Protein Carboxyl Research Articles

Concentration-dependent effect of norfloxacin on iron toxicity in the intestine of large yellow croaker Larimichthys crocea (Richardson, 1846)

This study explores the effects of norfloxacin (NOR) on oxidative damage, iron (Fe) transport, energy metabolism, and immunotoxicity in the intestine of large yellow croaker under Fe stress. The fish were subjected to Fe (180 μg/L), low-dose NOR (1.8 μg/L, LNOR), high-dose NOR (180 μg/L, HNOR), Fe plus LNOR, and Fe plus HNOR for 60 days. These results demonstrated that Fe alone exposure increased malondialdehyde (MDA), protein carboxylation (PC), and mortality rate, and impaired intestinal tissue, which was related to the increment of Fe accumulation. Compared to Fe alone exposure, Fe plus LNOR exposure decreased MDA, PC, and mortality rate, and alleviated intestinal malformations by improving Fe transport, energy metabolism, anti-inflammatory response, and protein folding protective effect, and reducing pro-inflammatory response, indicating that LNOR had an antagonistic effect on Fe toxicity. Compared to Fe alone exposure, Fe plus HNOR exposure elevated MDA, PC, and mortality rate, and deteriorated intestinal malformations by inhibiting Fe excretion, energy metabolism, anti-inflammatory response, and protein folding protective effect, and enhancing pro-inflammatory response, indicating a synergetic effect between HNOR and Fe stress. These findings suggested that NOR had a dose-dependent effect on Fe-toxicity to large yellow croaker, which contributes to revealing the molecular mechanisms behind their interaction and its ecological implications.

Dynamic responses and adsorption mechanisms of Chlamydomonas reinhardtii extracellular polymeric substances under Cd, Cu, Pb, and Zn exposure.

Extracellular polymeric substances (EPS) can effectively attenuate heavy metal mobility in aquatic ecosystems and reduce metal toxicity to cells. However, a systematic study of microalgae EPS responses and their adsorption behaviors, characteristics, and mechanisms under different heavy metal exposures has not been performed. In this study, EPS extracted from Chlamydomonas reinhardtii CC-125 was analyzed for compositional changes (monosaccharides and proteins) under Cd, Cu, Pb, and Zn treatments. The EPS adsorption capacities and mechanisms for the four metal ions were also investigated. Cd (10 mg/L), Cu (5 mg/L), and Zn (5 mg/L) exposure induced changes in the microalgal EPS composition and structure, and a protein/polysaccharide ratio of greater than 1 was found. This result indicated the crucial role of proteins in stress resistance. In contrast, Pb stress resulted in an increase of 532.64% and 117.48% in proteins and polysaccharides, respectively, with galactose and glucose playing key roles in this process. A fluorescence analysis revealed that Cd/Pb exposure reduced the tryptophan and tyrosine levels in the EPS, while Cu/Zn only weakened tryptophan. As a biosorbent, the adsorption capacity of the EPS for the four metals followed the order of Pb > Cd > Cu > Zn. The fluorescence quenching titration results revealed that fluorescent compounds in the EPS had the strongest complexation ability with Pb (logKSV: 8.16 × 103), followed by Cu (logKSV: 1.79 × 103), while their abilities for Cd and Zn were weaker. A spectroscopic analysis indicated that the primary functional groups involved in EPS binding with Pb/Cd and Cd/Zn were protein carboxyl groups (C=O/O-C=O) and glycosidic bonds (C-OH/C-O-C), respectively. This study elucidates the response strategies and adsorption mechanisms of the C. reinhardtii EPS to different metals and provides a basis for environmental heavy metal pollution bioremediation.

Mammalian Esterase Activity: Implications for Peptide Prodrugs.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M-1 s-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s-1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

Carboxy-terminal polyglutamylation regulates signaling and phase separation of the Dishevelled protein

Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.

Differential effects of oxytetracycline on detoxification and antioxidant defense in the hepatopancreas and intestine of Chinese mitten crab under cadmium stress

This study aims to evaluate the effects of oxytetracycline (OTC) on detoxification and oxidative defense in the hepatopancreas and intestine of Chinese mitten crab (Eriocheir sinensis) under cadmium (Cd) stress. The crab was exposed to 0.6 μM Cd, 0.6 μM OTC, and 0.6 μM Cd plus 0.6 μM OTC for 42 days. Our results showed that in the intestine, OTC alone enhanced protein carboxylation (PC) and malondialdehyde (MDA) contents, which was associated with the increased OTC accumulation. Compared to Cd alone, Cd plus OTC increased Cd and OTC contents, and reduced detoxification (i.e., glutathione (GSH) content, gene expressions of cytochrome P450 (CYP) isoforms, 7-ethoxyresorufin O-deethylase (EROD) activity, mRNA levels and activities of glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST)), and antioxidant defense (i.e., gene expressions and activities of catalase (CAT) and superoxide dismutase (SOD)) in the intestine, leading to the increased in PC and MDA contents, suggesting that OTC had a synergistic effect on Cd-induced oxidative damage. In the hepatopancreas, although OTC alone increased OTC accumulation, it did not affect PC and MDA contents. Compared to Cd alone, Cd plus OTC reduced MDA content, which was closely related to the improvement of detoxification (i.e., GSH content, mRNA levels of CYP isoforms, EROD activity, gene expressions and activities of GPx, GR and GST), and antioxidant defense (gene expressions and activities of CAT and SOD, metallothionein content). Aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2) transcriptional expressions were positively correlated with most detoxification- and antioxidant-related gene expressions, respectively, indicating that AhR and Nrf2 were involved in the regulation of these gene expressions. Our results unambiguously demonstrated that OTC had tissue-specific effects on Cd-induced toxicological effect in E. sinensis, which contributed to accurately evaluating Cd toxicity modulated by TCs in crab.

Dual-grating waveguide coupler biosensor for the real-time determination of human immunoglobulin M (IgM) antibodies

A dual-grating waveguide coupler biosensor was developed to detect human immunoglobulin M (IgM) antibodies with modification procedures using (3-aminopropyl)triethoxysilane (APTES) as the self-assembled monolayer were optimized. The average bulk sensitivity and optimal resolution were 43.84 ± 1.15 RIU−1 and 6.18 × 10−6 RIU, respectively. Due to the photocatalytic property of TiO2, ultraviolet treatment for 7.5 min was used to form the hydroxyl groups on TiO2 surface. APTES immobilized the biomolecules via chemical bonding; the optimal APTES modified parameters were 2% (v/v) for 0.5 h at 80 °C. Glutaraldehyde (GA) immobilized the protein A without activating carboxyl groups of protein A. The optimal GA modified conditions were 2.5% (v/v) for 48 min. Protein A immobilized the capture antibody directionally, the fragment antigen binding (Fab) region of the capture antibody was more completely exposed to improve the detection effect. Using the optimal modification conditions, the limit of detection for human IgM immunoassay was 2.67 × 10−8 ± 1.18 × 10−8 g/mL. Furthermore, this biosensor was demonstrated to determine the concentration of human IgM within 1 h and has potential for commercialization.

The Role of Vitamin K in the Development of Neurodegenerative Diseases.

Neurodegenerative diseases are a growing global health problem with enormous consequences for individuals and society. The most common neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, can be caused by both genetic factors (mutations) and epigenetic changes caused by the environment, in particular, oxidative stress. One of the factors contributing to the development of oxidative stress that has an important effect on the nervous system is vitamin K, which is involved in redox processes. However, its role in cells is ambiguous: accumulation of high concentrations of vitamin K increases the content of reactive oxygen species increases, while small amounts of vitamin K have a protective effect and activate the antioxidant defense systems. The main function of vitamin K is its involvement in the gamma carboxylation of the so-called Gla proteins. Some Gla proteins are expressed in the nervous system and participate in its development. Vitamin K deficiency can lead to a decrease or loss of function of Gla proteins in the nervous system. It is assumed that the level of vitamin K in the body is associated with specific changes involved in the development of dementia and cognitive abilities. Vitamin K also influences the sphingolipid profile in the brain, which also affects cognitive function. The role of vitamin K in the regulation of biochemical processes at the cellular and whole-organism levels has been studied insufficiently. Further research can lead to the discovery of new targets for vitamin K and development of personalized diets and therapies.

Exploiting sugarcane waste molasses and dephenolized cottonseed protein as the promising component for eco-friendly wood-based panel adhesive formulation

ABSTRACT In this study, sugarcane waste molasses (SWM) was utilized as a raw component to prepare an eco-friendly plywood adhesive. The optimization of preparation conditions, examination of thermal curing behavior, and exploration of the adhesive's curing mechanism were investigated. Based on the results, the optimal preparation conditions were determined as follows: the SWM is mixed with dephenolized cottonseed protein (DCP), a mass ratio of 2:1 between SWM and DCP, with a concentration of 75% and the addition of 7.5 wt% ammonium dihydrogen phosphate (ADP). Additionally, the optimal hot-pressing condition involved a temperature of 190°C for 10 min. Under these optimized conditions, the adhesive exhibited a maximum wet shear strength of 1.14 MPa, successfully meeting the China National Standard GB/T9846-2015. The curing behavior of the adhesive was investigated by analyzing thermal analysis and measuring the insoluble mass proportion. As the heating temperature and heating time increased, the insoluble mass proportion gradually rose. Thermogravimetric analysis (TG) showed that the thermal decomposition reaction temperature was 146.4°C and 202.05°C, differential scanning calorimetry (DSC) showed that endothermic reaction occurred at 135.8°C, exothermic reaction occurred at 227.8 °C. Through Fourier-transform infrared spectroscopy (FTIR) and pyrolysis gas chromatography-mass spectrometry (Py-GC/MS), it was observed that the addition of ADP promotes the conversion of sucrose hydrolyzed products into furan compounds and reacts with proteins. Moreover, the amino and carboxyl groups of proteins react with the phenol hydroxyl groups of lignin to form C–N and C–O bonds, a polymer with a cross-linked network is formed.

Beneficial effects of one-year menaquinone-7 supplementation on vascular stiffness and blood pressure in post-menopausal women

Abstract Background Cardiovascular disease (CVD) is responsible for 35% of total deaths worldwide. Sex hormones, such as oestrogen and progesterone, have a cardiovascular protective role in women. After menopause, women are more prone to hypertension and vascular stiffness, accelerated vascular aging and risk for CVD. Menaquinone-7 (MK-7) is a fat-soluble vitamin K2 and serves as cofactor for vitamin K-dependent protein carboxylation. The vitamin K-dependent matrix Gla protein (MGP) is synthesized in the vasculature and has shown to inhibit vascular calcification and stiffness. Purpose To investigate effects of MK-7 on vascular stiffness in pre/peri- versus post-menopausal women. Methods In a double-blind placebo-controlled clinical intervention trial (NCT02404519), 166 women (age 40-70; dephosphorylated-undercarboxylated MGP (dp-ucMGP) > 400 pmol/L at baseline) received daily 180 µg of MK-7 (n=83) or a matching placebo (n=83) for one year. Blood was sampled during intake (inclusion/exclusion), at baseline and at the end of the study to determine dp-ucMGP levels. Regional carotid-femoral (cf-PWV) and carotid-radial (cr-PWV) pulse wave velocities, vessel wall characteristics (e.g., intima-media thickness, arterial diameter, and local carotid artery PWV (caPWV), and arterial blood pressure were measured at baseline and after one year of vitamin K2 supplementation. Pre/peri- and post-menopausal women were sub-divided according to arterial stiffness (high b-stiffness index: > overall median 9.83). Results Post-hoc analyses showed a significant decrease in dp-ucMGP plasma levels of MK-7 supplemented pre/peri-menopausal (n=34; -8.58% ± 27.65; p=0.009) and post-menopausal women (n=48; -13.40% ± 27.45; p<0.001). At baseline, vascular parameters were significantly increased in post-menopausal women compared to pre/peri-menopausal women, e.g., intima media thickness (p=0.013), carotid artery diameter (p=0.007), and caPWV (p=0.012). MK-7 treatment significantly attenuated vascular stiffness in post-menopausal women (Young’s modulus; placebo +49.14% ± 77.38; MK-7 +9.38% ± 67.10; p=0.044). Post-menopausal women with a high stiffness index showed significantly improved vascular markers after MK-7 treatment, e.g., decreased blood pressure at brachialis (-3.03% ± 9.04; p=0.012), decreased blood pressure at carotid artery (-4.49% ± 11.66; p=0.008), increased distensibility coefficient (+13.25% ± 32.34; p=0.040), and increased compliance coefficient (±12.66% ± 32.64; p=0.040). Conclusions Our results confirm that hormonal changes affect the vasculature, and that post-menopausal women suffer from increased vascular stiffness reflected by functional vascular damage. Further, MK-7 supplementation significantly attenuates arterial stiffness in post-menopausal women, in which women with increased stiffness benefit most on blood pressure. Further research is needed to unravel the mechanisms by which MK-7 executes this beneficial role in post-menopausal women.

Maximizing the Clinical Value of Blood-Based Biomarkers for Mild Traumatic Brain Injury.

Mild traumatic brain injury (TBI) and concussion can have serious consequences that develop over time with unpredictable levels of recovery. Millions of concussions occur yearly, and a substantial number result in lingering symptoms, loss of productivity, and lower quality of life. The diagnosis may not be made for multiple reasons, including due to patient hesitancy to undergo neuroimaging and inability of imaging to detect minimal damage. Biomarkers could fill this gap, but the time needed to send blood to a laboratory for analysis made this impractical until point-of-care measurement became available. A handheld blood test is now on the market for diagnosis of concussion based on the specific blood biomarkers glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl terminal hydrolase L1 (UCH-L1). This paper discusses rapid blood biomarker assessment for mild TBI and its implications in improving prediction of TBI course, avoiding repeated head trauma, and its potential role in assessing new therapeutic options. Although we focus on the Abbott i-STAT TBI plasma test because it is the first to be FDA-cleared, our discussion applies to any comparable test systems that may become available in the future. The difficulties in changing emergency department protocols to include new technology are addressed.

E3 ubiquitin ligase HECTD3 is a tumor suppressor and mediates the polyubiquitination of SLC7A11 to promote ferroptosis in colon cancer

Homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3) has been reported to play an essential role in biological processes, including drug resistance, metastasis or apoptosis. However, the relationships between HECTD3 and Colorectal cancer (CRC) remain to be unclear. In this study, we discovered that HECTD3 expressed lowly in CRC compared with normal tissues and patients with low HECTD3 suffered from poorer survival outcomes relative to those with high HECTD3 levels. HECTD3 inhibition could significantly enhance proliferative, clone abilities and self-renewal capacities of CRC cells in vitro and in vivo. Mechanistically, our findings revealed that HECTD3 had endogenous interactions with SLC7A11 proteins. HECTD3 promoted the polyubiquitination of SLC7A11 to trigger the degradation of SLC7A11 proteins. Targeting HECTD3 could notably prolong the half-life period of SLC7A11 proteins, thereby promoting its stability. However, the cysteine mutation at amino acid 823 (ubiquitinase active site) of HECTD3 impaired the polyubiquitination of SLC7A11. HECTD3 deficiency depended on accumulated SLC7A11 proteins to accelerate malignant progression of CRC in vitro and in vivo. Thus, HECTD3 could suppress SLC7A11 levels to attenuate the SLC7A11-mediated cystine uptake, leading to enhanced CRC ferroptosis. SLC7A11 inhibition through polyubiquitination by HECTD3 increased ferroptosis, thereby inhibiting CRC tumor growth. Taken together, these results showed that HECTD3 controlled the stability of SLC7A11 and uncovered the function of HECTD3/SLC7A11 axis in regulating CRC progression.

Vitamin K and the Visual System-A Narrative Review.

Vitamin K occupies a unique and often obscured place among its fellow fat-soluble vitamins. Evidence is mounting, however, that vitamin K (VK) may play an important role in the visual system apart from the hepatic carboxylation of hemostatic-related proteins. However, to our knowledge, no review covering the topic has appeared in the medical literature. Recent studies have confirmed that matrix Gla protein (MGP), a vitamin K-dependent protein (VKDP), is essential for the regulation of intraocular pressure in mice. The PREDIMED (Prevención con Dieta Mediterránea) study, a randomized trial involving 5860 adults at risk for cardiovascular disease, demonstrated a 29% reduction in the risk of cataract surgery in participants with the highest tertile of dietary vitamin K1 (PK) intake compared with those with the lowest tertile. However, the specific requirements of the eye and visual system (EVS) for VK, and what might constitute an optimized VK status, is currently unknown and largely unexplored. It is, therefore, the intention of this narrative review to provide an introduction concerning VK and the visual system, review ocular VK biology, and provide some historical context for recent discoveries. Potential opportunities and gaps in current research efforts will be touched upon in the hope of raising awareness and encouraging continued VK-related investigations in this important and highly specialized sensory system.

Enhanced oral bioavailability from food protein nanoparticles: A mini review.

The oral route is the most desirable drug administration path. The oral bioavailability is always compromised from the poor physicochemical and/or biopharmaceutical properties of the active pharmaceutical ingredients. Food protein nanoparticles show promise for oral drug delivery, with improved biosafety and cost-effectiveness compared to polymeric nanoparticles. More importantly, diverse food proteins provide "choice and variety" to meet the challenges faced by different drugs in oral delivery resulting from low solubility, poor permeability, and gastrointestinal stability. The abundance of hydroxyl, amino, and carboxyl groups in food proteins allows easy surface modification of the nanoparticles to impart unique functions. Albeit being in its infancy, food protein nanoparticles exhibit high capability to enhance oral bioavailability of a wide range of drugs from small molecules to biomacromolecules. Considering the rapid growth of the field, the achievements and mechanisms of food protein nanoparticles in enhancing oral bioavailability are reviewed. Factors affecting the performance of food protein nanoparticles are discussed with the purpose to inspire the development of food protein nanoparticle-based oral drug delivery systems.

Glutaraldehyde fixation promotes palladium and gold nanoparticles formation in yeast and enhances their catalytic activity in 4-nitrophenol reduction

When observing biosynthesized metal nanoparticles in microorganisms, glutaraldehyde is commonly used as a fixative to prepare TEM ultra-thin sections. However, as a chemical reagent with aldehyde groups, its reduction potential on metal ions has yet to be studied elaborately. Herein, we explored the influences of glutaraldehyde on yeast-synthesized gold nanoparticles (AuNPs), palladium nanoparticles (PdNPs) and their catalytic performance. A modified method for ultra-thin section preparation without glutaraldehyde fixation was developed to exclude its influence on AuNPs/PdNPs observation. It was confirmed that glutaraldehyde could promote the biosynthesis of AuNPs and PdNPs extra- and intracellularly, without altering their crystal structure and chemical state. The adsorption and reduction of Au(III)/Pd(II) were attributed to the different components of the yeast cell. Specifically, the amines and carboxyl groups in proteins and polysaccharides were involved in adsorption, while the reducing sugars hydrolyzed from polysaccharides were responsible for Au(III)/Pd(II) reduction. After glutaraldehyde fixation, the catalytic activities of Au/Pd-loaded yeast in 4-nitrophenol reduction were enhanced as well. Therefore, the influence of chemical fixatives in biosynthesized metal nanoparticles should be taken into consideration in regard to SEM, TEM observation and catalytic performance.

Blood coagulation factors and platelet response to drug-induced hepatitis and hepatosis in rats.

Knowing the variability of blood coagulation responses to liver damage of different origins can provide a key to curing liver tissues or to mitigating treatment side effects. The aim of the present work was to compare the changes in the main components of hemostasis under experimental drug-induced hepatosis and hepatitis in rats. We modeled diclofenac-induced hepatitis and tetracycline-induced hepatosis. Hemostasis response was gauged by measuring fibrinogen, factor X, protein C (PC), and prothrombin in plasma. The decarboxylated form of prothrombin was detected by measuring prothrombin index and ecamulin index. Platelet reactivity was studied using aggregometry. Both hepatitis and hepatosis decreased the synthesis of fibrinogen, factor X, and prothrombin. However, protein carboxylation was not disrupted in hepatosis but was much impaired in hepatitis. PC decreased in both models as a consequence of its consumption possibly during inflammatory response. Platelet aggregation rate was lower in hepatosis but higher in hepatitis. Our findings imply the need for a thorough monitoring of the hemostasis system in liver diseases to avoid possible thrombotic complications. Its state indicates the disorder's rate and character.

HECTD3 regulates the tumourigenesis of glioblastoma by polyubiquitinating PARP1 and activating EGFR signalling pathway.

The E3 ubiquitin ligase HECTD3 is a homologue of the E6-related protein carboxyl terminus, which plays a crucial role in biological processes and tumourigenesis. However, the functional characterisation of HECTD3 in glioblastoma is still elusive. Determination of the functional role of HECTD3 in glioblastoma was made by a combination of HECTD3 molecular pattern analysis from human glioblastoma databases and subcutaneous and in situ injections of tumours in mice models. This study reports that the DOC domain of HECTD3 interacts with the DNA binding domain of PARP1, and HECTD3 mediated the K63-linked polyubiquitination of PARP1 and stabilised the latter expression. Moreover, the Cysteine (Cys) 823 (ubiquitin-binding site) mutation of HECTD3 significantly reduced PARP1 polyubiquitination and HECTD3 was involved in the recruitment of ubiquitin-related molecules to PARP1 ubiquitin-binding sites (Lysines 209 and 221, respectively). Lastly, activation of EGFR-mediated signalling pathways by HECTD3 regulates PARP1 polyubiquitination. Our results unveil the potential role of HECTD3 in glioblastoma and strongly preconise further investigation and consider HECTD3 as a promising therapeutic marker for glioblastoma treatment.

Elevated expression of protein-L-isoaspartate O-methyltransferase-1 (PCMT1) in cervical cancer.

BackgroundProtein-L-isoaspartate O-methyltransferase-1 (PCMT1) is a protein carboxyl methyltransferase enzyme, which has been found to play roles in cancers. However, no clinical information about the correlation between cervical cancer and PCMT1 expression has been reported.MethodsWe used immunohistochemistry (IHC) to characterize the protein level of PCMT1 in human cervical intraepithelial neoplasia and cervical cancer specimens. The mRNA expression profile of PCMT1 in cervical cancer was also analyzed by using Gene Expression Omnibus (GEO) databases. The prognostic value of PCMT1 in patients with cervical cancer was evaluated by using the Kaplan-Meier plotter. Gene set enrichment analysis (GSEA) was conducted by using The Cancer Genome Atlas (TCGA) cervical cancer dataset.ResultsThe protein level of PCMT1 was increased in cervical high-grade squamous intraepithelial lesion (HSIL) (7.40±0.42) and cervical cancer tissues (10.70±0.54), compared to normal cervix (5.00±0.86) and low-grade squamous intraepithelial lesion (LSIL) (6.22±0.57) (P<0.05). the immunoreactivity score (IRS) of PCMT1 was also higher in cervical cancer tissues than in paired adjacent non-cancerous cervical tissues (9.03±0.52 vs. 6.32±0.46) (P<0.05). High expression of PCMT1 was associated with decreased overall survival (OS) of patients with cervical cancer (P=0.0022). GSEA demonstrated that cervical cancer patients with high expression of PCMT1 were enriched in the various cancer-related signaling pathways.ConclusionsThese results suggest that PCMT1 might be a diagnostic and prognostic biomarker for cervical cancer, and further validation studies should be performed.

HECTD3 enhances cell radiation resistance and migration by regulating LKB1 mediated ZEB1 in glioma.

Homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3) has been reported to play a role in carcinogenesis. Here, we explored the role of HECTD3 in regulating the radiation resistance of glioma, and the underlying mechanism. HECTD3 expressions in glioma tissues were assessed using Western blotting, quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and immunohistochemistry. Glioma cells were exposed to 2-, 4-, 6- or 8-Gy X-ray to mimic the radiation treatment. Cell count kit-8 (CCK-8), clone formation assay, flow cytometry assay, transwell chambers and animal assay were used to test cell viability, apoptosis, migration, invasiveness and tumourigenesis, respectively. HECTD3 expression was increased in glioma tissues, especially from patients with radiation resistance. Knockdown of HECTD3 promoted cell apoptosis and inhibited cell viability under the condition of 8-Gy X-ray, as well as suppressed cell migration and invasiveness. In mechanism, HECTD3 positively regulated ZEB1 (zinc finger E-box binding hemeobox 1) expression through regulating the ubiquitination of liver kinase B1 (LKB1) protein. Overexpression of ZEB1 significantly abolished the effects of HECTD3 downregulation in inhibiting the radiation resistance and migration of glioma cells. Moreover, downregulation of HECTD3 further enhanced the anti-tumour effect of X-ray on glioma growth in vivo. In conclusion, HECTD3 was overexpressed in glioma patients with radiation resistance. Knockdown of HECTD3 sensitized glioma cells to radiation and inhibited cell migration by downregulating ZEB1 expression via regulating the ubiquitination of LKB1 protein. This study reveals that HECTD3 might be a potent target to enhance the radiation sensitivity of glioma.

GGCX mutants that impair hemostasis reveal the importance of processivity and full carboxylation to VKD protein function

Abstractγ-Glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K–dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M and S300F) whose combined heterozygosity in a patient causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation. Complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of the VKD protein in the complex. The VKD protein in the complex with wild-type carboxylase was carboxylated before challenge protein carboxylation occurred and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time and did not completely carboxylate FIX in the complex. S300F carboxylation was poor with both FIX and MGP. Additional studies analyzed FIX- and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The total amount of carboxylated peptide generated by the V255M mutant was higher than that of wild-type GGCX; however, the individual peptides were partially carboxylated. Analysis of the V255M mutant in FIX HEK293 cells lacking endogenous GGCX revealed poor FIX clotting activity. This study shows that disrupted processivity causes disease and explains the defect in the patient. Kinetic analyses also suggest that disrupted processivity may occur in wild-type carboxylase under some conditions (eg, warfarin therapy or vitamin K deficiency).

Chlorogenic acid improves growth performance and intestinal health through autophagy-mediated nuclear factor erythroid 2-related factor 2 pathway in oxidatively stressed broilers induced by dexamethasone

The effects of chlorogenic acid (CGA) on growth performance, intestinal morphology, antioxidant capacity, and the autophagy-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in oxidatively stressed broilers were investigated. A total of 400 one-day-old male Cobb broilers were divided randomly into 4 groups using a 2 × 2 factorial arrangement with 2 CGA supplemental levels (0 and 500 mg/kg) and 2 dexamethasone (DEX) challenge levels (0 and 3 mg/kg body weight). All the broilers were injected intraperitoneally with DEX or sterile saline beginning at the age of 15 d for 6 consecutive days. The experiment lasted for 21 d. The CGA increased average daily gain (ADG), villus height, villus height/crypt depth (V/C) value, and the protein expressions of Occludin and ZO-1 in the ileum and decreased the feed:gain (F:G) ratio, which were impaired by the DEX challenge. Superoxide dismutase (SOD), catalase (CAT), gutathione S-transferase (GST), and heme oxygenase-1 (HO-1) activities in the serum and ileum were increased by CGA, whereas protein carboxyl (PCO) level in the serum and ileum, and malondialdehyde (MDA) level in the ileum were decreased of the DEX challenged broilers. The DEX challenge decreased microtubule-associated protein 1 light chain 3 (LC3)-II, Beclin1, and autophagy-related gene (ATG) 7 mRNA expressions, and the LC3-II/LC3-I value and increased LC3-I, cysteinyl aspartate specific proteinase (Caspase)-3 and Caspase-9 mRNA expressions in the ileum, which were improved by CGA. DEX also decreased the protein expressions of Kelch-like ECH-associated protein-1 (Keap1), Nrf2, HO-1, NADPH quinone oxidoreductase-1(NQO-1) and increased sequestosome 1 (p62) in the ileum, which were improved by CGA. Interactions occurred between DEX and CGA for the ADG, F:G ratio, villus height, crypt depth, V/C value, and SOD, CAT, GST, and HO-1 activities, MDA and PCO levels, LC3-II/LC3-I value, and expressions of LC3-I, LC3-II, Beclin1, ATG7, Caspase-3, Caspase-9, Occludin, ZO-1, Keap1, Nrf2, HO-1, NQO-1, and p62. In conclusion, CGA improved the growth performance and intestinal health of oxidatively stressed broilers by activating the autophagy-mediated Nrf2 pathway.