Formation of fatty alcohols-components of meibum lipids-by the fatty acyl-CoA reductase FAR2 is essential for dry eye prevention.

Various lipids (mainly meibum lipids secreted by the meibomian glands) are present in the tear film lipid layer and play important roles in tear stability and the health of the cornea and conjunctiva. Many meibum lipids contain fatty alcohols (FAls) with chain lengths ≥C24, but the fatty acyl-CoA reductases (FARs) that produce them remain unclear. Here, using cell-based assays, we found that the two FAR isozymes (FAR1 and FAR2) show different substrate specificities: FAR1 and FAR2 are involved in the production of C16-C18 and ≥C20 FAls, respectively. Next, we generated Far2 knockout (KO) mice and examined their dry eye phenotype and meibum lipid composition. These mice showed a severe dry eye phenotype, characterized by plugged meibomian gland orifices, corneal damage, and tear film instability. The plugging was attributed to an increase in the melting point of the meibum lipids. Liquid chromatography coupled with tandem mass spectrometry revealed that FAl-containing meibum lipids (wax monoesters and types 1ω, 2α, and 2ω wax diesters) with a hydroxyl group at position 1 were almost completely absent in Far2 KO mice. The levels of di-unsaturated (O-acyl)-ω-hydroxy fatty acids were higher in Far2 KO mice than in wild type mice, but those of tri-unsaturated ones were comparable, suggesting the presence of two synthesis pathways for type 1ω wax diesters. These results indicate the importance of FAl-containing meibum lipids in the formation of a functional tear film lipid layer. In addition, our study provides clues to the molecular mechanism of the biosynthesis of meibum lipids.

Diagnostic test property of transcription-reverse transcription concerted reaction reagent TRCReady® SARS-CoV-2 i using nasopharyngeal swab samples.

TRCReady® SARS-CoV-2 i is a reagent for transcription-reverse transcription concerted reaction (TRC) to detect SARS-CoV-2 N2 gene, used with the automated rapid isothermal nucleic acid amplification test (NAAT) analyzer TRCReady®-80. Sensitivity and specificity of TRCReady® SARS-CoV-2 i was assessed by comparison with the results of real-time reverse transcription-polymerase chain reaction (RT-PCR) using nasopharyngeal swab samples. From November 2020 to March 2021, a total of 441 nasopharyngeal swabs were obtained and analyzed both with TRCReady® SARS-CoV-2 i and RT-PCR. Sensitivity and specificity of TRCReady® SARS-CoV-2 i were 94.6% (53/56) and 99.2% (382/385), respectively. Reaction time to positivity of TRCReady® SARS-CoV-2 i ranged from 1.166 to 9.805 (median: 2.887) min, and minimum detection sensitivity of TRCReady® SARS-CoV-2 i was 9 copies per test, with reaction time as 5.014 min. Detection of SARS-CoV-2 gene from nasopharyngeal swab sample using TRCReady® SARS-CoV-2 i shows comparative diagnostic test accuracy with RT-PCR, and can be used as a useful test to diagnose SARS-CoV-2 infection.

Prevalence, Temporal Change, and Determinants of Anxiety and Depression in Hospitalized Patients With Heart Failure.

BACKGROUND: Anxiety and depression may be under-recognized in patients with heart failure (HF). We therefore investigated the prevalence and temporal change of these symptoms in hospitalized patients with HF. METHODS AND RESULTS: We prospectively evaluated consecutive hospitalized patients with HF using the Hospital Anxiety and Depression Scale (HADS) on admission and at discharge. The HADS-A (anxiety) and HADS-D (depression) scores were categorized as follows; 0-7, no symptoms; 8-10, mild; and 11-21, significant anxiety or depression. Symptom worsening was defined as the HADS category at discharge being poorer than that on admission. Of 224 patients (mean age 77.5 years), 35 (16%) and 62 (28%) had significant symptoms of anxiety and depression, respectively. During hospitalization, the HADS-A significantly decreased (on admission; median 6 [interquartile range (IQR) 3-9] vs at discharge; median 4 [IQR 2-7], P < .01), whereas the HADS-D did not improve (on admission; median 8 [IQR 5-11] vs at discharge; median 8 [IQR 4-11], P =.82). Anxiety and depression worsened during hospitalization in 19 (10%) and 40 (21%) patients, respectively. Advanced age, higher natriuretic peptide levels, and acute-on-chronic HF were associated with worsening anxiety, and longer hospitalization length was associated with worsening depression. CONCLUSIONS: Anxiety and depression were common and depression persisted during HF hospitalization.

Characterization of bacteriophages Cp1 and Cp2, the strain-typing agents for Xanthomonas axonopodis pv. citri.

The strains of Xanthomonas axonopodis pv. citri, the causative agent of citrus canker, are historically classified based on bacteriophage (phage) sensitivity. Nearly all X. axonopodis pv. citri strains isolated from different regions in Japan are lysed by either phage Cp1 or Cp2; Cp1-sensitive (Cp1(s)) strains have been observed to be resistant to Cp2 (Cp2(r)) and vice versa. In this study, genomic and molecular characterization was performed for the typing agents Cp1 and Cp2. Morphologically, Cp1 belongs to the Siphoviridae. Genomic analysis revealed that its genome comprises 43,870-bp double-stranded DNA (dsDNA), with 10-bp 3'-extruding cohesive ends, and contains 48 open reading frames. The genomic organization was similar to that of Xanthomonas phage phiL7, but it lacked a group I intron in the DNA polymerase gene. Cp2 resembles morphologically Escherichia coli T7-like phages of Podoviridae. The 42,963-bp linear dsDNA genome of Cp2 contained terminal repeats. The Cp2 genomic sequence has 40 open reading frames, many of which did not show detectable homologs in the current databases. By proteomic analysis, a gene cluster encoding structural proteins corresponding to the class III module of T7-like phages was identified on the Cp2 genome. Therefore, Cp1 and Cp2 were found to belong to completely different virus groups. In addition, we found that Cp1 and Cp2 use different molecules on the host cell surface as phage receptors and that host selection of X. axonopodis pv. citri strains by Cp1 and Cp2 is not determined at the initial stage by binding to receptors.

Epigenetic targets of Janus kinase inhibitors are linked to genetic risks of rheumatoid arthritis.

BACKGROUND: Current strategies that target cytokines (e.g., tumor necrosis factor (TNF)-α), or signaling molecules (e.g., Janus kinase (JAK)) have advanced the management for allergies and autoimmune diseases. Nevertheless, the molecular mechanism that underpins its clinical efficacy have largely remained elusive, especially in the local tissue environment. Here, we aimed to identify the genetic, epigenetic, and immunological targets of JAK inhibitors (JAKis), focusing on their effects on synovial fibroblasts (SFs), the major local effectors associated with destructive joint inflammation in rheumatoid arthritis (RA). METHODS: SFs were activated by cytokines related to inflammation in RA, and were treated with three types of JAKis or a TNF-α inhibitor (TNFi). Dynamic changes in transcriptome and chromatin accessibility were profiled across samples to identify drug targets. Furthermore, the putative targets were validated using luciferase assays and clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing. RESULTS: We found that both JAKis and the TNFi targeted the inflammatory module including IL6. Conversely, specific gene signatures that were preferentially inhibited by either of the drug classes were identified. Strikingly, RA risk enhancers for CD40 and TRAF1 were distinctively regulated by JAKis and the TNFi. We performed luciferase assays and CRISPR-based genome editing, and successfully fine-mapped the single causal variants in these loci, rs6074022-CD40 and rs7021049-TRAF1. CONCLUSIONS: JAKis and the TNFi had a direct impact on different RA risk enhancers, and we identified nucleotide-resolution targets for both drugs. Distinctive targets of clinically effective drugs could be useful for tailoring the application of these drugs and future design of more efficient treatment strategies.

X-ray structure determination and deuteration of nattokinase.

Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D2O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D2O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.

Synovial Tissue Heterogeneity in Japanese Patients With Rheumatoid Arthritis Elucidated Using a Cell-Type Deconvolution Approach.

OBJECTIVE: Recent advances in single-cell RNA sequencing technology have improved our understanding of the immunological landscape of rheumatoid arthritis (RA). We aimed to stratify the synovium from East Asian patients with RA by immune cell compositions and gain insight into the inflammatory drivers of each synovial phenotype. METHODS: Synovial tissues were obtained from East Asian patients in Japan with RA (n = 41) undergoing articular surgery. The cellular composition was quantified by a deconvolution approach using a public single-cell-based reference. Inflammatory pathway activity was calculated by gene set variation analysis, and chromatin accessibility was evaluated using assay of transposase accessible chromatin-sequencing. RESULTS: We stratified RA synovium into three distinct subtypes based on the hierarchical clustering of cellular composition data. One subtype was characterized by abundant HLA-DRAhigh synovial fibroblasts, autoimmune-associated B cells, GZMK+ GZMB+ CD8+ T cells, interleukin (IL)1-ß+ monocytes, and plasmablasts. In addition, tumor necrosis factor (TNF)-α, interferons (IFNs), and IL-6 signaling were highly activated in this subtype, and the expression of various chemokines was significantly enhanced. Moreover, we found an open chromatin region overlapping with RA risk locus rs9405192 near the IRF4 gene, suggesting the genetic background influences the development of this inflammatory synovial state. The other two subtypes were characterized by increased IFNs and IL-6 signaling, and expression of molecules associated with degeneration, respectively. CONCLUSION: This study adds insights into the synovial heterogeneity in East Asian patients and shows a promising link with predominant inflammatory signals. Evaluating the site of inflammation has the potential to lead to appropriate drug selection that matches the individual pathology.

Comprehensive symptom assessment using Integrated Palliative care Outcome Scale in hospitalized heart failure patients.

AIMS: Patients with heart failure (HF) may have variable unrecognized symptom burdens. We sought to investigate the details, determinants, and prognostic significance of symptom burden in hospitalized patients with HF. METHODS AND RESULTS: We prospectively evaluated consecutive hospitalized patients with HF as primary diagnosis at our institution using the Integrated Palliative care Outcome Scale (IPOS) both on admission and at discharge. The IPOS, which is a well-validated multi-dimensional symptom assessment scale among advanced illness, consists of 17 questions for enquiring about physical symptoms (10 items), emotional symptoms (4 items) and communication and practical issues (3 items) using a 5-point Likert scale (0 [best]-4 [worst] points). Clinically relevant symptoms were defined as ≥2 points for each IPOS item. Worsening symptom burden was defined as the total IPOS score at discharge being poorer than that on admission. Of 294 patients (mean age: 77.5 ± 12.0 years, male: 168 patients, New York Heart Association class IV: 96 patients, mean left ventricular ejection fraction [LVEF]: 44%, and median N-terminal pro B-type natriuretic peptide [NT-proBNP] level: 4418 ng/L), the median (IQR) total IPOS score on admission was 19 (12, 27) and they were widely distributed (minimum: 0 - maximum: 52). The total IPOS score on admission was not correlated with the HF severity, including LVEF (Spearman's ρ = -0.05, P = 0.43), NT-proBNP levels (Spearman's ρ = 0.08, P = 0.20) or in-hospital mortality prediction model (GWTG-HF risk score) (Spearman's ρ = 0.01, P = 0.90). Total IPOS scores significantly decreased during hospitalization as a whole (median [IQR]: 13 [6, 21] at discharge; P < 0.001 vs. those on admission). All of the four emotional symptoms (patient anxiety, depression, family anxiety and feeling at peace) remained in the top 5 of clinically relevant symptoms at discharge, whereas none of 10 physical symptoms were nominated. Worsening symptom burden was noted in 28% of the patients during hospitalization, and was independently associated with higher all-cause mortality after discharge (hazard ratio: 2.28, 95% confidence interval: 1.02-5.09; P = 0.044) even after adjustment by age and HF mortality prediction model (MAGGIC risk score). CONCLUSIONS: We revealed that hospitalized patients with HF had multi-dimensional symptom burdens which varied among individuals and were not correlated with the disease severity. Emotional symptoms, such as anxiety and depression, were the main clinically relevant symptoms at discharge. A worsening IPOS score was noted in a quarter of patients with HF and was associated with a poor prognosis, suggesting the importance of holistic symptom assessment during the course of hospitalization for HF.

Advanced Diagnostic System and Introduction of Newborn Screening of Adrenoleukodystrophy and Peroxisomal Disorders in Japan.

We established a diagnostic system for adrenoleukodystrophy (ALD) and peroxisomal disorders (PD) over 35 years ago in Japan, and have diagnosed 237 families with ALD and more than 100 cases of PD other than ALD using biochemical and molecular analyses. In particular, since the only treatment for the cerebral form of ALD is hematopoietic stem cell transplantation at an early stage of onset, we have developed a protocol for the rapid diagnosis of ALD that can provide the measurements of the levels of very-long-chain fatty acids in the serum and genetic analysis within a few days. In addition, to improve the prognosis of patients with ALD, we are working on the detection of pre-symptomatic patients by familial analysis from the proband, and the introduction of newborn screening. In this review, we introduce the diagnostic and newborn screening approaches for ALD and PD in Japan.

[Preparation of a brochure for patients undergoing FOLFIRI chemotherapy based on survey of adverse reactions].

We have retrospectively been collecting data on adverse reactions to FOLFIRI chemotherapy in our hospital from September 2005 to August 2006, by electronic medical records. The retrospective study was of 67 patients who received FOLFIRI for advanced colorectal cancer. Survey results showed high incidences of vomiting(58.2%), anorexia(91.4%), constipation (47.8%), diarrhea (61.2%) and alopecia(71.6%). The first cycle using FOLFIRI therapy, vomiting (20.9%), anorexia(53.7%), constipation ( 14.9%), and diarrhea (23.9%)were observed. We sought to minimize inter-individual differences in pharmaceutical care and drug consultation by clinical pharmacists and to ensure the accurate understanding of patients. We are sure that this kind of activity will help us to provide better pharmaceutical care for patients.

A case of catastrophic antiphospholipid syndrome presenting with acute respiratory distress syndrome as the initial manifestation.

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by a combination of arterial or venous thrombosis and recurrent fetal loss, accompanied by elevated titers of antiphospholipid antibodies (aPL). Catastrophic antiphospholipid syndrome (CAPS) is a small subset of APS characterized by widespread systemic thrombotic disease with multiorgan failure. We herein describe an autopsy case of CAPS who developed severe respiratory failure due to acute respiratory distress syndrome (ARDS) as the initial manifestation. Patients with APS may exhibit a broad spectrum of pulmonary diseases. ARDS is the common pulmonary complication in CAPS, although it rarely occurs in APS. Some mechanisms of ARDS in CAPS have been postulated but the precise mechanism is still not clearly understood. It is important to understand that APS or CAPS could be a cause of ARDS since ARDS might develop as the initial manifestation of APS or CAPS as seen in our case. Our case is interesting in that severe respiratory failure due to ARDS was the initial presentation of CAPS.