Spatial multiomic profiling reveals the novel polarization of foamy macrophages within necrotic granulomatous lesions developed in lungs of C3HeB/FeJ mice infected with Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis leads to the development of tuberculosis (TB) with the formation of granulomatous lesions. Foamy macrophages (FM) are a hallmark of TB granulomas, because they provide the primary platform of M. tuberculosis proliferation and the main source of caseous necrosis. In this study, we applied spatial multiomic profiling to identify the signatures of FM within the necrotic granulomas developed in a mouse model resembling human TB histopathology. C3HeB/FeJ mice were infected with M. tuberculosis to induce the formation of necrotic granulomas in the lungs. Using laser microdissection, necrotic granulomas were fractionated into three distinct regions, including the central caseous necrosis, the rim containing FM, and the peripheral layer of macrophages and lymphocytes, and subjected to proteomic and transcriptomic analyses. Comparison of proteomic and transcriptomic analyses of three distinct granulomatous regions revealed that four proteins/genes are commonly enriched in the rim region. Immunohistochemistry confirmed the localization of identified signatures to the rim of necrotic granulomas. We also investigated the localization of the representative markers for M1 macrophages in granulomas because the signatures of the rim included M2 macrophage markers. The localization of both macrophage markers suggests that FM in necrotic granulomas possessed the features of M1 or M2 macrophages. Gene set enrichment analysis of transcriptomic profiling revealed the upregulation of genes related to M2 macrophage activation and mTORC1 signaling in the rim. These results will provide new insights into the process of FM biogenesis, leading to further understanding of the pathophysiology of TB granulomas.

Viral Production in Seawater Filtered Through 0.2-µm Pore-Size Filters: A Hidden Biogeochemical Cycle in a Neglected Realm.

Viral production is a key parameter for assessing virus-mediated biogeochemical cycles. One widely used method for the determination of viral production, called the virus reduction assay, reduces viral abundance, while maintaining bacterial abundance, using 0.2-µm pore-size filters. Viral production is estimated from the increase of viral abundance during incubation. We hypothesized that small-cell-sized bacterial communities can pass through 0.2-µm filters and drive viral production, representing a missing fraction of viral production that is missed by the virus reduction assay. Coastal seawater was filtered through 0.2-µm filters and diluted with virus-free seawater. Viral production in the <0.2-µm filtrate was estimated from changes in viral abundance determined through flow cytometry. We found that viruses were produced in the <0.2-µm communities, which were strongly enriched with low nucleic acid content bacteria. Estimated viral production in the <0.2-µm filtrates accounted for up to 43% of total viral production and 10% of dissolved organic carbon production mediated by viral lysis of bacterial cells. By not considering viral production in these <0.2-µm communities, the virus reduction assay may underestimate viral production. Virus-bacteria interactions in <0.2-µm communities may represent a significant and overlooked role of viruses in marine food webs and carbon fluxes.

New Algorithm by Maximizing Mutual Information for Correction of Frequency Drifts Arising from One-Dimensional NMR Spectroscopic Data Acquisition.

Benchtop nuclear magnetic resonance (NMR) instruments are getting popular these days. However, the obtained spectra sometimes suffer from significant frequency drifts, which cause difficulty in accumulating the raw data. In this paper, a new algorithm for correction of frequency drifts is proposed, which operates by maximizing mutual information between the obtained spectroscopic data. The algorithm worked well for both 1H and 19F NMR spectroscopic data, even in the case of very noisy ones. In comparison with the previously reported algorithms, the present algorithm has an advantage that NMR spectra complicated by signal overlapping and spin coupling can be handled without difficulty. This makes the present algorithm particularly advantageous for application of benchtop NMR spectrometers in organic chemistry.

Transparent Exopolymer Particles in Deep Oceans: Synthesis and Future Challenges.

Transparent exopolymer particles (TEP) are a class of abundant gel-like particles that are omnipresent in seawater. While versatile roles of TEP in the regulation of carbon cycles have been studied extensively over the past three decades, investigators have only recently begun to find intriguing features of TEP distribution and processes in deep waters. The emergence of new research reflects the growing attention to ecological and biogeochemical processes in deep oceans, where large quantities of organic carbon are stored and processed. Here, we review recent research concerning the role of TEP in deep oceans. We discuss: (1) critical features in TEP distribution patterns, (2) TEP sources and sinks, and (3) contributions of TEP to the organic carbon inventory. We conclude that gaining a better understanding of TEP-mediated carbon cycling requires the effective application of gel theory and particle coagulation models for deep water settings. To achieve this goal, we need a better recognition and determination of the quantities, turnover, transport, chemical properties, and microbial processing of TEP.

Perception of mutual aid and its related factors: a study of Japanese high school students.

Japan is a super-ageing country. Constructing the community-based integrated care system in local communities is urgently needed. Mutual aid in local communities is critical for this system. In order to clarify the status of perception of mutual aid in Japanese high school students and to clarify the factors related to the formation of the perception, we conducted a questionnaire study of high school students in a city in Japan (n = 8,687). The results indicate that Japanese high school students show a tendency to have perception of mutual aid for local people (70.8%) rather than the local area (38.9%). Significantly fewer male students have perception of mutual aid than female students (p < 0.01). Factors that affected the perception significantly (p < 0.05) were: i) willingness to stay in the local area for 10 more years, ii) recognition of persons in need of care in the local area, iii) memories of experiencing communication with handicapped and/or elderly people, and iv) experience of taking care of local children. It is important to create opportunities for high school students to communicate with local residents, especially handicapped and/or elderly people in order to foster students' perception of mutual aid.

Distribution of Dimethylsulfoniopropionate Degradation Genes Reflects Strong Water Current Dependencies in the Sanriku Coastal Region in Japan: From Mesocosm to Field Study.

Dimethyl sulfide (DMS) is an important component of the global sulfur cycle as it is the most abundant sulfur compound that is emitted via the ocean surface to the atmosphere. Dimethylsulfoniopropionate (DMSP), the precursor of DMS, is mainly produced by phytoplankton and is degraded by marine bacteria. To reveal the role of bacteria in the regulation of DMSP degradation and DMS production, mesocosm and field studies were performed in the Sanriku Coast on the Pacific Ocean in northeast Japan. The responsible bacteria for the transformation of DMSP to DMS and the assimilation of DMSP were monitored, and the genes encoding DMSP lyase (dddD and dddP) and DMSP demethylase (dmdA) were analyzed. The mesocosm study showed that the dmdA subclade D was the dominant DMSP degradation gene in the free-living (FL) and particle-associated (PA) fractions. The dddD gene was found in higher abundance than the dddP gene in all the tested samples. Most importantly, DMS concentration was positively correlated with the abundance of the dddD gene. These results indicated that bacteria possessing dmdA and dddD genes were the major contributors to the DMSP degradation and DMS production, respectively. The genes dmdA subclade D and dddP were abundant in the Tsugaru Warm (TW) Current, while the dmdA subclade C/2 and dddD genes were dominant in the Oyashio (OY) Current. Functional gene network analysis also showed that the DMSP degradation genes were divided into OY and TW Current-related modules, and genes sharing similar functions were clustered in the same module. Our data suggest that environmental fluctuations resulted in habitat filtering and niche partitioning of bacteria possessing DMSP degradation genes. Overall, our findings provide novel insights into the distribution and abundance of DMSP degradation genes in a coastal region with different water current systems.

Development of a novel T cell-oriented vaccine using CTL/Th-hybrid epitope long peptide and biodegradable microparticles, against an intracellular bacterium.

Antigen-specific CD8+ T-lymphocytes (cytotoxic T-lymphocytes: CTL), as well as CD4+ T-lymphocytes (helper T-lymphocytes: Th), simultaneously play an important role in the elimination of intracellular bacteria such as Mycobacterium tuberculosis and Listeria monocytogenes. Administration of T-cell epitope short peptide needs large numbers of peptides for effective vaccination due to its easily degradable nature in vivo. In this respect, biocompatible and biodegradable microparticles combined with CTL/Th-hybrid epitope long peptide (long peptide) have been used to diminish the degradation of loaded peptide. The aim of this study is to develop a novel T cell-oriented vaccine against intracellular bacteria that is composed of long peptide and poly (lactic-co-glycolic acid) (PLGA) microparticles. Mouse bone marrow-derived dendritic cells (BMDCs) were loaded with L. monocytogenes listeriolysin O (LLO)-derived or ovalbumin (OVA)-derived long peptide/PLGA or other comparative antigens. The antigen-loaded BMDCs were injected subcutaneously into the flank of mice twice, and then, the spleens were collected and lymphocyte proliferation and interferon-γ production were evaluated. The median diameter of the PLGA spheres was 1.38 µm. Both LLO- and OVA-long peptide/PLGA showed significantly more robust CTL and Th proliferations with higher interferon-γ production than the long peptide alone or CTL and Th short peptides/PLGA vaccination. Furthermore, the LLO-long peptide/PLGA vaccination showed a significantly lower bacterial burden in spleens compared with the long peptide alone or the CTL and Th short peptides/PLGA vaccination after the challenge of lethal amounts of L. monocytogenes. These results suggest that the novel vaccine taking advantages of CTL/Th-hybrid epitope long peptide and PLGA microparticle is effective for protection against intracellular bacteria.

Tracking long-distance migration of marine fishes using compound-specific stable isotope analysis of amino acids.

The long-distance migrations by marine fishes are difficult to track by field observation. Here, we propose a new method to track such migrations using stable nitrogen isotopic composition at the base of the food web (δ15 NBase ), which can be estimated by using compound-specific isotope analysis. δ15 NBase exclusively reflects the δ15 N of nitrate in the ocean at a regional scale and is not affected by the trophic position of sampled organisms. In other words, δ15 NBase allows for direct comparison of isotope ratios between proxy organisms of the isoscape and the target migratory animal. We initially constructed a δ15 NBase isoscape in the northern North Pacific by bulk and compound-specific isotope analyses of copepods (n = 360 and 24, respectively), and then we determined retrospective δ15 NBase values of spawning chum salmon (Oncorhynchus keta) from their vertebral centra (10 sections from each of two salmon). We then estimated the migration routes of chum salmon during their skeletal growth by using a state-space model. Our isotope tracking method successfully reproduced a known chum salmon migration route between the Okhotsk and Bering seas, and our findings suggest the presence of a new migration route to the Bering Sea Shelf during a later growth stage.

Proteomic Profiling Reveals the Architecture of Granulomatous Lesions Caused by Tuberculosis and Mycobacterium avium Complex Lung Disease.

Tuberculosis (TB) and Mycobacterium avium complex lung disease (MAC-LD) are both characterized pathologically by granuloma lesions, which are typically composed of a necrotic caseum at the center surrounded by fibrotic cells and lymphocytes. Although the histological characterization of TB and MAC-LD granulomas has been well-documented, their molecular signatures have not been fully evaluated. In this research we applied mass spectrometry-based proteomics combined with laser microdissection to investigate the unique protein markers in human mycobacterial granulomatous lesions. Comparing the protein abundance between caseous and cellular sub-compartments of mycobacterial granulomas, we found distinct differences. Proteins involved in cellular metabolism in transcription and translation were abundant in cellular regions, while in caseous regions proteins related to antimicrobial response accumulated. To investigate the determinants of their heterogeneity, we compared the protein abundance in caseous regions between TB and MAC-LD granulomas. We found that several proteins were significantly abundant in the MAC-LD caseum of which proteomic profiles were different from those of the TB caseum. Immunohistochemistry demonstrated that one of these proteins, Angiogenin, specifically localized to the caseous regions of selected MAC-LD granulomas. We also detected peptides derived from mycobacterial proteins in the granulomas of both diseases. This study provides new insights into the architecture of granulomatous lesions in TB and MAC-LD.

Building a Low-cost Standalone Electrochemical Instrument Based on a Credit Card-sized Computer.

A low-cost, standalone electrochemical instrument was built from a credit card-sized computer and inexpensive A/D and D/A converter chips. The instrument is capable of cyclic voltammetry and constant potential electrolysis, with the potential range of -4 to +4 V and the current range of 1 µA to 20 mA.

Improved Method for Isolation and Purification of Underivatized Amino Acids for Radiocarbon Analysis.

We have improved a method for isolation and purification of individual amino acids for compound-specific radiocarbon analysis (CSRA). To remove high-performance liquid chromatography (HPLC) eluent blanks from isolated amino acid fractions prior to the radiocarbon (Δ14C) measurement, each fraction was filtered through a membrane filter and then washed with diethyl ether twice. Radiocarbon measurements on standard amino acids processed and purified with the above method using elemental analyzer-accelerator mass spectrometry resulted in Δ14C values that were in strong agreement ( R2 = 0.998) with the original Δ14C value of each amino acid standard. From these measurements, we calculate dead and modern carbon contamination contributions as 1.2 ± 0.2 and 0.3 ± 0.1 µgC, respectively, which are consistent with direct assessments of HPLC procedural blanks of 1.0 ± 0.8 µgC per sample. These contamination constraints allow correction of measured Δ14C values for accurate and precise CSRA and are widely applicable to future archeological and biogeochemical studies.

Cardioprotective and functional effects of levosimendan and milrinone in mice with cecal ligation and puncture-induced sepsis.

Levosimendan and milrinone may be used in place of dobutamine to increase cardiac output in septic patients with a low cardiac output due to impaired cardiac function. The effects of the two inotropic agents on cardiac inflammation and left ventricular (LV) performance were examined in mice with cecal ligation and puncture (CLP)-induced sepsis. CLP mice displayed significant cardiac inflammation, as indicated by highly increased pro-inflammatory cytokines and neutrophil infiltration in myocardial tissues. When continuously given, levosimendan prevented but milrinone exaggerated cardiac inflammation, but they significantly reduced the elevations in plasma cardiac troponin-I and heart-type fatty acid-binding protein, clinical markers of cardiac injury. Echocardiographic assessment of cardiac function showed that the effect of levosimendan, given by an intravenous bolus injection, on LV performance was impaired in CLP mice, whereas milrinone produced inotropic responses equally in sham-operated and CLP mice. A lesser effect of levosimendan on LV performance after CLP was also found in spontaneously beating Langendorff-perfused hearts. In ventricular myocytes isolated from control and CLP mice, levosimendan, but not milrinone, caused a large increase in the L-type calcium current. This study represents that levosimendan and milrinone have cardioprotective properties but provide different advantages and drawbacks to cardiac inflammation/dysfunction in sepsis.

Photoreduction of quinones by thiols sensitized by phthalocyanines.

Under the irradiation of red light (690 nm), quinones were converted to hydroquinones by thiols in the presence of metallophthalocyanines. The reaction proceeded via the charge separation between the triplet state of phthalocyanine and the quinone. The product determining step was protonation of the quinone anion radical, as indicated by the fact that the reaction was accelerated by the use of more acidic thiols or addition of an acid.

Digoxin Attenuates Murine Experimental Colitis by Downregulating Th17-related Cytokines.

BACKGROUND: Digoxin, a cardiac glycoside used for the treatment of heart failure, was reported to inhibit the retinoid-related orphan receptor gamma t (RORγt) and attenuate the severity of experimental autoimmune encephalomyelitis and arthritis in mice. However, the effects of digoxin in a mice model of inflammatory bowel disease have not been elucidated. METHODS: Colitis was induced in severe combined immunodeficiency mice by adoptive transfer of CD45RB CD4 T cells. Digoxin or a vehicle was injected into mice with colitis intraperitoneally every other day and changes in body weight were evaluated. After 6 to 8 weeks, the treated mice were killed and evaluated for histological score, T-cell subset, and cytokine messenger RNA (mRNA) expression in the colonic tissue. RESULTS: Wasting disease and histological damage were significantly attenuated in digoxin-treated mice with colitis compared with those in the vehicle-treated mice. In addition, the mRNAs of Th17-related cytokines were downregulated, whereas those of interleukin-10 were upregulated in the colonic mucosa of digoxin-treated mice. However, unexpectedly, the mRNA expression level of tumor necrosis factor alpha did not decrease in the colonic mucosa of digoxin-treated mice with colitis. This observation suggests that digoxin may ameliorate colitis by a tumor necrosis factor alpha-independent pathway. CONCLUSIONS: This study has shown for the first time that treatment with digoxin can ameliorate murine experimental colitis. This finding suggests that the suppression of Th17 using reagents such as digoxin could be effective in treating Crohn's disease refractory to anti-tumor necrosis factor alpha therapy.

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T-cell proliferation.

Bone-marrow-derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2-polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone-marrow-derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone-marrow-derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon-γ (IFN-γ) and interleukin-4 (IL-4)/IL-13, respectively. Following in vitro stimulation with lipopolysaccharide, M2-polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL-4, IL-13, transforming growth factor-ß and IL-10, whereas M1-polarized peritoneal macrophages expressed negligible amounts of Th1 and pro-inflammatory cytokines. ELISA showed that M2-polarized peritoneal macrophages produced significantly more IL-10 than M1-polarized peritoneal macrophages. Notably, M2-polarized peritoneal macrophages contributed more to the suppression of T-cell proliferation than did M1-polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL-4 and IL-13, increased in T-cells co-cultured with M2-polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T-cell proliferation in vitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype ex vivo.

Nontypeable Haemophilus influenzae exploits the interaction between protein-E and vitronectin for the adherence and invasion to bronchial epithelial cells.

BACKGROUND: Nontypeable Haemophilus influenzae (NTHi) is one of the most common Gram-negative pathogens in otitis media and exacerbation of chronic obstructive pulmonary disease. NTHi has been reported to invade bronchial epithelial cells. This penetration enables NTHi to evade the host immune system and antibiotics, and it seems to be related to the intractable features of these diseases. However, the precise mechanism of the invasion has been unknown. We hypothesized that protein-E, an outer membrane protein of NTHi, plays a role in this penetration into bronchial epithelial cells. RESULTS: We utilized two NTHi strains. NTHi efficiently attached to plate-bound vitronectin (254-309/field at 1,000× magnification) and this attachment was blocked by pretreatment with protein-E peptide (PE84-108). The blockade of adhesion was dependent on the concentration of PE84-108. NTHi strains invaded bronchial epithelial cells and the intracellular bacteria were localized in early endosomes. Furthermore, intracellular invasion of NTHi was also blocked by PE84-108, but not by Arg-Gly-Asp (RGD) peptide. Pretreatment with PE84-108 significantly prevented cells from being invaded by both NTHi strains, which was confirmed by fluorescent microscope observation. In addition, pretreatment with PE84-108 significantly reduced percentages of CFU after gentamicin treatment of cells per input CFU. CONCLUSIONS: These results suggest that NTHi does not directly bind to the cell surface, but binds to host vitronectin that is bound to the cell surface, via bacterial protein-E. Bacterial protein-E and host vitronectin play a role in the attachment to bronchial epithelial cells and is also involved in the subsequent intracellular invasion of NTHi. A novel vaccine or treatment strategy targeting the protein-E-vitronectin axis may prevent respiratory intracellular infection of NTHi and may lead to better clinical outcomes.

Three-year prospective, observational study of central line-associated bloodstream infections in a 600-bed Japanese acute care hospital.

BACKGROUND: Central line-associated bloodstream infection (CLABSI) is an important concern associated with central venous catheter (CVC) use. The objective of this study was to determine the influences of CVC access sites, CVC types, and presumed causative microorganisms on CLABSI occurrence in an acute care hospital. METHODS: We conducted a prospective, observational study of CLABSI occurrence for 3 consecutive years in a 600-bed Japanese acute care hospital. Data collected included patient characteristics, CVC access sites, CVC types, and microorganisms isolated by blood culture. RESULTS: For 1,650 CVCs used for 1,237 patients, 39 cases of infection were identified. Most infections had occurred within 1 month of CVC insertion. Maximal sterile barrier precautions had been used for most cases (97.3%). The average CLABSI occurrence days with internal jugular vein access were shorter than those with subclavian vein access and femoral vein access. CLABSI rates were 1.1 and 0.7 for single- and multilumen CVCs, respectively. CLABSI occurrence tended to be shorter when gram-positive cocci were isolated and tended to be longer when fungi (Candida spp) were isolated. CONCLUSION: Most CLABSI cases had occurred within 1 month of CVC insertion. Longer CVC duration increased chance of fungal infection.

[Molecular dissection of Mycobacterium tuberculosis-containing phagosomes].

Mycobacterium tuberculosis is an intracellular bacterium that can proliferate within phagocytosed macrophages. M. tuberculosis gains this ability by inhibiting phagolysosome biogenesis. On the other hand, autophagy induction can eliminate infected mycobacteria in macrophages. Numerous reports have demonstrated the mechanism of membrane trafficking in macrophages infected with mycobacteria to elucidate how M. tuberculosis proliferates within macrophages. In this review, we make a commentary on the molecular dissection of M. tuberculosis-containing phagosomes demonstrating which host factors constitute the replication niche for mycobacteria, and approach the real images of mycobacterial phagosomes.

Switching of single-molecule magnetic properties of TbIII -porphyrin double-decker complexes and observation of their supramolecular structures on a carbon surface.

Double-decker complexes based on single-molecule magnets (SMMs) are a class of highly promising molecules for applications in molecular spintronics, wherein control of both the ligand oxidative states and the 2D supramolecular structure on carbon materials is of great importance. This study focuses on the synthesis and study of 2,3,7,8,12,13,17,18-octaethylporphyrin (OEP)-Tb(III) double-decker complexes with different electronic structures comprising protonated, anionic, and radical forms. Magnetic susceptibility measurements revealed that only the anionic and radical forms of the OEP-Tb(III) double-decker complexes exhibited SMM properties. The barrier heights for magnetic moment reversal were estimated to be 207 and 215 cm(-1) for the anionic and radical forms, respectively. Scanning tunneling microscopy (STM) investigations revealed that these OEP-Tb(III) complexes form well-ordered monolayers upon simple dropcasting from dilute dichloromethane solutions. All three complexes form an isomorphic pseudo-hexagonal 2D pattern, regardless of the differences in the electronic structures of their porphyrin-Tb cores. This finding is of interest for SMM technology as ultrathin films of these materials undergoing chemical transformations will not require any detrimental reorganization. Finally, we demonstrate self-assembly of the protonated 5,15-bisdodecylporphyrin (BDP)-Tb(III) double-decker complex as an example of successful supramolecular design to achieve controlled alignment of SMM-active sites.

Yellow NIR dye: π-fused bisbenzoBODIPYs with electron-withdrawing groups.

Bicyclo[2.2.2]octadiene-fused (BCOD-fused) bis(benzoborondipyrromethene)s (bisbenzoBODIPYs) bearing electron-withdrawing groups such as fluorine and cyano groups were prepared either by incorporating tetrafluoroisoindole moieties into BODIPY chromophores or by introducing cyano or ethoxycarbonyl groups at the 3,5-positions. The BCOD-fused bisbenzoBODIPYs were quantitatively converted to the corresponding benzene-fused bisbenzoBODIPYs by a retro-Diels-Alder reaction. The π-fused bisbenzoBODIPYs were found to have intense absorption in the near-infrared region and not to have any strong absorption bands in the visible region. Moreover, the bisbenzoBODIPYs were stable under atmospheric conditions.