GC-MS analysis of exhaled gas for fine detection of inflammatory diseases.

Exhaled gas analysis is a non-invasive test ideal for continuous monitoring of biological metabolic information. We analyzed the exhaled gas of patients with inflammatory diseases for trace gas components that could serve as biomarkers that enable early detection of inflammatory diseases and assessment of treatment efficacy. Furthermore, we examined the clinical potential of this method. We enrolled 34 patients with inflammatory disease and 69 healthy participants. Volatile components from exhaled gas were collected and analyzed by a gas chromatography-mass spectrometry system, and the data were examined for gender, age, inflammatory markers, and changes in markers before and after treatment. The data were tested for statistical significance through discriminant analysis by Volcano plot, Analysis of variance test, principal component analysis, and cluster analysis comparing healthy and patient groups. There were no significant differences in the trace components of exhaled gas by gender or age. However, we found differences in some components of the exhaled gas between healthy and untreated patients. In addition, after treatment, gas patterns including the patient-specific components changed to a state closer to the inflammation-free status. We identified trace components in the exhaled gas of patients with inflammatory diseases and found that some of these regressed after treatment.

Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease.

Fabry disease (FD) is an X-linked inherited lysosomal metabolism disorder in which globotriaosylceramide (Gb3) accumulates in various organs resulting from a deficiency in alpha-galactosidase A. The clinical features of FD include progressive impairments of the renal, cardiac, and peripheral nervous systems. In addition, patients with FD often develop neuropsychiatric symptoms, such as depression and dementia, which are believed to be induced by the cellular injury of cerebrovascular and partially neuronal cells due to Gb3 accumulation. Although the analysis of autopsy brain tissue from patients with FD showed no accumulation of Gb3, abnormal deposits of Gb3 were found in the neurons of several brain areas, including the hippocampus. Therefore, in this study, we generated induced pluripotent stem cells (iPSCs) from patients with FD and differentiated them into neuronal cells to investigate pathological and biological changes in the neurons of FD. Neural stem cells (NSCs) and neurons were successfully differentiated from the iPSCs we generated; however, cellular damage and morphological changes were not found in these cells. Immunostaining revealed no Gb3 accumulation in NSCs and neurons. Transmission electron microscopy did not reveal any zebra body-like structures or inclusion bodies, which are characteristic of FD. These results indicated that neuronal cells derived from FD-iPSCs exhibited normal morphology and no Gb3 accumulation. It is likely that more in vivo environment-like cultures are needed for iPSC-derived neurons to reproduce disease-specific features.

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.

A new method for measuring cholesterol efflux capacity uses stable isotope-labeled, not radioactive-labeled, cholesterol.

The incidence of cardiovascular events correlates inversely with cholesterol efflux capacity (CEC) more than with HDL-cholesterol level. The measurement of CEC is used to qualify cardiovascular disease risk and is conventionally performed with radioisotope (RI)-labeled cholesterol. Here, we established a CEC measurement technique using stable isotope-labeled cholesterol as an alternative, and we compared the new method with RI and fluorescence (boron dipyrromethene difluoride-cholesterol) methods in cells and in patient serum. We incubated J774 cells labeled with [d7]cholesterol ([d7]C) with patient serum depleted of apoB, and [d7]C extracted from the culture medium was quantified by liquid chromatography/quadrupole time-of-flight mass spectrometry. [d7]C efflux increased with greater apoB-depleted serum concentration and longer incubation time. The assay coefficient of variation (CV) of five consecutive measurements of three sets of samples ranged from 7.3% to 9.5%, and the interassay CV determined by measuring three samples four times ranged from 4.1% to 8.5%, both indicating good precision. We then measured CEC levels of 41 outpatients with serum HDL-cholesterol levels between 36 and 94 mg/dl (mean: 61.7 ± 18.0 mg/dl); in the presence of cAMP, we observed a significant, positive correlation between CEC levels determined with the stable isotope and RI methods that was stronger than the correlation between measurements obtained by the fluorescence and RI methods (r = 0.73, P < 0.0001 vs. r = 0.55, P < 0.001). Therefore, our stable isotope method can be considered useful as a non-RI method and thus deserves evaluation in future clinical studies.

Characteristics of PPT1 and TPP1 enzymes in neuronal ceroid lipofuscinosis (NCL) 1 and 2 by dried blood spots (DBS) and leukocytes and their application to newborn screening.

We first characterized PPT1 and TPP1 enzymes in dried blood spots (DBS), plasma/serum, and leukocytes/lymphocytes using neuronal ceroid lipofuscinosis (NCL) 1 and 2 patients and control subjects. PPT1 enzyme had only one acid form in control DBS, plasma/serum, and leukocytes/lymphocytes and showed deficient activities in these samples from NCL 1 patients. Conversely, TPP1 enzymes in control DBS and leukocytes/lymphocytes consisted of two forms, an acidic form and a neutral form, whereas serum TPP1 enzyme had only a neutral form. In control subjects, the optimal pH of PPT1 enzyme in DBS, plasma/serum, and leukocytes/lymphocytes was 4.5 to 5.0 in the acidic form, whereas TPP1 enzyme in control DBS and leukocytes/lymphocytes was pH 4.5 and 6.5, respectively. In NCL 1 and 2, both PPT1 and TPP1 enzyme activities in DBS, plasma, and leukocytes/lymphocytes were markedly reduced in acidic pH, whereas heterozygotes of NCL 1 and 2 in the acidic form showed intermediate activities between patients and control subjects. In neutral conditions, pH 6.0, the PPT1 enzyme activities in NCL 1 patients showed rather higher residual activities and intermediate activities in heterozygotes in NCL 1, which was probably caused by mutated proteins in three cases with NCL 1 patients. TPP1 enzyme activities at neutral pH 6.5 to 7.0 in DBS and leukocytes/lymphocytes showed higher enzyme activities in NCL 2 patients and heterozygotes. The reason for the increases of neutral TPP1 enzyme activities at pH 6.5 to 7.0 in NCL 2 DBS and leukocytes/lymphocytes, is obscure, but possibly caused by secondary activation of neutral TPP1 enzyme due to the absence of the acidic form. Interestingly, TPP1 activity in serum only consisted of a neutral form, no acidic form, and was not deficient in any NCL 2 patient. Therefore, we can diagnose NCL 1 patients by plasma/serum enzyme assay of PPT1, but not diagnose NCL 2 by serum TPP1 enzyme assay. A pilot study of newborn screening of NCL 1 and 2 has been established by more than 1000 newborn DBS assays. Using this assay system, we will be able to perform newborn screening of NCL 1 and 2 by DBS.

The severe clinical phenotype for a heterozygous Fabry female patient correlates to the methylation of non-mutated allele associated with chromosome 10q26 deletion syndrome.

Heterozygous Fabry females usually have an attenuated form of Fabry disease, causing them to be symptomatic; however, in rare cases, they can present with a severe phenotype. In this study, we report on a 37-year-old woman with acroparesthesia, a dysmorphic face, left ventricular hypertrophy, and intellectual disability. Her father had Fabry disease and died due to chronic renal and congestive cardiac failure. Her paternal uncle had chronic renal failure and intellectual disability, and her paternal aunt was affected with congestive cardiac failure. The patient has two sisters with no significant medical illness. However, her nephew has acroparesthesia, anhidrosis, and school phobia, and her niece shows mild phenotypes. The patient's enzyme analysis showed very low α-galactosidase A (α-gal A) activity in dried blood spot (DBS), lymphocytes, and skin fibroblasts with massive excretion of Gb3 and Gb2 in urine and lyso-Gb3 in DBS and plasma. Electron microscopic examination showed a large accumulation of sphingolipids in vascular endothelial cells and keratinocytes. Chromosomal analysis and comparative genomic hybridization microarray showed 10q26 terminal deletion. Molecular data showed a novel heterozygous stop codon mutation in exon 1 of the GLA gene in her sisters and niece, and a hemizygous state in her nephew. When we checked the methylation status, we found her non-mutated allele in the GLA gene was methylated. However, the non-mutated alleles of her sisters were non-methylated, and those of her niece were partially methylated. The chromosomal and methylation study may speculate the severity of her clinical phenotypes.

Near-Infrared Photochemoimmunotherapy by Photoactivatable Bifunctional Antibody-Drug Conjugates Targeting Human Epidermal Growth Factor Receptor 2 Positive Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a new class of molecular targeted cancer therapy based on antibody-photoabsorber conjugates and NIR light irradiation. Recent studies have shown effective tumor control, including that of human epidermal growth factor receptor 2 (HER2)-positive cancer, by selective molecular targeting with NIR-PIT. However, the depth of NIR light penetration limits its use. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent maytansinoid DM1. Here, we developed bifunctional antibody-drug-photoabsorber conjugates, T-DM1-IR700, that can work as both NIR-PIT and chemoimmunotherapy agents. We evaluated the feasibility of T-DM1-IR700-mediated NIR light irradiation by comparing the in vitro and in vivo cytotoxic efficacy of trastuzumab-IR700 (T-IR700)-mediated NIR light irradiation in HER2-expressing cells. T-IR700 and T-DM1-IR700 showed almost identical binding to HER2 in vitro and in vivo. Owing to the presence of internalized DM1 in the target cells, NIR-PIT using T-DM1-IR700 tended to induce greater cytotoxicity than that of NIR-PIT using T-IR700 in vitro. In vivo NIR-PIT using T-DM1-IR700 did not show a superior antitumor effect to NIR-PIT using T-IR700 in subcutaneous small-tumor models, which could receive sufficient NIR light. In contrast, NIR-PIT using T-DM1-IR700 tended to reduce the tumor volume and showed significant prolonged survival compared to NIR-PIT using T-IR700 in large-tumor models that could not receive sufficient NIR light. We successfully developed a T-DM1-IR700 conjugate that has a similar immunoreactivity to the parental antibody with increased cytotoxicity due to DM1 and potential as a new NIR-PIT agent for targeting tumors that are large and inaccessible to sufficient NIR light irradiation to activate the photoabsorber IR700.

Polyamine regulating protein antizyme binds to ATP citrate lyase to accelerate acetyl-CoA production in cancer cells.

Antizyme (AZ) regulates cellular polyamines (i.e., putrescine, spermidine, and spermine) through binding to ornithine decarboxylase and subsequent ubiquitin-independent degradation of the enzyme protein by the 26S proteasome. Screening for AZ-binding proteins using a yeast two-hybrid system identified ATP citrate lyase (ACLY), a cytosolic enzyme which catalyzes the production of acetyl-CoA that is used for lipid anabolism or acetylation of cellular components. We confirmed that both AZ1 and AZ2 bind to ACLY and AZ colocalizes with ACLY to the cytoplasm. Unexpectedly, neither AZ1 nor AZ2 accelerated ACLY degradation. Additionally, purified AZ, particularly AZ1, increased the activity of purified ACLY in a dose-dependent manner in vitro, suggesting that AZ activates ACLY through protein-protein interaction. Polyamines themselves had no effect on the ACLY activity in vitro. Knockdown of AZ1 and/or AZ2 in human cancer cells significantly decreased the ACLY activity as well as cellular levels of acetyl-CoA and cholesterol. Our results are the first to show the crosstalk between polyamine and acetyl-CoA metabolism. We hypothesize that AZ may promote acetyl-CoA synthesis to downregulate spermidine and spermine through acetylation.

Associations among serum trimethylamine-N-oxide (TMAO) levels, kidney function and infarcted coronary artery number in patients undergoing cardiovascular surgery: a cross-sectional study.

BACKGROUND: Trimethylamine-N-oxide (TMAO) is a metabolite of phosphatidylcholine generated by gut microbiota and liver enzymes, and has recently been recognized as contributing to atherosclerosis. Elevated serum TMAO levels have been shown to increase the risk of cardiovascular disease (sudden death, myocardial infarction, or stroke) in patients undergoing elective coronary angiography. We aimed to clarify whether TMAO levels are associated with the number of infarcted coronary arteries as a measure of the severity of atherosclerosis, with adjustment using a priori-defined covariates such as kidney function. METHODS: By conducting a cross-sectional study of 227 patients who underwent cardiovascular surgery for coronary artery disease, valvular heart disease, or aortic disease, the association between serum TMAO levels as measured by HPLC-APCI-MS/MS and the number of infarcted coronary arteries was evaluated using ordered logistic regression models with adjustment of 10 covariates, including chronic kidney disease (CKD) stage. Unadjusted and adjusted odds ratios (ORs) and 95 % confidence intervals (95 % CIs) were determined. RESULTS: Significantly higher TMAO levels were observed in advanced-stage CKD (p ≤ 0.001). In fully adjusted models with the 10 covariates, a significantly increased number of infarcted coronary arteries was identified in the highest quartile and quintile of TMAO compared to the lowest quartile (OR 11.9; 95 % CI 3.88-36.7, p ≤ 0.001) and quintile (OR 14.1; 95 % CI 3.88-51.2; p ≤ 0.001), respectively, independent of dyslipidemia. CONCLUSIONS: Higher serum TMAO levels may be associated with advanced CKD stages and with an increased number of infarcted coronary arteries in patients who undergo cardiovascular surgery.

The Powdering Process with a Set of Ceramic Mills for Green Tea Promoted Catechin Extraction and the ROS Inhibition Effect.

For serving green tea, there are two prominent methods: steeping the leaf or the powdered leaf (matcha style) in hot water. The purpose of the present study was to reveal chemical and functional differences before and after the powdering process of green tea leaf, since powdered green tea may contribute to expanding the functionality because of the different ingesting style. In this study, we revealed that the powdering process with a ceramic mill and stirring in hot water increased the average extracted concentration of epigallocatechin gallate (EGCG) by more than three times compared with that in leaf tea using high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass Spectrometry (LC-MS/MS) analyses. Moreover, powdered green tea has a higher inhibition effect of reactive oxygen species (ROS) production in vitro compared with the same amount of leaf tea. Our data suggest that powdered green tea might have a different function from leaf tea due to the higher catechin contents and particles.

Branched amphiphilic peptide capsules: cellular uptake and retention of encapsulated solutes.

Branched amphiphilic peptide capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble to form bilayer delimited capsules. In two recent publications we described the lipid analogous characteristics of our BAPCs, examined their initial assembly, mode of fusion, solute encapsulation, and resizing and delineated their capability to be maintained at a specific size by storing them at 4°C. In this report we describe the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs in vivo. The ability of our constructs to retain alpha particle emitting radionuclides without any apparent leakage and their persistence in the peri-nuclear region of the cell for extended periods of time, coupled with their ease of preparation and potential tune-ability, makes them attractive as biocompatible carriers for targeted cancer therapy using particle emitting radioisotopes. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Branched amphiphilic cationic oligopeptides form peptiplexes with DNA: a study of their biophysical properties and transfection efficiency.

Over the past decade, peptides have emerged as a new family of potential carriers in gene therapy. Peptides are easy to synthesize and quite stable. Additionally, sequences shared by the host proteome are not expected to be immunogenic or trigger inflammatory responses, which are commonly observed with viral approaches. We recently reported on a new class of branched amphiphilic peptide capsules (BAPCs) that self-assemble into extremely stable nanospheres. These capsules are capable of retaining and delivering alpha-emitting radionuclides to cells. Here we report that, in the presence of double stranded plasmid DNA, BAPCs are unable to form. Instead, depending of the peptide/DNA ratios, the peptides either coat the plasmid surface forming nanofibers (high peptide to DNA ratio) or condense the plasmid into nanometer-sized compacted structures (at low peptide to DNA ratios). Different gene delivery efficiencies are observed for the two types of assemblies. The compacted nanometer-sized structures display much higher transfection efficiencies in HeLa cells. This level of transfection is greater than that observed for a lipid-based reagent when the total number of viable transfected cells is taken into account.

An efficient system for secretory production of fibrinogen using a hepatocellular carcinoma cell line.

AIM: Despite an increasing demand, blood products are not always safe because most are derived from blood donations. One possible solution is the development and commercialization of recombinant fibrinogen, but this process remains poorly developed. This study aimed to develop an effective production system for producing risk-free fibrinogen using human hepatocellular cell lines and serum-free media. METHODS: Three human liver cancer cell lines (HepG2, FLC-4 and FLC-7) were cultivated in a serum-supplemented medium or two serum-free media (ASF104N and IS-RPMI) to compare their fibrinogen secretion abilities. Fibrinogen subunit gene expression was estimated by quantitative polymerase chain reaction. Massive fibrinogen production was induced using a 5-mL radial flow bioreactor (RFB) while monitoring glucose metabolism. Subsequently, fibrinogen's biochemical characteristics derived from these cells were analyzed. RESULTS: FLC-7 cell culture combined with IS-RPMI resulted in significantly better fibrinogen production (21.6 µg/10(7) cells per day). ASF104N had more positive effects on cell growth compared with IS-RPMI, whereas fibrinogen production was more efficient with IS-RPMI than with ASF104N. Changing the medium from ASF104N to IS-RPMI led to significantly increased fibrinogen gene expression and glucose consumption. In the RFB culture, the fibrinogen secretion rate of FLC-7 cells reached 0.73 µg/mL per day during a 42-day cultivation period. The subunit composition and clot formation activity of FLC-7 cell-derived fibrinogen corresponded to those of plasma fibrinogen. CONCLUSION: The FLC-7 cell culture system is suitable for large-scale fibrinogen preparation production.

A practical fluorometric assay method to measure lysosomal acid lipase activity in dried blood spots for the screening of cholesteryl ester storage disease and Wolman disease.

Fluorometric measurements of 4-methylumbelliferone (4-MU) are generally used to screen lysosomal storage diseases (LSDs) using dried blood spots (DBSs). However, in DBS, it is difficult to measure lysosomal acid lipase (LAL) activity due to the influence of other lipases in whole blood. Recently, Hamilton used a fluorometric enzyme assay with 4-MU derivatives to measure the LAL activity in DBS. This method requires mercury chloride as stopping reagent, and the fluorescence intensity of 4-MU was measured at an acidic pH. We report a revised method to measure the LAL activity without using toxic mercury chloride and to measure the fluorescence intensity of 4-MU at a basic pH. For this measurement, we established a more practical method that does not require mercury chloride. The LAL activity in DBS was measured in 51 normal controls, seven obligate carriers and seven patients with CESD. The average LAL activities ± SD in the DBS from the normal, obligate carriers and CESD patients were 0.68 ± 0.2 (range: 0.3-1.08), 0.21 ± 0.1 (range: 0.11-0.41) and 0.02 ± 0.02 (range: 0-0.06) nmol/punch/h, respectively. There was a significant difference between the normal and the CESD. Our method does not require toxic mercury chloride and is an appropriate revised enzyme assay using DBS for screening patients with CESD.

Sphingosine-1-phosphate induces differentiation of cultured renal tubular epithelial cells under Rho kinase activation via the S1P2 receptor.

BACKGROUND: Sphingosine-1-phosphate (S1P) is reportedly involved in the pathogenesis of kidney disease; however, the precise role played by S1P in renal disorders still remains controversial. Rho kinase plays an important role in the development of diabetic nephropathy by inducing glomerular and tubulointerstitial fibrosis. Rho kinase is known to be stimulated by S1P through its specific receptor, S1P2 receptor (S1P2). Hence, we investigated whether S1P-S1P2 signaling plays a role in the epithelial-mesenchymal transition (EMT) through Rho kinase activation in renal tubules. METHOD: To characterize the distribution of the S1P2, an immunohistochemical examination of the receptor was performed in the kidney of the non-diabetic and diabetic mice. Next, we examined Rho kinase activity as well as E-cadherin and alpha-smooth muscle actin (α-SMA) expression by real-time RT-PCR and western blotting in cultured rat tubular epithelial cells under S1P stimulation with and without a Rho kinase inhibitor and an S1P2 blocker. In addition, the distribution of E-cadherin and α-SMA was examined by immunocytochemistry. RESULT: S1P2 was expressed mainly in the renal tubules; expression was intense in collecting ducts and distal tubules compared to other segments. S1P induced activation of Rho kinase through the S1P2, which changed the distribution of E-cadherin and increased the expression of α-SMA. CONCLUSION: Rho kinase activation by S1P via S1P2 initiated EMT changes in cultured renal tubular cells. Our results suggest that excessive stimulation of S1P might facilitate renal fibrosis via activation of Rho kinase through S1P2.

Fibrinopeptide A-induced blood-feeding arrest in the yellow fever mosquito Aedes aegypti.

Female mosquitoes engage in blood feeding from their hosts to facilitate egg maturation but cease feeding once a sufficient blood meal has been acquired. Abdominal distention has been proposed as a contributing factor; however, it has also been suggested that there are chemical controls. In this study, we focus on negative chemical regulators of blood feeding, particularly those present in the host blood. Serum derived from animal blood inhibits the feeding of ATP, a phagostimulant of blood feeding in Aedes aegypti. Fibrinopeptide A (FPA), a 16-amino acid peptide cleaved from fibrinogen during blood coagulation, serves as an inhibitory factor in the serum. Our findings suggest that blood-feeding arrest in female mosquitoes is triggered by the detection of FPA in the host blood, which increases as blood coagulation proceeds in the mosquito's midgut, highlighting the role of host-derived substances as negative regulators of mosquito behavior.

Staphylococcus epidermidis Esp degrades specific proteins associated with Staphylococcus aureus biofilm formation and host-pathogen interaction.

Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (Esp(S235A)) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction.

Branched oligopeptides form nanocapsules with lipid vesicle characteristics.

In a recent article (Gudlur et al. PLOS ONE, 2012, 7 (9) e45374), we described the special properties of a mixed branched peptide assembly in which equimolar bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK self-associate to form bilayer delimited capsules capable of trapping solutes. These polycationic vesicle-like capsules are readily taken up by epithelial cells in culture, escape or evade the endocytic pathway, and accumulate in the perinuclear region where they persist without any apparent degradation. In this report, we examine the lipidlike properties of this system including initial assembly; solute encapsulation and washing; fusion and resizing by membrane extrusion through polycarbonate filters with defined pore sizes. The resized peptide capsules have uniform diameters in nm size ranges. Once resized, the capsules can be maintained at the new size by storing them at 4 °C. Having the ability to prepare stable uniform nanoscale capsules of desired sizes makes them potentially attractive as biocompatible delivery vehicles for various solutes/drugs.

Objective display and discrimination of floral odors from Amorphophallus titanum, bloomed on different dates and at different locations, using an electronic nose.

As olfactory perceptions vary from person to person, it is difficult to describe smells objectively. In contrast, electronic noses also detect smells with their sensors, but in addition describe those using electronic signals. Here we showed a virtual connection method between a human nose perceptions and electronic nose responses with the smell of standard gases. In this method, Amorphophallus titanum flowers, which emit a strong carrion smell, could objectively be described using an electronic nose, in a way resembling the skill of sommeliers. We could describe the flower smell to be close to that of a mixture of methyl mercaptan and propionic acid, by calculation of the dilution index from electronic resistances. In other words, the smell resembled that of "decayed cabbage, garlic and pungent sour" with possible descriptors. Additionally, we compared the smells of flowers which bloomed on different dates and at different locations and showed the similarity of odor intensities visually, in standard gas categories. We anticipate our assay to be a starting point for a perceptive connection between our noses and electronic noses.

Generation and characterization of motor neuron progenitors and motor neurons using metachromatic leukodystrophy-induced pluripotent stem cells.

The pathological consequences leading to primary storage, autophagy impairment, impaired mitochondrial dynamics, and endoplasmic reticulum (ER) stress on neural cell dysfunction and apoptosis in metachromatic leukodystrophy (MLD) have been poorly elucidated. In the present study, we generated 2 cell lines of patient-specific-induced pluripotent stem cells (iPSCs) and modeled the progression of pathological events during the differentiation of iPSCs to motor neuron progenitors (MNPs) and mature motor neurons (MNs). The iPS cells were generated from two late-infantile MLD patient-derived skin fibroblasts using electroporation or the Sendai virus. Olig2+ MNPs were generated from both iPSC lines using a combination of small molecules in a chemically defined neural medium. Furthermore, the MNPs could be differentiated into mature MNs, which was confirmed by RT-PCR and MN markers, including SMI32 and ChAT. The population of MNs was approximately 50% under the culture conditions. Pathological observation of MLD patient-derived iPSCs revealed lysosomal accumulation and impaired autophagy. In addition, both MNPs and MNs derived from MLD-iPSCs showed increased lysosomal accumulation, dysfunctional autophagy, impaired mitophagy, endoplasmic reticulum (ER) stress or unfolded protein response (UPR) activation, and premature cellular death.