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Objective The current guidelines and canonical norms of diagnosis or treatment for Post-traumatic stress disorder (PTSD) with sleep disorder are still conflicting and have not yet reached a consensus. This study aimed to unravel the most effective countermeasures between two categories (psychotherapy and pharmacotherapy) put forward by the National Institute for Health and Clinical Excellence (NICE) and World Federation of Societies of Biological Psychiatry (WFSBP) respectively to treat PTSD individuals co-exist with sleep disorders.Methods Four databases, including PubMed, EMBASE, Cochrane Library, and APA PsyNet, were searched from inception to February 02, 2023.Results Twenty articles with 24 Randomized controlled trials (RCTs) and a total number of 1,647 participants were included. As demonstrated in the network meta-analysis comparison results, CBT-I (standardized mean differences (SMD) = –1.51,95% confidence interval (CI):–2.55 to −0.47), CBT-I plus IRT (SMD = –1.71, 95%CI:–3.39, −0.03), prazosin (SMD = –0.87,95%CI:–1.59 to −0.16) and hydroxyzine (SMD = –1.06, 95%CI: −1.94 to −0.19) significantly reduced PTSD symptoms compared with placebo. In contrast to placebo, CBT-I (SMD = –5.61,95%CI:-8.82 to –2.40) significantly improved sleep quality. For nightmare severity, IRT (SMD =–0.65, 95%CI:-1.00 to −0.31), prazosin (SMD = –1.20,95%CI:–1.72 to −0.67) and hydroxyzine (SMD = –0.98,95%CI:-1.58 to −0.37) significantly reduced nightmare severity in comparison with placebo.Conclusions This study suggested that under most circumstances, psychotherapy namely CBT-I had a favorable profile, but pharmacotherapy with prazosin was effective in managing nightmare severity. The sole avail of CBT-I was recommended to improving sleep quality while CBT-I and CBT-I plus IRT showed excellent management of PTSD symptom severity. Exposure to CBT-I isrecommended for depression. The relevant clinical guidelines for the management of individuals with PTSD and sleep disorders may regard this as a reference.PROSPERO CRD42023415240.

Background Systemic lupus erythematosus (SLE), an extensive autoimmune disorder, compromises viral resistance and alters immune responses post respiratory virus vaccines. This study aims to assess immune response levels and safety in SLE patients following respiratory virus vaccines.Methods Extensive searches, until 1 March 2024, were conducted using PubMed, EMBASE, and Cochrane Library. Outcomes, encompassing seroconversion rate (SCR), antibody and IgG titers, neutralizing antibodies, anti-spike antibodies, anti-receptor binding domain (RBD) IgG, and adverse events, were appraised.Results Sixteen articles, comprising 25 observational studies, were included. SLE patients exhibited lower SCR (OR = 0.42, 95%CI: 0.26 to 0.69), antibody titers (SMD=-2.84, 95%CI: −3.36 to −1.61), and neutralizing antibodies (OR = 0.27, 95%CI: 0.13 to 0.56) compared to the healthy population post respiratory virus vaccines. Notably, differences were statistically insignificant for anti-RBD IgG (OR = 1.75, 95%CI: 0.10 to 29.42), IgG titers (SMD=-2.54, 95%CI: −5.57 to −0.49), anti-spike antibodies (OR = 0.35, 95%CI: 0.08 to 1.53), injection site discomfort (OR = 1.03, 95%CI: 0.52 to 2.06), fatigue (OR = 1.23, 95%CI: 0.74 to 2.03), fever (OR = 1.02, 95%CI: 0.64 to 1.63), localized reactions (OR = 0.69, 95%CI: 0.37 to 1.30), systemic reactions (OR = 1.00, 95%CI: 0.59 to 1.69), allergic reactions (OR = 5.11, 95%CI: 0.24 to 107.10), self-reported vaccination-related adverse events (OR = 1.61, 95%CI: 0.56 to 4.63), and disease flares after vaccination (OR = 1.00, 95%CI: 0.14 to 7.28).Conclusion Despite the reduced immune response and host protection in SLE patients post-Corona Virus Disease 2019 (COVID-19) and influenza vaccines compared to the healthy population, safety profiles are comparable. Therefore, it is recommended that SLE patients receive COVID-19 and influenza viral vaccines to fortify their resistance.

Abstract 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 passion fruit, Passiflora edulis, recognized for its rich nutritional properties, has long been used for its varied ethnobotanical applications. This study investigates the therapeutic potential of P. edulis var. Tainung No. 1 rind extracts by examining their polyphenolic content (TPC), total flavonoid content (TFC), anti-skin aging activities against key enzymes such as elastase, tyrosinase, and hyaluronidase, and their ability to inhibit bacterial growth, single-stranded DNA-binding protein (SSB), and their cytotoxic effects on oral carcinoma cells. The acetone extract from the rind exhibited the highest levels of TPC, TFC, anti-SSB, and antibacterial activities. The antibacterial effectiveness of the acetone-extracted rind was ranked as follows: Escherichia coli > Pseudomonas aeruginosa > Staphylococcus aureus. A titration curve for SSB inhibition showed an IC50 value of 313.2 μg/mL, indicating the potency of the acetone extract in inhibiting SSB. It also significantly reduced the activity of enzymes associated with skin aging, particularly tyrosinase, with a 54.5% inhibition at a concentration of 100 μg/mL. Gas chromatography–mass spectrometry (GC–MS) analysis tentatively identified several major bioactive compounds in the acetone extract, including stigmast-5-en-3-ol, vitamin E, palmitic acid, stigmasterol, linoleic acid, campesterol, and octadecanoic acid. Molecular docking studies suggested some of these compounds as potential inhibitors of tyrosinase and SSB. Furthermore, the extract demonstrated anticancer potential against Ca9-22 oral carcinoma cells by inhibiting cell survival, migration, and proliferation and inducing apoptosis. These results underscore the potential of P. edulis (Tainung No. 1) rind as a promising candidate for anti-skin aging, antibacterial, and anticancer applications, meriting further therapeutic investigation.

The tamarillo, or Solanum betaceum, recognized for its comprehensive nutritional profile, has long been valued for its diverse ethnobotanical uses. This study delves into the potential therapeutic applications of S. betaceum by analyzing its polyphenolic content (TPC), total flavonoid content (TFC), anti-skin aging activities against key enzymes like elastase, tyrosinase, and hyaluronidase, and its cytotoxic effects on oral carcinoma cells. Extracts from the seeds, pulp, and peel of red and yellow fruits were prepared using methanol, ethanol, and acetone. The highest TPC was found in the methanol extract from red fruit seeds (9.89 mg GAE/g), and the highest TFC was found in the methanol extract of yellow fruit peel (3.02 mg QUE/g). Some of these extracts significantly inhibited skin aging-associated enzymes with the red fruit seed extract (100 μg/mL) showing up to 50.4% inhibition of tyrosinase. Additionally, the red fruit seed extract obtained using methanol demonstrated potential anticancer effects against Ca9-22 oral carcinoma cells by inhibiting cell survival, migration, and proliferation as well as inducing apoptosis. These results underscore the potential of S. betaceum fruit extracts, especially from red fruit seeds, as promising agents for anti-skin aging and anticancer applications, meriting further exploration for therapeutic uses.

Abstract 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.

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.

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.

BackgroundOsteoporosis 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.MethodsThis 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.ResultsAfter 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).ConclusionSOT 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.

Aims: To evaluate the efficacy of different pharmacologic treatment for severe hypertension during pregnancy.Methods: Two reviewers searched Ovid MEDLINE, Ovid EMbase, and the Cochrane Library for randomized clinical trials from the establishment of the database to 15 July 2021 that were eligible for inclusion and analyzed the pharmaceuticals used for severe hypertension in pregnancy.Results: 29 relevant trials with 2,521 participants were involved. Compared with diazoxide in rate of achieving target blood pressure, other pharmaceuticals, including epoprostenol (RR:1.58, 95%CI:1.01–2.47), hydralazine\dihydralazine (RR:1.57, 95%CI:1.07–2.31), ketanserin (RR:1.67, 95%CI:1.09–2.55), labetalol (RR:1.54, 95%CI:1.04–2.28), nifedipine (RR:1.54, 95%CI:1.04–2.29), and urapidil (RR:1.57, 95%CI:1.00–2.47), were statistically significant in the rate of achieving target blood pressure. According to the SUCRA, diazoxide showed the best therapeutic effect, followed by nicardipine, nifedipine, labetalol, and nitroglycerine. The three pharmaceuticals with the worst therapeutic effect were ketanserin, hydralazine, and urapidil. It is worth noting that the high ranking of the top two pharmaceuticals, including diazoxide and nicardipine, comes from extremely low sample sizes. Other outcomes were reported in the main text.Conclusion: This comprehensive network meta-analysis demonstrated that the nifedipine should be recommended as a strategy for blood pressure management in pregnant women with severe hypertension. Moreover, the conventional pharmaceuticals, including labetalol and hydralazine, showed limited efficacy. However, it was important to note that the instability of hydralazine reducing blood pressure and the high benefit of labetalol with high dosages intakes should also be of concern to clinicians.

ObjectiveTo 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.MethodsData 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.ResultsThe 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).ConclusionThe 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.

Plinia cauliflora has been associated with numerous ethnobotanical applications. In this study, we uncovered that the fruit extract of P. cauliflora, obtained using 50% ethanol, possesses inhibition activity against the Klebsiella pneumoniae single-stranded DNA-binding protein (KpSSB). SSB plays a critical role in cell survival, making it an attractive target for the development of anti-infective drugs. The inhibition activity against KpSSB by the P. cauliflora extract demonstrated an IC50 value of 73 ± 8 μg/mL. By using gas chromatography–mass spectrometry, the chemical content of this extract was tentatively determined. The top 15 compounds (>0.7%) were as follows: 5-hydroxymethylfurfural, 2,3-dihydro-3,5-dihydroxy-6-methyl-4h-pyran-4-one, 2,5-diformylfuran, furfural, ace-tic acid, citraconic anhydride, formic acid, ethyl 4-hydroxy-3-methylbut-2-enoate, furfuryl alcohol, furyl hydroxymethyl ketone, 3-acetyl-3-hydroxyoxolane-2-one, 2,3-dihydro-5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, 2(3H)-furanone, dihy-dro-4-hydroxy-, hydroxyacetone, and 1-hydroxybut-3-en-2-one. To analyze the possible binding modes, the three most abundant compounds were then subjected to docking analysis. We also investigated whether the P. cauliflora extract exhibited any cytotoxic and antiproliferative effects on the survival of B16F10 melanoma cells. Additionally, we found that the extract of P. cauliflora could inhibit the migration and induce apoptosis of B16F10 cells. The results of this study collectively suggest that P. cauliflora holds potential pharmacological benefits, warranting further exploration for therapeutic applications.

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

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.

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.

Dihydropyrimidinase (DHPase) is a key enzyme for pyrimidine degradation. DHPase contains a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHPase catalyzes the hydrolysis of dihydrouracil to N-carbamoyl-β-alanine. Whether 5-aminouracil (5-AU), a thymine antagonist and an anticancer drug that can block DNA synthesis and induce replication stress, can interact with DHPase remains to be investigated. In this study, we determined the crystal structure of Pseudomonas aeruginosa DHPase (PaDHPase) complexed with 5-AU at 2.1 Å resolution (PDB entry 7E3U). This complexed structure revealed that 5-AU interacts with Znα (3.2 Å), Znβ (3.0 Å), the main chains of residues Ser289 (2.8 Å) and Asn337 (3.3 Å), and the side chain of residue Tyr155 (2.8 Å). These residues are also known as the substrate-binding sites of DHPase. Dynamic loop I (amino acid residues Pro65-Val70) in PaDHPase is not involved in the binding of 5-AU. The fluorescence quenching analysis and site-directed mutagenesis were used to confirm the binding mode revealed by the complexed crystal structure. The 5-AU binding mode of PaDHPase is, however, different from that of 5-fluorouracil, the best-known fluoropyrimidine used for anticancer therapy. These results provide molecular insights that may facilitate the development of new inhibitors targeting DHPase and constitute the 5-AU interactome.

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.

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.

Few studies have comprehensively investigated the occurrence of Pneumocystis jirovecii pneumonia (PJP) among solid organ transplant (SOT) recipients. This study investigated the risk of PJP after organ transplantation. Each patient who underwent SOT was propensity-score-matched with four non-SOT individuals in terms of sex, age, insured salary, urbanization of residence, comorbidities, and year of enrollment. When considering the 3-year follow-up, the patients who had undergone SOT were at higher risk of PJP, with the adjusted odds ratio (aOR) being 17.18 (95% confidence interval (CI): 8.80–33.53). Furthermore, SOT recipients were also at higher PJP risk than the patients without SOT at 6 months, 1 year, and 2 years, with the aOR being 22.64 (95% CI: 7.53–68.11), 26.19 (95% CI: 9.89–69.37), and 23.06 (95% CI: 10.23–51.97), respectively. Patients comorbid with HIV infection, hematological malignancies, or vasculitis were at higher risk (aOR = 59.08, 95% CI = 20.30–171.92), (aOR = 11.94, 95% CI = 5.36–26.61), and (aOR = 21.72, 95% CI = 2.41–195.81), respectively. The recipients of SOT were at higher risk of PJP, and PJP can develop at any stage after transplantation. SOT recipients comorbid with HIV, hematologic malignancies, or vasculitis were at higher PJP risk.

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, β-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.

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.

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.

Dihydroorotase (DHOase) possesses a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHOase catalyzes the reversible cyclization of N-carbamoyl aspartate (CA-asp) to dihydroorotate (DHO) in the third step of the pathway for the biosynthesis of pyrimidine nucleotides and is an attractive target for potential anticancer and antimalarial chemotherapy. Crystal structures of ligand-bound DHOase show that the flexible loop extends toward the active site when CA-asp is bound (loop-in mode) or moves away from the active site, facilitating the product DHO release (loop-out mode). DHOase binds the product-like inhibitor 5-fluoroorotate (5-FOA) in a similar mode to DHO. In the present study, we report the crystal structure of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with 5-FOA at 2.5 Å resolution (PDB entry 7CA0). ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). However, our complexed structure revealed that ScDHOase bound 5-FOA differently from EcDHOase. 5-FOA ligated the Zn atoms in the active site of ScDHOase. In addition, 5-FOA bound to ScDHOase through the loop-in mode. We also characterized the binding of 5-FOA to ScDHOase by using the site-directed mutagenesis and fluorescence quenching method. Based on these lines of molecular evidence, we discussed whether these different binding modes are species- or crystallography-dependent.

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.

Dihydropyrimidinase, a dimetalloenzyme containing a carboxylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. Unlike all known dihydropyrimidinases, which are tetrameric, pseudomonal dihydropyrimidinase forms a dimer at neutral pH. In this paper, we report the crystal structure of P. aeruginosa dihydropyrimidinase at pH 5.9 (PDB entry 5YKD). The crystals of P. aeruginosa dihydropyrimidinase belonged to space group C2221 with cell dimensions of a = 108.9, b = 155.7, and c = 235.6 Å. The structure of P. aeruginosa dihydropyrimidinase was solved at 2.17 Å resolution. An asymmetric unit of the crystal contained four crystallographically independent P. aeruginosa dihydropyrimidinase monomers. Gel filtration chromatographic analysis of purified P. aeruginosa dihydropyrimidinase revealed a mixture of dimers and tetramers at pH 5.9. Thus, P. aeruginosa dihydropyrimidinase can form a stable tetramer both in the crystalline state and in the solution. Based on sequence analysis and structural comparison of the dimer-dimer interface between P. aeruginosa dihydropyrimidinase and Thermus sp. dihydropyrimidinase, different oligomerization mechanisms are proposed.

The pulse oximeter is a popular instrument to monitor the arterial oxygen saturation (SPO2). Although a fingertip-type pulse oximeter is the mainstream one on the market at present, it is still inconvenient for long-term monitoring, in particular, with respect to motion. Therefore, the development of a wearable pulse oximeter, such as a finger base-type pulse oximeter, can effectively solve the above issue. However, the tissue structure of the finger base is complex, and there is lack of detailed information on the effect of the light source and detector placement on measuring SPO2. In this study, the practicability of a ring-type pulse oximeter with a multi-detector was investigated by optical human tissue simulation. The optimal design of a ring-type pulse oximeter that can provide the best efficiency of measuring SPO2 was discussed. The efficiency of ring-type pulse oximeters with a single detector and a multi-detector was also discussed. Finally, a wearable and wireless ring-type pulse oximeter was also implemented to validate the simulation results and was compared with the commercial fingertip-type pulse oximeter.

Single-stranded DNA-binding protein (SSB) and PriA helicase play important roles in bacterial DNA replication restart process. The mechanism by which PriA helicase is bound and stimulated by SSB in Escherichia coli (Ec) has been established, but information on this process in Gram-positive bacteria are limited. We characterized the properties of SSB from Staphylococcus aureus (SaSsbA, a counterpart of EcSSB) and analyzed its interaction with SaPriA. The gel filtration chromatography analysis of purified SaSsbA showed a stable tetramer in solution. The crystal structure of SaSsbA determined at 1.82 Å resolution (PDB entry 5XGT) reveals that the classic oligonucleotide/oligosaccharide-binding folds are formed in the N-terminal DNA-binding domain, but the entire C-terminal domain is disordered. Unlike EcSSB, which can stimulate EcPriA via a physical interaction between EcPriA and the C-terminus of EcSSB (SSB-Ct), SaSsbA does not affect the activity of SaPriA. We also found that SaPriA can be bound by SaSsbA, but not by SaSsbA-Ct. Although no effect was found with SaSsbA, SaPriA can be significantly stimulated by the Gram-negative Klebsiella pneumoniae SSB (KpSSB). In addition, we found that the conserved SSB-Ct binding site of KpPriA (Trp82, Tyr86, Lys370, Arg697, and Gln701) is not present in SaPriA. Arg697 in KpPriA is known to play a critical role in altering the SSB35/SSB65 distribution, but this corresponding residue in SaPriA is Glu767 instead, which has an opposite charge to Arg. SaPriA E767R mutant was constructed and analyzed; however, it still cannot be stimulated by SaSsbA. Finally, we found that the conserved MDFDDDIPF motif in the Gram-negative bacterial SSB is DISDDDLPF in SaSsbA, i.e., F172 in EcSSB and F168 in KpSSB is S161 in SaSsbA, not F. When acting with SaSsbA S161F mutant, the activity of SaPriA was dramatically enhanced elevenfold. Overall, the conserved binding sites, both in EcPriA and EcSSB, are not present in SaPriA and SaSsbA, thereby no stimulation occurs. Our observations through structure-sequence comparison and mutational analyses indicate that the case of EcPriA-EcSSB is not applicable to SaPriA-SaSsbA because of inherent differences among the species.

The role of DnaD in the recruitment of replicative helicase has been identified. However, knowledge of the DNA, PriA, and DnaA binding mechanism of this protein for the DnaA- and PriA-directed replication primosome assemblies is limited. We characterized the DNA-binding properties of DnaD from Staphylococcus aureus (SaDnaD) and analyzed its interactions with SaPriA and SaDnaA. The gel filtration chromatography analysis of purified SaDnaD and its deletion mutant proteins (SaDnaD1-195, SaDnaD1-200 and SaDnaD1-204) showed a stable tetramer in solution. This finding indicates that the C-terminal region aa 196-228 is not crucial for SaDnaD oligomerization. SaDnaD forms distinct complexes with ssDNA of different lengths. In fluorescence titrations, SaDnaD bound to ssDNA with a binding-site size of approximately 32 nt. A stable complex of SaDnaD1-195, SaDnaD1-200, and SaDnaD1-204 with ssDNA dT40 was undetectable, indicating that the C-terminal region of SaDnaD (particularly aa 205-228) is crucial for ssDNA binding. The SPR results revealed that SaDnaD1-195 can interact with SaDnaA but not with SaPriA, which may indicate that DnaD has different binding sites for PriA and DnaA. Both SaDnaD and SaDnaDY176A mutant proteins, but not SaDnaD1-195, can significantly stimulate the ATPase activity of SaPriA. Hence, the stimulation effect mainly resulted from direct contact within the protein-protein interaction, not via the DNA-protein interaction. Kinetic studies revealed that the SaDnaD-SaPriA interaction increases the Vmax of the SaPriA ATPase fivefold without significantly affecting the Km. These results indicate that the conserved C-terminal region is crucial for ssDNA and PriA helicase binding, but not for DnaA protein-binding and self-tetramerization.

Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. These metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. However, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit dihydropyrimidinase remains unclear. This study investigated the inhibition of dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. Allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit dihydropyrimidinase, and the IC50 values of these compounds were within the millimolar range. The inhibition of dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. Some of these compounds are known as inhibitors of allantoinase and dihydroorotase. Although the inhibitory effects of these flavonoids on dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited dihydropyrimidinase with IC50 values of 48 and 40 μM for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. The results from the Lineweaver-Burk plot indicated that dihydromyricetin was a competitive inhibitor. Results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with dihydropyrimidinase with the K(d) value of 22.6 μM. A structural study using PatchDock showed that dihydromyricetin was docked in the active site pocket of dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. This study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude). These flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for multiple targets) for designing compounds that target several cyclic amidohydrolases.