Substrate Promiscuity of Thermoplasma acidophilum Malic Enzyme for CO2 Fixation Reaction.

CO2 fixation technology has gained attention as a method to effectively utilize the abundant CO2 in the atmosphere by converting it into useful chemicals. However, since CO2 is a highly stable molecule, many of the currently developed methods for chemical CO2 fixation require harsh conditions and reactive reagents. The establishment of efficient and sustainable processes is eagerly awaited. In this study, we investigated a biocatalytic process and achieved a carboxylation reaction under mild conditions (37 °C, 0.1 MPa CO2) using a biocatalyst, Thermoplasma acidophilum NADP+-malic enzyme (TaME), and gaseous CO2 by coupling enzymatic coenzyme regeneration. We also demonstrated for the first time that the carboxylation reaction by ME proceeds not only with pyruvate, a natural substrate, but also with 2-ketoglutarate.

Immobilization of Thermoplasma acidophilum Glucose Dehydrogenase and Isocitrate Dehydrogenase Through Enzyme-Inorganic Hybrid Nanocrystal Formation.

The development of green catalysts, specifically biocatalysts, is crucial for building a sustainable society. To enhance the versatility of biocatalysts, the immobilization of enzymes plays a vital role as it improves their recyclability and robustness. As target enzymes to immobilize, glucose dehydrogenases and carboxylases are particularly important among various kinds of enzymes due to their involvement in two significant reactions: regeneration of the reduced form of coenzyme required for various reactions, and carboxylation reactions utilizing CO2 as a substrate, respectively. In this study, we immobilized Thermoplasma acidophilum glucose dehydrogenase (TaGDH) and T. acidophilum isocitrate dehydrogenase (TaIDH) using a previously reported method involving the formation of enzyme-inorganic hybrid nanocrystals, in the course of our continuing study focusing on carboxylation catalyzed by the free form of TaGDH and TaIDH. Subsequently, we investigated the properties of the resulting immobilized enzymes. Our results indicate the successful immobilization of TaGDH and TaIDH through the formation of hybrid nanocrystals utilizing Mn2+. The immobilization process enhanced TaIDH activity, up to 211%, while TaGDH retained 71% of its original activity. Notably, the immobilized TaGDH exhibited higher activity at temperatures exceeding 87 °C than the free TaGDH. Moreover, these immobilized enzymes could be recycled. Finally, we successfully utilized the immobilized enzymes for the carboxylation of 2-ketoglutaric acid under 1 MPa CO2. In conclusion, this study represents the first immobilization of TaGDH and TaIDH using the hybrid nanocrystal forming method. Furthermore, we achieved significant activity enhancement of TaIDH through immobilization and demonstrated the recyclability of the immobilized enzymes.

Similarities and differences in autoinflammatory diseases with urticarial rash, cryopyrin-associated periodic syndrome and Schnitzler syndrome.

Cryopyrin-associated periodic syndromes (CAPS) and Schnitzler syndrome (SchS) are autoinflammatory diseases that present with urticaria-like rashes. CAPS is characterized by periodic or persistent systemic inflammation caused by the dysfunction of the NLRP3 gene. With the advent of IL-1-targeted therapies, the prognosis of CAPS has improved remarkably. SchS is considered an acquired form of autoinflammatory syndrome. Patients with SchS are adults of relatively older age. The pathogenesis of SchS remains unknown and is not associated with the NLRP3 gene. Previously, the p.L265P mutation in the MYD88 gene, which is frequently detected in Waldenström macroglobulinemia (WM) with IgM gammopathy, was identified in several cases of SchS. However, because persistent fever and fatigue are symptoms of WM that require therapeutic intervention, it is a challenge to determine whether these patients truly had SchS or whether advanced WM was misidentified as SchS. There are no established treatments for SchS. The treatment algorithm proposed with the diagnostic criteria is to use colchicine as first-line treatment, and systemic administration of steroids is not recommended due to concerns about side effects. In difficult-to-treat cases, treatment targeting IL-1 is recommended. If targeted IL-1 treatment does not improve symptoms, the diagnosis should be reconsidered. We hope that the efficacy of IL-1 therapy in clinical practice will serve as a stepping stone to elucidate the pathogenesis of SchS, focusing on its similarities and differences from CAPS.

The protein-stabilizing effects of TMAO in aqueous and non-aqueous conditions.

We present a new water-dependent molecular mechanism for the widely-used protein stabilizing osmolyte, trimethylamine N-oxide (TMAO), whose mode of action has remained controversial. Classical interpretations, such as osmolyte exclusion from the vicinity of protein, cannot adequately explain the behavior of this osmolyte and were challenged by recent data showing the direct interactions of TMAO with proteins, mainly via hydrophobic binding. Solvent effect theories also fail to propose a straightforward mechanism. To explore the role of water and the hydrophobic association, we disabled osmolyte-protein hydrophobic interactions by replacing water with hexane and using lipase enzyme as an anhydrous-stable protein. Biocatalysis experiments showed that under this non-aqueous condition, TMAO does not act as a stabilizer, but strongly deactivates the enzyme. Molecular dynamics (MD) simulations reveal that TMAO accumulates near the enzyme and makes many hydrogen bonds with it, like denaturing osmolytes. Some TMAO molecules even reach the active site and interact strongly with the catalystic traid. In aqueous solvent, the enzyme functions well: the extent of TMAO interactions is reduced and can be divided into both polar and non-polar terms. Structural analysis shows that in water, some TMAO molecules bind to the enzyme surface like a surfactant. We show that these interactions limit water-protein hydrogen bonds and unfavorable water-hydrophobic surface contacts. Moreover, a more hydrophobic environment is formed in the solvation layer, which reduces water dynamics and subsequently, rigidifies the backbone in aqueous solution. We show that osmolyte amphiphilicity and protein surface heterogeneity can address the weaknesses of exclusion and solvent effect theories about the TMAO mechanism.

Potential Benefits of TNF Targeting Therapy in Blau Syndrome, a NOD2-Associated Systemic Autoinflammatory Granulomatosis.

Blau syndrome is a systemic autoinflammatory granulomatous disease caused by mutations in the nucleotide-binding oligomerization domain 2 (NOD2) gene. NOD2 is an intracellular pathogen recognition receptor. Upon binding to muramyl dipeptide (MDP), NOD2 activates the NF-κB pathway, leading to the upregulation of proinflammatory cytokines. Clinical manifestations of Blau syndrome appear in patients before the age of four. Skin manifestations resolve spontaneously in some cases; however, joint and eye manifestations are progressive, and lead to serious complications, such as joint contracture and blindness. Currently, there is no specific curative treatment for the disease. Administration of high-dose oral steroids can improve clinical manifestations; however, treatments is difficult to maintain due to the severity of the side effects, especially in children. While several new therapies have been reported, including JAK inhibitors, anti-IL-6 and anti-IL-1 therapies, anti-TNF therapy plays a central role in the treatment of Blau syndrome. We recently performed an ex vivo study, using peripheral blood and induced pluripotent stem cells from patients. This study demonstrated that abnormal cytokine expression in macrophages from untreated patients requires IFNγ stimulation, and that anti-TNF treatment corrects the abnormalities associated with Blau syndrome, even in the presence of IFNγ. Therefore, although the molecular mechanisms by which the genetic mutations in NOD2 lead to granuloma formation remain unclear, it is possible that prior exposure to TNFα combined with IFNγ stimulation may provide the impetus for the clinical manifestations of Blau syndrome.

Immobilization of Baeyer-Villiger monooxygenase from acetone grown Fusarium sp.

OBJECTIVE: A novel biocatalyst for Baeyer-Villiger oxidations is necessary for pharmaceutical and chemical industries, so this study aims to find a Baeyer-Villiger monooxygenase (BVMO) and to improve its stability by immobilization. RESULTS: Acetone, the simplest ketone, was selected as the only carbon source for the screening of microorganisms with a BVMO. A eukaryote, Fusarium sp. NBRC 109816, with a BVMO (FBVMO), was isolated from a soil sample. FBVMO was overexpressed in E. coli and successfully immobilized by the organic-inorganic nanocrystal formation method. The immobilization improved the thermostability of FBVMO. Substrate specificity investigation revealed that both free and immobilized FBVMO were found to show catalytic activities not only for Baeyer-Villiger oxidation of ketones to esters but also for oxidation of sulfides to sulfoxides. Furthermore, a preparative scale reaction using immobilized FBVMO was successfully conducted. CONCLUSIONS: FBVMO was discovered from an environmental sample, overexpressed in E. coli, and immobilized by the organic-inorganic nanocrystal formation method. The immobilization successfully improved its thermostability.

Patterns of reproductive isolation in a haplodiploid mite, Amphitetranychus viennensis: prezygotic isolation, hybrid inviability and hybrid sterility.

BACKGROUND: Evolution of reproductive isolation is an important process, generating biodiversity and driving speciation. To better understand this process, it is necessary to investigate factors underlying reproductive isolation through various approaches but also in various taxa. Previous studies, mainly focusing on diploid animals, supported the prevalent view that reproductive barriers evolve gradually as a by-product of genetic changes accumulated by natural selection by showing a positive relationship between the degree of reproductive isolation and genetic distance. Haplodiploid animals are expected to generate additional insight into speciation, but few studies investigated the prevalent view in haplodiploid animals. In this study, we investigate whether the relationship also holds in a haplodiploid spider mite, Amphitetranychus viennensis (Zacher). RESULTS: We sampled seven populations of the mite in the Palaearctic region, measured their genetic distance (mtDNA) and carried out cross experiments with all combinations. We analyzed how lack of fertilization rate (as measure of prezygotic isolation) as well as hybrid inviability and hybrid sterility (as measures of postzygotic isolation) varies with genetic distance. We found that the degree of reproductive isolation varies among cross combinations, and that all three measures of reproductive isolation have a positive relationship with genetic distance. Based on the mtDNA marker, lack of fertilization rate, hybrid female inviability and hybrid female sterility were estimated to be nearly complete (99.0-99.9% barrier) at genetic distances of 0.475-0.657, 0.150-0.209 and 0.145-0.210, respectively. Besides, we found asymmetries in reproductive isolation. CONCLUSIONS: The prevalent view on the evolution of reproductive barriers is supported in the haplodiploid spider mite we studied here. According to the estimated minimum genetic distance for total reproductive isolation in parent population crosses in this study and previous work, a genetic distance of 0.15-0.21 in mtDNA (COI) appears required for speciation in spider mites. Variations and asymmetries in the degree of reproductive isolation highlight the importance of reinforcement of prezygotic reproductive isolation through incompatibility and the importance of cytonuclear interactions for reproductive isolation in haplodiploid spider mites.

Geotrichum candidum aldehyde dehydrogenase-inorganic nanocrystal with enhanced activity.

A novel Geotrichum candidum aldehyde dehydrogenase (GcALDH) effectively oxidized aldehydes to carboxylic acids under mild conditions. Nonetheless, the free form of GcALDH still had a limitation in stability and recyclability. Herein, to overcome these limitations, this study achieved the immobilization of the GcALDH by the organic-inorganic nanocrystal formation. The GcALDH nanocrystal exhibited 261 ± 40 % of activity comparing to the free enzyme. It also exhibited improved thermostability and recyclability as well as remained excellent substrate promiscuity, which suggested its potential use in green industries. To the best of our knowledge, this is the first time to report the ALDH-inorganic nanocrystal formation with superior properties.

Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing.

Large volumes of high-throughput sequencing data have been submitted to the Sequencing Read Archive (SRA). The lack of experimental metadata associated with the data makes reuse and understanding data quality very difficult. In the case of RNA sequencing (RNA-Seq), which reveals the presence and quantity of RNA in a biological sample at any moment, it is necessary to consider that gene expression responds over a short time interval (several seconds to a few minutes) in many organisms. Therefore, to isolate RNA that accurately reflects the transcriptome at the point of harvest, raw biological samples should be processed by freezing in liquid nitrogen, immersing in RNA stabilization reagent or lysing and homogenizing in RNA lysis buffer containing guanidine thiocyanate as soon as possible. As the number of samples handled simultaneously increases, the time until the RNA is protected can increase. Here, to evaluate the effect of different lag times in RNA protection on RNA-Seq data, we harvested CHO-S cells after 3, 5, 6, and 7 days of cultivation, added RNA lysis buffer in a time course of 15, 30, 45, and 60 min after harvest, and conducted RNA-Seq. These RNA samples showed high RNA integrity number (RIN) values indicating non-degraded RNA, and sequence data from libraries prepared with these RNA samples was of high quality according to FastQC. We observed that, at the same cultivation day, global trends of gene expression were similar across the time course of addition of RNA lysis buffer; however, the expression of some genes was significantly different between the time-course samples of the same cultivation day; most of these differentially expressed genes were related to apoptosis. We conclude that the time lag between sample harvest and RNA protection influences gene expression of specific genes. It is, therefore, necessary to know not only RIN values of RNA and the quality of the sequence data but also how the experiment was performed when acquiring RNA-Seq data from the database.

A phase III, 52-week, open-label study to evaluate the safety and efficacy of 5% sofpironium bromide (BBI-4000) gel in Japanese patients with primary axillary hyperhidrosis.

A long-term study was conducted in Japanese patients with primary axillary hyperhidrosis who completed the preceding 6-week phase III, confirmatory study of 5% sofpironium bromide gel (hereinafter referred to as sofpironium) to evaluate the safety and efficacy of 52-week treatment with sofpironium. In the long-term study, 185 patients who completed the confirmatory study (94 and 91 patients in the vehicle and sofpironium groups, respectively) started to receive sofpironium (switching and extension groups, respectively), and all these patients were included in both the full analysis set (FAS) and the safety analysis set (SAF). In the FAS, there were more females than males (73.0% vs. 27.0%), and median age was 38.0 years. A total of 161 patients (86 and 75 patients in the switching and extension groups, respectively) completed the study at week 52. The proportions of patients with hyperhidrosis disease severity score of 1 or 2 and a 50% or more reduction in total gravimetric weight of sweat were 57.4% in the switching group and 58.2% in the extension group at week 52. The proportions of patients who achieved this efficacy end-point in the long-term study were similar to that (53.9%) in the sofpironium group in the confirmatory study. In the SAF, the incidences of adverse events (AEs) were 80.9% in the switching group and 83.5% in the extension group, and the incidences of adverse drug reactions were 39.4% and 45.1%, respectively. AEs that occurred in at least 20% of patients in both treatment groups were application site dermatitis (25.5% and 33.0%, respectively) and nasopharyngitis (31.9% and 23.1%, respectively). Reported AEs were generally mild, and there were no deaths. Serious AEs occurred in three patients, but none were considered related to the study drug. In this study, the efficacy of sofpironium was maintained during 52-week treatment, and no new safety risk was observed.

A phase 3, multicenter, randomized, double-blind, vehicle-controlled, parallel-group study of 5% sofpironium bromide (BBI-4000) gel in Japanese patients with primary axillary hyperhidrosis.

A phase 3 study was conducted to verify the efficacy and safety of 5% sofpironium bromide (BBI-4000) gel (hereinafter referred to as sofpironium) administrated for 6 weeks in Japanese patients with primary axillary hyperhidrosis. The primary efficacy end-point was the proportion of patients who satisfied both criteria of a Hyperhidrosis Disease Severity Score (HDSS) of 1 or 2 at the end of 6-week treatment and a 50% or more reduction in total gravimetric weight of sweat at the end of treatment relative to baseline. A total of 281 patients were randomized to receive 5% sofpironium (141 patients) or vehicle (140 patients), and all patients were included in the full analysis set (FAS). In the FAS, 70.1% of patients were female, and the median age was 35.0 years. The proportion of patients who achieved the primary efficacy end-point was 53.9% in the sofpironium group and 36.4% in the vehicle group, with a statistically significant difference of 17.5% (95% confidence interval, 6.02-28.93) between these two groups (P = 0.003). The incidence of adverse events was 44.0% in the sofpironium group and 30.7% in the vehicle group, and the incidence of adverse drug reactions was 16.3% in the sofpironium group and 5.0% in the vehicle group. Reported adverse events were generally mild or moderate in severity. In the sofpironium group, common events (incidence, ≥5%) were nasopharyngitis (14.2%) and dermatitis/erythema at the application site (8.5%/5.7%), with no serious adverse events reported. This study demonstrated the efficacy and safety of 5% sofpironium.

Single-cell transcriptome analyses reveal heterogeneity in suspension cultures and clonal markers of CHO-K1 cells.

Cell-to-cell variability in cell populations arises from a combination of intrinsic factors and extrinsic factors related to the milieu. However, the heterogeneity of high cell density suspension cultures for therapeutic protein production remains unknown. Here, we illustrate the increasing heterogeneity in the cellular transcriptome of serum-free adapted CHO K1 cells during high cell density suspension culture over time without concomitant changes in the genomic sequence. Cell cycle-dependent subpopulations and cell clusters, which typically appear in other single-cell transcriptome analyses, were not found in these suspension cultures. Our results indicate that cell division changes the intracellular microenvironment and leads to cell cycle-dependent heterogeneity. Whole mitochondrial single-cell genome sequencing showed cell-to-cell mitochondrial genome variation and heteroplasmy within cells. The mitochondrial genome sequencing method developed here is potentially useful for the validation of cell clonality. The culture time-dependent increase in cellular heterogeneity observed in this study did not show any attenuation in this increasing heterogeneity. Future advances in bioengineering such as culture upscaling, prolonged culturing, and complex culture systems will be confronted with the need to assess and control cellular heterogeneity, and the method described here may prove useful for this purpose.

Complementary description of Panonychus caricae Hatzinikolis, 1984, with the resurrection of the genus Sasanychus Ehara, 1978 (Acari, Prostigmata, Tetranychidae).

A complementary description of Panonychus caricae Hatzinikolis, 1984, is presented based on the morphology of adult female and male individuals collected from fig trees (Ficus sp., Moraceae) in Greece. Morphological differences between Panonychus caricae and two closely related species, Panonychus ulmi (Koch, 1836) and Panonychus hadzhibejliae (Reck, 1947), are discussed. Panonychus caricae can be separated from two other Panonychus species using the length of the female dorsal setae in combination with the ratio between the length of female dorsal opisthosomal setae f2 and h1, and the ratio between the length of dorsal setae sc1 and h1. A phylogenetic maximum likelihood tree was constructed based on the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA (mtDNA) from 10 species of the subgenus Panonychus s.str. (including the re-described species P. caricae) and the only two species of the subgenus Sasanychus. The phylogenetic tree indicates that these 12 species are clearly separated from each other. The two subgenera, Panonychus s.str. and Sasanychus, comprise strongly supported monophyletic clades with 98% bootstrap values. The convergence of molecular and morphological data (dorsal setae set on tubercles or not, number of tactile setae on tibiae I and II, and patterns of the dorsocentral striae) suggests that Sasanychus should not be classified under the genus Panonychus. Consequently, molecular and morphological evidence supports the resurrection of the genus Sasanychus, which contains two species, S. akitanus (Ehara) and S. pusillus Ehara Gotoh, as distinct from Panonychus. A key to the world species of Panonychus and Sasanychus is also provided.

Clinical characteristics and treatment of 50 cases of Blau syndrome in Japan confirmed by genetic analysis of the NOD2 mutation.

OBJECTIVES: To collect clinical information and NOD2 mutation data on patients with Blau syndrome and to evaluate their prognosis. METHODS: Fifty patients with NOD2 mutations were analysed. The activity of each NOD2 mutant was evaluated in HEK293 cells by reporter assay. Clinical information was collected from medical records through the attending physicians. RESULTS: The study population comprised 26 males and 24 females aged 0-61 years. Thirty-two cases were sporadic, and 18 were familial from 9 unrelated families. Fifteen different mutations in NOD2 were identified, including 2 novel mutations (p.W490S and D512V); all showed spontaneous nuclear factor kappa B activation, and the most common mutation was p.R334W. Twenty-six patients had fever at relatively early timepoints in the disease course. Forty-three of 47 patients had a skin rash. The onset of disease in 9 patients was recognised after BCG vaccination. Forty-five of 49 patients had joint lesions. Thirty-eight of 50 patients had ocular symptoms, 7 of which resulted in blindness. After the diagnosis of Blau syndrome, 26 patients were treated with biologics; all were antitumour necrosis factor agents. Only 3 patients were treated with biologics alone; the others received a biologic in combination with methotrexate and/or prednisolone. None of the patients who became blind received biologic treatment. CONCLUSIONS: In patients with Blau syndrome, severe joint contractures and blindness may occur if diagnosis and appropriate treatment are delayed. Early treatment with a biologic agent may improve the prognosis.

Tea extract increases cell fusion via regulation of cell surface DC-STAMP.

Mononuclear osteoclast precursor cells fuse with each other to become mature multinucleated osteoclasts, which is regulated by dendritic cell-specific transmembrane protein (DC-STAMP). We evaluated the effects of tea extract and catechins on cell-cell fusion and DC-STAMP expression to elucidate their relationship with osteoclast development. When tea extract or epigallocatechin gallate (EGCg) was applied to RAW264.7 cells, multinucleated cells were increased significantly, while tartrate-resistant acid phosphatase (TRAP) activity was hardly upregulated. Flow cytometric analysis revealed that EGCg suppressed DC-STAMP expression on the cell surface, which is similar to osteoclast development. These observations suggest that TRAP activity is not activated even when suppression of both surface DC-STAMP expression and multinucleation occurs, which might be mediated by another pathway.

Impact and relevance of alcohol dehydrogenase enantioselectivities on biotechnological applications.

Alcohol dehydrogenases (ADHs) catalyze the reversible reduction of a carbonyl group to its corresponding alcohol. ADHs are widely employed for organic synthesis due to their lack of harm to the environment, broad substrate acceptance, and high enantioselectivity. This review focuses on the impact and relevance of ADH enantioselectivities on their biotechnological application. Stereoselective ADHs are beneficial to reduce challenging ketones such as ketones owning two bulky substituents or similar-sized substituents to the carbonyl carbon. Meanwhile, in cascade reactions, non-stereoselective ADHs can be utilized for the quantitative oxidation of racemic alcohol to ketone and dynamic kinetic resolution.

Organic-inorganic nanocrystal reductase to promote green asymmetric synthesis.

An acetophenone reductase from Geotrichum candidum (GcAPRD) was immobilized by the organic-inorganic nanocrystal method. The GcAPRD nanocrystal presented improved stability and recyclability compared with those of the free GcAPRD. Moreover, the GcAPRD nanocrystal reduced broad kinds of ketones with excellent enantioselectivities to produce beneficial chiral alcohols such as (S)-1-(3',4'-dichlorophenyl)ethanol with >99% yield and >99% ee. The robust and versatile properties of the GcAPRD nanocrystal demonstrated an approach to promote green asymmetric synthesis and sustainable chemistry.

Reversible control of enantioselectivity by the length of ketone substituent in biocatalytic reduction.

Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.

Correction to: Structural basis for a highly (S)-enantioselective reductase towards aliphatic ketones with only one carbon difference between side chain.

The original version of this article contains error for some of the authors corrections were not included during correction stage.