Inhibition of SARS-CoV-2 Nsp9 ssDNA-Binding Activity and Cytotoxic Effects on H838, H1975, and A549 Human Non-Small Cell Lung Cancer Cells: Exploring the Potential of Nepenthes miranda Leaf Extract for Pulmonary Disease Treatment.

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.

Cytotoxicity and Multi-Enzyme Inhibition of Nepenthes miranda Stem Extract on H838 Human Non-Small Cell Lung Cancer Cells and RPA32, Elastase, Tyrosinase, and Hyaluronidase Proteins.

The carnivorous pitcher plants of the genus Nepenthes have long been known for their ethnobotanical applications. In this study, we prepared various extracts from the pitcher, stem, and leaf of Nepenthes miranda using 100% ethanol and assessed their inhibitory effects on key enzymes related to skin aging, including elastase, tyrosinase, and hyaluronidase. The cytotoxicity of the stem extract of N. miranda on H838 human lung carcinoma cells were also characterized by effects on cell survival, migration, proliferation, apoptosis induction, and DNA damage. The cytotoxic efficacy of the extract was enhanced when combined with the chemotherapeutic agent 5-fluorouracil (5-FU), indicating a synergistic effect. Flow cytometry analysis suggested that the stem extract might suppress H838 cell proliferation by inducing G2 cell cycle arrest, thereby inhibiting carcinoma cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 15 most abundant compounds in the stem extract of N. miranda. Notably, the extract showed a potent inhibition of the human RPA32 protein (huRPA32), critical for DNA replication, suggesting a novel mechanism for its anticancer action. Molecular docking studies further substantiated the interaction between the extract and huRPA32, highlighting bioactive compounds, especially the two most abundant constituents, stigmast-5-en-3-ol and plumbagin, as potential inhibitors of huRPA32's DNA-binding activity, offering promising avenues for cancer therapy. Overall, our findings position the stem extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and anti-skin-aging treatments, warranting further investigation into its molecular mechanisms and potential clinical applications.

Binding Pattern and Structural Interactome of the Anticancer Drug 5-Fluorouracil: A Critical Review.

5-Fluorouracil (5-FU) stands as one of the most widely prescribed chemotherapeutics. Despite over 60 years of study, a systematic synopsis of how 5-FU binds to proteins has been lacking. Investigating the specific binding patterns of 5-FU to proteins is essential for identifying additional interacting proteins and comprehending their medical implications. In this review, an analysis of the 5-FU binding environment was conducted based on available complex structures. From the earliest complex structure in 2001 to the present, two groups of residues emerged upon 5-FU binding, classified as P- and R-type residues. These high-frequency interactive residues with 5-FU include positively charged residues Arg and Lys (P type) and ring residues Phe, Tyr, Trp, and His (R type). Due to their high occurrence, 5-FU binding modes were simplistically classified into three types, based on interactive residues (within <4 Å) with 5-FU: Type 1 (P-R type), Type 2 (P type), and Type 3 (R type). In summary, among 14 selected complex structures, 8 conform to Type 1, 2 conform to Type 2, and 4 conform to Type 3. Residues with high interaction frequencies involving the N1, N3, O4, and F5 atoms of 5-FU were also examined. Collectively, these interaction analyses offer a structural perspective on the specific binding patterns of 5-FU within protein pockets and contribute to the construction of a structural interactome delineating the associations of the anticancer drug 5-FU.

Novel enzyme-linked aptamer-antibody sandwich assay and hybrid lateral flow strip for SARS-CoV-2 detection.

We have successfully generated oligonucleotide aptamers (Apts) and monoclonal antibodies (mAbs) targeting the recombinant nucleocapsid (N) protein of SARS-CoV-2. Apts were obtained through seven rounds of systematic evolution of ligands by exponential enrichment (SELEX), while mAbs were derived from the 6F6E11 hybridoma cell line. Leveraging these Apts and mAbs, we have successfully devised two innovative and remarkably sensitive detection techniques for the rapid identification of SARS-CoV-2 N protein in nasopharyngeal samples: the enzyme-linked aptamer-antibody sandwich assay (ELAAA) and the hybrid lateral flow strip (hybrid-LFS). ELAAA exhibited an impressive detection limit of 0.1 ng/mL, while hybrid-LFS offered a detection range of 0.1 - 0.5 ng/mL. In the evaluation using ten nasopharyngeal samples spiked with known N protein concentrations, ELAAA demonstrated an average recovery rate of 92%. Additionally, during the assessment of five nasopharyngeal samples from infected individuals and ten samples from healthy volunteers, hybrid-LFS displayed excellent sensitivity and specificity. Our study introduces a novel and efficient on-site approach for SARS-CoV-2 detection in nasopharyngeal samples. The reliable hybrid Apt-mAb strategy not only advances virus diagnostic methods but also holds promise in combating the spread of related diseases.

The complexed crystal structure of dihydropyrimidinase reveals a potential interactive link with the neurotransmitter γ-aminobutyric acid (GABA).

Dihydropyrimidinase (DHPase) plays a crucial role in pyrimidine degradation, showcasing a broad substrate specificity that extends beyond pyrimidine catabolism, hinting at additional roles for this ancient enzyme. In this study, we solved the crystal structure of Pseudomonas aeruginosa DHPase (PaDHPase) complexed with the neurotransmitter γ-aminobutyric acid (GABA) at a resolution of 1.97 Å (PDB ID 8WQ9). Our structural analysis revealed two GABA binding sites in each monomer of PaDHPase. Interactions between PaDHPase and GABA molecules, involving residues within a contact distance of <4 Å, were examined. In silico analyses via PISA and PLIP software revealed hydrogen bonds formed between the side chain of Cys318 and GABA 1, as well as the main chains of Ser333, Ile335, and Asn337 with GABA 2. Comparative structural analysis between GABA-bound and unbound states unveiled significant conformational changes at the active site, particularly within dynamic loop I, supporting the conclusion that PaDHPase binds GABA through the loop-out mechanism. Building upon this molecular evidence, we discuss and propose a working model. The study expands the GABA interactome by identifying DHPase as a novel GABA-interacting protein and provides structural insight into the interaction between a dimetal center in the protein's active site and GABA. Further investigations are warranted to explore potential interactions of GABA with other DHPase-like proteins and to understand whether DHPase may have additional regulatory and physiological roles in the cell, extending beyond pyrimidine catabolism.

Risk of herpes simplex virus infection in solid organ transplant recipients: A population-based cross-sectional study.

BACKGROUND: Herpes simplex virus (HSV) is an opportunistic infection antigen in solid organ transplant (SOT) recipients. However, this phenomenon has received limited attention from epidemiologists. Our study aims to determine the HSV infection risk in SOT recipients. METHODS: This was a nationwide population-based cross-sectional study based on the National Health Insurance Research Database from 2002 to 2015. We used propensity score matching to avoid selection bias and analyzed the association between HSV infection and SOT recipients with multiple logistic regression analysis. RESULTS: At a 3-year follow-up, SOT recipients had a higher risk of developing HSV, with an adjusted odds ratio (aOR) of 3.28 (95% confidence interval (CI), 2.51-4.29). Moreover, at 6-month, 1-year, and 2-year follow-ups, SOT recipients also had an increased risk of HSV than general patients with aORs of 3.85 (95% CI, 2.29-6.49), 4.27 (95% CI, 2.86-6.36), and 3.73 (95% CI, 2.74-5.08), respectively. In the subgroup analysis, lung transplant recipients (aOR = 8.01; 95% CI, 2.39-26.88) exhibited a significantly higher chance of HSV among SOT recipients, followed by kidney transplant recipients (aOR = 3.33; 95% CI, 2.11-5.25) and liver transplant recipients (aOR = 3.15; 95% CI, 2.28-4.34). CONCLUSION: HSV can develop at any time after organ transplantation. SOT recipients had a higher risk of HSV infection than the general population at 6 months, 1 year, 2 years, and 3 years after transplantation, with the highest chance at 1 year after. In addition, the patients who underwent lung transplantion were at higher risk for HSV infection than liver or kidney transplant recipients.

Crystal Structure of DNA Replication Protein SsbA Complexed with the Anticancer Drug 5-Fluorouracil.

Single-stranded DNA-binding proteins (SSBs) play a crucial role in DNA metabolism by binding and stabilizing single-stranded DNA (ssDNA) intermediates. Through their multifaceted roles in DNA replication, recombination, repair, replication restart, and other cellular processes, SSB emerges as a central player in maintaining genomic integrity. These attributes collectively position SSBs as essential guardians of genomic integrity, establishing interactions with an array of distinct proteins. Unlike Escherichia coli, which contains only one type of SSB, some bacteria have two paralogous SSBs, referred to as SsbA and SsbB. In this study, we identified Staphylococcus aureus SsbA (SaSsbA) as a fresh addition to the roster of the anticancer drug 5-fluorouracil (5-FU) binding proteins, thereby expanding the ambit of the 5-FU interactome to encompass this DNA replication protein. To investigate the binding mode, we solved the complexed crystal structure with 5-FU at 2.3 Å (PDB ID 7YM1). The structure of glycerol-bound SaSsbA was also determined at 1.8 Å (PDB ID 8GW5). The interaction between 5-FU and SaSsbA was found to involve R18, P21, V52, F54, Q78, R80, E94, and V96. Based on the collective results from mutational and structural analyses, it became evident that SaSsbA's mode of binding with 5-FU diverges from that of SaSsbB. This complexed structure also holds the potential to furnish valuable comprehension regarding how 5-FU might bind to and impede analogous proteins in humans, particularly within cancer-related signaling pathways. Leveraging the information furnished by the glycerol and 5-FU binding sites, the complexed structures of SaSsbA bring to the forefront the potential viability of several interactive residues as potential targets for therapeutic interventions aimed at curtailing SaSsbA activity. Acknowledging the capacity of microbiota to influence the host's response to 5-FU, there emerges a pressing need for further research to revisit the roles that bacterial and human SSBs play in the realm of anticancer therapy.

Correction: Chen et al. Cytotoxic, Antibacterial, and Antioxidant Activities of the Leaf Extract of Sinningia bullata. Plants 2023, 12, 859.

The authors would like to supplement some methodology information including the controls in the originally published version of this manuscript [...].

COVID-19 vaccination effectiveness and safety in vulnerable populations: a meta-analysis of 33 observational studies.

Background: Even 3 years into the COVID-19 pandemic, questions remain about how to safely and effectively vaccinate vulnerable populations. A systematic analysis of the safety and efficacy of the COVID-19 vaccine in at-risk groups has not been conducted to date. Methods: This study involved a comprehensive search of PubMed, EMBASE, and Cochrane Central Controlled Trial Registry data through 12 July 2022. Post-vaccination outcomes included the number of humoral and cellular immune responders in vulnerable and healthy populations, antibody levels in humoral immune responders, and adverse events. Results: A total of 23 articles assessing 32 studies, were included. The levels of IgG (SMD = -1.82, 95% CI [-2.28, -1.35]), IgA (SMD = -0.37, 95% CI [-0.70, -0.03]), IgM (SMD = -0.94, 95% CI [-1.38, -0.51]), neutralizing antibodies (SMD = -1.37, 95% CI [-2.62, -0.11]), and T cells (SMD = -1.98, 95% CI [-3.44, -0.53]) were significantly lower in vulnerable than in healthy populations. The positive detection rates of IgG (OR = 0.05, 95% CI [0.02, 0.14]) and IgA (OR = 0.03, 95% CI [0.01, 0.11]) antibodies and the cellular immune response rates (OR = 0.20, 95% CI [0.09, 0.45]) were also lower in the vulnerable populations. There were no statistically significant differences in fever (OR = 2.53, 95% CI [0.11, 60.86]), chills (OR = 2.03, 95% CI [0.08, 53.85]), myalgia (OR = 10.31, 95% CI [0.56, 191.08]), local pain at the injection site (OR = 17.83, 95% CI [0.32, 989.06]), headache (OR = 53.57, 95% CI [3.21, 892.79]), tenderness (OR = 2.68, 95% CI [0.49, 14.73]), and fatigue (OR = 22.89, 95% CI [0.45, 1164.22]) between the vulnerable and healthy populations. Conclusion: Seroconversion rates after COVID-19 vaccination were generally worse in the vulnerable than healthy populations, but there was no difference in adverse events. Patients with hematological cancers had the lowest IgG antibody levels of all the vulnerable populations, so closer attention to these patients is recommended. Subjects who received the combined vaccine had higher antibody levels than those who received the single vaccine.

High risk of osteoporosis and fracture following solid organ transplantation: a population-based study.

Background: Osteoporosis and fractures increase morbidity and mortality rates after solid organ transplantation (SOT), but few studies have analyzed the risk of osteoporosis and related fractures after SOT. In this retrospective cohort study, we investigated the risk of osteoporosis and fractures in different SOT recipients. Methods: This study was a retrospective cohort study using a nationally representative database in Taiwan. We collected the data of SOT recipients and used the propensity score matching method to obtain a comparison cohort. To reduce bias, we excluded patients who had been diagnosed with osteoporosis or fracture before inclusion. All participants were followed up until the date of diagnosis as having a pathological fracture, death, or the end of 2018, whichever occurred first. The Cox proportional hazards model was used to investigate the risk of osteoporosis and pathological fracture in SOT recipients. Results: After adjustment for the aforementioned variables, SOT recipients were observed to have a higher risk of osteoporosis (hazard ratio (HR) = 1.46, 95% confidence interval (CI): 1.29-1.65) and fracture (HR: 1.19, 95% CI: 1.01-1.39) than the general individuals. Among the different SOT recipients, the highest risk of fractures was noted in heart or lung transplant recipients, with a HR of 4.62 (95% CI: 2.05-10.44). Among the age groups, patients aged >61 years had the highest HRs for osteoporosis (HR: 11.51; 95% CI, 9.10-14.56) and fracture (HR: 11.75, 95% CI: 8.97-15.40). Conclusion: SOT recipients had a higher risk of osteoporosis and related fractures than the general population, with the highest risks observed in patients receiving heart or lung transplants, older patients, and patients with CCI scores of >3.

The Inhibitory Effects and Cytotoxic Activities of the Stem Extract of Nepenthes miranda against Single-Stranded DNA-Binding Protein and Oral Carcinoma Cells.

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.

Global, regional, and national burdens of myocarditis, 1990-2019: systematic analysis from GBD 2019 : GBD for myocarditis.

OBJECTIVES: Myocarditis, a health-threatening heart disease, is attracting increasing attention. This systematic study was conducted to study the prevalence of disease through the trends of incidence, mortality, disability-adjusted life years (DALYs) over the last 30 years, which would be helpful for the policymakers to better the choices for reasonable decisions. METHODS: The global, regional, and national burdens of myocarditis from 1990-2019 were analyzed by using the 2019 Global Burden of Disease (GBD) database. This study on myocarditis produced new findings according to age, sex, and Social-Demographic Index (SDI) by investigating DALYs, age-standardized incidence rate (ASIR), age-standardized death rate (ASDR), and corresponding estimated annual percentage change (EAPC). RESULTS: The number of myocarditis incidence increased by 62.19%, from 780,410 cases in 1990 to 1,265,770 cases in 2019. The ASIR decreased by 4.42% (95%CI, from -0.26% to -0.21%) over the past 30 years. The number of deaths from myocarditis increased by 65.40% from 19,618 in 1990 to 324,490 in 2019, but the ASDR was relatively stable over the investigated period. ASDR increased in low-middle SDI regions (EAPC=0.48; 95%CI, 0.24 to 0.72) and decreased in low SDI regions (EAPC=-0.97; 95%CI, from -1.05 to -0.89). The age-standardized DALY rate decreased by 1.19% (95%CI, from -1.33% to -1.04%) per year. CONCLUSIONS: Globally, the ASIR and DALY for myocarditis decreased and the ASDR was stable over the past 30 years. The risk of incidences and death cases increased with age. Measures should be taken to control the risk of myocarditis in high-burden regions. Medical supplies should be improved in the high-middle SDI regions and middle SDI regions to reduce the deaths from myocarditis in these regions.

Cytotoxic, Antibacterial, and Antioxidant Activities of the Leaf Extract of Sinningia bullata.

Sinningia bullata is a tuberous member of the flowering plant family Gesneriaceae. Prior to this work, the antibacterial, antioxidant, and cytotoxic properties of S. bullata were undetermined. Here, we prepared different extracts from the leaf, stem, and tuber of S. bullata and investigated their pharmacological activities. The leaf extract of S. bullata, obtained by 100% acetone (Sb-L-A), had the highest total flavonoid content, antioxidation capacity, and cytotoxic and antibacterial activities. Sb-L-A displayed a broad range of antibacterial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The inhibition zones of Sb-L-A ranged from 8 to 30 mm and were in the following order: S. aureus > E. coli > P. aeruginosa. Incubation of B16F10 melanoma cells with Sb-L-A at a concentration of 80 µg/mL caused deaths at the rate of 96%, reduced migration by 100%, suppressed proliferation and colony formation by 99%, and induced apoptosis, which was observed in 96% of the B16F10 cells. In addition, the cytotoxic activities of Sb-L-A were synergistically enhanced when coacting with the antitumor drug epothilone B. Sb-L-A was also used to determine the cytotoxic effects against 4T1 mammary carcinoma cells. Sb-L-A of 60 µg/mL boosted the distribution of the G2 phase from 1.4% to 24.4% in the B16F10 cells. Accordingly, Sb-L-A might suppress melanoma cell proliferation by inducing G2 cell-cycle arrest. The most abundant compounds in Sb-L-A were identified using gas chromatography-mass spectrometry. Overall, the collective data in this study may indicate the pharmacological potentials of Sb-L-A for possible medical applications.

Complexed Crystal Structure of the Dihydroorotase Domain of Human CAD Protein with the Anticancer Drug 5-Fluorouracil.

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.

Crystal Structure of Allantoinase from Escherichia coli BL21: A Molecular Insight into a Role of the Active Site Loops in Catalysis

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.

The Inhibitory Effects and Cytotoxic Activities of the Stem Extract of Sarracenia purpurea against Melanoma Cells and the SsbA Protein.

The Staphylococcus aureus SsbA protein (SaSsbA) is a single-stranded DNA-binding protein (SSB) that is categorically required for DNA replication and cell survival, and it is thus an attractive target for potential antipathogen chemotherapy. In this study, we prepared the stem extract of Sarracenia purpurea obtained from 100% acetone to investigate its inhibitory effect against SaSsbA. In addition, the cytotoxic effects of this extract on the survival, apoptosis, proliferation, and migration of B16F10 melanoma cells were also examined. Initially, myricetin, quercetin, kaempferol, dihydroquercetin, dihydrokaempferol, rutin, catechin, ß-amyrin, oridonin, thioflavin T, primuline, and thioflavin S were used as possible inhibitors against SaSsbA. Of these compounds, dihydrokaempferol and oridonin were capable of inhibiting the ssDNA-binding activity of SaSsbA with respective IC50 values of 750 ± 62 and 2607 ± 242 µM. Given the poor inhibition abilities of dihydrokaempferol and oridonin, we screened the extracts of S. purpurea, Nepenthes miranda, and Plinia cauliflora for SaSsbA inhibitors. The stem extract of S. purpurea exhibited high anti-SaSsbA activity, with an IC50 value of 4.0 ± 0.3 µg/mL. The most abundant compounds in the stem extract of S. purpurea were identified using gas chromatography−mass spectrometry. The top five most abundant contents in this extract were driman-8,11-diol, deoxysericealactone, stigmast-5-en-3-ol, apocynin, and α-amyrin. Using the MOE-Dock tool, the binding modes of these compounds, as well as dihydrokaempferol and oridonin, to SaSsbA were elucidated, and their binding energies were also calculated. Based on the S scores, the binding capacity of these compounds was in the following order: deoxysericealactone > dihydrokaempferol > apocynin > driman-8,11-diol > stigmast-5-en-3-ol > oridonin > α-amyrin. Incubation of B16F10 cells with the stem extract of S. purpurea at a concentration of 100 µg/mL caused deaths at the rate of 76%, reduced migration by 95%, suppressed proliferation and colony formation by 99%, and induced apoptosis, which was observed in 96% of the B16F10 cells. Overall, the collective data in this study indicate the pharmacological potential of the stem extract of S. purpurea for further medical applications.

Global, regional, and national burden of uterine fibroids in the last 30 years: Estimates from the 1990 to 2019 Global Burden of Disease Study.

Objective: To study specific information on trends in incidence, mortality, disability-adjusted life years (DALY) and the corresponding trends among five sociodemographic index regions, 21 regions, and 204 countries for decision-making, which would enable policymakers to distribute limited resources and devise policies more rationally. Methods: Data on uterine fibroids (UNs) from 1990 to 2019, including incidence, mortality, and DALYs, were obtained from the 2019 Global Burden of Disease Study. An estimated annual percentage change (EAPC) was calculated to assess morbidity, mortality, and DALY trends. Results: The incident cases of UFs increased from 5,769,658 (95%UI, 7,634,3995-4,274,824) incidences in 1990 to 9,643,336 (95%UI, 7,178,053-12,714,741) incidences in 2017, and the age-standardized incidence rate was steady at 225.67/100,000 persons (95%UI, 167.33-298.87) in 1990 to 241.18/100,000 persons (95%UI, 179,45-318.02) in 2019. The incidence ratio in the high sociodemographic index (SDI) region showed a unimodal distribution, with peaks in 2005. Between 2009 and 2017, the age-standardized death rate of UFs declined globally, especially in low-SDI and low-middle SDI regions. In contrast with 860,619 DALYs (95%UI, 473,067-1,505,289) in 1990, the number of DALYs was 1,378,497 (95%UI, 710,915-2,475,244) in 2019, which had increased significantly, whereas the age-standardized DALY rate decreased expressively with an EAPC of -1.93 (95%CI, from -2.16 to -1.71). Conclusion: The global burden of UFs increased between 1990 and 2019, and the incidences and DALYs increased prominently worldwide, while the deaths from UFs had no evident growth. Lower SDI regions carried an incremental burden of UFs, while disease reduction was observed in higher SDI regions. It is high time we paid attention to the underprivileged regional quality of life and health protection.

Cytotoxic Activities and the Allantoinase Inhibitory Effect of the Leaf Extract of the Carnivorous Pitcher Plant Nepenthes miranda.

Nepenthes are carnivorous pitcher plants that have several ethnobotanical uses, such as curing stomachache and fever. Here, we prepared different extracts from the stem, leaf, and pitcher of Nepenthes miranda to further investigate their pharmacological potential. The leaf extract of N. miranda obtained by 100% acetone (N. miranda-leaf-acetone) was used in this study to analyze the cytotoxic activities, antioxidation capacity, antibacterial activity, and allantoinase (ALLase) inhibitory effect of this plant. The cytotoxic effects of N. miranda-leaf-acetone on the survival, apoptosis, and migration of the cancer cell lines PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were demonstrated. Based on collective data, the cytotoxic activities of N. miranda-leaf-acetone followed the order: B16F10 > 4T1 > PC-9 cells. In addition, the cytotoxic activities of N. miranda-leaf-acetone were synergistically enhanced when co-acting with the clinical anticancer drug 5-fluorouracil. N. miranda-leaf-acetone could also inhibit the activity of ALLase, a key enzyme in the catabolism pathway for purine degradation. Through gas chromatography−mass spectrometry, the 16 most abundant ingredients in N. miranda-leaf-acetone were identified. The top six compounds in N. miranda-leaf-acetone, namely, plumbagin, lupenone, palmitic acid, stigmast-5-en-3-ol, neophytadiene, and citraconic anhydride, were docked to ALLase, and their docking scores were compared. The docking results suggested plumbagin and stigmast-5-en-3-ol as potential inhibitors of ALLase. Overall, these results may indicate the pharmacological potential of N. miranda for further medical applications.

Anticancer and Antioxidant Activities of the Root Extract of the Carnivorous Pitcher Plant Sarracenia purpurea.

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.

Crystal Structure of an SSB Protein from Salmonella enterica and Its Inhibition by Flavanonol Taxifolin.

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 ß-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.