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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.

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: Osimertinib, a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), shows superior lung cancer treatment efficacy. However, osimertinib-induced severe hypersensitivity, including Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN), is frequently observed in Asian populations and hinders cancer treatment., Objective: We investigated the genetic HLA predisposition and immune pathomechanism of osimertinib-induced hypersensitivity., Methods: We enrolled 17 patients with osimertinib-induced delayed hypersensitivity (seven with severe SJS/TEN and 10 with mild maculopapular exanthema), 98 osimertinib-tolerant subjects, and 2,123 general population controls. We performed HLA genotyping, drug-induced lymphocyte activation test, and surface plasmon resonance assay., Results: HLA-B∗51:02 was present in 83.3% of osimertinib-induced SJS/TEN patients but in only 3.3% of the general population controls (P = 2.8 × 10 -7 ; corrected P = 6.9 × 10 -6 ; odds ratio [OR] = 146), and 0% of osimertinib-tolerant controls (P = 6.5 × 10 -8 ; corrected P = 1.6 × 10 -6 ; OR = 707). The association of HLA-B∗51:01 and HLA-A∗24:02 with osimertinib-induced maculopapular exanthema patients, rather than with osimertinib-tolerant subjects (P = .002, OR = 15.7 for HLA-B∗51:01; and P = .003, OR = 9.5 for HLA-A∗24:02), was identified as a phenotype-specific association. Granulysin, the SJS/TEN-specific cytotoxic protein, was significantly higher in plasma of SJS/TEN patients (39.8 ± 4.5 ng/mL; P < .001) and in in vitro lymphocyte activation test (sensitivity = 83.3%; P < .01) compared with tolerant controls. Patients with osimertinib-induced hypersensitivity appeared to tolerate alternative EGFR-TKIs. Surface plasmon resonance results also confirmed that HLA-B∗51:02 protein has a higher binding affinity for osimertinib and lower or no affinity for other EGFR-TKIs., Conclusions: HLA-B∗51:02 frequently occurs in Asian populations and is strongly associated with osimertinib-induced SJS/TEN. Our findings suggest HLA-B∗51:02 screening as a preemptive test to reduce osimertinib-induced severe hypersensitivity., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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.

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 IC 50 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.

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.

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.

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. Funders had no role in the development or publication of this manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Yang, Yao, Huang, Yin, Xian-Yu, Zhang and Chen.)

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 IC 50 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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Lai, Yang, Gau, Huang, Tsai, Huang and Lee.)

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., (© 2023. The Author(s).)

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.

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.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Deng, Xian-Yu, Han, Huang, Ma, Li, Gao, Liu and Zhang.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cheng, Li, Gong, Huang, Zhang and Yan.)

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 IC 50 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.

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.

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 Zn α (3.2 Å), Zn β (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., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this paper., (Copyright © 2022 En-Shyh Lin et al.)

Single-stranded DNA (ssDNA)-binding protein (SSB) plays a crucial role in DNA replication, repair, and recombination as well as replication fork restarts. SSB is essential for cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. Whether naturally occurring products can inhibit SSB remains unknown. In this study, the effect of the flavonols myricetin, quercetin, kaempferol, and galangin on the inhibition of Pseudomonas aeruginosa SSB (PaSSB) was investigated. Furthermore, SSB was identified as a novel quercetin-binding protein. Through an electrophoretic mobility shift analysis, myricetin could inhibit the ssDNA binding activity of PaSSB with an IC 50 of 2.8 ± 0.4 μM. The effect of quercetin, kaempferol, and galangin was insignificant. To elucidate the flavonol inhibition specificity, the crystal structure of PaSSB complexed with the non-inhibitor quercetin was solved using the molecular replacement method at a resolution of 2.3 Å (PDB entry 7VUM) and compared with a structure with the inhibitor myricetin (PDB entry 5YUN). Although myricetin and quercetin bound PaSSB at a similar site, their binding poses were different. Compared with myricetin, the aromatic ring of quercetin shifted by a distance of 4.9 Å and an angle of 31 o for hydrogen bonding to the side chain of Asn108 in PaSSB. In addition, myricetin occupied and interacted with the ssDNA binding sites Lys7 and Glu80 in PaSSB whereas quercetin did not. This result might explain why myricetin could, but quercetin could not, strongly inhibit PaSSB. This molecular evidence reveals the flavonol inhibition specificity and also extends the interactomes of the natural anticancer products myricetin and quercetin to include the OB-fold protein SSB.

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.

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.

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., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this study., (Copyright © 2021 Hong-Hsiang Guan et al.)

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda , was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides and considered an attractive target for potential antimalarial, anticancer, and antipathogen chemotherapy. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to DHOase remains unknown. Here, we report the crystal structures of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with malate, 5-FU, and 5-aminouracil (5-AU). ScDHOase shares structural similarity with Escherichia coli DHOase. We also characterized the binding of 5-FU and 5-AU to ScDHOase by using the fluorescence quenching method. These complexed structures revealed that residues Arg18, Asn43, Thr106, and Ala275 of ScDHOase were involved in the 5-FU (PDB entry 6L0B) and 5-AU binding (PDB entry 6L0F). Overall, these results provide structural insights that may facilitate the development of new inhibitors targeting DHOase and constitute the 5-FU and 5-AU interactomes for further clinical chemotherapies., Competing Interests: Declaration of competing interest The author has no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Co-trimoxazole, a sulfonamide antibiotic, is used to treat a variety of infections worldwide, and it remains a common first-line medicine for prophylaxis against Pneumocystis jiroveci pneumonia. However, it can cause severe cutaneous adverse reaction (SCAR), including Stevens-Johnson syndrome, toxic epidermal necrolysis, and drug reaction with eosinophilia and systemic symptoms. The pathomechanism of co-trimoxazole-induced SCAR remains unclear., Objective: We aimed to investigate the genetic predisposition of co-trimoxazole-induced SCAR., Methods: We conducted a multicountry case-control association study that included 151 patients with of co-trimoxazole-induced SCAR and 4631 population controls from Taiwan, Thailand, and Malaysia, as well as 138 tolerant controls from Taiwan. Whole-genome sequencing was performed for the patients and population controls from Taiwan; it further validated the results from Thailand and Malaysia., Results: The whole-genome sequencing study (43 case patients vs 507 controls) discovered that the single-nucleotide polymorphism rs41554616, which is located between the HLA-B and MICA loci, had the strongest association with co-trimoxazole-induced SCAR (P = 8.2 × 10 -9 ; odds ratio [OR] = 7.7). There were weak associations of variants in co-trimoxazole-related metabolizing enzymes (CYP2D6, GSTP1, GCLC, N-acetyltransferase [NAT2], and CYP2C8). A replication study using HLA genotyping revealed that HLA-B∗13:01 was strongly associated with co-trimoxazole-induced SCAR (the combined sample comprised 91 case patients vs 2545 controls [P = 7.2 × 10 -21 ; OR = 8.7]). A strong HLA association was also observed in the case patients from Thailand (P = 3.2 × 10 -5 ; OR = 3.6) and Malaysia (P = .002; OR = 12.8), respectively. A meta-analysis and phenotype stratification study further indicated a strong association between HLA-B∗13:01 and co-trimoxazole-induced drug reaction with eosinophilia and systemic symptoms (P = 4.2 × 10 -23 ; OR = 40.1)., Conclusion: This study identified HLA-B∗13:01 as an important genetic factor associated with co-trimoxazole-induced SCAR in Asians., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Single-stranded DNA (ssDNA)-binding protein (SSB) is essential for DNA metabolic processes. SSB also binds to many DNA-binding proteins that constitute the SSB interactome. The mechanism through which PriA helicase, an initiator protein in the DNA replication restart process, is stimulated by SSB in Escherichia coli (EcSSB) has been established. However, some Gram-positive bacterial SSBs such as Bacillus subtilis SsbA (a counterpart of EcSSB), Staphylococcus aureus SsbA, SsbB, and SsbC do not activate PriA helicase. Here, we describe some of the methods used in our laboratory to compare SSB-PriA functional and physical interactions in Gram-positive and -negative bacteria.

Single-stranded DNA-binding proteins (SSBs) are essential to cells because they participate in DNA metabolic processes, such as DNA replication, repair, and recombination. Some bacteria possess more than one paralogous SSB. Three similar SSBs, namely, SsbA, SsbB, and SsbC, are found in Staphylococcus aureus. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to SSBs remains unknown. In this study, we found that 5-FU could form a stable complex with S. aureus SsbB (SaSsbB). We cocrystallized 5-FU with SaSsbB and solved complex structures to assess binding modes. Two complex forms of the structures were determined, namely, the individual asymmetric unit (two SaSsbB monomers) containing one (PDB entry 7D8J) or two 5-FU molecules (PDB entry 7DEP). The locations of 5-FU in these two SaSsbB complexes were similar regardless of the binding ratio. The structures revealed that residues T12, K13, T30, F48, and N50 of SaSsbB were involved in 5-FU binding. The mutations of T12, K13, and F48 caused the low 5-FU binding activity of SaSsbB, a result consistent with the structural analysis results. Taken together, the complexed structure and the binding mode analysis of SaSsbB extended the anticancer drug 5-FU interactome to include the oligonucleotide/oligosaccharide-binding fold protein., Competing Interests: Declaration of competing interest The author has no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. This enzyme is important in pyrimidine metabolism, and blocking its activity would be detrimental to cell survival. This study investigated the dihydropyrimidinase inhibition by plumbagin isolated from the extract of carnivorous plant Nepenthes miranda (Nm). Plumbagin inhibited dihydropyrimidinase with IC 50 value of 58 ± 3 μM. Double reciprocal results of Lineweaver-Burk plot indicated that this compound is a competitive inhibitor of dihydropyrimidinase. Fluorescence quenching analysis revealed that plumbagin could form a stable complex with dihydropyrimidinase with the K d value of 37.7 ± 1.4 μM. Docking experiments revealed that the dynamic loop crucial for stabilization of the intermediate state in dihydropyrimidinase might be involved in the inhibition effect of plumbagin. Mutation at either Y155 or K156 within the dynamic loop of dihydropyrimidinase caused low plumbagin binding affinity. In addition to their dihydropyrimidinase inhibition, plumbagin and Nm extracts also exhibited cytotoxicity on melanoma cell survival, migration, and proliferation. Further research can directly focus on designing compounds that target the dynamic loop in dihydropyrimidinase during catalysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Dihydropyrimidinase catalyzes the reversible hydrolytic ring opening of dihydrouracil and dihydrothymine to N-carbamoyl-β-alanine and N-carbamyl-β-aminoisobutyrate, respectively. Dihydropyrimidinase from microorganisms is normally known as hydantoinase because of its role as a biocatalyst in the synthesis of d- and l-amino acids for the industrial production of antibiotic precursors and its broad substrate specificity. Dihydropyrimidinase belongs to the cyclic amidohydrolase family, which also includes imidase, allantoinase, and dihydroorotase. Although these metal-dependent enzymes share low levels of amino acid sequence homology, they possess similar active site architectures and may use a similar mechanism for catalysis. By contrast, the five human dihydropyrimidinase-related proteins possess high amino acid sequence identity and are structurally homologous to dihydropyrimidinase, but they are neuronal proteins with no dihydropyrimidinase activity. In this chapter, we summarize and discuss current knowledge and the recent advances on the structure, catalytic mechanism, and inhibition of dihydropyrimidinase., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Single-stranded DNA (ssDNA)-binding proteins (SSBs) play an important role in all DNA-dependent cellular processes, such as DNA replication, recombination, repair, and replication restart. The N-terminal domain of SSBs forms an oligonucleotide/oligosaccharide-binding (OB) fold for ssDNA binding. The SSB-dC35 complex structure has revealed how an Escherichia coli SSB (EcSSB) tetramer binds to 65-nucleotide (nt)-long ssDNA, namely, the (SSB) 65 binding mode. Knowledge on whether the ssDNA-binding mode for EcSSB is typical for all SSBs or is bacterial strain and length dependent is limited. Here, we studied the ssDNA-binding properties of a Pseudomonas aeruginosa SSB (PaSSB) and investigated its interaction mode through crystallographic analysis. The complex crystal structure containing a PaSSB tetramer with two ssDNA chains was solved at a resolution of 1.91 Å (PDB entry 6IRQ). Results revealed that each bound ssDNA dT25 adopts an S-shaped conformation. This binding mode, as shown by the complex structure of PaSSB, differs significantly from (SSB) 65 . ssDNA-binding contributions from aromatic residues in PaSSB, except the contribution of Trp54, were not significant. Using electrophoretic mobility shift analysis, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers. The minimal length of ssDNA required for PaSSB tetramer binding and the size of the ssDNA-binding site were 25 and 29 nt, respectively. These observations through structure-function analysis suggested that only two OB folds rather than four OB folds in PaSSB are enough for the formation of a stable complex with ssDNA. The PaSSB noninteracting OB folds proposed here may allow sliding via reptation in a dynamic ssDNA binding process., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Single-stranded DNA-binding protein (SSB) is essential to cells as it participates in DNA metabolic processes, such as DNA replication, repair, and recombination. Escherichia coli SSB (EcSSB) tetramer cooperatively binds and wraps ssDNA in two major binding modes. In this study, we report the complex crystal structure of Pseudomonas aeruginosa SSB (PaSSB) with ssDNA dT20 at 2.39 Å resolution (PDB entry 6JDG) that revealed a new binding mode, namely, (SSB) 3:1 . In the (SSB) 65 mode revealed by the EcSSB-dC35 complex structure, all four subunits fully participate in the binding to ssDNA. However, only three subunits in the PaSSB tetramer can participate in wrapping ssDNA in the (SSB) 3:1 mode. The bound ssDNA in the PaSSB-ssDNA complex adopts an Ω-shaped conformation rather than a χ-shaped conformation in the (SSB) 65 mode possibly due to the disability of Phe60. Phe60 is known to play a critical role in defining DNA-binding paths and promoting the wrapping of ssDNA around SSB tetramers. However, it is not important in the (SSB) 3:1 mode. The ssDNA binding path revealed by our structural evidence suggests that ssDNA occupies half of the binding sites of the two subunits and slightly comes into contact with the ssDNA binding sites of the third subunit. Accordingly, we propose and sketch a possible wrapping mechanism of SSB via this novel ssDNA-binding mode, (SSB) 3:1 ., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Dihydropyrimidinase (DHPase) catalyzes the reversible cyclization of dihydrouracil to N-carbamoyl-β-alanine in the second step of the pyrimidine degradation pathway. Whether 5-fluorouracil (5-FU), the best-known fluoropyrimidine that is used to target the enzyme thymidylate synthase for anticancer therapy, can bind to DHPase remains unknown. In this study, we found that 5-FU can form a stable complex with Pseudomonas aeruginosa DHPase (PaDHPase). The crystal structure of PaDHPase complexed with 5-FU was determined at 1.76 Å resolution (PDB entry 6KLK). Various interactions between 5-FU and PaDHPase were examined. Six residues, namely, His61, Tyr155, Asp316, Cys318, Ser289 and Asn337, of PaDHPase were involved in 5-FU binding. Except for Cys318, these residues are also known as the substrate-binding sites of DHPase. 5-FU interacts with the main chains of residues Ser289 (3.0 Å) and Asn337 (3.2 Å) and the side chains of residues Tyr155 (2.8 Å) and Cys318 (2.9 Å). Mutation at either Tyr155 or Cys318 of PaDHPase caused a low 5-FU binding activity of PaDHPase. This structure and the binding mode provided molecular insights into how the dimetal center in DHPase undergoes a conformational change during 5-FU binding. Further research can directly focus on revisiting the role of DHPase in anticancer therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

DnaT is a replication restart primosomal protein required for re-initiating chromosomal DNA replication in bacteria. DnaT can be a monomer, dimer, trimer, tetramer, or pentamer. The oligomerization and disassembly of DnaT oligomers are critical in primosome assembly. Prior to this work, only the ssDNA-bound structure of the pentameric DnaT truncated protein (aa 84-153; DnaT84-153) was available. The mechanism by which DnaT oligomerizes as different states is unclear. In this paper, we report the crystal structure of the C-terminal domain of DnaT (aa 84-179; DnaTc) at 2.30 Å resolution (PDB entry 6AEQ). DnaTc forms a dimer both in the crystalline state and in solution. As compared with the ssDNA-bound structure of the pentameric DnaT84-153, their subunit-subunit interfaces significantly differ. The different oligomeric architecture suggests a strong conformational change possibly induced by ssDNA. Superposition analysis further indicated that the monomer of a DnaTc dimer shifted away by a distance of 7.5 Å and rotated by an angle of 170° for binding to ssDNA. Basing from these molecular evidence, we discussed and proposed a working model to explain how DnaTc oligomerizes through residue R146 mediation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Dihydropyrimidinase (DHPase) is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase (DHOase), hydantoinase, and imidase. Almost all of these zinc metalloenzymes possess a binuclear metal center in which two metal ions are bridged by a post-translational carbamylated Lys. Crystal structure of Tetraodon nigroviridis DHPase reveals that one zinc ion is sufficient to stabilize Lys carbamylation. In this study, we found that one metal coordination was not sufficient to fix CO 2 to the Lys in bacterial DHPase. We prepared and characterized mono-Zn DHPase from Pseudomonas aeruginosa (PaDHPase), and the catalytic activity of mono-Zn PaDHPase was not detected. The crystal structure of mono-Zn PaDHPase determined at 2.23 Å resolution (PDB entry 6AJD) revealed that Lys150 was no longer carbamylated. This finding indicated the decarbamylation of the Lys during the metal chelating process. To confirm the state of Lys carbamylation in mono-Zn PaDHPase in solution, mass spectrometric (MS) analysis was carried out. The MS result was in agreement with the theoretical value for uncarbamylated PaDHPase. Crystal structure of the human DHOase domain (huDHOase) K1556A mutant was also determined (PDB entry 5YNZ), and the structure revealed that the active site of huDHOase K1556A mutant contained one metal ion. Like mono-Zn PaDHPase, oxygen ligands of the carbamylated Lys were not required for Znα binding. Considering the collective data from X-ray crystal structure and MS analysis, mono-Zn PaDHPase in both crystalline state and solution was not carbamylated. In addition, structural evidences indicated that post-translational carbamylated Lys was not required for Znα binding in PaDHPase and in huDHOase., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Single-stranded DNA-binding protein (SSB) is essential for all DNA-dependent cellular processes. The mechanism through which PriA helicase, an initiator protein in the DNA replication restart process, is stimulated by SSB in Escherichia coli (Ec) has been established. Nevertheless, whether or not PriA stimulated by SSB is conserved among Gram-negative bacteria remains unclear, and the SSB specificity for the stimulation effect on PriA is unknown. In this study, three similar SSBs from Klebsiella pneumoniae (KpSSB), Salmonella enterica (StSSB), and Pseudomonas aeruginosa (PaSSB) were used to analyze the stimulation effect. Two chimeric proteins, namely, KpSSBn-PaSSBc and KpSSBn-StSSBc, were also used. KpSSB, StSSB, and KpSSBn-StSSBc can stimulate KpPriA activity, but PaSSB and KpSSBn-PaSSBc cannot. The crystal structure of PaSSB solved at 2.04 Å resolution (PDB entry 5YUO) reveals the classic OB fold structure, similar to that of EcSSB. Comparison of SSBs through sequence analysis showed that the typical glycine-rich flexible region in PaSSB contains very few glycine residues. Through analyses of protein chimeragenesis, structure-sequence, and ATPase stimulation effects, we concluded that the inherent difference in the glycine-rich flexible region among SSB species is a determinant of PriA stimulation. Further research can directly focus on determining the type of glycine-rich hinge that can stimulate PriA and the reason why bacterial SSBs need to evolve different C-terminal domains during evolution., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Single-stranded DNA-binding protein (SSB) is essential for all DNA-dependent cellular processes. SSB inhibitors have been recently suggested as broad-spectrum antibacterial agents in antibiotic development. In this paper, we report the first inhibitor-complexed crystal structure of SSB from Pseudomonas aeruginosa PAO1 (PaSSB) at 2.68 Å resolution (PDB entry 5YUN). The inhibitor, myricetin, is a flavonol that possesses many pharmacological activities, such as anticancer, anti-inflammatory, and antibacterial properties, and is beneficial for humans. Four monomers of PaSSB and two of myricetins were found per asymmetric unit. Various interactions between myricetin and PaSSB were examined. Among these, four residues in PaSSB, Lys7, Arg62, Glu80, and Gly107 were found crucial for forming hydrogen bond to myricetin. These two myricetins occupy the grooves for ssDNA-binding of SSB that may prevent ssDNA-wrapping and ssDNA-binding stably from SSB. In addition to explaining how SSB can be inhibited, the myricetin-SSB interaction modes in this paper may also provide insights into how myricetin can bind and inhibit proteins on cancer-signaling pathways., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Single-stranded DNA-binding proteins (SSBs) are essential to cells as they participate in DNA metabolic processes, such as DNA replication, repair, and recombination. The functions of SSBs have been studied extensively in Escherichia coli . Unlike E. coli , which contains only one type of SSB (EcSSB), some bacteria have more than one paralogous SSB. In Staphylococcus aureus , three SSBs are found, namely, SsbA, SaSsbB, and SsbC. While EcSSB can significantly stimulate EcPriA helicase, SaSsbA does not affect the SaPriA activity. It remains unclear whether SsbBs can participate in the PriA-directed DNA replication restart process. In this study, we characterized the properties of SaSsbBs through structural and functional analyses. Crystal structure of SaSsbB determined at 2.9 Å resolution (PDB entry 5YYU) revealed four OB folds in the N-terminal DNA-binding domain. DNA binding analysis using EMSA showed that SaSsbB binds to ssDNA with greater affinity than SaSsbA does. Gene map analysis demonstrated that SAAV0835 encoding SaSsbB is flanked by unknown genes encoding hypothetical proteins, namely, putative Sipho&#95;Gp157, ERF, and HNHc&#95;6 gene products. Structure-based mutational analysis indicated that the four aromatic residues (Phe37, Phe48, Phe54, and Tyr82) in SaSsbB are at positions that structurally correspond to the important residues of EcSSB for binding to ssDNA and are also critical for SaSsbB to bind ssDNA. Similar to EcSSB and other SSBs such as SaSsbA and SaSsbC, SaSsbB also exhibited high thermostability. However, unlike EcSSB, which can stimulate EcPriA, SaSsbB did not affect the activity of SaPriA. Based on results in this study and previous works, we therefore established that SsbA and SsbB, as well as SsbC, do not stimulate PriA activity., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, repair, and recombination. Unlike E. coli , which contains only one type of SSB (EcSSB), some bacteria have two paralogous SSBs, namely, SsbA and SsbB. In this study, we found the third SSB-like protein in Staphylococcus aureus , SAAV2152, which was designated as SaSsbC. SaSsbC is a protein of 131 amino acids and shares 38%, 36%, and 33% sequence identity to SaSsbB, SaSsbA, and EcSSB, respectively. Gene map analysis showed that unlike the E. coli ssb gene, which is adjacent to uvrA gene, the S. aureus ssb gene SAAV2152 is flanked by the putative SceD , the putative YwpF , and fabZ genes. A homology model showed that SaSsbC consists of the classic oligonucleotide/oligosaccharide-binding fold at the N-terminus. At the C-terminus, SaSsbC did not exhibit sequence similarity to that of EcSSB. Electrophoretic mobility shift analysis showed that SaSsbC formed a single complex with ssDNA of different lengths. Mutational analysis revealed that Tyr36, Tyr47, Phe53, and Tyr81 in SaSsbC are at positions that structurally correspond to the important residues of EcSSB for binding to ssDNA and are also critical for SaSsbC to bind ssDNA. Unlike EcSSB, which can stimulate EcPriA, SaSsbC did not affect the activity of SaPriA. In addition, SaSsbA inhibitor 9-methyl-2,3,7-trihydroxy-6-fluorone (NSC5426) could inhibit the ssDNA-binding activity of SaSsbC with IC 50 of 78 μM. In conclusion, this study has identified and characterized SAAV2152 as a kind of SSB, and further research can directly focus on determining its actual physiological role in S. aureus ., Competing Interests: CONFLICTS OF INTEREST The author has no conflicts of interest.

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 C 222 1 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.

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.