Additive impact of soy protein dietary intake and exercise on visceral fat mass reduction and mitochondrial complex I activation in skeletal muscle.

Soy protein has shown remarkable effectiveness in reducing fat mass compared with other protein sources, and exercise has the potential to further enhance this fat loss effect. Previous studies have demonstrated that soy protein intake leads to decreased fatty acid synthesis, which contributes to its fat-loss properties. However, the exact mechanism by which these lipids are consumed remains unclear. To investigate this, we conducted a comprehensive study using C57/BL6 male mice, comparing the effects of soy and casein proteins with and without exercise (Casein-Sed, Casein-Ex, Soy-Sed, and Soy-Ex groups) under high- and low-protein conditions (14% or 40% protein). Our findings revealed that combining soy protein intake with exercise significantly reduced epididymal white adipose tissue (eWAT) weight, particularly in the high-protein diet group. Further analysis revealed that exercise increased the expression of lipid oxidation-regulatory proteins, including mitochondrial oxidative phosphorylation protein (OXPHOS) complexes, in the plantaris muscle regardless of the protein source. Although soy protein intake did not directly affect muscle mitochondrial protein expression, the activity of OXPHOS complex I was additively enhanced by exercise and soy protein under the 40% protein condition. Notably, complex I activity inversely correlated with eWAT weight in the soy protein diet group. These results highlight the potential link between improved complex I activity induced by soy protein and fat mass reduction, which emphasizes the promising benefits of combining soy protein with exercise in promoting fat loss.NEW & NOTEWORTHY The findings revealed that soy protein intake combined with exercise resulted in reduced adipose tissue weight compared with that obtained with casein protein intake. Furthermore, the joint impact of exercise and soy protein consumption resulted in enhanced activity of oxidative phosphorylation protein (OXPHOS) complex I in fast-twitch muscles, which appears to be associated with fat mass reduction. These findings elucidate the potential additive effects of soy protein and exercise on body weight management.

Intrinsic clarithromycin heteroresistance in Mycobacterium avium.

BACKGROUND: Mycobacterium avium is associated with pulmonary disease in otherwise healthy adults. Several clarithromycin-refractory cases have been reported, including some cases caused by clarithromycin-susceptible strains. OBJECTIVES: To characterize the reason for the discrepancy between clinical response and antibiotic susceptibility results. METHODS: We conducted population analysis of clarithromycin-tolerant and heteroresistant subpopulations of M. avium cultured in vitro and in homogenates of infected lungs of mice. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for 28 M. avium and two M. kansasii strains. Mice were intranasally infected with M. avium and treated with or without clarithromycin (100 mg/kg) thrice weekly. They were sacrificed on day 35 and the bacteria in lung homogenates were tested for clarithromycin resistance. Population analysis assays were performed based on colony growth on plates containing two-fold dilutions of clarithromycin. RESULTS: The MBC/MIC ratios were ≥8 in all 28 strains of M. avium tested. In the population analysis assay, several colonies were observed on the plates containing clarithromycin concentrations above the MIC (2-64 mg/L). No growth of M. kansasii colonies was observed on the plates containing clarithromycin concentrations ≥2 mg/L. M. avium in the homogenates of infected lungs showed clearer clarithromycin-resistant subpopulations than in vitro, regardless of clarithromycin exposure. CONCLUSION: M. avium shows intrinsic heterogeneous resistance (heteroresistance) to clarithromycin. This may explain the observed discrepancies between clarithromycin susceptibility testing results and clinical response to clarithromycin treatment. Further studies are needed to confirm a link between heteroresistance and clinical outcomes.

Detection of parechovirus-A in hospitalized children with acute lower respiratory infection in Myanmar, 2017-2018.

Parechovirus-A (PeV-A) causes emerging infection in children, and clinical presentation depends on genotype. The virus has been investigated mainly in developed countries; however, data from developing countries, especially in Asia, are sparse. This study investigated whether PeV-A circulated in children in Myanmar. This retrospective study evaluated PeV-A in nasopharyngeal samples from children aged 1 month to 12 years who were hospitalized with acute lower respiratory infection at Yankin Children Hospital, Yangon, Myanmar, during the period from May 2017 to April 2019. Real-time polymerase chain reaction (PCR) was used to detect PeV-A, and PCR-positive samples were used for genotyping and phylogenetic analysis. In total, 11/570 (1.9%) of samples were positive for PeV-A; 7 were successfully genotyped by sequencing the VP3/VP1 region, as follows: PeV-A1 (n = 4), PeV-A5 (n = 1), PeV-A6 (n = 1), and PeV-A14 (n = 1). Median age was 10.0 months (interquartile range 4.0-12.0 months), and other respiratory viruses were detected in all cases. Phylogenetic analysis showed that all detected PeV-A1 strains were in clade 1 A, which was a minor clade worldwide. Four PeV-A genotypes were detected in Myanmar. The clinical impact of PeV-A in children should be evaluated in future studies.

Structure and Additive-free Transamidation of Planar N-Cyano Amides.

Although transamidation of amides generally requires metals, additives, or harsh conditions, we present here a facile transamidation of N-cyano amides with various amines at ambient temperature without any additive. N-cyano amides preferred the trans conformation and have a reduced double bond character revealed by crystal analysis. The DFT study indicates that the transamidation reaction proceeds through the direct attack of amine on the amide carbonyl since the LUMO (or LUMO+1) is located at the carbonyl moiety.

Synthesis and Conformational Analysis of N-Aromatic Acetamides Bearing Thiophene: Effect of Intramolecular Chalcogen-Chalcogen Interaction on Amide Conformational Stability.

The conformations of aromatic amides bearing an N-(2-thienyl) or N-(3-thienyl) group were investigated in solution and in the crystal state. NMR spectral data indicate that the conformational preferences of these amides in solution are dependent not only on the relative π-electron densities of the N-aromatic moieties, but also on the three-dimensional relationship between carbonyl oxygen and the N-aromatic moieties. A comparison of the conformational preferences of N-(2-thienyl)amides and N-(3-thienyl)amides revealed that the Z-conformers of N-(2-thienyl)acetamides are stabilized by 1,5-type intramolecular S···O═C interactions between amide carbonyl and thiophene sulfur. The crystal structures of these compounds were similar to the solution structures. The stabilization energy due to 1,5-type intramolecular S···O═C interaction in N-aryl-N-(2-thienyl)acetamides and N-methyl-N-(2-thienyl)acetamide was estimated to be ca. 0.74 and 0.93 kcal/mol, respectively.

Structures and Reactivities of N-Alkenyl-Substituted Anilides: The "Magic" Methyl Effect on Alkene.

Methyl substitution at the double bond of N-alkenyl anilides influences both the preferred conformation and the susceptibility to acidic hydrolysis. The R1-substituted amide favors the trans conformation, whereas amides substituted at R2 or R3 favor the cis conformation. Substitution at the R1 and R3 positions increases the ratio of the trans conformer. DFT study indicated that these conformational preferences can be explained in terms of substituent-induced torsion twisting of the N-alkenyl moiety relative to the amide plane. R1 substitution enhances the susceptibility to acidic hydrolysis, whereas R2 or R3 substitution increases the stability. The effect of the double bond on the conformational effect was showcased by contrasting the preferred conformation of R1-substituted anilide (trans) and hydrogenated N-isopropyl amide (cis).

The neuronal nitric oxide synthase expression increases during satellite cell-derived primary myoblasts differentiation.

The neuronal nitric oxide synthase (nNOS; encoded by NOS1)-derived nitric oxide (NO) plays an important role in maintaining skeletal muscle mass. In adult skeletal muscle, nNOS localizes to the cell membrane, cytosol, and nucleus, and regulates muscle hypertrophy and atrophy in various subcellular fractions. However, its role in muscle stem cells (also known as muscle satellite cells), which provide myonuclei for postnatal muscle growth, maintenance, and regeneration, remains unclear. The present study aimed to determine nNOS expression in muscle satellite cell-derived primary myoblasts during differentiation and its DNA methylation levels, an epigenetic modification that controls gene expression. Undifferentiated and differentiated satellite cell-derived primary myoblasts were found to express nNOS. Immunohistochemical analysis revealed that nNOS colocalized with Pax7 (satellite cell marker) only in the undifferentiated myoblasts. Furthermore, nNOS immunoreactivity spread to the cytosol of Pax7-negative differentiated myotube-like cells. The level of Nos1µ mRNA, the main isoform of skeletal muscle nNOS, was increased in differentiated satellite cell-derived primary myoblasts compared to that in the undifferentiated cells. However, Nos1 methylation levels remained unchanged during differentiation. These findings suggest that nNOS induction and the appropriate transition of its subcellular localization may contribute to muscle differentiation.

The differences in renal hemodynamic response following high-intensity exercise between younger and older males.

BACKGROUND: Renal blood flow (RBF) decreases with exercise, but this change is only temporary, and habitual exercise may be an effective method to improve renal function. The kidney shows structural and functional changes with aging, but it is unclear how aging affects the hemodynamic response of the kidneys to exercise. Therefore, we evaluated the differences in the hemodynamic response of the kidneys to high-intensity exercise between younger and older men. METHODS: Sixteen men (8 young and 8 older) underwent an incremental exercise test using a cycle ergometer with a 1-min warm up followed by exercise at 10-20 W/min until the discontinuation criteria were met. Renal hemodynamics were assessed before exercise, immediately after exercise, and at 60-min after exercise using ultrasound echo. RESULTS: High-intensity exercise significantly reduced RBF in both groups (younger: ∆ - 53 ± 16%, p = 0.0005; older: ∆ - 53 ± 19%, p = 0.0004). In the younger group, RBF returned to the pre-exercise level 60-min after exercise (∆ - 0.4 ± 5.7%, p > 0.9999). In contrast, RBF 60-min after exercise was significantly lower than that before exercise in the older group (∆ - 24 ± 19%, p = 0.0006). The older group had significantly lower RBF than younger adults 60-min after exercise (423 ± 32 vs. 301 ± 98 mL/min, p = 0.0283). CONCLUSIONS: Our findings demonstrate that RBF following high-intensity exercise recovered 60-min after exercise in younger group, whereas RBF recovery was delayed in the older group.

Short-term running exercise alters DNA methylation patterns in neuronal nitric oxide synthase and brain-derived neurotrophic factor genes in the mouse hippocampus and reduces anxiety-like behaviors.

Running exercise has beneficial effects on brain health. However, the effects of relatively short-term running exercise (STEx) on behavior, and its underlying signaling pathways, are poorly understood. In this study, we evaluated the possibility that the regulation by STEx of brain-derived neurotrophic factor (BDNF) and neuronal nitric oxide synthase (nNOS, encoded by NOS1), which are important molecules for anxiety regulation, might involve mechanisms of epigenetic modification, such as DNA methylation. C57BL/6J male mice were divided into sedentary (SED, n = 12) and STEx (EX, n = 15) groups; STEx was conducted with the mice for a duration of 11 days. STEx reduced anxiety-like behaviors, and STEx reduced Nos1α and increased Bdnf exon I and IV mRNA levels in the hippocampus. Interestingly, behavioral parameters were associated with Bdnf exon I and IV and Nos1α mRNA levels in the ventral, but not dorsal, hippocampal region. However, STEx had no effect on peroxisome proliferator-activated receptor-γ coactivator 1α (Pgc-1α) or fibronectin type III domain-containing 5 (Fndc5) mRNA levels, which are relatively long-term exercise-induced upstream regulators of BDNF. In parallel with gene expression changes, we found, for the first time, that STEx downregulated Bdnf promoter IV and upregulated Nos1 DNA methylation levels in the hippocampus, and these patterns were partially different between the dorsal and ventral regions. These findings suggest that the beneficial effects of running exercise on mood regulation may be controlled by alterations in epigenetic mechanisms, especially in the ventral hippocampus. These effects occur even after a relatively short-term period of exercise.

Conformational Switch of Benzanilide Derivative Induced by Acid; Effect of Pentafluorobenzoyl Group.

Amide-based molecular switches had its limitation on structural diversities. In this work, we designed and synthesized a series of pentafluorobenzoyl-based benzanilide compounds. The conformational ratio of these compounds in solution was correlated linearly with Hammett's σp value of the substituent on the anilide ring, reflecting the repulsive interaction between the carbonyl group and the electron-rich aryl group. The addition of acid into the solution of 6, bearing pentafluorobenzoyl group, switched the stable amide conformation. In addition, the sizeable rotational barrier of 6 induced by the pentafluorobenzoyl moiety enabled us to monitor the conformational transition by means of 1H NMR spectroscopy.

Clinical manifestations and outcome of viral acute lower respiratory infection in hospitalised children in Myanmar.

BACKGROUND: Acute lower respiratory infection (ALRI) remains the leading cause of death in children worldwide, and viruses have been the major cause of ALRI. In Myanmar, ALRI is associated with high morbidity and mortality in children, and detailed information on ALRI is currently lacking. METHODS: This prospective study investigated the viral aetiologies, clinical manifestations, and outcomes of ALRI in hospitalised children aged 1 month to 12 years at the Yankin Children Hospital, Yangon, Myanmar from May 2017 to April 2019. The sample size was set to 300 patients for each year. Two nasopharyngeal swabs were obtained for the patients with suspected viral ALRI; one for rapid tests for influenza and respiratory syncytial virus (RSV), and the other for real-time PCR for the 16 ALRI-causing viruses. Pneumococcal colonization rates were also investigated using real-time PCR. Clinical information was extracted from the medical records, and enrolled patients were categorised by age and severity for comparison. RESULTS: Among the 5463 patients admitted with a diagnosis of ALRI, 570 (10.4%) were enrolled in this study. The median age of the patients was 8 months (interquartile range, 4-15 months). The most common symptoms were cough (93%) and difficulty in breathing (73%), while the most common signs of ALRI were tachypnoea (78%) and chest indrawing (67%). A total of 16 viruses were detected in 502 of 570 patients' samples (88%), with RSV B (36%) and rhinovirus (28%) being the most commonly detected. Multiple viruses were detected in 221 of 570 samples (37%) collected from 570 patients. Severe ALRI was diagnosed in 107 of 570 patients (19%), and RSV B and human rhinovirus were commonly detected. The mortality rate was 5%; influenza virus A (29%) and RSV B (21%) were commonly detected, and stunting and lack of immunization were frequently observed in such cases. Additionally, 45% (259/570) of the patients had pneumococcal colonization. CONCLUSIONS: Viral ALRI in hospitalised children with a median of 8 months has significant morbidity and mortality rates in Myanmar. RSV and rhinovirus were the most commonly detected from nasopharyngeal swabs, while influenza virus and RSV were the most frequently associated with fatal cases.

Neurolymphomatosis in follicular lymphoma: an autopsy case report.

Neurolymphomatosis is a neurological manifestation of lymphoma that involves the cranial or spinal peripheral nerves, nerve roots, and plexus with direct invasion of neoplastic cells. Neurolymphomatosis is rare among patients with low-grade lymphoma. We report an autopsied case of neurolymphomatosis that arose from follicular lymphoma. A 49-year-old woman who presented with pain of her neck and shoulder and numbness of her chin. Computed tomography revealed enlarged lymph nodes in her whole body, and biopsy from the axillary lymph node revealed grade 2 follicular lymphoma. Although the patient underwent chemotherapy, she gradually developed muscle weakness in the upper limbs and sensory disturbances of the trunk and limbs. 18 F-fluorodeoxyglucose positron emission tomography (FDG-PET) revealed increased tracer uptake of the cervical nerve roots. Repeated FDG-PET after additional therapy revealed progression of disease within the nerve roots and brachial plexus, whereas gadolinium-contrast magnetic resonance imaging (MRI) showed weak enhancement of the cervical nerve roots without formation of mass lesions. She died after a total disease duration of 12 months. Postmortem observations revealed invasion of lymphoma cells into the cervical nerve roots, dorsal root ganglia, and subarachnoid spaces of the spinal cord. Neurolymphomatosis was prominent at the segments of C6-Th2. Combined loss of axons and myelin sheaths was observed in the cervical nerve roots and posterior columns. Lymphoma cells also invaded the cranial nerves. The subarachnoid and perivascular spaces of the brain demonstrated focal invasion of the lymphoma. Mass lesions were not observed in the central nervous system. The lymphoma cells did not show histological transformation to higher grades, and the density of the centroblasts remained at grade 2. Our report clarifies that low-grade follicular lymphoma can manifest as neurolymphomatosis and central nervous system invasion in the absence of transformation toward higher histological grades. FDG-PET may be more sensitive to non-mass-forming lesions, including neurolymphomatosis, than gadolinium-contrast MRI.

Utility of emergent plain X-ray for childhood acute leukemia with bone pain.

BACKGROUND: We aimed to evaluate the utility of plain X-ray radiograph (PXR) findings in suggesting a diagnosis of acute leukemia in children presenting with bone pain in the emergency department (ED) of a children's hospital. METHODS: Using our radiology reporting system and registered data for childhood acute leukemia, we collected data regarding patients who underwent musculoskeletal PXR examinations in the ED due to bone pain in their extremities, from March 1, 2002 to June 30, 2015. We retrospectively reviewed their PXR findings and clinical information from the electronic medical records. RESULTS: A total of 1,331 patients underwent PXR examinations and in 12 PXR findings showed suspected acute leukemia. From the registered data we found 12 acute leukemia patients who underwent emergency extremity PXR. Ten patients were finally confirmed to have acute leukemia by bone marrow examinations. The most common finding was lucent metaphyseal bands, demonstrated in seven cases, whereas six patients did not show any abnormalities in their peripheral blood cell counts. Sensitivity and specificity values of PXR for acute leukemia diagnosis were 90.0% and 99.8%, respectively. Positive predictive value and negative predictive values were 75.0% and 99.9%, respectively. CONCLUSIONS: Plain X-ray radiograph is a useful diagnostic tool to detect possible acute leukemia in patients presenting with bone pain, earlier than abnormalities of their peripheral blood cell counts. Our results implied the possibility of re-examining PXRs in acute leukemia more carefully, even when there are no abnormalities in blood cell counts.

Metolazone upregulates mitochondrial chaperones and extends lifespan in Caenorhabditis elegans.

Accumulating studies have argued that the mitochondrial unfolded protein response (UPRmt) is a mitochondrial stress response that promotes longevity in model organisms. In the present study, we screened an off-patent drug library to identify compounds that activate UPRmt using a mitochondrial chaperone hsp-6::GFP reporter system in Caenorhabditis elegans. Metolazone, a diuretic primarily used to treat congestive heart failure and high blood pressure, was identified as a prominent hit as it upregulated hsp-6::GFP and not the endoplasmic reticulum chaperone hsp-4::GFP. Furthermore, metolazone specifically induced the expression of mitochondrial chaperones in the HeLa cell line. Metolazone also extended the lifespan of worms in a atfs-1 and ubl-5-dependent manner. Notably, metolazone failed to increase lifespan in worms with knocked-down nkcc-1. These results suggested that metolazone activates the UPRmt across species and prolongs the lifespan of C. elegans.

Bronchial Brushing and Diagnosis of Pulmonary Nontuberculous Mycobacteria Infection.

BACKGROUND: The optimal bronchoscopy procedure for diagnosis of pulmonary nontuberculous mycobacteria (NTM) infection is unclear. OBJECTIVE: This study investigated the usefulness of bronchial brushing in bronchoscopy for diagnosis of pulmonary NTM infection in patients with suspected NTM lung disease and nodular bronchiectasis on chest computed tomography (CT) images. METHODS: Bronchoscopy was prospectively performed for 69 patients with clinically suspected pulmonary NTM infection on chest CT from December 2017 through December 2019. Before and after bronchial brushing, bronchial washing was performed with 20 or 40 mL of normal sterile saline at the same segmental or subsegmental bronchi. Before and after bronchial brushing, samples of the washing fluid (pre- and postbrushing samples) and brush deposits (brush samples) were obtained and cultured separately. RESULTS: NTM was detected in 37 of the 69 (53.6%) patients (Mycobacterium avium in 27, Mycobacterium intracellulare in 7, M. abscessus in 2, and M. kansasii in 2). NTM was detected in 34 (49.3%) prebrushing samples, in 27 (39.1%) postbrushing samples, and in 20 (29.0%) brush samples from the 69 patients. In 2 (2.9%) patients, NTM was detected only in postbrushing samples; in 1 (1.4%) patient, NTM was detected only in a brush sample. As compared with bronchial washing only, additional bronchial brushing increased the NTM culture-positive rate by 4.3% (3/69). Bronchial brushing caused bleeding, requiring hemostasis in 5 (7.2%) patients. CONCLUSION: Additional bronchial brushing increased the NTM culture-positive rate by only 4.3% (3/69), as compared with bronchial washing alone. Thus, the usefulness of brushing appears to be limited.

SYNTHESIS OF PROBE MOLECULES, 6-(DIMETHYLAMINO)-2-PHENYLISOINDOLIN-1-ONES, FOR MECHANISTIC STUDIES OF FIREFLY LUCIFERASE INHIBITION.

Firefly luciferase is used in high-throughput screening based on the detection of chemiluminescence. It catalyzes an esterification reaction of luciferin with adenosine 5'-triphosphate (ATP) followed by decarbonylation with oxygen and concomitance of light. Previously, we reported that firefly luciferase also possesses acyl-CoA synthetase activity and catalyzes an aromatic carboxylic acid group of F-53, using ATP, Mg2+ and coenzyme A (CoA), to produce F-53 covalently attached to active-site lysine-529 residue of firefly luciferase through the formation of an amide group. The amidation of lysine-529 resulted in a deactivation of luciferase. In order to probe firefly luciferase inhibition's mechanism, we synthesized two probe molecules 1 and 2, mimicking F-53. Molecule 1 contains an azido-appended side chain in the aromatic ring of F-53, while 2 possesses an azido and a carboxylic acid group appended side chains. Both synthetic schemes are readily amenable to large-scale syntheses. Molecule 1 was made from 2-allylaniline, which was derived from a thermal-induced aromatic-Claisen rearrangement of N-allylaniline. The azido-appended side chain of 2 was installed from a Horner-Wadsworth-Emmons reaction and the carboxylic acid side chain from a Sonogashira reaction.

Cortical Microinfarcts Detected by 3-Tesla Magnetic Resonance Imaging: Differentiation Between Cerebral Amyloid Angiopathy and Embolism.

Background and Purpose- Cortical microinfarcts (CMIs) are small ischemic lesions found in cerebral amyloid angiopathy (CAA) and embolic stroke. This study aimed to differentiate CMIs caused by CAA from those caused by microembolisms, using 3-Tesla magnetic resonance imaging. Methods- We retrospectively investigated 70 patients with at least 1 cortical infarct <10 mm on 3-dimensional double inversion recovery imaging. Of the 70 patients, 43 had an embolic stroke history (Emboli-G) while 27 had CAA-group. We compared the size, number, location, and distribution of CMIs between groups and designed a radiological score for differentiation based on the comparisons. Results- CAA-group showed significantly more lesions <5 mm, which were restricted to the cortex (P<0.01). Cortical lesion number was significantly higher in Emboli-G than in CAA-group (4 versus 2; P<0.01). Lesions in CAA-group and Emboli-G were disproportionately located in the occipital lobe (P<0.01) and frontal or parietal lobe (P=0.04), respectively. In radiological scoring, ≥3 points strongly predicted microembolism (sensitivity, 63%; specificity, 92%) or CAA (sensitivity, 63%; specificity, 91%). The areas under the receiver operating characteristic curve were 0.85 and 0.87 for microembolism and CAA, respectively. Conclusions- Characteristics of CMIs on 3T-magnetic resonance imaging may differentiate CMIs due to CAA from those due to microembolisms.

IgG4 plasma cell myeloma: Clinicopathological characteristics and diagnosis.

Plasma cell myeloma (PCM) is usually associated with the presence of M-protein in the serum and urine of patients, and about half of the PCM cases exhibit the IgG M-protein and increased gamma-globulin fraction on membrane electrophoresis. The IgG4 subclass is located in the beta-2 fraction on membrane electrophoresis. The aim of this study was to develop a method to evaluate IgG4-producing PCM (IgG4-PCM) and its clinicopathological characteristics. We found three cases of IgG4-PCM among 80 cases of IgG-producing PCM by membrane electrophoresis, which were confirmed by IgG4 immunostaining. None of the cases had a clinical history of IgG4-related disease, although they exhibited high levels of serum IgG4. A bone marrow aspiration specimen had an increased number of plasma cells with a relatively mature morphology. No cases exhibited lymphoplasmacytic inflammation, obliterative phlebitis or fibrosis. Immunohistochemistry revealed that tumor cells expressed CD138 and IgG4 and showed monoclonal expression of kappa. We revealed that IgG4-PCM might not be associated with IgG4-related disease and that the detection of M-protein with beta-globulin fraction by electrophoresis may be useful for screening IgG4-PCM.

[Leukemic pulmonary infiltration diagnosed by sputum Giemsa-staining].

A 54-year-old man with acute myeloid leukemia (AML) underwent allogeneic bone marrow transplantation from a human leukocyte antigen-matched unrelated donor in nonremission status. Bone marrow aspiration performed on day 14 showed that the patient had achieved complete remission; however, he relapsed on day 28. The patient developed a wet cough, and chest computed tomography performed on day 27 revealed pneumonia. Because pneumonia developed along with the leukemic relapse, we suspected that it was due to pulmonary leukemic infiltration (PLI). Giemsa-stained sputum showed some blast cells and fluorescence in situ hybridization indicated that the patient had monosomy 7, which was also detected in bone marrow blasts. Though we prescribed hydroxycarbamide and decreased tacrolimus rapidly, AML progressed and led to the patient's death on day 45. Histopathological findings of the autopsy performed the next day showed diffuse alveolar damage in both lungs. The blast cells were packed in blood vessels of alveolar septa and were also seen in alveoli. PLI was diagnosed pathologically. In conclusion, our case demonstrates that Giemsa stain of sputum is useful in quick diagnosis of PLI without invasive examination.

One week, but not 12 hours, of cast immobilization alters promotor DNA methylation patterns in the nNOS gene in mouse skeletal muscle.

KEY POINTS: DNA methylation may play an important role in regulating gene expression in skeletal muscle to adapt to physical activity and inactivity. Neuronal nitric oxide synthase (nNOS) in skeletal muscle is a key regulator of skeletal muscle mass; however, it is unclear whether nNOS expression is regulated by DNA methylation. We found that 1 week of cast immobilization increased nNOS DNA methylation levels and downregulated nNOS gene expression in atrophic slow-twitch soleus muscle from the mouse leg. These changes were not detected in non-atrophic fast-twitch extensor digitorum longus muscle. Twelve hours of cast immobilization decreased nNOS gene expression, whereas nNOS DNA methylation levels were unchanged, suggesting that downregulation of nNOS gene expression by short-term muscle inactivity is independent of the DNA methylation pattern. These findings contribute to a better understanding of the maintenance of skeletal muscle mass and prevention of muscle atrophy by epigenetic mechanisms via the nNOS/NO pathway. ABSTRACT: DNA methylation is a mechanism that controls gene expression in skeletal muscle under various environmental stimuli, such as physical activity and inactivity. Neuronal nitric oxide synthase (nNOS) regulates muscle atrophy in skeletal muscle. However, the mechanisms regulating nNOS expression in atrophic muscle remain unclear. We hypothesized that nNOS expression in atrophic muscle is regulated by DNA methylation of the nNOS promotor in soleus (Sol; slow-twitch fibre dominant) and extensor digitorum longus (EDL; fast-twitch fibre dominant) muscles. One week of cast immobilization induced significant muscle atrophy in Sol but not in EDL. We showed that 1 week of cast immobilization increased nNOS DNA methylation levels in Sol, although only a minor change was detected in EDL. Consistent with the increased DNA methylation levels in atrophic Sol, the gene expression levels of total nNOS and nNOSµ (i.e. the major splicing variant of nNOS in skeletal muscle) decreased. The abundance of the nNOS protein and cell membrane (especially type IIa fibre) immunoreactivity also decreased in atrophic Sol. These changes were not observed in EDL after 1 week of cast immobilization. Furthermore, despite the lack of significant atrophy, 12 h of cast immobilization decreased gene expression levels of total nNOS and nNOSµ in Sol. However, no association was detected between nNOS DNA methylation and gene expression. The expression of the nNOSß gene, another splicing variant of nNOS, in EDL was unchanged by cast immobilization, whereas its expression was not detected in Sol. We concluded that chronic adaptation of nNOS gene expression in cast immobilized muscle may involve nNOS DNA methylation.