α,ß-trehalose, an intracellular substance in resting cyst of colpodid ciliates as a key to environmental tolerances.

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,ß-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,ß-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance (1H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,ß-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment.

Comparison of pneumonia severity scores for COVID-19 patients with the Omicron variant.

INTRODUCTION: We demonstrated that there was a significant relationship between the severity measured using the A-DROP scoring system and the mortality rate in patients with COVID-19 community-acquired pneumonia (CAP) in the ancestral strain, Alpha variant, and Delta variant. We investigated the usefulness of the A-DROP scoring system in SARS-CoV-2 Omicron variant CAP and compared it with severity scores, the Pneumonia Severity Index (PSI) and CURB-65 score. METHODS: We analyzed a total of 547 patients with COVID-19 CAP Omicron variant; 198 cases were the BA.1 subvariant, 127 cases were the BA.2 subvariant, and 222 cases were the BA.5 subvariant, respectively. RESULTS: The mortality rates in patients with COVID-19 CAP among the three Omicron subvariants were identical in each pneumonia severity group. The mortality rate in patients with the Omicron variant was 0 % in patients classified with mild disease, 0.6 % in those with moderate disease, 10.4 % in those with severe disease, and 34.8 % in those with extremely severe disease. The mortality rate in patients with COVID-19 CAP increased depending on the severity classified according to the A-DROP system in each of the Omicron subvariants (Cochran-Armitage trend test; p < 0.001). The values of the area under the curve in Receiver Operating Characteristic analysis for prediction of 30-day mortality was 0.881, 0.879, and 0.863 for A-DROP, PSI, and CURB-65, respectively. There were no significant differences in the predictive ability of each pneumonia severity score. CONCLUSIONS: Our results demonstrated that the A-DROP scoring system is useful for predicting mortality in patients with COVID-19 CAP.

In situ characterization of the agglutination of lectins via cross-linking of carbohydrates by time-resolved measurement of forward static light scattering.

We present real-time observations of a structurally variable process for cross-linking agglutination between multivalent lectins and glycoclusters using a small-angle forward static light scattering (F-SLS) technique. In this study, a cross-linking agglutination reaction was carried out using a tetravalent Neu5Acα2,6LacNAc-glycocluster and Sambucus sieboldiana agglutinin (SSA). The scattering intensity of time-resolved F-SLS increased with formation of the Neu5Acα2,6LacNAc-glycocluster-SSA cross-linked complex. Using this approach, fine sequential cross-linking agglutination between glycoclusters and lectins was observed in real-time. The rate of increase in the intensity of time-resolved F-SLS increased with the concentration of sialo-glycoclusters and SSA. Structural analysis based on the fractal dimension using time-resolved F-SLS patterns revealed that the density of the aggregates changed with progression of the cross-linking reaction until equilibrium was reached. This is the first report to evaluate the cross-linking agglutination reaction between glycoclusters and lectins and analysis of the subsequent structure of the obtained aggregates using time-resolved measurements of F-SLS.

Heterologous expression of the lectin CmRlec from Cordyceps militaris (Cordycipitaceae, Ascomycota) in Escherichia coli.

Ascomycete lectins may play an important role in their life cycle. In this report, we mined a ricin B-type lectin, named CmRlec, from the Cordyceps militaris genome by homology search. Furthermore, we succeeded in the soluble expression of CmRlec using ß-glucuronidase as a solubilization tag and demonstrated that this lectin is a novel chitin-recognizing lectin.

Assessment of the pneumonia severity score in community-acquired and nursing and healthcare-associated pneumonia due to COVID-19.

INTRODUCTION: The Japanese Respiratory Society (JRS) pneumonia guidelines recommend simple predictive rules, the A-DROP scoring system, for assessment of the severity of community-acquired pneumonia (CAP) and nursing and healthcare-associated pneumonia (NHCAP). We evaluated whether the A-DROP system can be adapted for assessment of the severity of coronavirus disease 2019 (COVID-19) pneumonia. METHODS: Data from 1141 patients with COVID-19 pneumonia were analyzed, comprising 502 patients observed in the 1st to 3rd wave period, 338 patients in the 4th wave and 301 patients in the 5th wave in Japan. RESULTS: The mortality rate and mechanical ventilation rate were 0% and 1.4% in patients classified with mild disease (A-DROP score, 0 point), 3.2% and 46.7% in those with moderate disease (1 or 2 points), 20.8% and 78.3% with severe disease (3 points), and 55.0% and 100% with extremely severe disease (4 or 5 points), indicating an increase in the mortality and mechanical ventilation rates in accordance with severity (Cochran-Armitage trend test; p = <0.001). This significant relationship between the severity in the A-DROP scoring system and either the mortality rate or mechanical ventilation rate was observed in patients with COVID-19 CAP and NHCAP. In each of the five COVID-19 waves, the same significant relationship was observed. CONCLUSIONS: The mortality rate and mechanical ventilation rate in patients with COVID-19 pneumonia increased depending on severity classified according to the A-DROP scoring system. Our results suggest that the A-DROP scoring system can be adapted for the assessment of severity of COVID-19 CAP and NHCAP.

Validation of JRS atypical pneumonia score in patients with community-acquired Chlamydia psittaci pneumonia.

INTRODUCTION: The Japanese Respiratory Society (JRS) atypical pneumonia score is a useful tool for the rapid presumptive diagnosis of atypical pneumonia. We investigated the clinical features of community-acquired pneumonia (CAP) due to Chlamydia psittaci and validated the JRS atypical pneumonia score in patients with C. psittaci CAP. METHODS: This study was conducted at 30 institutions and assessed a total of 72 sporadic cases with C. psittaci CAP, 412 cases with Mycoplasma pneumoniae CAP, and 576 cases with Streptococcus pneumoniae CAP. RESULTS: Sixty-two of 72 patients with C. psittaci CAP had a history of avian exposure. Among the six parameters of the JRS score, matching rates of four parameters were significantly lower in the C. psittaci CAP than the M. pneumoniae CAP in the following parameters: age <60 years, no or minor comorbid illness, stubborn or paroxysmal cough, and absence of chest adventitious sounds. The sensitivity of the diagnosis of atypical pneumonia in patients with C. psittaci CAP was significantly lower than the M. pneumoniae CAP (65.3% and 87.4%, p < 0.0001). When the diagnostic sensitivity was analyzed for different ages, the diagnostic sensitivities for the C. psittaci CAP were 90.5% for non-elderly patients and 30.0% for elderly patients. CONCLUSIONS: The JRS atypical pneumonia score is a useful tool for distinguishing between C. psittaci CAP and bacterial CAP in patients aged <60 years, but not in patients aged ≥60 years. A history of avian exposure in middle-aged patients with normal white blood cell count may be suggestive of C. psittaci pneumonia.

Clinical efficacy of casirivimab-imdevimab antibody combination treatment in patients with COVID-19 Delta variant.

INTRODUCTION: Casirivimab-imdevimab, an antibody cocktail containing two severe acute respiratory syndrome coronavirus 2 neutralizing antibodies, reduces the viral load and the risk of coronavirus disease 2019 (COVID-19)-related hospitalization or death. The objective of this study was to evaluate the clinical efficacy of casirivimab-imdevimab in patients with COVID-19 Delta variant in Japan. METHODS: This study was conducted at five institutions and assessed a total of 461 patients with COVID-19 who met the inclusion criteria. The treatment group received a dose of casirivimab-imdevimab consisting of a cocktail of two monoclonal antibodies, (casirivimab 600 mg and imdevimab 600 mg intravenously). The control consisted of age- and sex-matched COVID-19 patients (n = 461) who sufficed the inclusion criteria but did not receive casirivimab-imdevimab. The outcome was the requirement of oxygen therapy. RESULTS: In the treatment group, patients received oxygen therapy (n = 30), nasal canula (n = 23), high flow nasal cannula (n = 5), and mechanical ventilation (n = 2). In the control group, patients received oxygen therapy (n = 56), nasal canula (n = 45), high flow nasal cannula (n = 8), and mechanical ventilation (n = 3). The administration of oxygen therapy was significantly lower in the treatment group than the control group (6.5% vs. 12.1%, P = 0.0044). All these patients admitted to our hospitals and received additional therapy and recovered. CONCLUSIONS: Our results demonstrate that the casirivimab-imdevimab combination antibody treatment is associated with reduced rates of requiring oxygen therapy among high-risk patients with COVID-19 Delta variant.

Changes in diagnostic usefulness of the JRS scoring system in COVID-19 pneumonia by SARS-CoV-2 vaccination.

INTRODUCTION: The Japanese Respiratory Society (JRS) scoring system is a useful tool for the rapid presumptive diagnosis of atypical pneumonia in non-elderly (aged <60 years) patients. As SARS-CoV-2 vaccination progresses, COVID-19 in elderly people has markedly reduced. We investigated changes in diagnostic usefulness of the JRS scoring system in COVID-19 pneumonia between the Delta variant group (vaccination period) and non-Delta variant group (before the vaccination period). METHODS: This study was conducted at five institutions and assessed a total of 1121 patients with COVID-19 pneumonia (298 had the Delta variant). During the vaccination period, the Delta variant has spread and replaced the Alfa variant. We evaluated the vaccination period as the Delta variant group. RESULTS: Among the six parameters of the JRS scoring system, matching rates of two parameters were higher in the Delta variant group than the non-Delta variant group (pre-vaccination period): age <60 years (77.5% vs 42.2%, P < 0.0001) and no or minor comorbid illness (69.1% vs 57.8%, p = 0.0007). The sensitivity of the diagnosis of atypical pneumonia in patients with COVID-19 pneumonia was significantly higher in the Delta variant group compared with the non-Delta variant group (80.2% vs 58.3%, p < 0.0001). When the diagnostic sensitivity was analyzed for different ages, the diagnostic sensitivities for the Delta variant and non-Delta variant groups were 92.6% and 95.5% for non-elderly patients and 39.1% and 32.5% for elderly patients, respectively. CONCLUSIONS: Our results demonstrated that the JRS scoring system is a useful tool for distinguishing between COVID-19 pneumonia and bacterial pneumonia in the COVID-19 vaccination period, but not before the vaccination period.

Early identification of novel coronavirus (COVID-19) pneumonia using clinical and radiographic findings.

INTRODUCTION: The Japanese Respiratory Society (JRS) scoring system is a useful tool for identifying Mycoplasma pneumoniae pneumonia. Most COVID-19 pneumonia in non-elderly patients (aged <60 years) are classified as atypical pneumonia using the JRS scoring system. We evaluated whether physicians could distinguish between COVID-19 pneumonia and M. pneumoniae pneumonia using chest computed tomography (CT) findings. In addition, we investigated chest CT findings if there is a difference between the variant and non-variant strain. METHODS: This study was conducted at five institutions and assessed a total of 823 patients with COVID-19 pneumonia (335 had lineage B.1.1.7.) and 100 patients with M. pneumoniae pneumonia. RESULTS: In COVID-19 pneumonia, at the first CT examination, peripheral, bilateral ground-glass opacity (GGO) with or without consolidation or crazy-paving pattern was observed frequently. GGO frequently had a round morphology (39.2%). No differences were observed in the radiological findings between the non-B.1.1.7 groups and B.1.1.7 groups. The frequency of pleural effusion, lymphadenopathy, bronchial wall thickening and nodules (tree-in-bud and centrilobular) was low. In contrast to COVID-19 pneumonia, bronchial wall thickening (84%) was observed most frequently, followed by nodules (81%) in M. pneumoniae pneumonia. These findings were significantly higher in M. pneumoniae pneumonia than COVID-19 pneumonia. CONCLUSIONS: Our results demonstrated that a combination of the JRS scoring system and chest CT findings is useful for the rapid presumptive diagnosis of COVID-19 pneumonia in patients aged <60 years. However, this clinical and radiographic diagnosis is not adapted to elderly people.

Clinical features of nursing and healthcare-associated pneumonia due to COVID-19.

INTRODUCTION: The objective of this study was to clarify the clinical differences between nursing and healthcare-associated pneumonia (NHCAP) and community-acquired pneumonia (CAP) due to COVID-19. We also investigated the clinical characteristics to determine whether there is a difference between the variant and non-variant strain in patients with NHCAP due to COVID-19. In addition, we analyzed the clinical outcomes in NHCAP patients with mental disorders who were hospitalized in a medical institution for treatment of mental illness. METHODS: This study was conducted at five institutions and assessed a total of 836 patients with COVID-19 pneumonia (154 cases were classified as NHCAP and 335 had lineage B.1.1.7.). RESULTS: No differences in patient background, clinical findings, disease severity, or outcomes were observed in patients with NHCAP between the non-B.1.1.7 group and B.1.1.7 group. The median age, frequency of comorbid illness, rates of intensive care unit stay, and mortality rate were significantly higher in patients with NHCAP than in those with CAP. Among the patients with NHCAP, the mortality rate was highest at 37.5% in patients with recent cancer treatment, followed by elderly or disabled patients receiving nursing care (24.3%), residents of care facilities (23.0%), patients receiving dialysis (13.6%), and patients in mental hospitals (9.4%). CONCLUSIONS: Our results demonstrated that there were many differences in the clinical characteristics between NHCAP patients and CAP patients due to COVID-19. It is necessary to consider the prevention and treatment content depending on the presence or absence of applicable criteria for NHCAP.

Functional design of glycan-conjugated molecules using a chemoenzymatic approach.

Carbohydrates play important and diverse roles in the fundamental processes of life. We have established a method for accurately and a large-scale synthesis of functional carbohydrates with diverse properties using a unique enzymatic method. Furthermore, various artificial glycan-conjugated molecules have been developed by adding these synthetic carbohydrates to macromolecules and to middle- and low-molecular-weight molecules with different properties. These glycan-conjugated molecules have biological activities comparable to or higher than those of natural compounds and present unique functions. In this review, several synthetic glycan-conjugated molecules are taken as examples to show design, synthesis, and function.

Detection of Mycoplasma pneumoniae using a highly sensitive rapid diagnostic method with silver amplification technology.

To prevent an increase in the frequency of antimicrobial resistance (AMR), the specific and rapid diagnosis of causative pathogens is important, as the results can be used to initiate appropriate antibiotics treatment. Recently, the highly sensitive rapid immunochromatographic assay of silver amplification technology was developed for the detection of Mycoplasma pneumoniae. We investigated the sensitivity and specificity of the silver amplification immunochromatographic assay in adolescent and adult patients. A total of 767 patients with respiratory tract infection (RTI) and 605 with pneumonia were assessed by the silver amplification assay and real-time polymerase chain reaction (PCR). M. pneumoniae was identified by PCR in 95 patients with RTI and in 30 patients with community-acquired pneumonia (CAP), but it was not identified in patients with nursing- and healthcare-associated pneumonia. Eighteen of the 95 RTI patients and 7 of the 30 CAP patients with PCR-positive M. pneumoniae were found to be infected with macrolide-resistant M. pneumoniae. When PCR was used as the control test, the sensitivity, specificity, and overall agreement with the silver amplification assay were 90.5%, 99.0%, and 97.9%, respectively, in RTI patients and 90.0%, 99.1%, and 98.7%, respectively, in pneumonia patients. Our results show that the silver amplification assay has excellent sensitivity and specificity compared with PCR despite being a rapid diagnostic method. The silver amplification assay may be helpful for initiating appropriate antibiotic treatment and preventing AMR.

Origins of Thermal Spin Depolarization in Half-Metallic Ferromagnet CrO_{2}.

Using high-resolution spin-resolved photoemission spectroscopy, we observe a thermal spin depolarization to which all spin-polarized electrons contribute. Furthermore, we observe a distinct minority spin state near the Fermi level and a corresponding depolarization that seldom contributes to demagnetization. The origin of this depolarization has been identified as the many-body effect characteristic of half-metallic ferromagnets. Our investigation opens an experimental field of itinerant ferromagnetic physics focusing on phenomena with sub-meV energy scale.

Neutrophil-to-lymphocyte ratio as an early marker of outcomes in patients with advanced non-small-cell lung cancer treated with nivolumab.

BACKGROUND: There is an unmet need to identify markers that predict the response to nivolumab in patients with non-small-cell lung cancer (NSCLC). The neutrophil-to-lymphocyte ratio (NLR) was recently recognized as an indicator of a poor prognosis in patients with various cancers. In the present study, we quantified the predictive impact of NLR in patients with NSCLC treated with nivolumab. METHODS: We retrospectively analyzed 101 patients with advanced NSCLC treated with nivolumab at Kansai Medical University Hospital from December 2015 to December 2016. Patients were administered nivolumab at a dose of 3 mg/kg every 2 weeks. The predictive value of NLR for disease progression before treatment and 2 and 4 weeks after nivolumab treatment was assessed. RESULTS: The median progression-free survival (PFS) of patients with an NLR of < 3 before treatment was 3.4 months, whereas that of patients with an NLR of ≥ 3 was 2.9 months (p = 0.484). The median PFS of patients with an NLR of < 3 at 2 weeks after treatment was 5.3 months, whereas that of patients with an NLR of ≥ 3 was 2.1 months (p = 0.00528). The median PFS of patients with an NLR of < 3 at 4 weeks after treatment was 5.3 months, whereas that of patients with an NLR of ≥ 3 was 2.0 months (p = 0.00515). CONCLUSION: The NLR at 2 and 4 weeks after treatment might be a useful marker for the prediction of the treatment response or disease progression in patients with advanced NSCLC receiving nivolumab.

A novel analytical procedure for assaying lysozyme activity using an end-blocked chitotetraose derivative as substrate.

An end-modified ß-d-galactosyl chitotetraose derivative [44-O-ß-d-galactosyl-ß-tri-N-acetylchitotriosyl 2-acetamide-2,3-dideoxy-glucopyranose; Gal(GlcN)3D] was designed and synthesized from chitin tetrasaccharide. The derivative was chemically modified by dehydration of the reducing end GlcN and enzymatic addition of a Gal group to the non-reducing end GlcN. Hydrolysis of Gal(GlcN)3D and related compounds using hen egg-white lysozyme was then examined. Gal(GlcN)3D was specifically cleaved to Gal(GlcN)2 and GlcND. Kinetic studies and docking simulations were further conducted to elucidate its mode of binding to lysozyme. These analyses revealed the binding of Gal(GlcN)3D to lysozyme is more favorable than that of (GlcN)4D. We conclude the 4-O-substituted Gal group at the non-reducing end of Gal(GlcN)3D does not prohibit the action of lysozyme, but gives some affinity to the subsite (i.e. equivalent to GlcN). From these results, a new assay method for quantifying lysozyme was established by utilizing the Morgan-Elson reaction based on the generation of product D (2-acetamide-2,3-dideoxy-glucopyranose), which serves as a chromophore, formed from Gal(GlcN)3D by lysozyme through a conjugated reaction involving ß-N-acetylhexosaminidase. The assay system gave a linear dose-response curve in the range of 2-31 µg of lysozyme during a 15 min incubation. This novel assay method for the quantification of lysozyme is highly specific, sensitive, accurate and reproducible.

Chemoenzymatic synthesis and characterization of N-glycolylneuraminic acid-carrying sialoglycopolypeptides as effective inhibitors against equine influenza virus hemagglutination.

A series of novel sialoglycopolypeptides carrying N-glycolylneuraminic acid (Neu5Gc)-containing trisaccharides having α(2 â†’ 3)- and α(2 â†’ 6)-linkages in the side chains of γ-polyglutamic acid (γ-PGA) were designed as competitive inhibitors against equine influenza viruses (EIV), which critically recognize the Neu5Gc residue for receptor binding. Using horse red blood cells (HRBC) we successfully evaluated the binding activity of the multivalent Neu5Gc ligands to both equine and canine influenza viruses in the hemagglutination inhibition (HI) assay. Our findings show the multivalent α2,3-linked Neu5Gc-ligands (3a-c and 7) selectively inhibit hemagglutination mediated by both influenza viruses and display a strong inhibitory activity. Our results indicate that the multivalent Neu5Gc-ligands can be used as novel probes to elucidate the mechanism of infection/adhesion of Neu5Gc-binding influenza viruses.

Synthesis of tetravalent LacNAc-glycoclusters as high-affinity cross-linker against Erythrina cristagalli agglutinin.

Four kinds of tetravalent double-headed glycoclusters [(LacNAc)4-DHGs] were designed with linkers of varying lengths consisting of alkanedioic carboxyamido groups (C6, C12, C18 and C24) between two bi-antennary LacNAc-glycosides. These glycoclusters served as high-affinity cross-linking ligands for the LacNAc-binding lectin Erythrina cristagalli agglutinin (ECA). The binding activity and cross-linking between each ligand and ECA were characterized by a hemagglutination inhibition (HI) assay, isothermal titration calorimetry (ITC), a quantitative precipitation assay and dynamic light scattering (DLS). For the precipitation assay and DLS measurement, the synthesized (LacNAc)4-DHGs were found to be capable of binding and precipitating the ECA as multivalent ligands. ITC analysis indicated the binding of (LacNAc)4-DHGs was driven by a favorable enthalpy change. Furthermore, the entropy penalty from binding (LacNAc)4-DHGs clearly decreased in a spacer length-dependent manner. The binding affinities of flexible (LacNAc)4-DHGs (C18 and C24) with long spacers were found to be more favorable than those of the clusters having short spacers (C6 and C12). These results were supported by molecular dynamics simulations with explicit water molecules for the tetravalent glycoclusters with ECA. We concluded that the subtle modification in the epitope-presenting scaffolds exerts the significant effect in the recognition efficiency involved in the LacNAc moieties by ECA.

The structure of the proteinaceous inhibitor PliI from Aeromonas hydrophila in complex with its target lysozyme.

Recent microbiological data have revealed that Gram-negative bacteria are able to protect themselves against the lytic action of host lysozymes by secreting proteinaceous inhibitors. Four distinct classes of such inhibitors have been discovered that specifically act against c-type, g-type and i-type lysozymes. Here, the 1.24 Šresolution crystal structure of the periplasmic i-type lysozyme inhibitor from Aeromonas hydrophila (PliI-Ah) in complex with the i-type lysozyme from Meretrix lusoria is reported. The structure is the first to explain the inhibitory mechanism of the PliI family at the atomic level. A distinct `ridge' formed by three exposed PliI loops inserts into the substrate-binding groove of the lysozyme, resulting in a complementary `key-lock' interface. The interface is principally stabilized by the interactions made by the PliI-Ah residues Ser104 and Tyr107 belonging to the conserved SGxY motif, as well as by the other conserved residues Ser46 and Asp76. The functional importance of these residues is confirmed by inhibition assays with the corresponding point mutants of PliI-Ah. The accumulated structural data on lysozyme-inhibitor complexes from several classes indicate that in all cases an extensive interface of either a single or a double `key-lock' type is formed, resulting in highly efficient inhibition. These data provide a basis for the rational development of a new class of antibacterial drugs.