Effects of the F-number on the generation condition of a diode-laser-sustained plasma.

A laser-sustained plasma (LSP) is a promising method for generating atmospheric-pressure plasmas. Although diode lasers are highly efficient, compact, and low-cost light sources for LSP, previous studies have reported that the laser power required for LSP generation is high, owing to the poor beam quality of diode lasers. In this study, we attempted to generate an LSP using a 4-kW-class diode laser with different F-numbers. The minimum laser power required for LSP generation strongly depends on the F-number. Comparing F1.2 and F2.0, F1.2 reduces the required power by approximately 2,840 W. The LSP temperature and length also strongly depend on the F-number, with the temperature increasing by approximately 5,000 K and the length increasing by approximately 1.4 times at F 1.2 compared with those at F 2.0.

Thermo-Responsive Polymer-siRNA Conjugates Enabling Artificial Control of Gene Silencing around Body Temperature.

PURPOSE: Controlling small interfering RNA (siRNA) activity by external stimuli is useful to exert a selective therapeutic effect at the target site. This study aims to develop a technology to control siRNA activity in a thermo-responsive manner, which can be utilized even at temperatures close to body temperature. METHODS: siRNA was conjugated with a thermo-responsive copolymer that was synthesized by copolymerization of N-isopropylacrylamide (NIPAAm) and hydrophilic N,N-dimethylacrylamide (DMAA) to permit thermally controlled interaction between siRNA and an intracellular gene silencing-related protein by utilizing the coil-to-globule phase transition of the copolymer. The composition of the copolymer was fine-tuned to obtain lower critical solution temperature (LCST) around body temperature, and the phase transition behavior was evaluated. The cellular uptake and gene silencing efficiency of the copolymer-siRNA conjugates were then investigated in cultured cells. RESULTS: The siRNA conjugated with the copolymer with LCST of 38.0°C exhibited ~ 11.5 nm of the hydrodynamic diameter at 37°C and ~ 9.8 nm of the diameter at 41°C, indicating the coil-globule transition above the LCST. In line with this LCST behavior, its cellular uptake and gene silencing efficiency were enhanced when the temperature was increased from 37°C to 41°C. CONCLUSION: By fine-tuning the LCST behavior of the copolymer that was conjugated with siRNA, siRNA activity could be controlled in a thermo-responsive manner around the body temperature. This technique may offer a promising approach to induce therapeutic effects of siRNA selectively in the target site even in the in vivo conditions.

Cancerous pH-responsive polycarboxybetaine-coated lipid nanoparticle for smart delivery of siRNA against subcutaneous tumor model in mice.

Lipid nanoparticles (LNPs) have been commonly used as a vehicle for nucleic acids, such as small interfering RNA (siRNA); the surface modification of LNPs is one of the determinants of their delivery efficiency especially in systemic administration. However, the applications of siRNA-encapsulated LNPs are limited due to a lack effective systems to deliver to solid tumors. Here, we report a smart surface modification using a charge-switchable ethylenediamine-based polycarboxybetaine for enhancing tumor accumulation via interaction with anionic tumorous tissue constituents due to selective switching to cationic charge in response to cancerous acidic pH. Our polycarboxybetaine-modified LNP could enhance cellular uptake in cancerous pH, resulting in facilitated endosomal escape and gene knockdown efficiency. After systemic administration, the polycarboxybetaine-modified LNP accomplished high tumor accumulation in SKOV3-luc and CT 26 subcutaneous tumor models. The siPLK-1-encapsulated LNP thereby accomplished significant tumor growth inhibition. This study demonstrates a promising potential of the pH-responsive polycarboxybetaine as a material for modifying the surface of LNPs for efficient nucleic acid delivery.

The ERM protein moesin regulates natural killer cell homeostasis in vivo.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins with actin filaments in the cell cortex. Hemizygous mutations in the X-linked moesin gene are associated with primary immunodeficiency with T and B cell lymphopenia, which also affects natural killer (NK) cells in most cases. We previously showed that moesin deficiency in mice substantially affects lymphocyte homeostasis, but its impact on NK cells remains unexplored. Here, we found that in moesin-deficient mice, NK cells were decreased in the peripheral blood and bone marrow but increased in the spleen. Analysis of female heterozygous mice showed a selective advantage for moesin-expressing NK cells in the blood. Moesin-deficient NK cells exhibited increased cell death and impaired signaling in response to IL-15, suggesting that moesin regulates NK cell survival through IL-15-mediated signaling. Our findings thus identify moesin as an NK cell homeostasis regulator in vivo.

Iron chelation cancer therapy using hydrophilic block copolymers conjugated with deferoxamine.

Cancer cells have high iron requirements due to their rapid growth and proliferation. Iron depletion using iron chelators has a potential in cancer treatment. Previous studies have demonstrated that deferoxamine (DFO) specifically chelates Fe(III) and exhibited antitumor activity in clinical studies. However, its poor pharmacokinetics has limited the therapeutic potential and practical application. Although polymeric iron chelators have been developed to increase the blood retention, none of previous studies has demonstrated their potential in iron chelation cancer therapy. Here, we developed polymeric DFO by the covalent conjugation of DFO to poly(ethylene glycol)-poly(aspartic acid) (PEG-PAsp) block copolymers. The polymeric DFO exhibited iron-chelating ability comparable with free DFO, thereby arresting cell cycle and inducing apoptosis and antiproliferative activity. After intravenous administration, the polymeric DFO showed marked increase in blood retention and tumor accumulation in subcutaneous tumor models. Consequently, polymeric DFO showed significant suppression of the tumor growth compared with free DFO. This study reveals the first success of the design of polymeric DFO for enhancing iron chelation cancer therapy.

Polymeric Iron Chelators Enhancing Pro-Oxidant Antitumor Efficacy of Vitamin C by Inhibiting the Extracellular Fenton Reaction.

Intravenously injected high-dose vitamin C (VC) induces extracellular H2O2, which can penetrate into the tumor cells and suppress tumor growth. However, extracellular labile iron ions in the tumor decompose H2O2 via the Fenton reaction, limiting the therapeutic effect. In this regard, we recently developed a polymeric iron chelator that can inactivate the intratumoral labile iron ions. Here, we examined the effect of our polymeric iron chelator on the high-dose VC therapy in in vitro and in vivo. In the in vitro study, the polymeric iron chelator could inactivate the extracellular labile iron ions and prevent the unfavorable decomposition of VC-induced H2O2, augmenting pro-oxidative damage to DNA and inducing apoptosis in cultured cancer cells. Even in the in vivo study, the polymeric iron chelator significantly improved the antitumor effect of VC in subcutaneous DLD-1 and CT26 tumors in mice, while conventional iron chelators could not. This work indicates the importance of modulating tumor-associated iron ions in the high-dose VC therapy and should contribute to a better understanding of its mechanism.

A novel Siglec-F+ neutrophil subset in the mouse nasal mucosa exhibits an activated phenotype and is increased in an allergic rhinitis model.

Neutrophils are important phagocytic cells for host defense against pathogens. They are rapidly recruited to the site of infection, release antimicrobial peptides and cytokines, and engulf and kill microbes. Neutrophils also accumulate in allergic inflammatory sites. Here we characterized neutrophil accumulation in the nasal mucosa using a mouse model of allergic rhinitis, in which mice were sensitized by intraperitoneal injection of ovalbumin (OVA) and then challenged by intranasal administration of OVA or PBS. In the nasal mucosa of both PBS- and OVA-challenged mice, we found a cell subset expressing the eosinophil marker Siglec-F in the Ly-6G+ neutrophil population. Morphological analysis of the sorted Ly-6G+Siglec-F+ cells revealed that they were devoid of eosinophilic granules in the cytosol and were apparently neutrophils, but compared to conventional Ly-6G+Siglec-F- neutrophils, they had a more lobulated, "botryoid" nucleus. Siglec-F+ neutrophils were barely found in the nasopharynx-associated lymphoid tissue, cervical lymph nodes, the spleen, or blood. Both Siglec-F+ neutrophils and conventional neutrophils showed increased numbers in the nasal mucosa of OVA-challenged mice. Compared to conventional Siglec-F- neutrophils, Siglec-F+ neutrophils exhibited an activated phenotype and enhanced effector functions. Taken together, our findings identify Siglec-F+ neutrophils as a novel neutrophil subset with an activated phenotype that resides specifically in the nasal mucosa.

Serum IgG anti-GD1a antibody and mEGOS predict outcome in Guillain-Barré syndrome.

OBJECTIVE: Approximately 15%-20% of patients with Guillain-Barré syndrome (GBS) are unable to walk independently at 6 months from the onset of neurological symptom. The modified Erasmus GBS outcome score (mEGOS) has been reported as a prognostic tool.Herein we investigated the association between a poor outcome, inability to walk independently at 6 months and presence of antiganglioside antibodies. METHODS: The clinical and serological data of 177 patients with GBS were retrospectively collected in Japan to assess the associations between a poor outcome and serum IgG antibodies against each ganglioside (GM1, GD1a, GalNAc-GD1a, GQ1b and GT1a). In addition, we investigated whether the combination of mEGOS and serum IgG antibodies against gangliosides is useful in predicting a poor outcome. RESULTS: The patients with IgG anti-GD1a antibodies more frequently showed poor outcomes than those without these antibodies (9 (36%) of 25 vs 8 (6%) of 127 patients, p<0.001). Particularly, 80% showed a poor outcome when they had both serum IgG anti-GD1a antibody and a high mEGOS of ≥10 on day 7 of admission. CONCLUSIONS: The combination of serum IgG anti-GD1a antibodies and a high mEGOS could help in making a more accurate prognosis of patients than mEGOS alone, especially for predicting poor outcomes.

Relationship Between Olfactory Disturbance After Acute Ischemic Stroke and Latent Thalamic Hypoperfusion.

Odor detection, recognition, and identification were assessed in 19 acute ischemic stroke patients who had no magnetic resonance imaging-detectable thalamic lesions but in whom technetium-99m ethyl cysteinate dimer single photon emission tomography revealed thalamic hypoperfusion. Although these patients were unaware of reduced olfactory function, they exhibited significantly lower scores in tests for odor identification and recognition threshold as compared with 9 ischemic stroke controls that had normal thalamic hypoperfusion. However, absolute odor detection thresholds were similar in the 2 groups. These results demonstrate the usefulness of cerebral perfusion scintigraphy in assessing sensory loss after ischemic stroke and provide further evidence for the role of the thalamus in olfaction.

Sequential Self-Assembly Using Tannic Acid and Phenylboronic Acid-Modified Copolymers for Potential Protein Delivery.

Tannic acid (TA) can form stable complexes with proteins, attracting significant attention as protein delivery systems. However, its systemic application has been limited due to nonspecific interaction. Here, we report a simple technique to prepare systemically applicable protein delivery systems using sequential self-assembly of a protein, TA, and phenylboronic acid-conjugated PEG-poly(amino acid) block copolymers in aqueous solution. Mixing the protein and TA in aqueous solution led to covering of the protein with TA, and subsequent addition of the copolymer resulted in the formation of boronate esters between TA and copolymers, constructing the core-shell-type ternary complex. The ternary complex covered with PEG exhibited a small hydrodynamic diameter of ∼10-20 nm and prevented an unfavorable interaction with serum components, thereby accomplishing significantly prolonged blood circulation and enhanced tumor accumulation in a subcutaneous tumor model. The technique utilizing supramolecular self-assembly may serve as a novel approach for designing protein delivery systems.

Efficient Suppression of Abdominal Aortic Aneurysm Expansion in Rats through Systemic Administration of Statin-Loaded Nanomedicine.

Abdominal aortic aneurysm (AAA) is a life-threatening disease. However, no systemically injectable drug has been approved for AAA treatment due to low bioavailability. Polymeric micelles are nanomedicines that have the potential to improve therapeutic efficacy by selectively delivering drugs into disease sites, and research has mainly focused on cancer treatments. Here, we developed a statin-loaded polymeric micelle to treat AAAs in rat models. The micelle showed medicinal efficacy by preventing aortic aneurysm expansion in a dose-dependent manner. Furthermore, the micelle-injected group showed decreased macrophage infiltration and decreased matrix metalloproteinase-9 activity in cases of AAA.

Pyruvate Responsiveness Based on α-Oxohydrazone Formation for Intracellular siRNA Release from Polyion Complex-Based Carriers.

Selective release of small interfering RNA (siRNA) payloads in response to intracellular substances is a prerequisite for the smart design of siRNA carriers. In this context, we developed a molecular program that allows reactivity with pyruvate for siRNA release in the cell on the basis of polyionic-complex- (PIC-) based siRNA carriers. Hydrazide can react with the α-keto acid structure of anionic pyruvate to form α-oxohydrazone, resulting in the reduction of the cationic net charge of the cationic polymer bearing a hydrazide moiety, which in turn leads to an inefficient electrostatic interaction with anionic siRNA and the consequent destabilization of the PIC (i.e., PGlu [DET/hydrazide]) in pyruvate-enriched environments, such as the cytoplasm, thus achieving effective siRNA release from the PIC and its associated gene-silencing activity. The present study provides the rationale for an α-oxohydrazone-formation-based smart design of pyruvate-responsive materials in the cell.

Poly( N-isopropylacrylamide)-Based Polymer-Inducing Isothermal Hydrophilic-to-Hydrophobic Phase Transition via Detachment of Hydrophilic Acid-Labile Moiety.

The polymerization of N-isopropylacrylamide (NIPAAm) with ionizable monomers results in pH-responsive lower critical solution temperature (LCST) polymer which works in an ionization-dependent manner. However, gradual ionization of the comonomer occurs at a broad pH range due to the electrostatic field generated by the polymers, limiting the extent of LCST shift in response to pH change. Furthermore, excess introduction of comonomer may dull phase transition behavior. Here, we report the development of an ionization-independent LCST polymer that exerts a sharp isothermal hydrophilic-to-hydrophobic phase transition in response to slight pH change. Our polymer has a poly(NIPAAm/2-aminoisoprpylacrylamide (AIPAAm)) (P(NIPAAm/AIPAAm)) backbone that retains the continuous structural similarity of N-alkyl groups for preserving phase transition sensitivity, and primary amine for forming hydrophilic acid-labile 2-propionic-3-methylmaleic (PMM) amide linkage. The PMM moiety improves the polymer's hydrophilicity and drastically increases the LCST. Detachment of the PMM moiety in response to mild acidic condition (pH < 6.8) lowers the LCST to that of original P(NIPAAm/AIPAAm), permitting isothermal pH-responsive phase transition. Utilizing this mechanism, P(NIPAAm/AIPAAm) modified with PMM amide linkage exhibits a sharp hydrophilic-to-hydrophobic transition at a physiological temperature (37 °C) and, strikingly, facilitates interaction with cultured cells. Most importantly, our polymer showed significantly higher accumulation within a solid tumor after systemic injection compared to conventional PNIPAAm, which may be due to its phase transition responding to slightly acidic tumor microenvironment. Thus, this study provides a novel polymer that offers delicate control of LCST and pH-responsiveness suitable for use in even fuzzy biological environments.

Comparison between different isoelectric points of biodegradable gelatin sponges incorporating ß-tricalcium phosphate and recombinant human fibroblast growth factor-2 for ridge augmentation: A preclinical study of saddle-type defects in dogs.

BACKGROUND AND OBJECTIVE: It is well known that recombinant human fibroblast growth factor-2 (rhFGF-2) signaling plays an important role in tissue repair and regeneration. rhFGF-2 strongly binds to acidic gelatin via ionic linkages and is gradually released upon gelatin decomposition. On the other hand, the linkage between rhFGF-2 and basic gelatin is so weak that most rhFGF-2 is rapidly released from basic gelatin by simple desorption. Gelatin/ß-tricalcium phosphate (ß-TCP) sponges, which comprise 50 wt% gelatin and 50 wt% ß-TCP in a cross-linked structure, can release rhFGF-2 gradually owing to their electrical features. In a previous study, we reported that new bone height in the test group using rhFGF-2 with acidic gelatin/ß-TCP sponges was significantly greater than that in the control group using acidic gelatin/ß-TCP sponges alone in a ridge augmentation model in dogs. However, whether these results depend on controlled release by the gelatin/ß-TCP sponges remains controversial. In this study, we evaluated the effects of controlled release by comparing acidic and basic gelatin/ß-TCP sponges with different isoelectric points (IEP) on ridge augmentation in dogs. MATERIALS AND METHODS: Twelve weeks after extraction of the maxillary second and third incisors of six dogs, critically sized saddle-type defects (8 mm length × 4 mm depth) were surgically created bilaterally 2 mm from the mesial side of the canine. Acidic gelatin/ß-TCP sponges (IEP 5.0) soaked with 0.3% rhFGF-2 were applied to the defect in the acidic group, whereas basic gelatin/ß-TCP sponges (IEP 9.0) soaked with 0.3% rhFGF-2 were applied to the defect in the basic group. Twelve weeks after surgery, biopsy specimens were obtained and subjected to microcomputed tomography (micro-CT) and histological analyses. RESULTS: New bone area detected by micro-CT analysis was significantly smaller in the basic group than in the acidic group. New bone height calculated by histologic sections was significantly lower in the basic group than in the acidic group. The total tissue height was lower in the basic group than in the acidic group. However, the differences between both sites were not significant. CONCLUSIONS: These findings suggest that in ridge augmentation of saddle-type defects, controlled release of rhFGF-2 induces notably more alveolar bone formation than does short-term application of rhFGF-2.

Search for transcription factors affecting productivity of the polyketide FR901512 in filamentous fungal sp. No. 14919 and identification of Drf1, a novel negative regulator of the biosynthetic gene cluster.

In order to increase secondary metabolite production in filamentous fungi, a transcription factor gene in the biosynthetic gene cluster and global regulator genes such as laeA are considered plausible as targets for overexpression by genetic modification. In this study, we examined these overexpression effect in fungal sp. No. 14919 that produces FR901512, an HMG-CoA reductase inhibitor. Resultantly, the productivity was improved at 1.7-1.8 fold by overexpressing frlE, a transcription factor gene in the biosynthetic gene cluster, whereas productivity did not change by overexpression of laeA and veA. Furthermore, we searched for extra transcription factors affecting the productivity by transcriptome analysis between wild-type strain and highly productive UV mutants. After verifying productivity decrease by overexpression, Drf1, a novel transcription factor encoded by drf1 was identified as the negative regulator. Because each frlE product (FrlE) and Drf1 worked on the same cluster in positive and negative regulatory manners, their network was analyzed.

Low-cost optical fiber temperature-sensing system employing optical transceivers for Ethernet and long-period fiber grating.

Optical fiber sensors have numerous advantages over electrical sensors. However, in some cases, sensing systems based on optical fiber sensors are inferior to electrical sensors in terms of cost because expensive instruments are required. Therefore, cost-effective options must be developed. Here, inexpensive optical transceivers (OTRs) for Ethernet were used instead of a broadband light source and optical spectrum analyzer, and a CO2 laser-induced long-period fiber grating (LPFG) was used as a temperature sensor. The sensing system was implemented introducing OTRs and LPFG in an experimental internet protocol (IP) over Ethernet. The results showed that a low-cost sensing system can estimate the ambient temperature of the LPFG based on the no-reply rate for the internet control message protocol echo request. Regarding the sensing performance, the measurable range was ∼100°C with ±4.5°C errors.

Rheumatoid meningitis developed in patient with stable rheumatoid arthritis and myasthenia gravis-detailed analysis of intracranial inflammation using flow cytometry.

BACKGROUND: Rheumatoid meningitis (RM) is a rare disorder that often develops during a remission phase of rheumatoid arthritis (RA). This is the first study to demonstrate differences in regard to immunological disturbance between blood and cerebrospinal fluid (CSF) samples obtained from a patient with RM using flow cytometry. CASE PRESENTATION: A 36-year-old woman with RA and generalized myasthenia gravis (MG) developed RM during a remission phase. Although both RA and MG were stable and well controlled, she noticed fever, headache, and transient sensory disturbance. Blood and CSF examination findings suggested aseptic meningitis, while brain magnetic resonance imaging revealed restricted portions of meningitis and associated cortical lesions, compatible with a diagnosis of RM. The dose of oral prednisolone was increased, which ameliorated the symptoms within 1 week along with improvement in CSF findings. This patient exhibited features of RM that were manifested in a manner independent of the activity of RA. An investigation of cellular immunity using CSF specimens with flow cytometry showed differences in regard to the pathogenesis of inflammation in the CSF as compared to outside of the central nervous system. In contrast to results obtained with paired blood samples, CSF cells at the peak stage of RM showed a marked increase in CCR3+ Th2 cells and marked decrease in CD8+ cells, suggesting an immunoregulatory disturbance in the CSF. Those findings indicated a CSF-specific activation of humoral immunity, resulting in augmentation of meningeal inflammation, as shown by excess synthesis of intrathecal IgG and markedly elevated interleukin-6 level. Results of the present detailed investigation of lymphocyte subsets revealed a discrepancy regarding the process of inflammation in this RM patient between CSF and blood samples. CONCLUSIONS: RM is not a simple reflection of the immune status of RA, as the pathogenesis seems related to, at least in part, CSF-specific immunological dysregulation.

Knockout of the SREBP system increases production of the polyketide FR901512 in filamentous fungal sp. No. 14919 and lovastatin in Aspergillus terreus ATCC20542.

In the production of useful microbial secondary metabolites, the breeding of strains is generally performed by random mutagenesis. However, because random mutagenesis introduces many mutations into genomic DNA, the causative mutations leading to increased productivity are mostly unknown. Therefore, although gene targeting is more efficient for breeding than random mutagenesis, it is difficult to apply. In this study, a wild-type strain and randomly mutagenized strains of fungal sp. No. 14919, a filamentous fungus producing the HMG-CoA reductase inhibitor polyketide FR901512, were subjected to point mutation analysis based on whole genome sequencing. Among the mutated genes found, mutation of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) had a positive effect on increasing FR901512 productivity. By complementing the SCAP gene in the SCAP-mutated strain, productivity was decreased to the level of the SCAP-intact strain. Conversely, when either the SCAP or SREBP gene was deleted, the productivity was significantly increased. By genomic transcriptional analysis, the expression levels of three enzymes in the ergosterol biosynthesis pathway were shown to be decreased by SCAP mutation. These findings led to the hypothesis that raw materials of polyketides, such as acetyl-CoA and malonyl-CoA, became more available for FR901512 biosynthesis due to depression in sterol biosynthesis caused by knockout of the SREBP system. This mechanism was confirmed in Aspergillus terreus producing the polyketide lovastatin, which is structurally similar to FR901512. Thus, knockout of the SREBP system should be considered significant for increasing the productivities of polyketides, such as HMG-CoA reductase inhibitors, by filamentous fungi.

An Ethylenediamine-based Switch to Render the Polyzwitterion Cationic at Tumorous pH for Effective Tumor Accumulation of Coated Nanomaterials.

Polyzwitterions are employed as coating polymers for biomaterials to induce an antifouling property on the surface. Fine-tuning the betaine structure switches the antifouling property to be interactive with anionic tissue constituents in response to a tumorous pH gradient. The ethylenediamine moiety in the carboxybetaine enabled stepwise protonation and initiated the di-protonation process around tumorous pH (6.5). The net charge of the developed polyzwitterion (PGlu(DET-Car)) was thus neutral at pH 7.4 for antifouling, but was cationic at pH 6.5 for interaction with anionic constituents. Quantum dots coated with PGlu(DET-Car) exhibited comparable stealth and enhanced tumor accumulation relative to the PEG system. The present study provides a novel design of smart switchable polyzwitterion based on a precise control of the net charge.

Markers for Guillain-Barré syndrome with poor prognosis: a multi-center study.

Guillain-Barré syndrome (GBS) is an acute monophasic neuropathy. Prognostic tools include the modified Erasmus GBS outcome score (mEGOS), Erasmus GBS respiratory insufficiency score (EGRIS), and the increase in serum IgG levels (ΔIgG) 2 weeks after intravenous immunoglobulin (IVIg) treatment. Given that proportions of GBS subtypes differ between Western countries and Japan, the usefulness of these tools in Japan or other countries remains unknown. We enrolled 177 Japanese patients with GBS from 15 university hospitals and retrospectively obtained mEGOS and EGRIS for all and ΔIgG status for 79 of them. High mEGOS scores on admission or on day 7 were significantly associated with poorer outcomes (unable to walk independently at 6 months). High EGRIS scores (≥5 points) were associated with an increased risk for mechanical ventilation. Patients with ΔIgG <1,108 mg/dl had significantly poorer outcomes. We suggest that mEGOS, EGRIS, and ΔIgG in GBS are clinically relevant in Japan.