-based self-assembled microsphere will have great application potential in pollution prevention, environmental protection, and energy fields.

Kynurenic acid protects against mastitis in mice by ameliorating inflammatory responses and enhancing blood-milk barrier integrity.

Mastitis is one of the most serious diseases in humans and animals, especially in the modern dairy industry. Seeking safe and effective mastitis prevention strategies is urgent since food safety and drug residues in milk remain an enormous concern, despite the contribution of antibiotics to control mastitis. Kynurenic acid (KYNA), derived from the kynurenine pathway of tryptophan metabolism, has been shown to exhibit anti-inflammatory and immunomodulatory effects in many diseases. Recently, it was reported that impaired KYNA levels were associated with mastitis. However, the physiological role of KYNA in mastitis has not yet been elucidated. Therefore, the aim of this study was to investigate the protective role of KYNA in pathogen-induced mastitis in mice, as well as the underlying mechanism of this effect. We first evaluated the effects of KYNA on LPS-induced mastitis in mice. Additionally, the underlying anti-inflammatory mechanism of KYNA was investigated in mammary epithelial cells (MMECs). Furthermore, we examined the effects of KYNA on S. aureus and E. coli induced mastitis in mice. Our results demonstrated that KYNA alleviated LPS-induced mastitis by reducing inflammatory responses and enhancing blood-milk barrier integrity. The fundamental mechanisms involved the inhibition of NF-κB and activation of Nrf2/Ho-1, which is probably mediated by G protein-coupled receptor 35 but not aryl hydrocarbon receptor. Notably, KYNA also protected against S. aureus and E. coli induced mastitis in mice. In conclusion, our results highlight the role of KYNA in mastitis and serve as a basis for using endogenous metabolite as a novel preventative or therapeutic strategy for disease intervention.

Endoplasmic reticulum stress-mediated autophagy activation is involved in cadmium-induced ferroptosis of renal tubular epithelial cells.

Acute cadmium (Cd) exposure is a significant risk factor for renal injury and lacks effective treatment strategies. Ferroptosis is a recently identified iron-dependent form of nonapoptotic cell death mediated by membrane damage resulting from lipid peroxidation, and it is implicated in many diseases. However, whether ferroptosis is involved in Cd-induced renal injury and, if so, how it operates. Here, we show that Cd can induce ferroptosis in kidney and renal tubular epithelial cells, as demonstrated by elevation of intracellular iron levels and lipid peroxidation, as well as impaired antioxidant production. Treatment with a ferroptosis inhibitor alleviated Cd-induced cell death. Intriguingly, we established that Cd-induced ferroptosis depended on endoplasmic reticulum (ER) stress, by demonstrating that Cd activated the PERK-eIF2α-ATF4-CHOP pathway and that inhibition of ER stress reduced ferroptosis caused by Cd. We further found that autophagy was required for Cd-induced ferroptosis because the inhibition of autophagy by chloroquine mitigated Cd-induced ferroptosis. Furthermore, we showed that iron dysregulation by ferritinophagy contributed to Cd-induced ferroptosis, by showing that the iron chelator desferrioxamine alleviated Cd-induced cell death and lipid peroxidation. In addition, ER stress is likely activated by MitoROS which trigger autophagy and ferroptosis. Collectively, our results indicate that ferroptosis is involved in Cd-induced renal toxicity and regulated by the MitoROS-ER stress-ferritinophagy axis.