[A Case of Intestinal Lymphoma with Two Perforation in a Short Period Time].

An 86 years old man was diagnosed with jejunum tumor by detail examination of anemia. Eight days after endoscopy he developed abdominal pain, an emergency surgery was performed by a diagnosis of gastrointestinal perforation. They detected perforation in jejunum and performed jejunum resection, small intestinal lymphoma was diagnosed by histopathological examination. We were talked about chemotherapy against lymphoma, he was transferred at our hospital. The first chemotherapy was provided, abdominal pain appeared at 2 days after chemotherapy. Computed tomography revealed air bubbles out of intestinal tract, we performed emergency operation. A perforation of jejunum in the part where is different from the last time was existed, we performed jejunum resection. Chemotherapy was performed postoperatively, resulted in successful progress. Lymphoma of gastrointestinal tract often causes intestinal perforations, but reports that perforations heterochrony in separate parts multiple tumors of intestine is rare. We report a case of intestinal lymphoma with 2 perforations in a short period time.

[A Case of Small Cell Carcinoma of Esophagus in Which Long-Term Survival Was Achieved with Multimodal Treatment].

A 79-year-old man presented to our hospital with 1-month history of discomfort on swallowing and pain in the thoracodorsal region. Image investigation revealed a tumor lesion affecting the area from the lower esophagus to the gastric antrum, and small cell carcinoma of esophagus was diagnosed based on biopsy. He was treated with resection of the lower esophagus and the upper part of the stomach and a postoperative chemoradiotherapy. The patient follow-upwas without recurrence until a malignant lymphoma developed 4 years and 5 months after the surgery. Chemotherapy was provided for the malignant lymphoma, however, the patient died 6 years and 4 months after the surgery for small cell carcinoma of esophagus. Small cell endocrine carcinoma of the esophagus is a relatively rare disease and its prognosis is poor. In our patient, long-term survival was achieved with multimodal treatment, however malignant lymphoma developed during the follow-upp eriod. This is the second case of metachronous cancer after the treatment for small cell carcinoma of the esophagus in Japan, and it is considered to be extremely rare.

Auroral molecular-emission effects on the atomic oxygen line at 777.4 nm.

One of the representative auroral emission lines that radiates from F-region heights and is measurable on the ground is the 777.4 nm line from excited atomic oxygen. This line has been adopted, along with another E-region emission line, for example 427.8 nm, to estimate the mean energy and total energy flux of precipitating auroral electrons. The influence of emissions from part of the molecular nitrogen band, which mainly radiate from E-region heights, should be carefully evaluated because it might overlap the 777.4 nm atomic oxygen line in the spectrum. We performed statistical analysis of auroral spectrograph measurements that were obtained during the winter of 2016-2017 in Tromsø, Norway, to derive the ratio of the intensity of the 777.4 nm atomic oxygen line to that of the net measurement through a typically used optical filter with a full width at half maximum of a few nm. The ratio had a negative trend against geomagnetic activity, with a primary distribution of 0.5-0.7 and a minimum value of 0.3 for the most active auroral condition in this study. This result suggests that the 30-50% emission intensities measured through the optical filter may be from the molecular nitrogen band.

Proposal of the Coagulation Score as a Predictor for Short-Term and Long-Term Outcomes of Patients with Resectable Gastric Cancer.

BACKGROUND: Systemic hemostasis and thrombosis activation has been implicated in tumor progression and metastasis. This study aimed to investigate the use of coagulation factors as a novel prediction method for postoperative outcomes after curative gastrectomy in patients with stage II/III gastric cancer (GC). METHODS: Overall, 126 patients with stage II/III GC who underwent gastrectomy between May 2003 and February 2016 were eligible for inclusion in the study. We retrospectively evaluated the predictive value of preoperative platelet count and plasma fibrinogen and d-dimer levels, and coagulation score (0: fibrinogen and d-dimer both below upper limits; 1: either fibrinogen or d-dimer over upper limits; 2: both fibrinogen and d-dimer over upper limits) for short- and long-term outcomes. RESULTS: Postoperative complications were significantly more frequent in patients with elevated preoperative d-dimer levels compared with those with normal d-dimer levels (26 vs. 10 %; p = 0.032). The prevalence of postoperative complications showed a stepwise increase in proportion to the coagulation score. Patients with a coagulation score of 2 had significantly larger tumors (p = 0.013) and significantly greater intraoperative blood loss (p = 0.004) than those who scored 0 or 1. Coagulation score showed the highest values distinguished high-risk patients in overall and disease-free survival, and a coagulation score of 2 was an independent prognostic factor for recurrence. Patients with a coagulation score of 2 experienced a significantly higher prevalence of liver metastasis as an initial recurrence than those who scored 0 or 1 (p = 0.019). CONCLUSIONS: The coagulation score is a simple and promising predictor for postoperative complications and recurrence after gastrectomy in stage II/III GC patients.

From Peptides to Peptidomimetics: A Strategy Based on the Structural Features of Cyclopropane.

Peptidomimetics, non-natural mimicries of bioactive peptides, comprise an important class of drug molecules. The essence of the peptidomimetic design is to mimic the key conformation assumed by the bioactive peptides upon binding to their targets. Regulation of the conformation of peptidomimetics is important not only to enhance target binding affinity and selectivity, but also to confer cell-membrane permeability for targeting protein-protein interactions in cells. The rational design of peptidomimetics with suitable three-dimensional structures is challenging, however, due to the inherent flexibility of peptides and their dynamic conformational changes upon binding to the target biomolecules. In this Minireview, a three-dimensional structural diversity-oriented strategy based on the characteristic structural features of cyclopropane to address this challenging issue in peptidomimetic chemistry is described.

Cyclopropane-Based Peptidomimetics Mimicking Wide-Ranging Secondary Structures of Peptides: Conformational Analysis and Their Use in Rational Ligand Optimization.

Detailed conformational analyses of our previously reported cyclopropane-based peptidomimetics and conformational analysis-driven ligand optimization are described. Computational calculations and X-ray crystallography showed that the characteristic features of cyclopropane function effectively to constrain the molecular conformation in a three-dimensionally diverse manner. Subsequent principal component analysis revealed that the diversity covers the broad chemical space filled by peptide secondary structures in terms of both main-chain and side-chain conformations. Based on these analyses, a lead stereoisomer targeting melanocortin receptors was identified, and its potency and subtype selectivity were improved by further derivatization. The presented strategy is effective not only for designing non-peptidic ligands from a peptide ligand but also for the rational optimization of these ligands based on the plausible target-binding conformation without requiring the three- dimensional structural information of the target and its peptide ligands.

Usefulness of preoperative estimated glomerular filtration rate to predict complications after curative gastrectomy in patients with clinical T2-4 gastric cancer.

BACKGROUND: Gastrectomy with systemic lymphadenectomy is the standard of care for resectable gastric cancer (GC), but it is sometimes associated with postoperative morbidity. Predicting complications is therefore an essential part of risk management in clinical practice. The renal function is routinely evaluated before surgery by blood examinations to determine dose of medication and infusion. However, the value of various parameters of renal function in prediction of postoperative complications remain unclear. METHODS: We included 315 patients who underwent curative D2 gastrectomy for clinical T2-T4 GC without preoperative treatment, and evaluated the correlation between the incidence of postoperative complications and the indicators of renal function. RESULTS: Forty-three patients experienced clinically relevant postoperative complications. Estimated glomerular filtration rate (eGFR) showed a higher area under the curve for predicting complications compared with urea nitrogen, creatinine, and creatinine clearance. The optimal eGFR cutoff value was 63.2 ml/min/1.73 m2, and eGFR < 63.2 was an independent risk factor for postoperative complications in multivariable analysis (odds ratio 4.67; 95 % confidence interval 2.16-10.5; p < 0.001). Particularly, the incidence of anastomotic leakage was significantly higher in patients with eGFR < 63.2 than those with eGFR ≥ 63.2 (9.4 % vs. 3.5 %). eGFR < 63.2 was also associated with a higher incidence of postoperative complications independent of age, body mass index, operative procedure, and clinical disease stage. Postoperative hospital stay was significantly longer in the eGFR < 63.2 group. CONCLUSIONS: Preoperative eGFR is a simple and useful predictor for complications after gastrectomy in patients with GC and may improve clinical care and the process of obtaining informed consent.

Destruction of biological particles using non-thermal plasma.

Mechanism of inactivation of bio-particles exposed to non-thermal plasma (NTP), namely, dielectric barrier discharge (DBD), and plasma jet (PJ), has been studied using E. coli, B. subtilis spore, S. cerevisiae and bacteriophages. States of different biological components were monitored during the course of inactivation. Analysis of green fluorescent protein, GFP, introduced into E. coli. or B. subtiles spore cells proved that radicals generated by NTP penetrate into microbes, destroying the cell membrane and finally damage the genes. We have evaluated the damage of the bacteriophages. Bacteriophage λ having double stranded DNA was exposed to DBD, then DNA was purified and subjected to in vitro DNA packaging reactions. The re-packaged phages consist of the DNA from discharged phages and brand-new coat proteins were proved to be active, indicating that the damage of coat proteins is responsible for inactivation. M13 phages having single stranded DNA were also examined with the same manner. In this case, damage to the DNA was as severe as that of the coat proteins. For practical applications, DBD showed very intense sterilization ability for B. Subtilis spore with the D-value of less than 10 s. This result indicates a possibility of application of NTP for quick sterilization.

Tumor Infiltrative Pattern Predicts Sites of Recurrence After Curative Gastrectomy for Stages 2 and 3 Gastric Cancer.

BACKGROUND: In East Asia, the tumor infiltrative pattern (INF) has been routinely evaluated by hematoxylin and eosin-stained sections as a pathologic characteristic of surgically resected specimens. METHODS: The infiltrative pattern of gastric cancer (GC) has been histopathologically classified as INFa (expansive growth), INFb (intermediate type) and INFc (infiltrative growth) according to the Japanese Classification of Gastric Carcinoma. The prognostic value and characteristics of the disease recurrence pattern for each INF type were assessed in 785 patients with various stages of GC and also in 243 patients with stages 2 and 3 GC. RESULTS: Comparison of the overall survival experienced by patients independently of stage showed that INF was significantly associated with prognosis. Specifically, peritoneal metastasis was present in 91 % of stage 4 patients in the INFc group, whereas hepatic metastasis was present in 39 % of stage 4 patients in the INFa and INFb group. After curative gastrectomy of patients with stages 2 or 3 GC, INF was not significantly associated with survival. The prevalence of peritoneal recurrence was significantly higher in the INFc group than in the INFa and INFb group, whereas the prevalence of hepatic recurrence was significantly higher in the INFa and INFb group than in the INFc group. Multivariate analysis identified INFc as an independent risk factor for peritoneal recurrence after curative gastrectomy. The association of the INF type with the incidence of peritoneal recurrence was observed with all disease stages regardless whether the patient was given adjuvant chemotherapy or not. CONCLUSIONS: Evaluation of the INF type shows promise for its role as a predictor of postoperative recurrence sites in patients with GC.

Predictive value of drain amylase content for peripancreatic inflammatory fluid collections after laparoscopic (assisted) distal gastrectomy.

BACKGROUND: Laparoscopic (assisted) distal gastrectomy (LDG) with radical lymphadenectomy for gastric cancer has been widely conducted, particularly in the Far East. Peripancreatic inflammatory fluid collection (PIFC) is a serious and frequent postoperative complication after LDG for gastric cancer. The aim of this study was to evaluate the diagnostic performance of drain amylase content (D-AMY) for clinically relevant PIFC after LDG. METHODS: Two hundred and sixty-four patients who underwent LDG with prophylactic drains were enrolled. The predictive value of D-AMY on postoperative day (POD) 1 and POD 3 in the diagnosis of PIFC was evaluated. RESULTS: Twenty (7.6 %) patients experienced postoperative PIFC. Area under the curve in terms of receiver operating characteristics curve analysis of D-AMY on POD 1 was 0.801, and the optimal cutoff value for prediction of PIFC was 904 IU/l, with 98.2 % negative predictive value. Another cutoff was proposed as 4078 IU/l, with 92.2 % specificity. Multivariable analyses identified D-AMY on POD 1 ≥900 and ≥4000 IU/l as independent diagnostic factors for PIFC. Among patients at high risk of PIFC (D-AMY on POD 1 ≥900 IU/l), those who on POD 3 retained D-AMY value in excess of 31.2 % of the D-AMY value on POD 1 were more likely to experience PIFC compared with those with a pronounced decrease in D-AMY. CONCLUSIONS: D-AMY on POD 1 serves as a predictive factor for clinically relevant PIFC after LDG. Time-dependent changes in D-AMY can also be used for determining management of drains in patients at high risk of PIFC.

Adverse Effects of Intraoperative Blood Loss on Long-Term Outcomes after Curative Gastrectomy of Patients with Stage II/III Gastric Cancer.

BACKGROUND/AIMS: Gastrectomy with systemic lymphadenectomy sometimes causes excessive bleeding even by experienced surgeons. The aim of this study was to evaluate how intraoperative estimated blood loss (EBL) affected the long-term outcomes after curative surgery of patients with stage II/III gastric cancer (GC). METHODS: This study included 203 patients with stage II/III GC who did not receive perioperative blood transfusion between 1999 and 2015. The optimal cutoff and the prognostic significance of EBL were determined retrospectively. RESULTS: The median EBL was 285 ml. Receiver operating characteristic curve analysis identified 400 ml as an optimal cutoff. Patients with EBL ≥400 ml were more likely to have hepatic relapse and worse prognosis compared to those with EBL <400 ml. EBL ≥400 ml was identified as an independent prognostic factor for mortality by multivariable analysis. When patients were subdivided according to administration of adjuvant chemotherapy, there was a significant difference between the EBL ≥400 and <400 ml groups in patients who underwent surgery alone, whereas the prognosis was similar for patients of both groups who received adjuvant chemotherapy. CONCLUSION: EBL serves as a useful predictor for risk stratification after curative gastrectomy in patients with stage II/III GC.

Direct single-molecule observations of local denaturation of a DNA double helix under a negative supercoil state.

Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA.

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel.

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.

A new direct single-molecule observation method for DNA synthesis reaction using fluorescent replication protein A.

Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (-flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (-flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (-flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (-flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3'-5'exo-) was promoted by DNA stretching with buffer flow.

[Bacteria isolated from surgical infections and its susceptibilities to antimicrobial agents--special references to bacteria isolated between April 2010 and March 2011].

Bacteria isolated from surgical infections during the period from April 2010 to March 2011 were investigated in a multicenter study in Japan, and the following results were obtained. In this series, 631 strains including 25 strains of Candida spp. were isolated from 170 (81.7%) of 208 patients with surgical infections. Four hundred and twenty two strains were isolated from primary infections, and 184 strains were isolated from surgical site infection. From primary infections, anaerobic Gram-negative bacteria were predominant, followed by aerobic Gram-negative bacteria, while from surgical site infection aerobic Gram-positive bacteria were predominant, followed by anaerobic Gram-negative bacteria. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus spp. such as Enterococcus faecalis, Enterococcus faecium, and Enterococcus avium was highest, followed by Streptococcus spp. such as Streptococcus anginosus and Staphylococcus spp. such as Staphylococcus aureus, in this order, from primary infections, while Enterococcus spp. such as E. faecalis and E. faecium was highest, followed by Staphylococcus spp. such as S. aureus from surgical site infection. Among aerobic Gram-negative bacteria, Escherichia coli was the most predominantly isolated from primary infections, followed by Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Pseudomonas aeruginosa in this order, and from surgical site infection, E. coli and R aeruginosa were most predominantly isolated, followed by E. cloacae and K. pneumoniae. Among anaerobic Gram-positive bacteria, the isolation rates of Parvimonas micra, Eggerthella lenta, Streptococcus constellatus, Gemella morbillorum, and Collinsella aerofaciens were the highest from primary infections, and the isolation rate from surgical site infection was generally low. Among anaerobic Gram-negative bacteria, the isolation rate of Bilophila wadsworthia was the highest from primary infections, followed by, Bacteroides fragilis and Bacteroides ovatus, and from surgical site infection, B. fragilis was most predominantly isolated, followed by Bacteroides thetaiotaomnicron, in this order. In this series, vancomycin-resistant MRSA (methicillin-resistant S. aureus), vancomycin-resistant Enterococcus spp. and multidrug-resistant P. aeruginosa were not observed.

[Bacteria isolated from surgical infections and its susceptibilities to antimicrobial agents - Special references to bacteria isolated between April 2011 and March 2012].

Bacteria isolated from surgical infections during the period from April 2011 to March 2012 were investigated in a multicenter study in Japan, and the following results were obtained. In this series, 785 strains including 31 strains of Candida spp. were isolated from 204 (78.8%) of 259 patients with surgical infections. Five hundred and twenty three strains were isolated from primary infections, and 231 strains were isolated from surgical site infection. From primary infections, anaerobic Gram-negative bacteria were predominant, followed by aerobic Gram-negative bacteria, while from surgical site infection aerobic Gram-positive bacteria were predominant, followed by anaerobic Gram-negative bacteria. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus spp. was highest, followed by Streptococcus spp. and Staphylococcus spp., in this order, from primary infections, while Enterococcus spp. was highest, followed by Staphylococcus spp. from surgical site infection. Among aerobic Gram-negative bacteria, Escherichia coli was the most predominantly isolated from primary infections, followed by Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterobacter cloacae, in this order, and from surgical site infection, E. coli was most predominantly isolated, followed by P. aeruginosa, K. pneumoniae, and E. cloacae. Among anaerobic Gram-positive bacteria, the isolation rate of Eggerthella lenta was the highest from primary infections, followed by Parvimonas micra, Collinsella aerofaciens, Lactobacillus acidophilus and Finegoldia magna, and from surgical site infection, E. lenta was most predominantly isolated, followed by P micra and L. acidophilus, in this order. Among anaerobic Gram-negative bacteria, the isolation rate of Bacteroidesfragilis was the highest from primary infections, followed by Bilophila wadsworthia, Bacteroides thetaiotaomicron, Bacteroides uniformis and Bacteroides vulgatus, and from surgical site infection, B. fragilis was most predominantly isolated, followed by Bacteroides caccae, B. thetaiotaomicron, Bacteroides ovatus and B. wadsworthia, in this order. In this series, vancomycin-resistant MRSA (methicillin-resistant Staphylococcus aureus), vancomycin-resistant Enterococcus spp. and multidrug-resistant P. aeruginosa were not observed. We should carefully follow up B. wadsworthia which was resistant to various antimicrobial agents, and also Bacteroides spp. which was resistant to many ß-lactams.

GRL-04810 and GRL-05010, difluoride-containing nonpeptidic HIV-1 protease inhibitors (PIs) that inhibit the replication of multi-PI-resistant HIV-1 in vitro and possess favorable lipophilicity that may allow blood-brain barrier penetration.

We designed, synthesized, and identified two novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-04810 and GRL-05010, containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, bis-tetrahydrofuranylurethane (bis-THF), and a difluoride moiety, both of which are active against the laboratory strain HIV-1LAI (50% effective concentrations [EC50s], 0.0008 and 0.003 µM, respectively) with minimal cytotoxicity (50% cytotoxic concentrations [CC50s], 17.5 and 37.0 µM, respectively, in CD4(+) MT-2 cells). The two compounds were active against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to various antiviral regimens. GRL-04810 and GRL-05010 also blocked the infectivity and replication of each of the HIV-1NL4-3 variants selected by up to 5 µM lopinavir (EC50s, 0.03 and 0.03 µM, respectively) and atazanavir (EC50s, 0.02 and 0.04 µM, respectively). Moreover, they were active against darunavir (DRV)-resistant variants (EC50 in 0.03 to 0.034 µM range for GRL-04810 and 0.026 to 0.043 µM for GRL-05010), while DRV had EC50s between 0.02 and 0.174 µM. GRL-04810 had a favorable lipophilicity profile as determined with the partition (log P) and distribution (log D) coefficients of -0.14 and -0.29, respectively. The in vitro blood-brain barrier (BBB) permeability assay revealed that GRL-04810 and GRL-05010 may have a greater advantage in terms of crossing the BBB than the currently available PIs, with apparent penetration indexes of 47.8 × 10(-6) and 61.8 × 10(-6) cm/s, respectively. The present data demonstrate that GRL-04810 and GRL-05010 exert efficient activity against a wide spectrum of HIV-1 variants in vitro and suggest that two fluorine atoms added to their bis-THF moieties may well enhance their penetration across the BBB.

Preparation of chiral bromomethylenecyclopropane and its use in Suzuki-Miyaura coupling: synthesis of the arylmethyl-(Z)-cyclopropane structure core.

A preparative method for an optically active bromomethylenecyclopropane unit and its practical conversion to (Z)-cyclopropane-containing chiral compounds via Suzuki-Miyaura coupling were established.

Direct observation of fluorescently labeled single-stranded λDNA molecules in a micro-flow channel.

We developed two labeling methods for the direct observation of single-stranded DNA (ssDNA), using a ssDNA binding protein and a ssDNA recognition peptide. The first approach involved protein fusion between the 70-kDa ssDNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). The second method used the ssDNA binding peptide of Escherichia coli RecA labeled with Atto488 (ssBP-488; Atto488-IRMKIGVMFGNPETTTGGNALKFY). The labeled ssλDNA molecules were visualized over time in micro-flow channels. We report substantially different dynamics between these two labeling methods. When ssλDNA molecules were labeled with RPA-YFP, terminally bound fusion proteins were sheared from the free ends of the ssλDNA molecules unless 25-mer oligonucleotides were annealed to the free ends. RPA-YFP-ssλDNA complexes were dissociated by the addition of 0.2 M NaCl, although complex reassembly was possible with injection of additional RPA-YFP. In contrast to the flexible dynamics of RPA-YFP-ssλDNA complexes, the ssBP-488-ssλDNA complexes behaved as rigid rods and were not dissociated even in 2 M NaCl.

Novel Virus Air Sampler Based on Electrostatic Precipitation and Air Sampling of SARS-CoV-2.

The assessment of airborne viruses in air is a critical step in the design of appropriate prevention and control measures. Hence, herein, we developed a novel wet-type electrostatic air sampler using a viral dissolution buffer containing a radical scavenging agent, and verified the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the air of hospital rooms inhabiting coronavirus disease 2019 (COVID-19) patients and public areas. RNA damage caused by corona discharge was negligible when Buffer AVL was used as the collecting electrode. The viral RNA concentration in the air of the room varied by patient: 3.9 × 103 copy/m3 on the 10th day after onset in a mild case and 1.3 × 103 copy/m3 on the 18th day in a severe case. Viral RNA levels were 7.8 × 102 and 1.9 × 102 copy/m3 in the air of the office and food court, respectively, where people removed their masks when eating and talking, but it remained undetected in the station corridor where all the people were wearing masks. The assessment of airborne SARS-CoV-2 RNA using the proposed sampler can serve as a basis for the safe discontinuation of COVID-19 isolation precautions to identify exposure hotspots and alert individuals at increased infection risks.