Identification of an endonuclease and N6-adenine methyltransferase from Ureaplasma parvum SV3F4 strain.

Here, we report a novel endonuclease and N6-adenine DNA methyltransferase (m6A methyltransferase) in the Ureaplasma parvum SV3F4 strain. Our previous study found that the SV3F4 strain carries 17 unique genes, which are not encoded in the two previously reported U. parvum serovar 3 strain, OMC-P162 and ATCC 700970. Of these 17 unique genes, UP3_c0261 and UP3_c0262, were originally annotated as encoding hypothetical proteins. Comparative genomics analyses more recently indicated they encode a Type II restriction endonuclease and an m6A methyltransferase, respectively. The UP3_c0261 and UP3_c0262 genes were individually expressed and purified in Escherichia coli. The UP3_c0261 recombinant protein showed endonuclease activity on the pT7Blue vector, recognizing and cleaving a GTNAC motif, resulting in a 5 base 5' extension. The UP3_c0261 protein digested a polymerase chain reaction (PCR) product harboring the GTNAC motif. The endonuclease UP3_c0261 was designated as UpaF4I. Treatment of the PCR product with the recombinant protein UP3_c0262 completely blocked the restriction enzyme activity of UpaF4I. Analysis of the treated PCR product harboring a modified nucleotide by UP3_c0262 with HPLC-MS/MS and MS/MS showed that UP3_c0262 was an m6A methyltransferase containing a methylated A residue in both DNA strands of the GTNAC motif. Whole genome methylation analysis of SV3F4 showed that 99.9 % of the GTNAC motif was m6A modified. These results suggest the UP3_c0261 and UP3_c0262 genes may act as a novel Type II restriction-modification system in the Ureaplasma SV3F4 strain.

Functional redundancy of ubiquitin-like sulfur-carrier proteins facilitates flexible, efficient sulfur utilization in the primordial archaeon Thermococcus kodakarensis.

Ubiquitin-like proteins (Ubls) in eukaryotes and bacteria mediate sulfur transfer for the biosynthesis of sulfur-containing biomolecules and form conjugates with specific protein targets to regulate their functions. Here, we investigated the functions and physiological importance of Ubls in a hyperthermophilic archaeon by constructing a series of deletion mutants. We found that the Ubls (TK1065, TK1093, and TK2118) in Thermococcus kodakarensis are conjugated to their specific target proteins, and all three are involved in varying degrees in the biosynthesis of sulfur-containing biomolecules such as tungsten cofactor (Wco) and tRNA thiouridines. TK2118 (named UblB) is involved in the biosynthesis of Wco in a glyceraldehyde 3-phosphate:ferredoxin oxidoreductase, which is required for glycolytic growth, whereas TK1093 (named UblA) plays a key role in the efficient thiolation of tRNAs, which contributes to cellular thermotolerance. Intriguingly, in the presence of elemental sulfur (S0) in the culture medium, defective synthesis of these sulfur-containing molecules in Ubl mutants was restored, indicating that T. kodakarensis can use S0 as an alternative sulfur source without Ubls. Our analysis indicates that the Ubl-mediated sulfur-transfer system in T. kodakarensis is important for efficient sulfur assimilation, especially under low S0 conditions, which may allow this organism to survive in a low sulfur environment.IMPORTANCESulfur is a crucial element in living organisms, occurring in various sulfur-containing biomolecules including iron-sulfur clusters, vitamins, and RNA thionucleosides, as well as the amino acids cysteine and methionine. In archaea, the biosynthesis routes and sulfur donors of sulfur-containing biomolecules are largely unknown. Here, we explored the functions of Ubls in the deep-blanched hyperthermophilic archaeon, Thermococcus kodakarensis. We demonstrated functional redundancy of these proteins in the biosynthesis of tungsten cofactor and tRNA thiouridines and the significance of these sulfur-carrier functions, especially in low sulfur environments. We propose that acquisition of a Ubl sulfur-transfer system, in addition to an ancient inorganic sulfur assimilation pathway, enabled the primordial archaeon to advance into lower-sulfur environments and expand their habitable zone.

No improvement in mortality among critically ill patients with carbapenems as initial empirical therapy and more detection of multi-drug resistant pathogens associated with longer use: a post hoc analysis of a prospective cohort study.

Whether empirical therapy with carbapenems positively affects the outcomes of critically ill patients with bacterial infections remains unclear. This study aimed to investigate whether the use of carbapenems as the initial antimicrobial administration reduces mortality and whether the duration of carbapenem use affects the detection of multidrug-resistant (MDR) pathogens. This was a post hoc analysis of data acquired from Japanese participating sites from a multicenter, prospective observational study [Determinants of Antimicrobial Use and De-escalation in Critical Care (DIANA study)]. A total of 268 adult patients with clinically suspected or confirmed bacterial infections from 31 Japanese intensive care units (ICUs) were analyzed. The patients were divided into two groups: patients who were administered carbapenems as initial antimicrobials (initial carbapenem group, n = 99) and those who were not administered carbapenems (initial non-carbapenem group, n = 169). The primary outcomes were mortality at day 28 and detection of MDR pathogens. Multivariate logistic regression analysis revealed that mortality at day 28 did not differ between the two groups [18 (18%) vs 27 (16%), respectively; odds ratio: 1.25 (95% confidence interval (CI): 0.59-2.65), P = 0.564]. The subdistribution hazard ratio for detecting MDR pathogens on day 28 per additional day of carbapenem use is 1.08 (95% CI: 1.05-1.13, P < 0.001 using the Fine-Gray model with death regarded as a competing event). In conclusion, in-hospital mortality was similar between the groups, and a longer duration of carbapenem use as the initial antimicrobial therapy resulted in a higher risk of detection of new MDR pathogens.IMPORTANCEWe found no statistical difference in mortality with the empirical use of carbapenems as initial antimicrobial therapy among critically ill patients with bacterial infections. Our study revealed a lower proportion of inappropriate initial antimicrobial administrations than those reported in previous studies. This result suggests the importance of appropriate risk assessment for the involvement of multidrug-resistant (MDR) pathogens and the selection of suitable antibiotics based on risk. To the best of our knowledge, this study is the first to demonstrate that a longer duration of carbapenem use as initial therapy is associated with a higher risk of subsequent detection of MDR pathogens. This finding underscores the importance of efforts to minimize the duration of carbapenem use as initial antimicrobial therapy when it is necessary.

Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized reagent for recombinase polymerase amplification.

BACKGROUND: Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility. METHODS: Our hypothesis centered on the C-terminal region of uvsY influencing solubility. To test this, we generated a site-saturation mutagenesis library for amino acid residues Lys91-Glu134 of the N-terminal (His)6-tagged uvsY. RESULTS: Screening 480 clones identified A116H as the variant with superior solubility. Lyophilized RPA reagents featuring the uvsY variant A116H demonstrated enhanced performance compared to those with wild-type uvsY. CONCLUSIONS: The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.

Expression of a recombinant protein by an acetic acid bacterial host.

We evaluated the suitability of Komagataeibacter europaeus, a vinegar production organism adept at synthetic media growth, as a host for heterologous gene expression. Cryptic plasmids (pGE1 and pGE2 derivatives) from K. europaeus strain KGMA0119 were employed as vectors for heterologous gene expression. The focus was placed on the groES promoter as a potential inducible switch. The groES promoter was fused with the EGFP gene and introduced into a pGE1 derivative to assess its suitability. Ethanol, acetic acid, and heat stresses were examined under various conditions for induction. EGFP transcription surged 600-fold when late logarithmic phase K. europaeus cells, cultured at 30 °C, endured heat stress at 40 °C, coupled with 20% acetic acid and 30% ethanol stress after an additional 6-hour cultivation. This robust induction system was then applied to express two proteins, Tth pol from the thermophilic bacterium Thermus thermophilus strain M1 and UPV230, a restriction enzyme from the acid-tolerant microorganism Ureaplasma parvum, known to cause vaginal infections and miscarriages. Both Tth pol and UPV230 were successfully expressed in K. europaeus cells and purified. The recovery of Tth pol from K. europaeus cells (480 µg protein per liter culture) was approximately half that from E. coli (960 µg protein per liter culture). In contrast, UPV230 recovery from K. europaeus cells (640 µg protein per liter culture) was nearly 10 times higher than that from Escherichia coli (66 µg protein per liter). The data highlights the potential of acetic acid bacteria as a host for producing acidophilic proteins. The shift in recognition from a 6-base sequence to a 4-base sequence of UPV230 was observed, accompanied by a change in structure as the pH transitioned from acidic pH to near-neutral pH.

Application of recombinant human pyruvate kinase in recombinase polymerase amplification.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells. RPA with PKM was performed at 41°C with the in vitro synthesized urease subunit ß (ureB) DNA from Ureaplasma parvum serovar 3 as a standard DNA. The optimal concentrations of PKM and phosphoenolpyruvate were 20 ng/µL and 10 mM, respectively. The RPA reaction with PKM was more sensitive than that with CK. PKM exhibited higher thermostability than CK, suggesting that the RPA reagents with PKM are preferable to those with CK for onsite use.

Recombinase polymerase amplification using novel thermostable strand-displacing DNA polymerases from Aeribacillus pallidus and Geobacillus zalihae.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), and strand-displacing DNA polymerase (Pol). Component instability and the need to store commercial kits in a deep freezer until use are some limitations of RPA. In a previous study, Bacillus stearothermophilus Pol (Bst-Pol) was used as a thermostable strand-displacing DNA polymerase in RPA. Here, we attempted to optimize the lyophilization conditions for RPA with newly isolated thermostable DNA polymerases for storage at room temperature. We isolated novel two thermostable strand-displacing DNA polymerases, one from a thermophilic bacterium Aeribacillus pallidus (H1) and the other from Geobacillus zalihae (C1), and evaluated their performances in RPA reaction. Urease subunit ß (UreB) DNA from Ureaplasma parvum serovar 3 was used as a model target for evaluation. The RPA reaction with H1-Pol or C1-Pol was performed at 41 °C with the in vitro synthesized standard UreB DNA. The minimal initial copy numbers of standard DNA from which the amplified products were observed were 600, 600, and 6000 copies for RPA with H1-Pol, C1-Pol, and Bst-Pol, respectively. Optimization was carried out using RPA components, showing that the lyophilized RPA reagents containing H1-Pol exhibited the same performance as the corresponding liquid RPA reagents. In addition, lyophilized RPA reagents with H1-Pol showed almost the same activity after two weeks of storage at room temperature as the freshly prepared liquid RPA reagents. These results suggest that lyophilized RPA reagents with H1-Pol are preferable to liquid RPA reagents for onsite use.

Immobilization of a Broad Range of Polypeptides on the Frustule of the Diatom Thalassiosira pseudonana.

Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. One recently developed technique, living diatom silica immobilization (LiDSI), has made it possible to immobilize proteins, including multimeric and redox enzymes, via a cellular excretion system onto the silica frustule of the marine diatom Thalassiosira pseudonana. However, the number of application examples so far is limited, and the type of proteins appropriate for the technique is still enigmatic. Here, we applied LiDSI to six industrially relevant polypeptides, including protamine, metallothionein, phosphotriesterase, choline oxidase, laccase, and polyamine synthase. Protamine and metallothionein were successfully immobilized on the frustule as protein fusions with green fluorescent protein (GFP) at the N terminus, indicating that LiDSI can be used for polypeptides which are rich in arginine and cysteine. In contrast, we obtained mutants for the latter four enzymes in forms without green fluorescent protein. Immobilized phosphotriesterase, choline oxidase, and laccase showed enzyme activities even after the purification of frustule in the presence of 1% (wt/vol) octylphenoxy poly(ethyleneoxy)ethanol. An immobilized branched-chain polyamine synthase changed the intracellular polyamine composition and silica nanomorphology. These results illustrate the possibility of LiDSI for industrial applications. IMPORTANCE Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. Living diatom silica immobilization (LiDSI) is a recently developed technique for in vivo protein immobilization on the diatom frustule. We aimed to explore the possibility of using LiDSI for industrial applications by successfully immobilizing six polypeptides: (i) protamine (Oncorhynchus keta), a stable antibacterial agent; (ii) metallothionein (Saccharomyces cerevisiae), a metal adsorption molecule useful for bioremediation; (iii) phosphotriesterase (Sulfolobus solfataricus), a scavenger for toxic organic phosphates; (iv) choline oxidase (Arthrobacter globiformis), an enhancer for photosynthetic activity and yield of plants; (v) laccase (Bacillus subtilis), a phenol oxidase utilized for delignification of lignocellulosic materials; and (vi) branched-chain polyamine synthase (Thermococcus kodakarensis), which produces branched-chain polyamines important for DNA and RNA stabilization at high temperatures. This study provides new insights into the field of applied biological materials.

Use of broad-spectrum antimicrobials for more than 72 h and the detection of multidrug-resistant bacteria in Japanese intensive care units: a multicenter retrospective cohort study.

BACKGROUND: Large multicenter studies reporting on the association between the duration of broad-spectrum antimicrobial administration and the detection of multidrug-resistant (MDR) bacteria in the intensive care unit (ICU) are scarce. We evaluated the impact of broad-spectrum antimicrobial therapy for more than 72 h on the detection of MDR bacteria using the data from Japanese patients enrolled in the DIANA study. METHODS: We analyzed the data of ICU patients in the DIANA study (a multicenter international observational cohort study from Japan). Patients who received empirical antimicrobials were divided into a broad-spectrum antimicrobial group and a narrow-spectrum antimicrobial group, based on whether they received broad-spectrum antimicrobials for more or less than 72 h, respectively. Differences in patient characteristics, background of infectious diseases and empirical antimicrobial administration, and outcomes between the two groups were compared using the chi-square tests (Monte Carlo method) for categorical variables and the Mann-Whitney U-test for continuous variables. We also conducted a logistic regression analysis to investigate the factors associated with the detection of new MDR bacteria. RESULTS: A total of 254 patients from 31 Japanese ICUs were included in the analysis, of whom 159 (62.6%) were included in the broad-spectrum antimicrobial group and 95 (37.4%) were included in the narrow-spectrum antimicrobial group. The detection of new MDR bacteria was significantly higher in the broad-spectrum antimicrobial group (11.9% vs. 4.2%, p = 0.042). Logistic regression showed that broad-spectrum antimicrobial continuation for more than 72 h (OR [odds ratio] 3.09, p = 0.047) and cerebrovascular comorbidity on ICU admission (OR 2.91, p = 0.041) were associated with the detection of new MDR bacteria. CONCLUSIONS: Among Japanese ICU patients treated with empirical antimicrobials, broad-spectrum antimicrobial usage for more than 72 h was associated with the increased detection of new MDR bacteria. Antimicrobial stewardship programs in ICUs should discourage the prolonged use of empirical broad-spectrum antimicrobial therapy. Trial registration ClinicalTrials.gov, NCT02920463, Registered 30 September 2016, https://clinicaltrials.gov/ct2/show/NCT02920463.

Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA.

BACKGROUND: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA. METHODS: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels. RESULTS: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs. CONCLUSIONS: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses.

Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of 'sample-in and result-out' with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/µL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/µL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/µL with a total assay time of 15-30 min.

Leucine-Responsive Regulatory Protein in Acetic Acid Bacteria Is Stable and Functions at a Wide Range of Intracellular pH Levels.

Acetic acid bacteria grow while producing acetic acid, resulting in acidification of the culture. Limited reports elucidate the effect of changes in intracellular pH on transcriptional factors. In the present study, the intracellular pH of Komagataeibacter europaeus was monitored with a pH-sensitive green fluorescent protein, showing that the intracellular pH decreased from 6.3 to 4.7 accompanied by acetic acid production during cell growth. The leucine-responsive regulatory protein of K. europaeus (KeLrp) was used as a model to examine pH-dependent effects, and its properties were compared with those of the Escherichia coli ortholog (EcLrp) at different pH levels. The DNA-binding activities of EcLrp and KeLrp with the target DNA (Ec-ilvI and Ke-ilvI) were examined by gel mobility shift assays under various pH conditions. EcLrp showed the highest affinity with the target at pH 8.0 (Kd [dissociation constant], 0.7 µM), decreasing to a minimum of 3.4 µM at pH 4.0. Conversely, KeLrp did not show significant differences in binding affinity between pH 4 and 7 (Kd, 1.0 to 1.5 µM), and the highest affinity was at pH 5.0 (Kd, 1.0 µM). Circular dichroism spectroscopy revealed that the α-helical content of KeLrp was the highest at pH 5.0 (49%) and was almost unchanged while being maintained at >45% over a range of pH levels examined, while that of EcLrp decreased from its maximum (49% at pH 7.0) to its minimum (36% at pH 4.0). These data indicate that KeLrp is stable and functions over a wide range of intracellular pH levels. IMPORTANCE Lrp is a highly conserved transcriptional regulator found in bacteria and archaea and regulates transcriptions of various genes. The intracellular pH of acetic acid bacteria (AAB) changes accompanied by acetic acid production during cell growth. The Lrp of AAB K. europaeus (KeLrp) was structurally stable over a wide range of pH and maintained DNA-binding activity even at low pH compared with Lrp from E. coli living in a neutral environment. An in vitro experiment showed DNA-binding activity of KeLrp to the target varied with changes in pH. In AAB, change of the intracellular pH during a cell growth would be an important trigger in controlling the activity of Lrp in vivo.

Rapid response system in Japanese outpatient departments based on online registry: Multicentre observational study.

AIM: The rapid response system (RRS) has become well known as a patient safety system to reduce adverse in-patient events, and it is also required to respond to patients in the outpatient department. However, only few studies have reported on the RRS in the outpatient department. We analysed the current status of the RRS in the outpatient department based on a multicentre online registry in Japan. METHODS: This is a prospective multicentre observational study. Among the cases registered in the RRS online registry from January 2014 to March 2018, cases from the outpatient department, consisting of the general outpatient department, radiation department, dialysis department, endoscope department, rehabilitation department, and the surrounding areas were eligible for this study. RESULTS: A total of 6784 cases were registered, and 1022 cases were included. The main reason for activation was altered mental status (39.1%). Incomplete vital sign recording at activation was 67.0%, whereas body temperature (57.0%) and respiratory rate (36.4%) deficits were frequent. The most common intervention during RRS activation was fluid bolus (38.2%) and oxygen supplementation (30.9%). The general outpatient department accounted for nearly half of the activation locations. The 30-day mortality rate for the location was significantly higher in the dialysis department (P < 0.001). CONCLUSIONS: We have reported the first study of RRSs in outpatient departments at multicentre facilities in Japan. The difference in the mortality rate for the location was clarified. Future tasks will involve clarifying the RRS outcome indicators in the outpatient department and examining the effectiveness thereof.

Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection.

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method's sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method.

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.

The characteristics, types of intervention, and outcomes of postoperative patients who required rapid response system intervention: a nationwide database analysis.

PURPOSE: Improving the safety of general wards is a key to reducing serious adverse events in the postoperative period. We investigated the characteristics, treatment, and outcomes of postoperative patients managed by a rapid response system (RRS) in Japan to improve postoperative management. METHODS: This retrospective study analyzed cases requiring RRS intervention that were included in the In-Hospital Emergency Registry in Japan. We analyzed data reported by 34 Japanese hospitals between January 2014 and March 2018, mainly focusing on postoperative patients for whom the RRS was activated within 7 days of surgery. Non-postoperative patients, for whom the RRS was activated in all other settings, were used for comparison as necessary. RESULTS: There were 609 (12.7%) postoperative patients among the total patients in the registry. The major criteria were staff concerns (30.2%) and low oxygen saturation (29.7%). Hypotension, tachycardia, and inability to contact physicians were observed as triggers significantly more frequently in postoperative patients when compared with non-postoperative patients. Among RRS activations within 7 days of surgery, 68.9% of activations occurred within postoperative day 3. The ordering of tests (46.8%) and fluid bolus (34.6%) were major interventions that were performed significantly more frequently in postoperative patients when compared with non-postoperative patients. The rate of RRS activations resulting in ICU care was 32.8%. The mortality rate at 1 month was 16.2%. CONCLUSION: Approximately, 70% of the RRS activations occurred within postoperative day 3. Circulatory problems were a more frequent cause of RRS activation in the postoperative group than in the non-postoperative group.

Solvent engineering studies on recombinase polymerase amplification.

Recombinase polymerase amplification (RPA) is a technique that is used to specifically amplify a target nucleic acid sequence. Unlike the polymerase chain reaction (PCR), RPA is performed at a constant temperature between 37 and 42°C. Therefore, it can be potentially used for the onsite detection of various pathogens when combined with DNA extraction and amplicon detection techniques. In this study, we prepared recombinant recombinase and single-stranded DNA-binding protein from T4 phage and used them to examine the effects of reaction conditions and additives on the efficiency of RPA. The results revealed that the optimal pH was 7.5-8.0, optimal potassium acetate concentration was 40-80 mM, and optimal reaction temperature was 37-45°C although dimethyl sulfoxide at 5% v/v and formamide at 5% v/v inhibited the reaction. Our results suggest that RPA could be conducted using a wider range of optimal reaction conditions than those required for PCR and that RPA is highly suitable for point-of-care use.

Alteration of enzymes and their application to nucleic acid amplification (Review).

Since the discovery of polymerase chain reaction (PCR) in 1985, several methods have been developed to achieve nucleic acid amplification, and are currently used in various fields including clinical diagnosis and life science research. Thus, a wealth of information has accumulated regarding nucleic acid­related enzymes. In this review, some nucleic acid­related enzymes were selected and the recent advances in their modification along with their application to nucleic acid amplification were described. The discussion also focused on optimization of the corresponding reaction conditions. Using newly developed enzymes under well­optimized reaction conditions, the sensitivity, specificity, and fidelity of nucleic acid tests can be improved successfully.