The natural pesticide phenazine-1-carboxylic acid (PCA) is known to lack phloem mobility, whereas Metalaxyl is a representative phloem systemic fungicide. In order to endow PCA with phloem mobility and also enhance its antifungal activity, thirty-two phenazine-1-carboxylic acid-N-phenylalanine esters conjugates were designed and synthesized by conjugating PCA with the active structure N-acylalanine methyl ester of Metalaxyl. All target compounds were characterized by 1H NMR, 13C NMR and HRMS. The antifungal evaluation results revealed that several target compounds exhibited moderate to potent antifungal activities against Sclerotinia sclerotiorum, Bipolaris sorokiniana, Phytophthora parasitica, Phytophthora citrophthora. In particular, compound F7 displayed excellent antifungal activity against S. sclerotiorum with an EC50 value of 6.57 µg/mL, which was superior to that of Metalaxyl. Phloem mobility study in castor bean system indicated good phloem mobility for the target compounds F1-F16. Particularly, compound F2 exhibited excellent phloem mobility; the content of compound F2 in the phloem sap of castor bean was 19.12 μmol/L, which was six times higher than Metalaxyl (3.56 μmol/L). The phloem mobility tests under different pH culture solutions verified the phloem translocation of compounds related to the \"ion trap\" effect. The distribution of the compound F2 in tobacco plants further suggested its ambimobility in the phloem, exhibiting directional accumulation towards the apical growth point and the root. These results provide valuable insights for developing phloem mobility fungicides mediated by exogenous compounds.

BACKGROUND: Qualitative urinalysis using the Sternheimer stain is a common method in Japan for identifying bacteriuria, but there is a lack of studies examining its test characteristics. In this study, we aimed to investigate the sensitivity and specificity of the Sternheimer stain for urine culture results and compare it with the sensitivity and specificity of the Gram stain. Our goal was to determine the usefulness of the Sternheimer stain in identifying bacteriuria.
METHODS: Among 986 patients aged 16 years or older from whom samples for both urinalysis and urine culture were obtained at the emergency room of Tenri Hospital from January 2019 to December 2019, 342 patients with pyuria, defined as the presence of 10 or more white cells per cubic millimeter in a urine specimen, who had not received prior antimicrobial therapy were included. Urine cultures were used for comparison to determine the sensitivity and specificity of Sternheimer and Gram stain in this patient group. A positive Sternheimer stain result was defined as bacteriuria ≥ (1+), and that of Gram stain was defined as ≥ 1/1 field of high-power ( × 1000) oil immersion.
RESULTS: Using urine culture results for comparison, the sensitivity of Sternheimer stain was 92.2%, the specificity was 48.5%, the positive likelihood ratio was 1.79, and the negative likelihood ratio was 0.16.
CONCLUSIONS: Sternheimer stain is a rapid and useful method to exclude bacteriuria in a group of patients with pyuria in the emergency department.

Cancer as an acquired genetic disease is based on changes both in the genome itself and in transcription processes. Accordingly, it is at the DNA level that it makes sense to search for and design agents capable of effective and selective anticancer action. In this study, we used an iterative approach based on a molecular dynamics simulation to design a highly selective DNA-intercalating agent called HASDI. To confirm its selective affinity to DNA, we conducted two simulation experiments: HASDI in a complex with a DNA fragment of the EBNA1 gene (it targets 16 nucleotide pairs of this gene) and HASDI in a complex with a random DNA fragment of the KCNH2 gene. The molecular dynamics simulation was carried out in the GROMACS 2019 package. The binding energy was calculated by gmx_MMPBSA 1.5.2. The further analysis was performed using the built-in utilities of GROMACS, gmx_MMPBSA and also XMGRACE and Pymol 1.8. As a result, we determined that the EBNA1-50nt/HASDI complex was stable throughout the whole simulation trajectory. HASDI, due to the presence of a linker modified depending on a specific pair of nitrogenous bases, formed an average of 32 hydrogen bonds with a sequence of 16 nucleotide pairs. Phenazine rings were stably intercalated every 2 base pairs. The root-mean-square deviation of HASDI in such a complex fluctuated around the value of 6.5 Å and had no tendency to increase. The calculated value of the binding free energy was - 235.3 ± 7.77 kcal/mol. The KCNH2-50nt/HASDI complex, as an example of the intercalation of the designed structure into a random part of the human genome, maintained the stability of its position at a level comparable to the EBNA1-50nt/HASDI complex. The phenazine rings were constantly intercalated in their original positions, and the root-mean-square deviation fluctuated around one value, although it had a tendency to chaotic changes. At the same time, this complex was characterized by 17-19 hydrogen bonds, on average, and the binding free energy was - 193.47 ± 14.09 kcal/mol. Moreover, the DNA duplex had local single-nucleotide melting in the region of the 4th linker. According to a significant decrease in the number of hydrogen bonds, a decrease in energy gain, as well as a decrease in the stability of the DNA duplex characteristic of the KCNH2-50nt/HASDI complex compared to the target EBNA1-50nt/HASDI complex, the molecule we designed can be considered a potentially selective DNA polyintercalating agent capable of relatively accurate recognition of 16 base pairs.

Phenazines are an emerging class of organic compounds that have been recently utilized in aqueous redox flow batteries, a promising technology for large-scale energy storage. A virtual screening based on density functional theory calculations is used to investigate the redox potentials of around 100 phenazine derivatives in aqueous media containing various electron-donating or electron-withdrawing groups at different positions. The calculations identify the crucial positions that should be functionalized with multiple hydroxy groups to design new anolytes. The combined experimental-computational methodology reported herein guides the development of a new molecule with a record low reversible redox potential as a potential anolyte for aqueous redox flow batteries.

Pyocyanin, a redox-active phenazine pigment produced by Pseudomonas aeruginosa, inhibits 5-lipoxygenase (5-LOX) activity. However, whether pyocyanin can directly block the enzymatic activity by binding at the active site still remains a question because of its ability to produce superoxide radicals and H2O2. With the objective of characterizing this mechanism, we carried out molecular docking and molecular dynamics simulations and performed Molecular Mechanics Poisson-Boltzmann surface area (MMPBSA) binding energy studies. The results of the study revealed that pyocyanin is dynamically stable at the active site of 5-LOX and its MMPBSA binding energy (-84.720 kJ/mol) is comparable to that of the 5-LOX standard inhibitor zileuton (-72.729 kJ/mol). Similar studies using three other phenazine derivatives - 1-hydroxyphenazine, phenazine-1-carboxylic acid and phenazine-1-carboxamide - also showed encouraging results. In light of this evidence, we postulate as a proof of concept that pyocyanin and these phenazine derivatives have the potential to inhibit 5-LOX activity by directly binding at the active site and blocking enzymatic catalysis of the substrate. Considering the potential of 5-LOX inhibitors in inflammatory diseases such as asthma and rheumatoid arthritis, the findings of this study open up the exploration of phenazine derivatives in structure-based drug design against 5-LOX. Communicated by Ramaswamy H. Sarma.

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase\'s biology, with wide-reaching implications for drug development.

5-Methyl-2-phenyl-2-hexenal (MPH) has been used as a flavoring agent. In the present study, we performed a subchronic toxicity study in male and female F344 rats with oral administration of MPH by gavage at 0, 8, 24 and 70 mg/kg body weight (BW)/day for 90 days. No mortality or clinical signs were observed during the experimental period. Body weight and food consumption for all treated groups of both sexes were essentially the same as for the respective control groups. Hematologic examination demonstrated significant decreases in monocyte counts for females given 24 and 70 mg/kg BW/day. However, these changes were not substantial and no related histopathological changes were observed, suggesting that these changes were not toxicologically significant. Among organ weights, the absolute and/or relative weights of testes and liver were significantly increased in the 70 mg/kg BW/day groups of males and females, respectively, but no related histopathological changes were observed, suggesting that these changes did not reflect adverse effects. In addition, no treatment-related histopathological changes were observed for any of the tissues examined. Based on the overall data, the no-observed-adverse-effect level (NOAEL) for MPH was determined to be 70 mg/kg BW/day, the highest dose tested, in both male and female rats.

BACKGROUND: Gram stain of cerebrospinal fluid (CSF) is widely used in the diagnosis of acute meningitis, however, it is often conducted in the laboratory, as only some hospitals have access to point-of-care Gram stain (PCGS). The purpose of this study was to demonstrate the clinical impact and utility of PCGS in diagnosing and treating both bacterial and aseptic meningitis in adults.
METHODS: This was a hospital-based, retrospective observational study at a referral center in Okinawa, Japan. We reviewed the records of all patients aged 15 years or older who were admitted to the Division of Infectious Diseases between 1995 and 2015 and finally diagnosed with bacterial (n = 34) or aseptic meningitis (n = 97). For bacterial meningitis, we compared the treatments that were actually selected based on PCGS with simulated treatments that would have been based on the Japanese guidelines. For aseptic meningitis, we compared the rates of antibiotic use between real cases where PCGS was available and real cases where it was not.
RESULTS: PCGS was the most precise predictor for differentiating between bacterial and aseptic meningitis (sensitivity 91.2%, specificity 98.9%), being superior in this regard to medical histories, vital signs and physical examinations, and laboratory data available in the emergency room (ER). In bacterial meningitis, PCGS reduced the frequency of meropenem use (1/34 = 3.0%) compared with simulated cases in which PCGS was not available (19/34 = 55.9%) (p< 0.001). In aseptic meningitis cases, the rate of antibiotic administration was lower when PCGS was used (38/97 = 39.2%) than when it was not (45/74 = 60.8%) (p = 0.006).
CONCLUSIONS: PCGS of CSF distinguishes between bacterial and aseptic meningitis more accurately than other predictors available in the ER. Patients with bacterial meningitis are more likely to receive narrower-spectrum antimicrobials when PCGS is used than when it is not. PCGS of CSF thus can potentially suppress the empiric use of antimicrobials for aseptic meningitis.

BACKGROUND: Although Moraxella catarrhalis (M. catarrhalis) is a common cause of community-acquired pneumonia (CAP), studies investigating clinical manifestations of CAP due to M. catarrhalis (MC-CAP) in adults are limited. Since S. pneumoniae is the leading cause of CAP globally, it is important to distinguish between MC-CAP and CAP due to S. pneumoniae (SP-CAP) in clinical practice. However, no past study compared clinical characteristics of MC-CAP and SP-CAP by statistical analysis. We aimed to clarify the clinical characteristics of MC-CAP by comparing those of SP-CAP, as well as the utility of sputum Gram staining.
METHODS: This retrospective study screened CAP patients aged over 20 years visiting or admitted to Okinawa Miyako Hospital between May 2013 and April 2018. Among these, we included patients whom either M. catarrhalis alone or S. pneumoniae alone was isolated from their sputum by bacterial cultures.
RESULTS: We identified 134 MC-CAP and 130 SP-CAP patients. Although seasonality was not observed in SP-CAP, almost half of MC-CAP patients were admitted in the winter. Compared to those with SP-CAP, MC-CAP patients were older (p < 0.01) and more likely to have underlying pulmonary diseases such as asthma and bronchiectasis (p < 0.01). Approximately half of asthmatic MC-CAP and SP-CAP patients had asthma attacks. Although winter is an influenza season in Japan, co-infection with influenza virus was less common in MC-CAP compared to SP-CAP patients (3% vs. 15%, p < 0.01). Bronchopneumonia patterns on X-ray, as well as bronchial wall thickening, bilateral distribution, and segmental pattern on CT were more common in MC-CAP patients than in SP-CAP patients (p < 0.01). Sputum Gram stain was highly useful method for the diagnosis in both MC-CAP and SP-CAP (78.4% vs. 89.2%), and penicillins were most frequently chosen as an initial treatment for both pneumonias.
CONCLUSIONS: This is the first study to show that MC-CAP occurred in older people compared to SP-CAP, influenza virus co-infection was less common in MC-CAP than SP-CAP, and that MC-CAP frequently caused asthma attacks. Gram stain contributed for the appropriate treatment, resulting in conserving broad-spectrum antibiotics such as cephalosporins and fluoroquinolones in both MC-CAP and SP-CAP patients.

OBJECTIVE: The aim of this study was to evaluate the diagnostic value of renal pelvis urine Gram staining (RPUGS) in predicting postoperative fever and renal stone culture (RSC) positivity in percutaneous nephrolithotomy (PCNL).
METHODS: Totally 141 consecutive patients undergoing PCNL for renal stone were included between January 2018 and December 2019. The RPUGS and renal pelvis urine culture (RPUC) were performed using urine sample from renal collecting system, while RSC was performed using stone fragments. Patients were divided into two groups as Group 1 (n = 119) without postoperative fever (< 38 °C) and Group 2 (n = 22) with postoperative fever (≥ 38 °C). Stone culture and Gram staining models were created for predicting postoperative fever using constant covariates of the presence of residual stone, hydronephrosis, and stone burden.
RESULTS: A significantly higher number of patients in Group 2 had RPUGS, RSC, and RPUC positivity (p < 0.001, for each). The sensitivity, specificity, positive predictive value, and negative predictive value of RPUGS in predicting postoperative fever were 72.7%, 89.9%, 57.1%, and 94.7%, respectively. It was observed that both models had similar predictive values and diagnostic performances. Although RSC and RPUGS had a similar diagnostic value in predicting postoperative fever in univariable analysis, both were found to be independent predictors in multivariable analysis (OR: 10.6, 95% CI 4.07-27.9, p < 0.001 and OR: 15.0, 95% CI 5.4-41.2, p < 0.001, respectively).
CONCLUSIONS: In conclusion, RPUGS is as effective as RSC in predicting fever after PCNL. We recommend RPUGS during PCNL to manage post-PCNL infectious complications.