Outcomes of critically ill patients with liver failure who require mechanical ventilation: A retrospective, single-center study.

Background and Aims: Critically ill patients with liver failure have high mortality. Besides the management of organ-specific complications, liver transplantation constitutes a definitive treatment. However, clinicians may hesitate to introduce mechanical ventilation for patients on liver transplantation waitlists because of poor prognosis. This study investigated the outcomes of intensive care and ventilation support therapy effects in patients with liver failure. Methods: This single-center study retrospectively enrolled 32 consecutive patients with liver failure who were admitted to the intensive care unit from January 2014 to December 2020. The medical records were reviewed and analyzed retrospectively for Acute Physiologic and Chronic Health Evaluation (APACHE)-II. The model for end-stage liver disease scores, 90-day mortality, and survival was assessed using the Kaplan-Meier method. Results: The average patient age was 45.5 ± 20.1 years, and 53% of patients were women. On intensive care unit admission, APACHE-II and model for end-stage liver disease scores were 20 and 28, respectively. Among 13 patients considered for liver transplantation, 4 received transplants. Thirteen patients (40.6%) were intubated and mechanically ventilated in the intensive care unit. The 90-day mortality rate of patients with and without mechanical ventilation in the intensive care unit (13, 61.5% vs. 19, 47.4%, p = 0.4905) was similar. APACHE-II score >21 was an independent predictor of mechanical ventilation requirement in patients with liver failure during intensive care unit stay. Conclusion: Although critically ill patients with liver failure are at risk of multiorgan failure with poor outcomes, mechanical ventilation did not negatively affect the 90-day mortality or performance rates of liver transplantation. Clinicians should consider mechanical ventilation-based life support in critically ill patients with liver failure who are awaiting liver transplantation.

Novel ability of diflubenzuron as an inhibitor of mitochondrial function.

Compounds classified as benzoylphenylurea (BPU), such as diflubenzuron (DFB), are used as insecticides. Although BPU disrupts molting by inhibiting chitin biosynthesis and exhibits insecticidal activity, their exact mode of action remains unknown. Since epidermal cells proliferate and morphologically change from squamous to columnar cells during the early stages of insect molting, we speculate that a transition similar to that from epithelium to mesenchyme occurs and that BPU may inhibit this transition. Here, we addressed this possibility. We found that DFB decreases actin expression in insect cells (the tissue cultures of insect integument). Detailed analysis in Schneider S2 cells reveals that DFB inhibits the expression of actin isoforms (Act5C and Act42A) and the Drosophila ortholog of myocardin-related transcription factor (Mrtf), leading to cell growth suppression. Proteomics identified the Drosophila ortholog of prohibitin (Phb1D and Phb2E) as one of the DFB-binding proteins. DFB inhibits the interaction between Phb1D and Phb2E and induces mitochondrial dysfunction. The knock-down of Phb2E suppresses the expression of Act5C, Act42A, and Mrtf, leading to cell growth inhibition. Thus, the disruption of Phb function is a possible novel target of DFB.

Quantitative evaluation of the biological activity of various brassinosteroids using spiral root induction in rice seeds.

Spiral roots are induced in germinated rice seeds through treatment with nanomolar brassinosteroids (BRs) but not with other plant hormones. Here, we determined the minimum effective concentration (MEC) of various BRs to induce spiral roots in germinated rice seeds. The reciprocal logarithm of MEC, pMEC, was used as the BL-like activity index, which was linearly correlated with the reciprocal logarithm of a 50% effective dose (pED50) as evaluated in the rice lamina inclination assay. Furthermore, a ligand-receptor docking simulation was performed against the BL receptor complex, Arabidopsis thaliana BRI1/SERK1, and the binding free energy (ΔGbind) was calculated for the tested BRs. The ΔGbind calculation was performed using the molecular mechanics/generalized Born surface area method on an ensemble of uncorrelated snapshots collected via molecular dynamics to predict biological activity.

Gene-regulation modules in nonalcoholic fatty liver disease revealed by single-nucleus ATAC-seq.

We investigated the progression of nonalcoholic fatty liver disease from fatty liver to steatohepatitis using single-nucleus and bulk ATAC-seq on the livers of rats fed a high-fat diet (HFD). Rats fed HFD for 4 wk developed fatty liver, and those fed HFD for 8 wk further progressed to steatohepatitis. We observed an increase in the proportion of inflammatory macrophages, consistent with the pathological progression. Utilizing machine learning, we divided global gene regulation into modules, wherein transcription factors within a module could regulate genes within the same module, reaffirming known regulatory relationships between transcription factors and biological processes. We identified core genes-central to co-expression and protein-protein interaction-for the biological processes discovered. Notably, a large part of the core genes overlapped with genes previously implicated in nonalcoholic fatty liver disease. Single-nucleus ATAC-seq, combined with data-driven statistical analysis, offers insight into in vivo global gene regulation as a combination of modules and assists in identifying core genes of relevant biological processes.

Role of CRP2-MRTF interaction in functions of myofibroblasts.

Inflammatory response induces phenotypic modulation of fibroblasts into myofibroblasts. Although transforming growth factor-ßs (TGF-ßs) evoke such transition, the details of the mechanism are still unknown. Here, we report that a LIM domain protein, cysteine-and glycine-rich protein 2 (CSRP2 [CRP2]) plays a vital role in the functional expression profile in myofibroblasts and cancer-associated fibroblasts (CAFs). Knock-down of CRP2 severely inhibits the expression of smooth muscle cell (SMC) genes, cell motility, and CAF-mediated collective invasion of epidermoid carcinoma. We elucidate the following molecular bases: CRP2 directly binds to myocardin-related transcription factors (MRTF-A/B [MRTFs]) and serum response factor (SRF) and stabilizes the MRTF/SRF/CArG-box complex to activate SMC gene expression. Furthermore, a three-dimensional structural analysis of CRP2 identifies the amino acids required for the CRP2-MRTF-A interaction. Polar amino acids in the C-terminal half (serine-152, glutamate-154, serine-155, threonine-156, threonine-157, and threonine-159 in human CRP2) are responsible for direct binding to MRTF-A. On the other hand, hydrophobic amino acids outside the consensus sequence of the LIM domain (tryptophan-139, phenylalanine-144, leucine-153, and leucine-158 in human CRP2) play a role in stabilizing the unique structure of the LIM domain.Key words: CRP2, 3D structure, myocardin-related transcription factor, myofibroblast, cancer-associated fibroblasts.

A New Tool for the Flexible Genetic Manipulation of Geobacillus kaustophilus.

Geobacillus kaustophilus , a thermophilic Gram-positive bacterium, is an attractive host for the development of high-temperature bioprocesses. However, its reluctance against genetic manipulation by standard methodologies hampers its exploitation. Here, we describe a simple methodology in which an artificial DNA segment on the chromosome of Bacillus subtilis can be transferred via pLS20-mediated conjugation resulting in subsequent integration in the genome of G. kaustophilus. Therefore, we have developed a transformation strategy to design an artificial DNA segment on the chromosome of B. subtilis and introduce it into G. kaustophilus . The artificial DNA segment can be freely designed by taking advantage of the plasticity of the B. subtilis genome and combined with the simplicity of pLS20 conjugation transfer. This transformation strategy would adapt to various Gram-positive bacteria other than G. kaustophilus . Graphical abstract.

A novel method for transforming Geobacillus kaustophilus with a chromosomal segment of Bacillus subtilis transferred via pLS20-dependent conjugation.

BACKGROUND: Geobacillus kaustophilus is a thermophilic Gram-positive bacterium. Methods for its transformation are still under development. Earlier studies have demonstrated that pLS20catΔoriT mobilized the resident mobile plasmids from Bacillus subtilis to G. kaustophilus and transferred long segments of chromosome from one cell to another between B. subtilis. RESULTS: In this study, we applied mobilization of the B. subtilis chromosome mediated by pLS20catΔoriT to transform G. kaustophilus. We constructed a gene cassette to be integrated into G. kaustophilus and designed it within the B. subtilis chromosome. The pLS20catΔoriT-mediated conjugation successfully transferred the gene cassette from the B. subtilis chromosome into the G. kaustophilus allowing for the desired genetic transformation. CONCLUSIONS: This transformation approach described here will provide a new tool to facilitate the flexible genetic manipulation of G. kaustophilus.

Clinical Implication of Smoking-Related Aryl-Hydrocarbon Receptor Repressor (AHRR) Hypomethylation in Japanese Adults.

BACKGROUND: Tobacco smoking is a leading preventable cause of morbidity and mortality worldwide; still, the success rate of smoking cessation is low in general. From the viewpoint of public health and clinical care, an objective biomarker of long-term smoking behavior is sought.Methods and Results: This study assessed DNA methylation as a biomarker of smoking in a hospital setting through a combination of molecular approaches including genetic, DNA methylation and mRNA expression analyses. First, in an epigenome-wide association study involving Japanese individuals with chronic cardiovascular disease (n=94), genome-wide significant smoking association was identified at 2 CpG sites on chromosome 5, with the strongest signal at cg05575921 located in intron 3 of the aryl-hydrocarbon receptor repressor (AHRR) gene. Highly significant (P<1×10-27) smoking-cg05575921 association was validated in 2 additional panels (n=339 and n=300). For the relationship of cg05575921 methylation extent with time after smoking cessation and cumulative cigarette consumption among former smokers, smoking-related hypomethylation was found to remain for ≥20 years after smoking cessation and to be affected by multiple factors, such as cis-interaction of genetic variation. There was a significant inverse correlation (P=0.0005) between cg05575921 methylation extent and AHRR mRNA expression. CONCLUSIONS: The present study results support that reversion of AHRR hypomethylation can be a quantifiable biomarker for progress in and observance of smoking cessation, although some methodological points need to be considered.

Transcriptomic Response in the Heart and Kidney to Different Types of Antihypertensive Drug Administration.

Certain classes of antihypertensive drug may exert specific, blood pressure (BP)-independent protective effects on end-organ damages such as left ventricular hypertrophy, although the overall evidence has not been definitive in clinical trials. To unravel antihypertensive drug-induced gene expression changes that are potentially related to the amelioration of end-organ damages, we performed in vivo phenotypic evaluation and transcriptomic analysis on the heart and the kidney, with administration of antihypertensive drugs to two inbred strains (ie, hypertensive and normotensive) of rats. We chose 6 antihypertensive classes: enalapril (angiotensin-converting enzyme inhibitor), candesartan (angiotensin receptor blocker), hydrochlorothiazide (diuretics), amlodipine (calcium-channel blocker), carvedilol (vasodilating ß-blocker), and hydralazine. In the tested rat strains, 4 of 6 drugs, including 2 renin-angiotensin system inhibitors, were effective for BP lowering, whereas the remaining 2 drugs were not. Besides BP lowering, there appeared to be some interdrug heterogeneity in phenotypic changes, such as suppressed body weight gain and body weight-adjusted heart weight reduction. For the transcriptomic response, a considerable number of genes showed prominent mRNA expression changes either in a BP-dependent or BP-independent manner with substantial diversity between the target organs. Noticeable changes of mRNA expression were induced particularly by renin-angiotensin system blockade, for example, for genes in the natriuretic peptide system (Nppb and Corin) in the heart and for those in the renin-angiotensin system/kallikrein-kinin system (Ren and rat Klk1 paralogs) and those related to calcium ion binding (Calb1 and Slc8a1) in the kidney. The research resources constructed here will help corroborate occasionally inconclusive evidence in clinical settings.

Case Report of Left Ventricular Noncompaction Cardiomyopathy Characterized by Undulating Phenotypes in Adult Patients.

Left ventricular noncompaction cardiomyopathy (LVNC) is a heart muscle disorder morphologically characterized by reticulated trabeculations and intertrabecular recesses in the left ventricular (LV) cavity. LVNC is a genetically and phenotypically heterogeneous condition, which has been increasingly recognized with the accumulation of evidence provided by genotype-phenotype correlation analyses. Here, we report 2 sporadic adult cases of LVNC; both developed acute heart failure as an initial clinical manifestation and harbored causal sarcomere gene mutations. One case was a 57-year-old male with digenic heterozygote mutations, p.R1344Q in myosin heavy chain 7 (MYH7) and p.R144W in troponin T2, cardiac type (TNNT2), who showed morphological characteristics of LVNC in the lateral to apical regions of the LV together with a comorbidity of non-transmural myocardial infarction, resulting from a coronary artery stenosis. After the removal of ischemic insult and standard heart failure treatment, LVNC became less clear, and LV function gradually improved. The other case was a 36-year-old male with a heterozygote mutation, p.E334K in myosin binding protein C3 (MYBPC3), who exhibited cardiogenic shock on admission with morphological characteristics of LVNC being most prominent in the apical segment of the LV. The dosage of beta-blocker was deliberately increased in an outpatient clinic over 6 months following hospitalization, which remarkably improved the LV ejection fraction from 21% to 54.3%. Via a combination of imaging and histopathological and genetic tests, we have found that these cases are not compatible with a persistent phenotype of primary cardiomyopathy, but their morphological features are changeable in response to treatment. Thus, we point out phenotypic plasticity or undulation as a noticeable element of LVNC in this case report.

Assessment of Bacillus subtilis Plasmid pLS20 Conjugation in the Absence of Quorum Sensing Repression.

Bacillus subtilis conjugative plasmid pLS20 uses a quorum-sensing mechanism to control expression levels of its conjugation genes, involving the repressor RcopLS20, the anti-repressor RappLS20, and the signaling peptide Phr*pLS20. In previous studies, artificial overexpression of rappLS20 in the donor cells was shown to enhance conjugation efficiency. However, we found that the overexpression of rappLS20 led to various phenotypic traits, including cell aggregation and death, which might have affected the correct determination of the conjugation efficiency when determined by colony formation assay. In the current study, conjugation efficiencies were determined under different conditions using a two-color fluorescence-activated flow cytometry method and measuring a single-round of pLS20-mediated transfer of a mobilizable plasmid. Under standard conditions, the conjugation efficiency obtained by fluorescence-activated flow cytometry was 23-fold higher than that obtained by colony formation. Furthermore, the efficiency difference increased to 45-fold when rappLS20 was overexpressed.

Unexplained recurrent shock in peripheral T-cell lymphoma: A case report.

Malignant lymphoma sometimes manifests with septic-like shock symptoms. We report a case of peripheral T-cell lymphoma presenting with unexplained recurrent shock in absence of apparent lymphadenopathy. The patient also experienced varied symptoms, including severe chest and back pain, respiratory distress due to tracheobronchomalacia, skin rash, and fever.

On the Possibility of Developing Magnetostrictive Fe-Co/Ni Clad Plate with Both Vibration Energy Harvesting and Mass Sensing Elements.

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has spread rapidly around the world. In order to prevent the spread of infection, city blockades and immigration restrictions have been introduced in each country, but these measures have a severe serious impact on the economy. This paper examines the possibility of both harvesting vibration energy and detecting mass by using a magnetostrictive alloy. Few efforts have been made to develop new magnetostrictive biosensor materials. Therefore, we propose magnetostrictive Fe-Co/Ni clad steel vibration energy harvesters with mass detection, and we numerically and experimentally discuss the effect of the proof mass weight on the frequency shift and output voltage induced by bending vibration. The results reveal that the frequency and output voltage decrease significantly as the mass increases, indicating that the energy harvesting device is capable of mass detection. In the future, device miniaturization and the possibility of virus detection will be considered.

Multisystem Inflammatory Syndrome in Children　- A New Syndrome Complicated With Acute Heart Failure Following Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is a rare syndrome temporally related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). MIS-C shares similarities with Kawasaki disease, but left ventricular dysfunction is more common in MIS-C.Methods and Results:This study reports the case of a 16-year-old Japanese male patient with MIS-C. Although the initial presentation was severe with circulatory and respiratory failure, the patient recovered completely. Endomyocardial biopsy showed active myocarditis with fibrosis. Immunoglobulin treatment was useful for recovery. CONCLUSIONS: This is the first reported case of MIS-C in Japan. Cardiologists should be aware of MIS-C, a new disease, occurring during the global SARS-CoV-2 pandemic.

Development of a Genetically Stable Live Attenuated Influenza Vaccine Strain Using an Engineered High-Fidelity Viral Polymerase.

RNA viruses demonstrate a vast range of variants, called quasispecies, due to error-prone replication by viral RNA-dependent RNA polymerase. Although live attenuated vaccines are effective in preventing RNA virus infection, there is a risk of reversal to virulence after their administration. To test the hypothesis that high-fidelity viral polymerase reduces the diversity of influenza virus quasispecies, resulting in inhibition of reversal of the attenuated phenotype, we first screened for a high-fidelity viral polymerase using serial virus passages under selection with a guanosine analog ribavirin. Consequently, we identified a Leu66-to-Val single amino acid mutation in polymerase basic protein 1 (PB1). The high-fidelity phenotype of PB1-L66V was confirmed using next-generation sequencing analysis and biochemical assays with the purified influenza viral polymerase. As expected, PB1-L66V showed at least two-times-lower mutation rates and decreased misincorporation rates, compared to the wild type (WT). Therefore, we next generated an attenuated PB1-L66V virus with a temperature-sensitive (ts) phenotype based on FluMist, a live attenuated influenza vaccine (LAIV) that can restrict virus propagation by ts mutations, and examined the genetic stability of the attenuated PB1-L66V virus using serial virus passages. The PB1-L66V mutation prevented reversion of the ts phenotype to the WT phenotype, suggesting that the high-fidelity viral polymerase could contribute to generating an LAIV with high genetic stability, which would not revert to the pathogenic virus.IMPORTANCE The LAIV currently in use is prescribed for actively immunizing individuals aged 2 to 49 years. However, it is not approved for infants and elderly individuals, who actually need it the most, because it might prolong virus propagation and cause an apparent infection in these individuals, due to their weak immune systems. Recently, reversion of the ts phenotype of the LAIV strain currently in use to a pathogenic virus was demonstrated in cultured cells. Thus, the generation of mutations associated with enhanced virulence in LAIV should be considered. In this study, we isolated a novel influenza virus strain with a Leu66-to-Val single amino acid mutation in PB1 that displayed a significantly higher fidelity than the WT. We generated a novel LAIV candidate strain harboring this mutation. This strain showed higher genetic stability and no ts phenotype reversion. Thus, our high-fidelity strain might be useful for the development of a safer LAIV.

Nonsteroidal ecdysone receptor agonists use a water channel for binding to the ecdysone receptor complex EcR/USP.

The ecdysone receptor (EcR) possesses the remarkable capacity to adapt structurally to different types of ligands. EcR binds ecdysteroids, including 20-hydroxyecdysone (20E), as well as nonsteroidal synthetic agonists such as insecticidal dibenzoylhydrazines (DBHs). Here, we report the crystal structures of the ligand-binding domains of Heliothis virescens EcR/USP bound to the DBH agonist BYI09181 and to the imidazole-type compound BYI08346. The region delineated by helices H7 and H10 opens up to tightly fit a phenyl ring of the ligands to an extent that depends on the bulkiness of ring substituent. In the structure of 20E-bound EcR, this part of the ligand-binding pocket (LBP) contains a channel filled by water molecules that form an intricate hydrogen bond network between 20E and LBP. The water channel present in the nuclear receptor bound to its natural hormone acts as a critical molecular adaptation spring used to accommodate synthetic agonists inside its binding cavity.

Isoxaben analogs inhibit chitin synthesis in the cultured integument of the rice stem borer Chilo suppressalis.

Benzoylphenylureas (BPUs) were discovered as novel type insecticides about a half century ago; many analogs have been launched as insecticides and acaricides. BPUs are known to inhibit chitin synthesis in insects and other arthropods, but they have no effect against microorganisms such as fungi. We designed new chitin synthesis inhibitors based on the hypothesis that biomolecules that play important roles in cellulose and chitin biosynthesis are similar. In the full automatic modeling system (FAMS), the cellulose synthase was selected as a template three-dimensional structure. Thus, we focused on the structure of cellulose synthase inhibitor, isoxaben, to develop new chemistry. The 1,1-diethylethyl [-C(CH3)(CH2CH3)2] group of isoxaben was changed to a 4-substituted phenyl group bearing Cl, Et, or Ph. These compounds significantly inhibited chitin synthesis in the cultured integument of the rice stem borer Chilo suppressalis. The activity of the 4-ethylphenyl analog was enhanced 30-fold by adding piperonyl butoxide to the culture medium.

Nafamostat Mesylate Improved Survival Outcomes of Sepsis Patients Who Underwent Blood Purification: A Nationwide Registry Study in Japan.

Nafamostat mesylate (NM) is a synthetic serine protease inhibitor that can be used as an anticoagulant during blood purification in critically ill patients, as well as a treatment for disseminated intravascular coagulation. Although NM has been reported to reduce the risk of bleeding during blood purification, its effect on survival outcomes of patients who received blood purification treatments is unclear. We hypothesized that administration of NM during blood purification can reduce mortality in patients with sepsis. A post hoc analysis was conducted on a nationwide retrospective registry that included data from 3195 sepsis patients registered at 42 intensive care units throughout Japan. We evaluated the effect of NM on hospital mortality and bleeding complications using propensity score matching in 1216 sepsis patients who underwent blood purification in the intensive care unit (ICU). Two-hundred-and-sixty-eight pairs of propensity score-matched patients who received NM and conventional therapy were compared. Hospital and ICU mortality rates in the NM group were significantly lower than those in the conventional therapy group. However, rates of bleeding complications did not differ significantly between the two groups. These data suggest that administration of NM improved the survival outcomes of sepsis patients who underwent blood purification in the ICU.

Beneficial effect modification on survival outcome of sepsis between ART-123 and polymyxin B­immobilised haemoperfusion: a nationwide Japanese registry study.

BACKGROUND: Although recently published randomised controlled trials did not confirm significant positive effect of ART-123 or polymyxin B­immobilised haemoperfusion (PMX-HP) on survival outcome, previous studies using a dataset of 3195 patients with sepsis registered at 42 intensive care units throughout Japan revealed significantly reduced mortality following these treatments. A study has suggested the efficacy of combination therapy with ART-123 and PMX-HP; however, it did not evaluate the effect modification between them. We hypothesised that coadministration of ART-123 and PMX-HP has a significant positive effect modification on survival outcome. The purpose of this study was to evaluate the effect modification between ART-123 and PMX-HP treatment on the survival outcome of sepsis using post hoc analysis of the dataset of the Japan Septic Disseminated Intravascular Coagulation registry. RESULTS: Of the 3195 patients recorded in the registry, 2350 were analysed. The product term between ART-123 and PMX-HP was analysed by the Cox regression model to evaluate significance. The primary outcome of this study was hospital mortality. Although the administration of ART-123 was independently positively associated with survival outcome (adjusted hazard ratio [HR]: 0.834, 95% confidence interval [CI] 0.695-0.999; P = 0.049) in the model prior to the introduction of the product term, a significant effect modification on survival outcome was observed between the administration of ART-123 and PMX-HP treatment (adjusted HR: 0.667, 95% CI 0.462-0.961; P = 0.030). CONCLUSIONS: The main effect of the administration of ART-123 may be beneficial for survival outcome in patients with sepsis. In addition, a significant beneficial effect modification on survival outcome was observed between the administration of ART-123 and PMX-HP treatment.

Fabrication, Modeling and Characterization of Magnetostrictive Short Fiber Composites.

Magnetostrictive materials have a wide variety of applications due to their great capability as sensors and energy-harvesting devices. However, their brittleness inhibits their applications as magnetostrictive devices. Recently, we developed a continuous magnetostrictive Fe-Co-fiber-embedded epoxy matrix composite to increase the flexibility of the material. In this study, we fabricated random magnetostrictive Fe-Co short fiber/epoxy composite sheets. It was found that the discontinuous Fe-Co fiber composite sheet has the magnetostrictive properties along the orientation parallel to the length of the sheet. Finite element computations were also carried out using a coupled magneto-mechanical model, for the representative volume element (RVE) of unidirectional aligned magnetostrictive short fiber composites. A simple model of two-dimensional, randomly oriented, magnetostrictive short fiber composites was then proposed and the effective piezomagnetic coefficient was determined. It was shown that the present model is very accurate yet relatively simple to predict the piezomagnetic coefficient of magnetostrictive short fiber composites. This magnetostrictive composite sheet is expected to be used as a flexible smart material.