Search Results (25)

Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores the therapeutic potential of Nepenthes miranda leaf extract against nonstructural protein 9 (Nsp9) of SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) cell lines. Nsp9, essential for SARS-CoV-2 RNA replication, was expressed and purified, and its interaction with ssDNA was assessed. Initial tests with myricetin and oridonin, known for targeting ssDNA-binding proteins and Nsp9, respectively, did not inhibit the ssDNA-binding activity of Nsp9. Subsequent screenings of various N. miranda extracts identified those using acetone, methanol, and ethanol as particularly effective in disrupting Nsp9’s ssDNA-binding activity, as evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol and lupenone, major components in the leaf extract of N. miranda, as potential inhibitors. The cytotoxic properties of N. miranda leaf extract were examined across NSCLC lines H1975, A549, and H838, focusing on cell survival, apoptosis, and migration. Results showed a dose-dependent cytotoxic effect in the following order: H1975 > A549 > H838 cells, indicating specificity. Enhanced anticancer effects were observed when the extract was combined with afatinib, suggesting synergistic interactions. Flow cytometry indicated that N. miranda leaf extract could induce G2 cell cycle arrest in H1975 cells, potentially inhibiting cancer cell proliferation. Gas chromatography–mass spectrometry (GC–MS) enabled the tentative identification of the 19 most abundant compounds in the leaf extract of N. miranda. These outcomes underscore the dual utility of N. miranda leaf extract in potentially managing SARS-CoV-2 infection through Nsp9 inhibition and offering anticancer benefits against lung carcinoma. These results significantly broaden the potential medical applications of N. miranda leaf extract, suggesting its use not only in traditional remedies but also as a prospective treatment for pulmonary diseases. Overall, our findings position the leaf extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and antiviral therapies, warranting further investigation into its molecular mechanisms and potential clinical applications. Full article

Modern integrated circuits (ICs) take advantage of three-dimensional (3D) nanostructures in devices and interconnects to achieve high-speed and ultra-low-power performance. The choice of electrical insulation materials with excellent dielectric strength, electrical resistivity, strong mechanical strength, and high thermal conductivity becomes critical. Diamond possesses these properties and is recently recognized as a promising dielectric material for the fabrication of advanced ICs, which are sensitive to detrimental high-temperature processes. Therefore, a high-rate low-temperature deposition technique for large-grain, high-quality diamond films of the thickness of a few tens to a few hundred nanometers is desirable. The diamond growth rate by microwave plasma chemical vapor deposition (MPCVD) decreases rapidly with lowering substrate temperature. In addition, the thermal conductivity of non-diamond carbon is much lower than that of diamond. Furthermore, a small-grain diamond film suffers from poor thermal conductivity due to frequent phonon scattering at grain boundaries. This paper reports a novel MPCVD process aiming at high growth rate, large grain size, and high sp³/sp² ratio for diamond films deposited on silicon. Graphite paste containing nanoscale graphite and oxy-hydrocarbon binder and solvent vaporizes and mixes with gas feeds of hydrogen, methane, and carbon dioxide to form plasma. Rapid diamond growth of diamond seeds at 450 °C by the plasma results in large-grained diamond films on silicon at a high deposition rate of 200 nm/h. Full article

A rotator cuff tear is a prevalent ailment affecting the shoulder joint. The clinical efficacy of combined therapy remains uncertain for partial rotator cuff tears. In this study, we integrated extracorporeal shockwave therapy (ESWT) with platelet-rich plasma (PRP) injection, juxtaposed with PRP in isolation. Both cohorts exhibited significant improvements in visual analogue scale (VAS), Constant–Murley score (CMS), degrees of forward flexion, abduction, internal rotation, and external rotation, and the sum of range of motion (SROM) over the six-month assessment period. The application of ESWT in conjunction with PRP exhibited notable additional enhancements in both forward flexion (p = 0.033) and abduction (p = 0.015) after one month. Furthermore, a substantial augmentation in the range of shoulder motion (SROM) (p < 0.001) was observed after six months. We employed isobaric tag for relative and absolute quantitation (iTRAQ) to analyze the differential plasma protein expression in serum samples procured from the two groups after one month. The concentrations of S100A8 (p = 0.042) and S100A9 (p = 0.034), known to modulate local inflammation, were both lower in the ESWT + PRP cohort. These findings not only underscore the advantages of combined therapy but also illuminate the associated molecular changes. Full article

The coronavirus SARS-CoV-2 is the causative pathogen of the COVID-19 pandemic that has been causing global upheaval since 2019. The widespread administration of vaccines has partially deterred the spread of SARS-CoV-2, yet the virus is mutating its genome to reduce its antigenicity and evade the human herd immunity. It seems that SARS-CoV-2 will co-exist with the human population for many decades to come. While most infected individuals only experience mild to moderate symptoms, some develop severe pulmonary and systemic disease that can result in hospitalization or even death. The natural history model of SARS-CoV-2 infection has been proposed which includes three sequential stages: the early infection stage, pulmonary stage, and hyper-inflammatory stage. Recently, it has been observed that many people who recovered from an acute infection still experience persistent symptoms for weeks or months, a condition known as long COVID. Furthermore, some COVID-19 patients display escalated rates of both macro- and micro-thrombosis due to endotheliopathy. Hence, we added the thrombosis and convalescent stages to the natural history model, encompassing the entire period from early infection to long COVID. The early infection stage is characterized by symptomatic or asymptomatic elevation of viral titers. Some patients progress to the pulmonary stage characterized by opacities in chest X-rays and computed tomography. The thrombosis stage is characterized by heightened rates of pulmonary thrombosis and consistently elevated D-dimer levels. The hyper-inflammatory stage is characterized by storms of cytokines, such as IL-6, IL-17, and interferons, which is a systemic effect. In the convalescent stage, some people recover completely, while others suffer from long COVID with persistent symptoms such as fatigue, shortness of breath, or brain fog. The natural history model of SARS-CoV-2 infection can be used to elucidate treatment and care. Full article

The carnivorous pitcher plants of the genus Nepenthes exhibit many ethnobotanical uses, including treatments of stomachache and fever. In this study, we prepared different extracts from the pitcher, stem, and leaf extracts of Nepenthes miranda obtained using 100% methanol and analyzed their inhibitory effects on recombinant single-stranded DNA-binding protein (SSB) from Klebsiella pneumoniae (KpSSB). SSB is essential for DNA replication and cell survival and thus an attractive target for potential antipathogen chemotherapy. Different extracts prepared from Sinningia bullata, a tuberous member of the flowering plant family Gesneriaceae, were also used to investigate anti-KpSSB properties. Among these extracts, the stem extract of N. miranda exhibited the highest anti-KpSSB activity with an IC₅₀ value of 15.0 ± 1.8 μg/mL. The cytotoxic effects of the stem extract of N. miranda on the survival and apoptosis of the cancer cell lines Ca9-22 gingival carcinoma, CAL27 oral adenosquamous carcinoma, PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were also demonstrated and compared. Based on collective data, the cytotoxic activities of the stem extract at a concentration of 20 μg/mL followed the order Ca9-22 > CAL27 > PC9 > 4T1 > B16F10 cells. The stem extract of N. miranda at a concentration of 40 μg/mL completely inhibited Ca9-22 cell migration and proliferation. In addition, incubation with this extract at a concentration of 20 μg/mL boosted the distribution of the G2 phase from 7.9% to 29.2% in the Ca9-22 cells; in other words, the stem extract might suppress Ca9-22 cell proliferation by inducing G2 cell cycle arrest. Through gas chromatography–mass spectrometry, the 16 most abundant compounds in the stem extract of N. miranda were tentatively identified. The 10 most abundant compounds in the stem extract of N. miranda were used for docking analysis, and their docking scores were compared. The binding capacity of these compounds was in the order sitosterol > hexadecanoic acid > oleic acid > plumbagin > 2-ethyl-3-methylnaphtho[2,3-b]thiophene-4,9-dione > methyl α-d-galactopyranoside > 3-methoxycatechol > catechol > pyrogallol > hydroxyhydroquinone; thus, sitosterol might exhibit the greatest inhibitory capacity against KpSSB among the selected compounds. Overall, these results may indicate the pharmacological potential of N. miranda for further therapeutic applications. Full article

Numerous pathogenic CALR exon 9 mutations have been identified in myeloproliferative neoplasms (MPN), with type 1 (52bp deletion; CALR^DEL) and type 2 (5bp insertion; CALR^INS) being the most prevalent. Despite the universal pathobiology of MPN driven by various CALR mutants, it is unclear why different CALR mutations result in diverse clinical phenotypes. Through RNA sequencing followed by validation at the protein and mRNA levels, we found that S100A8 was specifically enriched in CALR^DEL but not in CALR^INS MPN-model cells. The expression of S100a8 could be regulated by STAT3 based on luciferase reporter assay complemented with inhibitor treatment. Pyrosequencing demonstrated relative hypomethylation in two CpG sites within the potential pSTAT3-targeting S100a8 promoter region in CALR^DEL cells as compared to CALR^INS cells, suggesting that distinct epigenetic alteration could factor into the divergent S100A8 levels in these cells. The functional analysis confirmed that S100A8 non-redundantly contributed to accelerated cellular proliferation and reduced apoptosis in CALR^DEL cells. Clinical validation showed significantly enhanced S100A8 expression in CALR^DEL-mutated MPN patients compared to CALR^INS-mutated cases, and thrombocytosis was less prominent in those with S100A8 upregulation. This study provides indispensable insights into how different CALR mutations discrepantly drive the expression of specific genes that contributes to unique phenotypes in MPN. Full article

Allantoinase (ALLase; EC 3.5.2.5) possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carbamylated lysine. ALLase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of allantoin into allantoate. Biochemically, ALLase belongs to the cyclic amidohydrolase family, which also includes dihydropyrimidinase, dihydroorotase, hydantoinase (HYDase), and imidase. Previously, the crystal structure of ALLase from Escherichia coli K-12 (EcALLase-K12) was reported; however, the two active site loops crucial for substrate binding were not determined. This situation would limit further docking and protein engineering experiments. Here, we solved the crystal structure of E. coli BL21 ALLase (EcALLase-BL21) at a resolution of 2.07 Å (PDB ID 8HFD) to obtain more information for structural analyses. The structure has a classic TIM barrel fold. As compared with the previous work, the two missed active site loops in EcALLase-K12 were clearly determined in our structure of EcALLase-BL21. EcALLase-BL21 shared active site similarity with HYDase, an important biocatalyst for industrial production of semisynthetic penicillin and cephalosporins. Based on this structural comparison, we discussed the functional role of the two active site loops in EcALLase-BL21 to better understand the substrate/inhibitor binding mechanism for further biotechnological and pharmaceutical applications. Full article

Dihydroorotase (DHOase) is the third enzyme in the pathway used for the biosynthesis of pyrimidine nucleotides. In mammals, DHOase is active in a trifunctional enzyme, CAD, which also carries out the activities of carbamoyl phosphate synthetase and aspartate transcarbamoylase. Prior to this study, it was unknown whether the FDA-approved clinical drug 5-fluorouracil (5-FU), which is used as an anticancer therapy, could bind to the DHOase domain of human CAD (huDHOase). Here, we identified huDHOase as a new 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. In order to investigate where 5-FU binds to huDHOase, we solved the complexed crystal structure at 1.97 Å (PDB ID 8GVZ). The structure of huDHOase complexed with malate was also determined for the sake of comparison (PDB ID 8GW0). These two nonsubstrate ligands were bound at the active site of huDHOase. It was previously established that the substrate N-carbamoyl-L-aspartate is either bound to or moves away from the active site, but it is the loop that is extended towards (loop-in mode) or moved away (loop-out mode) from the active site. DHOase also binds to nonsubstrate ligands via the loop-out mode. In contrast to the Escherichia coli DHOase model, our complexed structures revealed that huDHOase binds to either 5-FU or malate via the loop-in mode. We further characterized the binding of 5-FU to huDHOase using site-directed mutagenesis and the fluorescence quenching method. Considering the loop-in mode, the dynamic loop in huDHOase should be a suitable drug-targeting site for further designing inhibitors and clinical chemotherapies to suppress pyrimidine biosynthesis in cancer cell lines. Full article

A modified strain-hardening cementitious composite (SHCC) material, fabricated using corundum aggregates (SHCC-Cor), has been proposed for roadway applications as it offers high structural performance and high skid resistance. However, the acoustic performance of SHCC is unclear and has not been well studied in the past. Theoretically, SHCC may not provide the optimum solution in acoustic performance as it provides a low texture profile, high density, and low porosity. In this study, the acoustic performance of pavement slabs made of SHCC and modified SHCC-Cor are investigated using a nondestructive method to determine the surface roughness (macro texture) of slab surfaces. The pavement–tire noise level was then simulated using SPERoN software. As result, the noise level coming from the pavement made of SHCC could be up to 65 dB(A), while the noise level for SHCC-Cor increased up to 69.2 dB(A) because of the lower shape factor (G) due to a rougher surface as a result of the existence of corundum aggregate on the SHCC surface. The aeroacoustics were also increased compared to the SHCC slab. The modification of SHCC-Cor by introducing grooves (SHCC-Cor-Gro) successfully reduced the sound level coming from the vibration. Full article

To determine phenotype-related dupilumab response in adult patients with atopic dermatitis (AD), this multicenter, retrospective study included 111 adults with moderate-to-severe AD in Taiwan, with median age of 31.5 years (18–87) and 71 (64.0%) males. Patients received dupilumab 300 mg per two to three weeks up to 12 months. We found a significant improvement after 4 and 16 weeks of treatment in all patients for all the assessed scores, including eczema area and severity index (EASI) improvement ≥50% (EASI-50) and 75% (EASI-75), EASI reaching minimal clinically important difference (MCID), and Investigator’s Global Assessment (IGA) improvement ≥2. Importantly, prior to asthma, early AD onset and 3-week drug intervals were significantly associated with a high proportion of EASI-75 at month 12, while prurigo and lichenoid phenotypes were associated with a lower proportion of EASI-75 at month 12. However, the majority of adverse events were mild in severity. In conclusion, our study results identify phenotype-related dupilumab response at month 12 in adults with moderate-to-severe AD, and we suggest that treatment should not be discontinued until reaching a satisfactory clinical response. Full article

The carnivorous pitcher plant Sarracenia purpurea exhibits many ethnobotanical uses, including the treatments of type 2 diabetes and tuberculosis-like symptoms. In this study, we prepared different extracts from the leaves (pitchers), stems, and roots of S. purpurea and investigated their antioxidant and anticancer properties. To evaluate the extraction efficiency, we individually used different solvents, namely methanol, ethanol, acetone, and distilled water, for S. purpurea extract preparations. The root extract of S. purpurea, obtained by 100% acetone (S. purpurea-root-acetone), had the highest anticancer activities, antioxidation capacity (the DPPH activity with IC₅₀ of 89.3 ± 2.2 μg/mL), antibacterial activities, total phenolic content (33.4 ± 0.7 mg GAE/g), and total flavonoid content (107.9 ± 2.2 mg QUE/g). The most abundant compounds in S. purpurea-root-acetone were identified using gas chromatography–mass spectrometry; 7,8-Dihydro-α-ionone was the major compound present in S. purpurea-root-acetone. In addition, the co-cytotoxicity of S. purpurea-root-acetone (combined with the clinical anticancer drug 5-fluorouracil (5-FU) on the survival, apoptosis, proliferation, and migration of the 4T1 mammary carcinoma) was examined. The combination of 5-FU with S. purpurea-root-acetone could be highly efficient for anti-4T1 cells. We also found that S. purpurea-root-acetone could inhibit the enzymatic activity of human dihydroorotase (huDHOase), an attractive target for potential anticancer chemotherapy. The sic most abundant compounds in S. purpurea-root-acetone were tested using an in silico analysis via MOE-Dock software for their binding affinities. The top-ranked docking conformations were observed for 7,8-dihydro-α-ionone and stigmast-5-en-3-ol, suggesting the inhibition potential against huDHOase. Overall, the collective data in this study may indicate the pharmacological potentials of S. purpurea-root-acetone for possible medical applications. Full article

Single-stranded DNA (ssDNA)-binding proteins (SSBs) play a central role in cells by participating in DNA metabolism, including replication, repair, recombination, and replication fork restart. SSBs are essential for cell survival and thus an attractive target for potential anti-pathogen chemotherapy. In this study, we determined the crystal structure and examined the size of the ssDNA-binding site of an SSB from Salmonella enterica serovar Typhimurium LT2 (SeSSB), a ubiquitous opportunistic pathogen which is highly resistant to antibiotics. The crystal structure was solved at a resolution of 2.8 Å (PDB ID 7F25), indicating that the SeSSB monomer possesses an oligonucleotide/oligosaccharide-binding (OB) fold domain at its N-terminus and a flexible tail at its C-terminus. The core of the OB-fold in the SeSSB is made of a six-stranded β-barrel capped by an α-helix. The crystal structure of the SeSSB contained two monomers per asymmetric unit, which may indicate the formation of a dimer. However, the gel-filtration chromatography analysis showed that the SeSSB forms a tetramer in solution. Through an electrophoretic mobility shift analysis, we characterized the stoichiometry of the SeSSB complexed with a series of ssDNA dA homopolymers, and the size of the ssDNA-binding site was determined to be around 22 nt. We also found the flavanonol taxifolin, also known as dihydroquercetin, capable of inhibiting the ssDNA-binding activity of the SeSSB. Thus, this result extended the SSB interactome to include taxifolin, a natural product with a wide range of promising pharmacological activities. Full article

(20S) ginsenoside Rh2 (G-Rh2), a major bioactive metabolite of ginseng, effectively inhibits the survival and proliferation of human liver cancer cells. However, its molecular targets and working mechanism remain largely unknown. Excitingly, we screened out heat shock protein 90 alpha (HSP90A), a key regulatory protein associated with liver cancer, as a potential target of (20S) G-Rh2 by phage display analysis and mass spectrometry. The molecular docking and thermal shift analyses demonstrated that (20S) G-Rh2 directly bound to HSP90A, and this binding was confirmed to inhibit the interaction between HSP90A and its co-chaperone, cell division cycle control protein 37 (Cdc37). It is well-known that the HSP90A-Cdc37 system aids in the folding and maturation of cyclin-dependent kinases (CDKs). As expected, CDK4 and CDK6, the two G₀-G₁ phase promoting kinases as well as CDK2, a key G₁-S phase transition promoting kinase, were significantly downregulated with (20S) G-Rh2 treatment, and these downregulations were mediated by the proteasome pathway. In the same condition, the cell cycle was arrested at the G₀-G₁ phase and cell growth was inhibited significantly by (20S) G-Rh2 treatment. Taken together, this study for the first time reveals that (20S) G-Rh2 exerts its anti-tumor effect by targeting HSP90A and consequently disturbing the HSP90A-Cdc37 chaperone system. HSP90A is frequently overexpressed in human hepatoma cells and the higher expression is closely correlated to the poor prognosis of liver cancer patients. Thus, (20S) G-Rh2 might become a promising alternative drug for liver cancer therapy. Full article

Dihydroorotase (DHOase), a dimetalloenzyme containing a carbamylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase (ALLase), dihydropyrimidinase (DHPase), hydantoinase, and imidase. Unlike most known cyclic amidohydrolases, which are tetrameric, DHOase exists as a monomer or dimer. Here, we report and analyze two crystal structures of the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase) complexed with malate. The structures of different DHOases were also compared. An asymmetric unit of these crystals contained four crystallographically independent ScDHOase monomers. ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). Unlike EcDHOase, ScDHOase can form tetramers, both in the crystalline state and in solution. In addition, the subunit-interacting residues of ScDHOase for dimerization and tetramerization are significantly different from those of other DHOases. The tetramerization pattern of ScDHOase is also different from those of DHPase and ALLase. Based on sequence analysis and structural evidence, we identify two unique helices (α6 and α10) and a loop (loop 7) for tetramerization, and discuss why the residues for tetramerization in ScDHOase are not necessarily conserved among DHOases. Full article

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc. Full article