Tumor Location Is an Independent Prognostic Factor in Completely Resected Pathological Stage I Non-Small Cell Lung Cancer: A Multicenter Retrospective Study.

Previous studies suggested that the location of the primary tumor in non-small cell lung cancer (NSCLC) is associated with clinical features and prognosis, but results are conflicting. The purpose of this study was to explore tumor location as an independent risk factor of survival for patients with completely resected pathological stage I NSCLC. This was a multicenter retrospective study conducted in Taiwan. Included patients were diagnosed with stage I NSCLC and had undergone primary tumor resection. Variables including tumor location, pathological stage, histological differentiation, and International Association for the Study of Lung Cancer (IASLC) grade were evaluated for predictive ability for disease-free survival (DFS) and overall survival (OS). A total of 208 patients were included, with 123 (59.1%) patients having a primary tumor in the upper and middle lobes. The median duration of follow-up for survivors was 60.5 months. Compared to patients with IASLC Grade 3 disease, patients with Grade 1 disease had significantly longer DFS. Tumor location and IASLC grade were independent predictors for OS in multivariate analysis. Specifically, patients with NSCLC in the lower lobe and patients who are histologically classified as IASLC Grade 3 may have poorer prognosis and require greater attention to improve outcomes.

A Comparison of the Antioxidant Potential and Metabolite Analysis of Marine Fungi Associated with the Red Algae Pterocladiella capillacea from Northern Taiwan.

This study represents a primary investigation centered on screening six marine fungi, Emericellopsis maritima, Engyodontium album, Hypomontagnella monticulosa, Hortaea werneckii, Trichoderma harzianum, and Aspergillus sp.7, associated with the red algae Pterocladiella capillacea, which was collected from Chao-Jin Park in Keelung, Taiwan, as potential immunostimulants for shrimp aquaculture. Recognizing the imperative for novel strategies to combat pathogen resistance arising from the use of antibiotics and vaccines in aquaculture, this study aimed to evaluate the metabolomic profile, antioxidant capabilities, and antibacterial properties of marine fungi. The antibacterial activity of the fungal extract was evaluated against five major aquaculture pathogens: Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Enterobacter aeruginosa, and Vibrio parahaemolyticus. The viability and cytotoxicity of marine fungal extracts were preliminarily evaluated using brine shrimps before assessing cytotoxicity, growth performance, immune efficacy, and disease resistance in white shrimp. The present study demonstrated that total phytochemical analysis correlated with antioxidant activity. Emericellopsis maritima and Trichoderma harzianum exhibited the strongest DPPH antioxidant scavenging activities of half-maximal inhibitory concentration (IC50) 16.5 ± 1.2 and 12.2 ± 2.6, which are comparable to ascorbic acid. LC-HDMSE analysis of the marine fungal extracts identified more than 8000 metabolites mainly classified under the superclass level of organic oxygen compounds, Organoheterocyclic compounds, Phenylpropanoids and polyketides, alkaloid and derivatives, benzenoids, lignans and neolignans, lipid and lipid-like molecules, nucleotides and nucleosides, organic nitrogen compounds, and organic acids and derivatives. Overall, our study significantly contributes to the advancement of sustainable practices by exploring alternative antimicrobial solutions and harnessing the bioactive potential inherent in marine endophytic fungi. In conclusion, our study advances our comprehension of fungal communities and their applications and holds promise for the development of effective and environmentally friendly approaches for enhancing shrimp health and productivity.

Deciphering Early-Stage Molecular Mechanisms of Negative Pressure Wound Therapy in a Murine Model.

Negative Pressure Wound Therapy (NPWT) is a commonly employed clinical strategy for wound healing, yet its early-stage mechanisms remain poorly understood. To address this knowledge gap and overcome the limitations of human trials, we establish an NPWT C57BL/6JNarl mouse model to investigate the molecular mechanisms involved in NPWT. In this study, we investigate the intricate molecular mechanisms through which NPWT expedites wound healing. Our focus is on NPWT's modulation of inflammatory immune responses and the concurrent orchestration of multiple signal transduction pathways, resulting in shortened coagulation time and reduced inflammation. Notably, we observe a significant rise in dickkopf-related protein 1 (DKK-1) concentration during NPWT, promoting the differentiation of Hair Follicle Stem Cells (HFSCs) into epidermal cells, expediting wound closure. Under negative pressure, macrophages express and release DKK-1 cytokines, crucial for stimulating HFSC differentiation, as validated in animal experiments and in vitro studies. Our findings illuminate the inflammatory dynamics under NPWT, revealing potential signal transduction pathways. The proposed framework, involving early hemostasis, balanced inflammation, and macrophage-mediated DKK-1 induction, provides a novel perspective on enhancing wound healing during NPWT. Furthermore, these insights lay the groundwork for future pharmacological advancements in managing extensive wounds, opening avenues for targeted therapeutic interventions in wound care.

Core clock gene BMAL1 and RNA-binding protein MEX3A collaboratively regulate Lgr5 expression in intestinal crypt cells.

The intestinal epithelium is highly regenerative. Rapidly proliferating LGR5+ crypt base columnar (CBC) cells are responsible for epithelial turnover needed to maintain intestinal homeostasis. Upon tissue damage, loss of LGR5+ CBCs can be compensated by activation of quiescent +4 intestinal stem cells (ISCs) or early progenitor cells to restore intestinal regeneration. LGR5+ CBC self-renewal and ISC conversion to LGR5+ cells are regulated by external signals originating from the ISC niche. In contrast, little is known about intrinsic regulatory mechanisms critical for maintenance of LGR5+ CBC homeostasis. We found that LGR5 expression in intestinal crypt cells is controlled by the circadian core clock gene BMAL1 and the BMAL1-regulated RNA-binding protein MEX3A. BMAL1 directly activated transcription of Mex3a. MEX3A in turn bound to and stabilized Lgr5 mRNA. Bmal1 depletion reduced Mex3a and Lgr5 expression and led to increased ferroptosis, which consequently decreased LGR5+ CBC numbers and increased the number of crypt cells expressing +4 ISC marker BMI1. Together, these findings reveal a BMAL1-centered intrinsic regulatory pathway that maintains LGR5 expression in the crypt cells and suggest a potential mechanism contributing to ISC homeostasis.

Histone deacetylase III interactions with BK polyomavirus large tumor antigen may affect protein stability.

BACKGROUND: Human polyomavirus BK (BKPyV) causes associated nephropathy and contributes to urinary tract cancer development in renal transplant recipients. Large tumor antigen (LT) is an early protein essential in the polyomavirus life cycle. Protein acetylation plays a critical role in regulating protein stability, so this study investigated the acetylation of the BKPyV LT protein. METHODS: The BKPyV LT nucleotide was synthesized, and the protein was expressed by transfection into permissive cells. The BKPyV LT protein was immunoprecipitated and subjected to LC-MS/MS analysis to determine the acetylation residues. The relative lysine was then mutated to arginine in the LT nucleotide and BKPyV genome to analyze the role of LT lysine acetylation in the BKPyV life cycle. RESULTS: BKPyV LT acetylation sites were identified at Lys3 and Lys230 by mass spectrometry. HDAC3 and HDAC8 and their deacetylation activity are required for BKPyV LT expression. In addition, mutations of Lys3 and Lys230 to arginine increased LT expression, and the interaction of HDAC3 and LT was confirmed by coimmunoprecipitation. CONCLUSIONS: HDAC3 is a newly identified protein that interacts with BKPyV LT, and LT acetylation plays a vital role in the BKPyV life cycle.

Distinctive microbial community and genome structure in coastal seawater from a human-made port and nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean.

Pollution in human-made fishing ports caused by petroleum from boats, dead fish, toxic chemicals, and effluent poses a challenge to the organisms in seawater. To decipher the impact of pollution on the microbiome, we collected surface water from a fishing port and a nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean. By employing 16S rRNA gene amplicon sequencing and whole-genome shotgun sequencing, we discovered that Rhodobacteraceae, Vibrionaceae, and Oceanospirillaceae emerged as the dominant species in the fishing port, where we found many genes harboring the functions of antibiotic resistance (ansamycin, nitroimidazole, and aminocoumarin), metal tolerance (copper, chromium, iron and multimetal), virulence factors (chemotaxis, flagella, T3SS1), carbohydrate metabolism (biofilm formation and remodeling of bacterial cell walls), nitrogen metabolism (denitrification, N2 fixation, and ammonium assimilation), and ABC transporters (phosphate, lipopolysaccharide, and branched-chain amino acids). The dominant bacteria at the nearby offshore island (Alteromonadaceae, Cryomorphaceae, Flavobacteriaceae, Litoricolaceae, and Rhodobacteraceae) were partly similar to those in the South China Sea and the East China Sea. Furthermore, we inferred that the microbial community network of the cooccurrence of dominant bacteria on the offshore island was connected to dominant bacteria in the fishing port by mutual exclusion. By examining the assembled microbial genomes collected from the coastal seawater of the fishing port, we revealed four genomic islands containing large gene-containing sequences, including phage integrase, DNA invertase, restriction enzyme, DNA gyrase inhibitor, and antitoxin HigA-1. In this study, we provided clues for the possibility of genomic islands as the units of horizontal transfer and as the tools of microbes for facilitating adaptation in a human-made port environment.

Hypermethylation of ACADVL is involved in the high-intensity interval training-associated reduction of cardiac fibrosis in heart failure patients.

BACKGROUND: Emerging evidence suggests that DNA methylation can be affected by physical activities and is associated with cardiac fibrosis. This translational research examined the implications of DNA methylation associated with the high-intensity interval training (HIIT) effects on cardiac fibrosis in patients with heart failure (HF). METHODS: Twelve HF patients were included and received cardiovascular magnetic resonance imaging with late gadolinium enhancement for cardiac fibrosis severity and a cardiopulmonary exercise test for peak oxygen consumption ([Formula: see text]O2peak). Afterwards, they underwent 36 sessions of HIIT at alternating 80% and 40% of [Formula: see text]O2peak for 30 min per session in 3-4 months. Human serum from 11 participants, as a means to link cell biology to clinical presentations, was used to investigate the exercise effects on cardiac fibrosis. Primary human cardiac fibroblasts (HCFs) were incubated in patient serum, and analyses of cell behaviour, proteomics (n = 6) and DNA methylation profiling (n = 3) were performed. All measurements were conducted after completing HIIT. RESULTS: A significant increase (p = 0.009) in [Formula: see text]O2peak (pre- vs. post-HIIT = 19.0 ± 1.1 O2 ml/kg/min vs. 21.8 ± 1.1 O2 ml/kg/min) was observed after HIIT. The exercise strategy resulted in a significant decrease in left ventricle (LV) volume by 15% to 40% (p < 0.05) and a significant increase in LV ejection fraction by approximately 30% (p = 0.010). LV myocardial fibrosis significantly decreased from 30.9 ± 1.2% to 27.2 ± 0.8% (p = 0.013) and from 33.4 ± 1.6% to 30.1 ± 1.6% (p = 0.021) in the middle and apical LV myocardium after HIIT, respectively. The mean single-cell migration speed was significantly (p = 0.044) greater for HCFs treated with patient serum before (2.15 ± 0.17 µm/min) than after (1.11 ± 0.12 µm/min) HIIT. Forty-three of 1222 identified proteins were significantly involved in HIIT-induced altered HCF activities. There was significant (p = 0.044) hypermethylation of the acyl-CoA dehydrogenase very long chain (ACADVL) gene with a 4.474-fold increase after HIIT, which could activate downstream caspase-mediated actin disassembly and the cell death pathway. CONCLUSIONS: Human investigation has shown that HIIT is associated with reduced cardiac fibrosis in HF patients. Hypermethylation of ACADVL after HIIT may contribute to impeding HCF activities. This exercise-associated epigenetic reprogramming may contribute to reduce cardiac fibrosis and promote cardiorespiratory fitness in HF patients. TRIAL REGISTRATION: NCT04038723. Registered 31 July 2019, https://clinicaltrials.gov/ct2/show/NCT04038723 .

Subcellular Distribution of BALF2 and the Role of Rab1 in the Formation of Epstein-Barr Virus Cytoplasmic Assembly Compartment and Virion Release.

Epstein-Barr virus (EBV) replicates its genome in the nucleus and undergoes tegumentation and envelopment in the cytoplasm. We are interested in how the single-stranded DNA binding protein BALF2, which executes its function and distributes predominantly in the nucleus, is packaged into the tegument of virions. At the mid-stage of virus replication in epithelial TW01-EBV cells, a small pool of BALF2 colocalizes with tegument protein BBLF1, BGLF4 protein kinase, and the cis-Golgi marker GM130 at the perinuclear viral assembly compartment (AC). A possible nuclear localization signal (NLS) between amino acids 1100 and 1128 (C29), which contains positive charged amino acid 1113RRKRR1117, is able to promote yellow fluorescent protein (YFP)-LacZ into the nucleus. In addition, BALF2 interacts with the nucleocapsid-associated protein BVRF1, suggesting that BALF2 may be transported into the cytoplasm with nucleocapsids in a nuclear egress complex (NEC)-dependent manner. A group of proteins involved in intracellular transport were identified to interact with BALF2 in a proteomic analysis. Among them, the small GTPase Rab1A functioning in bi-directional trafficking at the ER-Golgi interface is also a tegument component. In reactivated TW01-EBV cells, BALF2 colocalizes with Rab1A in the cytoplasmic AC. Expression of dominant-negative GFP-Rab1A(N124I) diminished the accumulation of BALF2 in the AC, coupling with attenuation of gp350/220 glycosylation. Virion release was significantly downregulated by expressing dominant-negative GFP-Rab1A(N124I). Overall, the subcellular distribution of BALF2 is regulated through its complex interaction with various proteins. Rab1 activity is required for proper gp350/220 glycosylation and the maturation of EBV. IMPORTANCE Upon EBV lytic reactivation, the virus-encoded DNA replication machinery functions in the nucleus, while the newly synthesized DNA is encapsidated and transported to the cytoplasm for final virus assembly. The single-stranded DNA binding protein BALF2 executing functions within the nucleus was also identified in the tegument layer of mature virions. Here, we studied the functional domain of BALF2 that contributes to the nuclear targeting and used a proteomic approach to identify novel BALF2-interacting cellular proteins that may contribute to virion morphogenesis. The GTPase Rab1, a master regulator of anterograde and retrograde endoplasmic reticulum (ER)-Golgi trafficking, colocalizes with BALF2 in the juxtanuclear concave region at the midstage of EBV reactivation. Rab1 activity is required for BALF2 targeting to the cytoplasmic assembly compartment (AC) and for gp350/220 targeting to cis-Golgi for proper glycosylation and virion release. Our study hints that EBV hijacks the bi-directional ER-Golgi trafficking machinery to complete virus assembly.

Wnt3a Facilitates SARS-CoV-2 Pseudovirus Entry into Cells.

How ACE2 functions as the major host receptor of SARS-CoV-2 despite having low expression in the lungs is still unknown. To facilitate the development of therapeutic strategies against coronaviruses, gaining a deeper comprehension of the molecular mechanism of SARS-CoV-2 infection is imperative. In our previous study, we identified several potential host factors of SARS-CoV-2 using an shRNA arrayed screen, one of which was Wnt3a. Here, we validated the significance of Wnt3a, a potent activator of the Wnt/ß-catenin signaling pathway, for SARS-CoV-2 entry into cells by evaluating the effects of its knockdown and overexpression on SARS-CoV-2 pseudotyped virus entry. Further analysis revealed that SARS-CoV-2 pseudotyped virus infection activates the canonical Wnt/ß-catenin signaling pathway, which we found could subsequently stimulate ACE2 transcription. Collectively, our study identified Wnt3a as an important host factor that facilitates ACE2-mediated virus infection. Insight into the virus entry mechanism is impactful as it will aid in developing novel therapeutic strategies against current and future coronavirus pandemics.

Characterization and Potentiating Effects of the Ethanolic Extracts of the Red Seaweed Gracillaria sp. on the Activity of Carbenicillin against Vibrios.

ß-lactam-resistant Vibrio strains are a significant clinical problem, and ß-lactamase inhibitors are generally coadministered with ß-lactam drugs to control drug-resistant bacteria. Seaweed is a rich source of natural bioactive compounds; however, their potential as ß-lactamase inhibitors against bacterial pathogens remains unknown. Herein, we evaluated the potential ß-lactamase inhibitory effect of the ethanolic extracts of the red seaweed Gracilaria sp. (GE) against four Vibrio strains. The minimum inhibitory concentration, half-maximal inhibitory concentration, checkerboard assay results, and time-kill study results indicate that GE has limited antibacterial activity but can potentiate the activity of the ß-lactam antibiotic carbenicillin against Vibrio parahemolyticus and V. cholerae. We overexpressed and purified recombinant metallo-ß-lactamase, VarG, from V. cholerae for in vitro studies and observed that adding GE reduced the carbenicillin and nitrocefin degradation by VarG by 20% and 60%, respectively. Angiotensin I-converting enzyme inhibition studies demonstrated that GE did not inhibit VarG via metal chelation. Toxicity assays indicated that GE exhibited mild toxicity against human cells. Through gas chromatography and mass spectrometry, we showed that GE comprises alkaloids, phenolic compounds, terpenoids, terpenes, and halogenated aromatic compounds. This study revealed that extracts of the red seaweed Gracillaria sp. can potentially inhibit ß-lactamase activity.

Skin proteomic profiling of irradiation-induced fibrosis and its modulation by low molecular weight fucoidan via tight junction pathway.

Fucoidans, sulfated and fucosylated polysaccharides extracted from brown seaweed, were found to inhibit radiotherapy-induced cell damage and fibrosis through the TGF-ß1 pathway. However, the comprehensive molecular response during irradiation-induced fibrosis and fucoidan-assisted recovery still remain unclear. Rat hind limbs were irradiated and smeared with low molecular weight fucoidan (LMF). Protein profiles were examined by a mass spectrometry-based proteomics analysis. Out of a total of 4625 proteins, 233 were found to be significantly up-regulated after irradiation and down-regulated after LMF treatment. Pathway and protein-protein interaction network analyses further indicated that four proteins including Actb, Ezr, Msn and Cdc42 were clustered into the tight junction and regulation of actin cytoskeleton pathways. These four proteins may serve as biomarkers for the detection of skin fibrosis induced by irradiation or TGF-ß1, and for the recovery following LMF treatment.

Comparative Transcriptome Analysis of Organ-Specific Adaptive Responses to Hypoxia Provides Insights to Human Diseases.

The common carp is a hypoxia-tolerant fish, and the understanding of its ability to live in low-oxygen environments has been applied to human health issues such as cancer and neuron degeneration. Here, we investigated differential gene expression changes during hypoxia in five common carp organs including the brain, the gill, the head kidney, the liver, and the intestine. Based on RNA sequencing, gene expression changes under hypoxic conditions were detected in over 1800 genes in common carp. The analysis of these genes further revealed that all five organs had high expression-specific properties. According to the results of the GO and KEGG, the pathways involved in the adaptation to hypoxia provided information on responses specific to each organ in low oxygen, such as glucose metabolism and energy usage, cholesterol synthesis, cell cycle, circadian rhythm, and dopamine activation. DisGeNET analysis showed that some human diseases such as cancer, diabetes, epilepsy, metabolism diseases, and social ability disorders were related to hypoxia-regulated genes. Our results suggested that common carp undergo various gene regulations in different organs under hypoxic conditions, and integrative bioinformatics may provide some potential targets for advancing disease research.

Phenolic Compound Ethyl 3,4-Dihydroxybenzoate Retards Drug Efflux and Potentiates Antibiotic Activity.

The World Health Organization indicated that antibiotic resistance is one of the greatest threats to health, food security, and development in the world. Drug resistance efflux pumps are essential for antibiotic resistance in bacteria. Here, we evaluated the plant phenolic compound ethyl 3,4-dihydroxybenzoate (EDHB) for its efflux pump inhibitory (EPI) activity against drug-resistant Escherichia coli. The half-maximal inhibitory concentration, modulation assays, and time-kill studies indicated that EDHB has limited antibacterial activity but can potentiate the activity of antibiotics for drug-resistant E. coli. Dye accumulation/efflux and MALDI-TOF studies showed that EDHB not only significantly increases dye accumulation and reduces dye efflux but also increases the extracellular amount of antibiotics in the drug-resistant E. coli, indicating its interference with substrate translocation via a bacterial efflux pump. Molecular docking analysis using AutoDock Vina indicated that EDHB putatively posed within the distal binding pocket of AcrB and in close interaction with the residues by H-bonds and hydrophobic contacts. Additionally, EDHB showed an elevated postantibiotic effect on drug-resistant E. coli. Our toxicity assays showed that EDHB did not change the bacterial membrane permeability and exhibited mild human cell toxicity. In summary, these findings indicate that EDHB could serve as a potential EPI for drug-resistant E. coli.

Carbohydrate Ligands for COVID-19 Spike Proteins.

An outbreak of SARS-CoV-2 coronavirus (COVID-19) first detected in Wuhan, China, has created a public health emergency all over the world. The pandemic has caused more than 340 million confirmed cases and 5.57 million deaths as of 23 January 2022. Although carbohydrates have been found to play a role in coronavirus binding and infection, the role of cell surface glycans in SARS-CoV-2 infection and pathogenesis is still not understood. Herein, we report that the SARS-CoV-2 spike protein S1 subunit binds specifically to blood group A and B antigens, and that the spike protein S2 subunit has a binding preference for Lea antigens. Further examination of the binding preference for different types of red blood cells (RBCs) indicated that the spike protein S1 subunit preferentially binds with blood group A RBCs, whereas the spike protein S2 subunit prefers to interact with blood group Lea RBCs. Angiotensin converting enzyme 2 (ACE2), a known target of SARS-CoV-2 spike proteins, was identified to be a blood group A antigen-containing glycoprotein. Additionally, 6-sulfo N-acetyllactosamine was found to inhibit the binding of the spike protein S1 subunit with blood group A RBCs and reduce the interaction between the spike protein S1 subunit and ACE2.

Identified Seaweed Compound Diphenylmethane Serves as an Efflux Pump Inhibitor in Drug-Resistant Escherichia coli.

Drug efflux pumps are one of the major elements used by antibiotic-resistant bacteria. Efflux pump inhibitors (EPIs) are potential therapeutic agents for adjunctive therapy, which can restore the activity of antibiotics that are no longer effective against pathogens. This study evaluated the seaweed compound diphenylmethane (DPM) for its EPI activity. The IC50 and modulation results showed that DPM has no antibacterial activity but can potentiate the activity of antibiotics against drug-resistant E. coli. Time-kill studies reported that a combination of DPM and erythromycin exhibited greater inhibitory activity against drug-resistant Escherichia coli. Dye accumulation and dye efflux studies using Hoechst 33342 and ethidium bromide showed that the addition of DPM significantly increased dye accumulation and reduced dye efflux in drug-resistant E. coli, suggesting its interference with dye translocation by an efflux pump. Using MALDI-TOF, it was observed that the addition of DPM could continuously reduce antibiotic efflux in drug-resistant E. coli. Additionally, DPM did not seem to damage the E. coli membranes, and the cell toxicity test showed that it features mild human-cell toxicity. In conclusion, these findings showed that DPM could serve as a potential EPI for drug-resistant E. coli.

Hesperidin and Chlorogenic Acid Synergistically Inhibit the Growth of Breast Cancer Cells via Estrogen Receptor/Mitochondrial Pathway.

Breast cancer is the most common cancer in women worldwide. Hesperidin (Hes) and chlorogenic acid (CA) are traditional medicinal molecules that abundantly exist in natural plants or foods. These compounds have been shown to prevent and suppress various cancers and therefore can be utilized as adjunctive therapies to aid cancer treatment. Here, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays show a greater synergistic inhibitory effect on the growth of breast cancer cells, MCF-7, but not normal breast cells, MCF-10A, than hesperidin or chlorogenic acid alone. We present the possible molecular signaling pathways in MCF-7 cells with or without herbal molecule treatments via proteomic approaches. The data were further analyzed by Ingenuity Pathway Analysis (IPA) and confirmed by quantifying mRNA associated with the estrogen-receptor signaling pathway and mitochondrial functions. We demonstrated that the expression of CYC1, TFAM, ATP5PB, mtATP6, mtDNA, and NRF-1 were decreased upon 12 h treatment, and subsequent ATP production was also significantly decreased at 24 h. These results identified a synergistic effect induced by combinational treatment with hesperidin and chlorogenic acid, which can regulate mitochondria and ATP production through the estrogen receptor pathway in MCF-7 cells. However, none of the treatments induced the generation of reactive oxygen species (ROS), suggesting that ROS likely plays no role in the observed pharmacological activities. Overall, our study sheds light on the adequacy of hesperidin and chlorogenic acid to serve as an adjunctive therapy when co-administrated with chemotherapy drugs in breast cancer patients.

A Novel Cooperative Metallo-ß-Lactamase Fold Metallohydrolase from Pathogen Vibrio vulnificus Exhibits ß-Lactam Antibiotic-Degrading Activities.

Vibrio vulnificus is a pathogen that accounts for one of the highest mortality rates and is responsible for most reported seafood-related illnesses and deaths worldwide. Owing to the threats of pathogens with ß-lactamase activity, it is important to identify and characterize ß-lactamases with clinical significance. In this study, the protein sequence of the metallo-ß-lactamase (MBL) fold metallohydrolase from V. vulnificus (designated Vmh) was analyzed, and its oligomeric state, ß-lactamase activity, and metal binding ability were determined. BLASTp analysis indicated that the V. vulnificus Vmh protein showed no significant sequence identity with any experimentally identified Ambler class B MBLs or enzymes containing the MBL protein fold; it was also predicted to have a signal peptide of 19 amino acids at its N terminus and an MBL protein fold from amino acid residues 23 to 216. Recombinant V. vulnificus Vmh protein was overexpressed and purified. Analytical ultracentrifugation and electrospray ionization-mass spectrometry (MS) data demonstrated its monomeric state in an aqueous solution. Recombinant V. vulnificus Vmh protein showed broad degrading activities against ß-lactam antibiotics, such as penicillins, cephalosporins, and imipenems, with kcat/Km values ranging from 6.23 × 102 to 1.02 × 104 M-1 s-1. The kinetic reactions of this enzyme exhibited sigmoidal behavior, suggesting the possibility of cooperativity. Zinc ions were required for the enzyme activity, which was abolished by adding the metal chelator EDTA. Inductively coupled plasma-MS indicated that this enzyme might bind two zinc ions per molecule as a cofactor.

Differential effects of SUMO1 and SUMO2 on circadian protein PER2 stability and function.

Posttranslational modification (PTM) of core circadian clock proteins, including Period2 (PER2), is required for proper circadian regulation. PER2 function is regulated by casein kinase 1 (CK1)-mediated phosphorylation and ubiquitination but little is known about other PER2 PTMs or their interaction with PER2 phosphorylation. We found that PER2 can be SUMOylated by both SUMO1 and SUMO2; however, SUMO1 versus SUMO2 conjugation had different effects on PER2 turnover and transcriptional suppressor function. SUMO2 conjugation facilitated PER2 interaction with ß-TrCP leading to PER2 proteasomal degradation. In contrast, SUMO1 conjugation, mediated by E3 SUMO-protein ligase RanBP2, enhanced CK1-mediated PER2S662 phosphorylation, inhibited PER2 degradation and increased PER2 transcriptional suppressor function. PER2 K736 was critical for both SUMO1- and SUMO2-conjugation. A PER2K736R mutation was sufficient to alter PER2 protein oscillation and reduce PER2-mediated transcriptional suppression. Together, our data revealed that SUMO1 versus SUMO2 conjugation acts as a determinant of PER2 stability and function and thereby affects the circadian regulatory system and the expression of clock-controlled genes.

Toxic or Not Toxic, That Is the Carbon Quantum Dot's Question: A Comprehensive Evaluation with Zebrafish Embryo, Eleutheroembryo, and Adult Models.

Carbon quantum dots (CQDs) are emerging novel nanomaterials with a wide range of applications and high biocompatibility. However, there is a lack of in-depth research on whether CQDs can cause acute or long-term adverse reactions in aquatic organisms. In this study, two different types of CQDs prepared by ammonia citrate and spermidine, namely CQDAC and CQDSpd, were used to evaluate their biocompatibilities. In the fish embryo acute toxicity test (FET), the LD50 of CQDAC and CQDSpd was about 500 and 100 ppm. During the stage of eleutheroembryo, the LD50 decreased to 340 and 55 ppm, respectively. However, both CQDs were quickly eliminated from embryo and eleutheroembryo, indicating a lack of bioaccumulation. Long-term accumulation of CQDs was also performed in this study, and adult zebrafish showed no adverse effects in 12 weeks. In addition, there was no difference in the hatchability and deformity rates of offspring produced by adult zebrafish, regardless of whether they were fed CQDs or not. The results showed that both CQDAC and CQDSpd have low toxicity and bioaccumulation to zebrafish. Moreover, the toxicity assay developed in this study provides a comprehensive platform to assess the impacts of CQDs on aquatic organisms in the future.

ZNRF1 Mediates Epidermal Growth Factor Receptor Ubiquitination to Control Receptor Lysosomal Trafficking and Degradation.

Activation of the epidermal growth factor receptor (EGFR) is crucial for development, tissue homeostasis, and immunity. Dysregulation of EGFR signaling is associated with numerous diseases. EGFR ubiquitination and endosomal trafficking are key events that regulate the termination of EGFR signaling, but their underlying mechanisms remain obscure. Here, we reveal that ZNRF1, an E3 ubiquitin ligase, controls ligand-induced EGFR signaling via mediating receptor ubiquitination. Deletion of ZNRF1 inhibits endosome-to-lysosome sorting of EGFR, resulting in delayed receptor degradation and prolonged downstream signaling. We further demonstrate that ZNRF1 and Casitas B-lineage lymphoma (CBL), another E3 ubiquitin ligase responsible for EGFR ubiquitination, mediate ubiquitination at distinct lysine residues on EGFR. Furthermore, loss of ZNRF1 results in increased susceptibility to herpes simplex virus 1 (HSV-1) infection due to enhanced EGFR-dependent viral entry. Our findings identify ZNRF1 as a novel regulator of EGFR signaling, which together with CBL controls ligand-induced EGFR ubiquitination and lysosomal trafficking.