Isothermal nucleic-acid amplification relies on strand-displacing DNA polymerases that synthesize DNA while unwinding duplex templates. We previously identified two thermostable family-A polymerases, C1-Pol from Geobacillus zalihae and H1-Pol from Aeribacillus pallidus, which show a characteristic and reproducible trade-off between stability and activity. C1-Pol is more thermostable, whereas H1-Pol exhibits stronger strand-displacement activity and higher recombinase polymerase amplification (RPA) efficiency. Domain-swapping analysis indicated that a chimeric construct (C1-Pol#4), in which the C1-Pol 5'→3' exonuclease region was replaced with that of H1-Pol, enhanced strand-displacement and nucleotide-incorporation activities while maintaining parental-level stability, suggesting that this region modulates these important biochemical properties. However, C1-Pol#4 did not improve RPA efficiency. To clarify the functional contribution of this domain, we constructed Klenow-type deletion mutants lacking the 5'→3' exonuclease region. The H1-Pol large fragment (H1-PolLF) exhibited reduced thermostability but markedly enhanced polymerase and strand-displacement activities at 40 °C, the optimal temperature for RPA reactions. At this temperature, H1-PolLF showed ∼10-fold higher strand-displacement activity than Bst DNA polymerase 2.0 and 4.4-fold higher than full-length H1-Pol. H1-PolLF also accelerated RPA, producing equivalent amplicons in half the reaction time and improving detection sensitivity 100-fold (6 × 10³ → 6 × 10¹ copies) than parental H1-Pol. Coupling H1-PolLF with branched-chain polyamine (BCPA)-conjugated magnetic beads enabled reliable detection of ∼10² target molecules from 10 mL saline. These findings demonstrate that removing the 5'→3' exonuclease domain fine-tunes polymerase and strand-displacing functions, yielding an enzyme highly suited for rapid, ultrasensitive isothermal nucleic-acid detection when used in combination with BCPA beads.

Branched-chain polyamines (BCPAs), exemplified by N⁴-bis(aminopropyl)spermidine, are distinctive polycations that occur predominantly in thermophilic bacteria and euryarchaeal archaea. Their dedicated aminopropyltransferase, BpsA (EC 2.5.1.128), extends spermidine into branched architectures via sequential decarboxylated S-adenosylmethionine (dcSAM)-dependent reactions. Accumulated evidence demonstrates that BCPAs engage nucleic acids with substantially higher affinity than linear polyamines such as spermidine, and they uniquely induce strong DNA compaction accompanied by B→A→C structural transitions. These interactions greatly enhance the resistance of DNA to thermal, chemical, and physical damage. Genetic and physiological analyses in Thermococcus kodakarensis further show that loss of BCPA biosynthesis compromises growth at very high temperatures, disrupts temperature- and membrane-associated stress responses, and alters transcriptional and translational regulation; intriguingly, the linear tetraamine thermospermine can partially substitute for BCPA in several of these functions. Beyond cellular physiology, immobilized BCPAs enable sensitive nucleic-acid capture and direct PCR and isothermal DNA amplification from highly dilute solutions, demonstrating their potential utility in molecular diagnostics and environmental DNA workflows. This review synthesizes current knowledge of BCPA distribution, biosynthesis, structure–function relationships, cellular roles, and emerging biotechnological applications, and highlights key open questions in the field.

Agmatine, a natural polyamine generated from arginine by arginine decarboxylase (ADC), has attracted increasing attention because of its pleiotropic beneficial effects on neuroprotection, lifestyle-related diseases, and gut-brain axis-mediated pathways. Although mammals appear to possess only limited capacity to synthesize endogenous agmatine, accumulating evidence suggests that agmatine derived from diet and the gut microbiota contributes to systemic levels of this polyamine. Previous studies have revealed that Aspergillus oryzae, the filamentous fungus foundational to traditional Japanese cuisine and indispensable for starch saccharification in sake, miso, soy sauce, mirin, and other fermented foods, produces high levels of agmatine specifically under solid-state cultivation. Subsequent studies identified a novel pyruvoyl-dependent ADC (Ao-ADC1) responsible for this unique agmatine production. This mini-review summarizes current knowledge on solid-state cultivation-specific agmatine production by A. oryzae, with a particular focus on the discovery and biochemical characteristics of Ao-ADC1. These findings challenge the commonly accepted notion that ascomycetes lack ADC. Understanding the molecular rationale and physiological significance of this unique agmatine biosynthetic pathway provides a foundation for rational strategies to enhance agmatine production in A. oryzae and for the development of agmatine-enriched fermented foods and nutraceuticals. Furthermore, integrating this fungal pathway with emerging insights into microbe-host interactions may further illuminate how fermentation-derived agmatine contributes to human health through gut-brain axis mediated mechanisms.

ABSTRACT N 4 -bis(aminopropyl)spermidine (BCPA), a branched-chain polyamine, is uniquely found in bacterial and archaeal hyperthermophiles. In Thermococcus kodakarensis , BCPA is synthesized by BCPA synthase (BpsA), an aminopropyl transferase encoded by bpsA . This highly positively charged molecule is localized in both the nucleic acid and membrane fractions of T. kodakarensis cells. The bpsA deletion strain (DBP1), which lacks BCPA, failed to grow at 93°C and exhibited poor survival under repeated cold stress, indicating that BCPA is essential for membrane stability and function in vivo . Additionally, the expression of specific genes, including the cytoplasmic hydrogenase subunit hyhL , was absent in DBP1, suggesting a role for BCPA in gene regulation. To further investigate BCPA’s function, we replaced bpsA in T. kodakarensis with speE from the hyperthermophilic archaeon Pyrobaculum calidifontis , enabling the production of norspermine instead of BCPA. The resulting KPS strain accumulated thermospermine as its major polyamine. Growth at 93°C was partially restored in KPS, and cold-stress survival improved significantly. Additionally, KPS exhibited biosurfactant (sophorolipid) tolerance comparable to that of the parental T. kodakarensis strain KU216 under thermal conditions. Furthermore, hyhL expression was restored in KPS, as confirmed by immunoblotting with anti-HyhL antisera, suggesting that thermospermine can functionally compensate for BCPA. Notably, mutant DBP1 cells lacking both BCPA and thermospermine did not survive repeated cycles of cold and heat stress. This observation suggests that these polyamines play a crucial role in long-term survival, potentially facilitating hibernation-like states in natural environments where extreme temperature fluctuations occur. IMPORTANCE At the hot springs of Kodakarajima Island, surrounded by cold ocean water, diverse hyperthermophiles, including Thermococcus, Thermotoga, and Thermus species, naturally produce branched-chain polyamines (BCPAs) via a unique aminopropyltransferase BpsA, in addition to spermidine. In Pyrobaculum calidifontis , the Pc- SpeE enzyme produces norspermine in vivo . However, when the speE gene from P. calidifontis is introduced into Thermococcus kodakarensis , the transformant (Δ bpsA::Pc-speE ) produces thermospermine instead of norspermine. This shift suggests that the product specificity of Pc- SpeE is influenced by factors inherent to the host organism. Interestingly, thermospermine appears to functionally substitute for BCPA, potentially by forming BCPA-like structures with bent nitrogen atoms. This structural mimicry could contribute to cellular stability under both heat and cold stress, highlighting a potential mechanism for temperature and stress adaptation in T. kodakarensis . These findings further suggest that while BCPA and thermospermine are distinct, they may play similar roles in stress resilience.

Agmatine, produced from arginine by arginine decarboxylase (ADC), is an essential precursor for spermidine and agmatidine in hyperthermophilic archaea. In the hyperthermophilic archaeon Pyrobaculum calidifontis, native ADC activity was detectable but rapidly lost when extracts were kept on ice, suggesting cold sensitivity. To investigate this instability, the Pc-speA gene was cloned and expressed in Escherichia coli. The recombinant protein formed insoluble aggregates but could be solubilized and refolded at various temperatures. Enzymatic assays showed that activity was restored only after refolding at high temperature, with maximal activity at 90 °C. SDS-PAGE confirmed autocatalytic cleavage into α- and β-subunits, generating the pyruvoyl group required for catalysis, and substrate assays verified specificity for arginine but not ornithine. Notably, refolded Pc-SpeA was cold-labile: storage at ≤20 °C caused aggregation and loss of activity, whereas stability was maintained at 60-80 °C. These findings provide the first biochemical characterization of P. calidifontis ADC and reveal its unusual requirement for elevated temperature to achieve and preserve enzymatic function.

N4-bis(aminopropyl)spermidine (BCPA), a branched-chain polyamine, is uniquely found in hyperthermophiles that thrive above 80°C. Compared to linear polyamines such as spermidine and spermine, BCPA induces DNA compaction at significantly lower concentrations and precipitates DNA more efficiently. To harness these properties, BCPA was immobilized onto N-hydroxysuccinimide (NHS)-activated magnetic microbeads, and its DNA recovery efficiency was evaluated using Salmon sperm DNA. BCPA-conjugated beads exhibited superior DNA-binding capacity compared to spermidine-conjugated and conventional silica beads, with bound DNA remaining unreleased upon treatment with 0.5 % sodium dodecyl sulfate (SDS), 2 mM ATP, or 2 mM phosphate (Pi) at pH 8.8. However, efficient DNA release was achieved with 2 mM pyrophosphate (PPi) at pH 8.0 or 1 mM PPi at pH 10.3. This property enables direct DNA amplification without a separate release step, as dNTPs used in PCR generate PPi as a byproduct, facilitating DNA detachment. To assess the beads' applicability for low-copy DNA detection, plasmid DNA containing the Ureaplasma parvum 16S rRNA region was prepared in saline at varying concentrations. BCPA-conjugated beads successfully recovered and directly amplified as few as 10³ copies of plasmid DNA from a 10-mL saline solution, whereas the same amount remained undetectable using conventional magnetic beads ethanol precipitation. These findings demonstrate the potential of BCPA-conjugated beads for efficient DNA capture and direct amplification, with promising applications in clinical diagnostics and environmental DNA monitoring.

Agmatine, a natural polyamine also known as 4-aminobutyl-guanidine, is biosynthesized from arginine by decarboxylation. Aspergillus oryzae contains high amounts of agmatine, suggesting highly active arginine decarboxylase (ADC) in this organism. However, genome analysis revealed no ADC homolog in A. oryzae. A. oryzae strain RIB40 has six homologs of phosphatidylserine decarboxylase (PSD), an enzyme that synthesizes phosphatidyl ethanolamine from phosphatidylserine. We previously discovered that one of these homologs, AO090102000327, encodes arginine decarboxylase, which we named ADC1. In the present study, we determined the crystal structures of ligand-free, arginine-treated, and agmatine-treated ADC1 each at 1.9-2.15 Å resolution. Each structure contained four ADC1 molecules (chains A-D) in the asymmetric unit of the cell. Each ADC1 molecule is a heterodimer consisting of the N-terminal region (Asn60-Gly441) and C-terminal region (Ser442-Thr482). In the ligand-free ADC1, the N-terminus of Ser442 was modified to form a pyruvoyl group. In the arginine-treated ADC1, arginine was converted to agmatine, with the pyruvoyl group covalently bound to agmatine by forming a Schiff base. The same structure was observed in agmatine-treated ADC1. These results indicate that ADC1 is a pyruvoyl-dependent decarboxylase and unveils the reaction mechanism of ADC from A. oryzae.

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.

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.

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.

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 (S-0) in the culture medium, defective synthesis of these sulfur-containing molecules in Ubl mutants was restored, indicating that T. kodakarensis can use S-0 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 S-0 conditions, which may allow this organism to survive in a low sulfur environment. IMPORTANCE Sulfur 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.

ABSTRACT 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.

ABSTRACT 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. IMPORTANCE Gene 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.

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.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 degrees 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 poly-merases 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 b (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 degrees 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 perfor-mance 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. (c) 2023, The Society for Biotechnology, Japan. All rights reserved.

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 β (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.

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.

Objectives: This study aimed to study the characteristics of in-hospital deterioration in patients with congenital heart disease who required rapid response system activation and identify risk factors associated with 1-month mortality. Methods: We retrospectively analysed data from a Japanese rapid response system registry with 35 participating hospitals. We included consecutive patients with congenital heart disease who required rapid response system activation between January 2014 and March 2018. Logistic regression analyses were performed to examine the associations between 1-month mortality and other patient-specific variables. Results: Among 9,607 patients for whom the rapid response system was activated, only 82 (0.9%) had congenital heart disease. Only few patients with congenital heart disease were being treated at the cardiology and cardiovascular surgery departments (12.3% and 9.9%, respectively). Moreover, the incidences of rapid-response events after intensive care unit discharge or surgery were low (6.8% and 12.2%, respectively). The most common reason for rapid response system activation was respiratory dysfunction (desaturation: 35.4%, tachypnoea: 25.6%, and new dyspnoea: 195%). Rapid response system interventions and intensive care unit transfers were required for 65.9% and 20.7% of patients, respectively. The mortality rate was 1.2% at the end of the rapid response system intervention and 11.0% after 1 month. Moreover, decreased respiratory rate and decreased heart rate at rapid response system activation were associated with increased 1-month mortality. The adjusted odds ratio was 1.10 (95% confidence interval 1.02-1.19) and 1.02 (95% confidence interval, 1.00-1.04 for respiratory rate and heart rate, respectively. Conclusions: Rapid response systems were rarely activated after cardiac surgery and intensive care unit discharge, which were situations with a high risk of sudden deterioration in patients with congenital heart disease. Therefore, encouraging the use of the rapid response system in these departments will enable intervention by a third, specialised team for in-hospital emergencies and help provide comprehensive medical care to patients. Furthermore, 1-month mortality was associated with vital signs at rapid response system activation. These findings may guide treatment selection for patients with congenital heart disease showing deterioration.