Cryopreservation of Adherent Human Pluripotent Stem Cells via a Novel Dish Culture Preservation Method.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) are valuable resources for cell therapy and drug discovery. Cryopreservation is a key technique used to realize these applications, which require a large number of cells. However, standard protocols for the preservation of the adherent cells involve multiple complicated steps, which can lead to technical difficulties. OBJECTIVE: To develop a more efficient method for cryopreservation of adherent cells using culture dishes. MATERIALS AND METHODS: Ice-seeding treatment was employed to avoid intercellular freezing, and rapid warming used to improve cell viability. RESULTS: The immediate survival rate after thawing was 48%. The recovery period of cells cryopreserved by the dish culture method was shortened upon subsequent passage culture, and the time for re-cultured cells to reach the appropriate confluency was reduced by two days. CONCLUSION: The hiPSCs can be successfully cryopreserved in culture dishes with improved viability and faster recovery. The optimization of the ice-seeding temperature and cooling rate increased the survival rate.

Exposure to clinical X-ray radiation does not alter antibiotic susceptibility or genotype profile in gram-negative and gram-positive clinical pathogens.

Inadvertent exposure of bacterial pathogens to X-ray radiation may be an environmental stress, where the bacterium may respond by increasing mutational events, thereby potentially resulting in increased antibiotic resistance and alteration to genotypic profile. In order to examine this, four clinical pathogens, including the Gram-negative organisms Escherichia coli O157:H7 NCTC12900 and Pseudomonas aeruginosa NCTC10662, as well as the Gram-positive organisms Staphylococcus aureus NCTC6571 and Enterococcus faecium were exposed to X-rays (35,495 cGy/cm2) over a seven-day period. Antibiotic susceptibility was assessed before, during and after exposure by examining susceptibility, as quantified by E-test with six antibiotics, as well as to a further 11 antibiotics by measurement of susceptibility zone sizes (mm). Additionally, the DNA profile of each organism was compared before, during and after exposure employing the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC PCR). Results indicated that exposure of these organisms to this amount of X-ray radiation did not alter their antibiotic susceptibility, nor their genomic DNA profile. Overall, these data indicate that exposure of bacteria to X-ray radiation does not alter the test organisms' antibiotic susceptibility profiles, nor alter genomic DNA profiles of bacteria, which therefore does not compromise molecular epidemiological tracking of bacteria within healthcare environments in which patients have been exposed to X-ray radiation.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

Expression and analysis of a cytolethal distending toxin (cdt) gene operon in Campylobacter lari.

The present study examines the expression of cytolethal distending toxin (cdt) gene encoding a cytotoxin in Campylobacter lari (n=6 urease-negative [UN] C. lari; n=4 urease-positive thermophilic Campylobacter [UPTC]). When reverse transcription polymerase chain reaction (RT-PCR) was carried out with 10 C. lari isolates using a primer pair to amplify the cdtB gene transcript segment, an approximate 260 bp RT-PCR amplicon was generated with all the isolates. In addition, cdtA, cdtB and cdtC gene operon was identified to be polycistronicly transcribed in the C. lari cells. The cdtB gene translation in the C. lari cells was also confirmed by Western blot analysis. Thus, the cdt gene operon in C. lari organisms, including UN C. lari and UPTC, was expressed at the transcriptional and translational levels in the cells. The present results suggest that all three cdt genes may be functional in the cells.

Comparative analysis of Campylobacter lari cytolethal distending toxin (CDT) effect on HeLa cells.

We aimed to clarify if Campylobacter lari exerts a cytolethal distending toxin (CDT) effect on HeLa cells. Campylobacter cell lysates (CCLys) from C. jejuni 81-176 and urease-positive thermophilic Campylobacter (UPTC) CF89-12 and UPTC NCTC12893 isolates were shown to exert a CDT effect on HeLa cells with morphological changes examined by Giemsa staining and microscopy. However, Campylobacter lari JCM2530(T) isolate showed no effect. In addition, Campylobacter cell culture supernatant wash gave low or absent toxic effects with both C. jejuni and C. lari organisms. When western blot analysis was carried out to clarify if there was a CDTB effect in the CCLys and soluble fractions from Campylobacter isolates, which had a CDT effect on HeLa cells or did not have any effect, anti-recombinant CjCDTB antibodies identified an immunoreactively positive signal at around approximately 25 kDa on all the C. lari isolates examined, as well as the C. jejuni 81116 strain. Thus, all the Campylobacter isolates including those without any CDT effect were shown to express CDTB at the translational level.

Reliability of nucleotide sequence information of full-length flagellin A gene (flaA) and flaA short variable region (SVR) for molecular discrimination of Campylobacter lari organisms.

We aimed to clarify if the thermophilic Campylobacter lari organisms including urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC) can be differentiated at the species and/or subspecies levels by employing the full-length flaA gene and flaA short variable region (SVR) nucleotide sequence information or not. Thermophilic Campylobacter isolates (n = 45) including UN C. lari (n = 17), UPTC (n = 18), and Campylobacter jejuni (n = 10) were well discriminated at the isolate level by the unweighted pair group method using arithmetic means analysis and neighbor joining procedures constructed based on the full-length flaA gene and flaA SVR nucleotide sequence information. Thus, these procedures may possibly be useful for epidemimological studies for C. lari and C. jejuni.

Molecular analysis and characterization of a urease gene operon from Campylobacter sputorum biovar paraureolyticus.

When recombinant plasmid DNA from a genomic DNA library and inverse PCR products of Campylobacter sputorum biovar paraureolyticus LMG17591 strain were analyzed, an approximate 6.5-kb pair region, encoding a urease gene operon, was identified. Within the operon, seven closely spaced and putative open reading frames for ureG, ureH(D), ureA, ureB, ureC, ureE, and ureF were detected in order. A possible overlap was detected between ureG and ureH(D), ureH(D) and ureA, and ureE and ureF. In addition, two putative promoter structures, probable ribosome-binding sites and a putative ρ-independent transcriptional terminator structure were identified. The urease gene operon transcription in the cells was confirmed by the reverse transcription-PCR analysis. A neighbor-joining tree constructed based on the nucleotide sequence information of urease genes showed that C. sputorum biovar paraureolyticus formed a cluster with Arcobacter butzleri, urease-positive thermophilic Campylobacter and some Helicobacter spp., separating those from the other urease-producing bacteria, suggesting a commonly shared ancestry among these organisms.

Comparison between in vitro qualities of platelets washed with commercially available additive solutions and those washed with M-sol.

BACKGROUND AND OBJECTIVES: We previously developed a novel additive solution (M-sol) with a high ability to preserve the in vitro qualities of platelets (PLTs) in washed PLTs Here, we compared the ability of M-sol with that of commercially available additive solutions (ASs) to preserve the in vitro qualities (pH, mean PLT volume, %disc, P-selectin, %hypotonic shock response and aggregation) of PLTs at a low plasma concentration. MATERIALS AND METHODS: The platelet concentrate was divided into two equal aliquots (control group and test group). After centrifugation of both groups and removal of as much supernatant as possible, the pellet of the control group was resuspended in M-sol and those of the test group were resuspended in other ASs, and subsequently stored in polyolefin bags with agitating at 20-24 degrees C. RESULTS: Compared with those stored in M-sol, the qualities of PLTs stored in PAS-B (alternative name; PAS-II or T-sol), PAS- C (alternative name; PAS-III or Intersol) or Plasma Lyte were degraded as early as 24 h after washing. The qualities of PLTs stored in PAS-D (alternative name; Composol PS) or PAS-E (alternative name; PAS-IIIM or SSP+) were comparable to that of those stored in M-sol 24 h after washing; however, the qualities had deteriorated 72 h after washing. CONCLUSIONS: At a low plasma concentration (5% or less), the M-sol showed a higher ability to preserve PLTs than the five ASs studied here. Although PAS-D and PAS-E are available as an AS for short-term storage of washed PLTs, M-sol is thought to be preferable for longer storage.

Chromatin remodeling and circadian control: master regulator CLOCK is an enzyme.

The molecular machinery that governs circadian rhythmicity is based on clock gene products organized in regulatory feedback loops. Recently, we have shown that CLOCK, a master circadian regulator, has histone acetyltransferase activity essential for clock gene expression. The Lys-14 residue of histone H3 is a preferential target of CLOCK-mediated acetylation. As the role of chromatin remodeling in eukaryotic transcription is well recognized, this finding identified unforeseen links between histone acetylation and cellular physiology. Indeed, we have shown that the enzymatic function of CLOCK drives circadian control. We reasoned that CLOCK's acetyltransferase activity could also target nonhistone proteins, a feature displayed by other HATs. Indeed, CLOCK also acetylates a nonhistone substrate: its own partner, BMAL1. This protein undergoes rhythmic acetylation in the mouse liver, with a timing that parallels the down-regulation of circadian transcription of clock-controlled genes. BMAL1 is specifically acetylated on a unique, highly conserved Lys-537 residue. This acetylation facilitates recruitment of the repressor CRY1 to BMAL1, indicating that CLOCK may intervene in negative circadian regulation. Our findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery.

The influence of early ambulation and other factors on headache after lumbar myelography.

In order to determine the influence of early ambulation and other factors on headaches occurring after lumbar myelography we randomised 207 patients (127 men and 80 women) into two groups. Following the investigation, we allowed the 101 patients (65 men and 36 women) in group A to sit or stand freely, while we confined the 106 patients (62 men and 44 women) in group B to bed for 20 hours. The nine patients in group B who could not maintain bed rest were excluded. There was no significant difference between the two groups as regards the prevalence of spinal headache (8.9% in group A v 14.4% in group B). Patients who reported headaches, however, were significantly more likely to be women (18.7%) than men (73%), be younger (mean age 45 years v 56 years), have a higher cerebrospinal pressure before removal of fluid (mean values 172 v 137 mm H2O) and a lower systolic (mean values 120 v 134 mmHg) and diastolic blood pressure. We conclude that, although other factors may be associated with headaches, late ambulation is not effective in preventing spinal headaches after lumbar myelography.

Virus inactivation in hemoglobin solution by heat treatment.

To increase the safety of stroma-free hemoglobin solution (SFH) as an artificial oxygen carrier source, we investigated the effect of heat treatment on virus inactivation in hemoglobin solution. The hemoglobin solution spiked with vesicular stomatitis virus (VSV) was treated at 60 degrees C for 1 hr under either an air or CO atmosphere. VSV was inactivated at >5.8 log10 and >6.0 log10 under the air and CO atmosphere, respectively. Although the methemoglobin rate increased after the heat treatment under the air atmosphere, no methemoglobin formation was observed by the treatment under the CO atmosphere. Isoelectric focusing analysis revealed the denaturation of hemoglobin after the heat treatment under the air, while hemoglobin banding was not altered in the carbonylated condition. Some protein bands other than hemoglobin were weakened or disappeared on SDS-PAGE after the heat treatment under both conditions. In addition, the hemoglobin concentration in the SFH was higher after the heat treatment than before the treatment. These findings indicate that the heat treatment under the CO atmosphere inactivates viruses without hemoglobin denaturation, and hence, this method is a promising approach to prepare a safer SFH as artificial oxygen carriers.

Comparison of the effects of different antiviral treatments on the antioxidant systems of stroma-free hemoglobin.

The effect of virus inactivation by 1,9-dimethylmethylene blue (DMMB) phototreatment, methylene blue (MB) phototreatment or heat on the activities of antioxidant systems of stroma-free hemoglobin (SFH) was studied. DMMB photoinactivated human immunodeficiency virus by > 3.69 log10 under conditions that inactivated 3.33 log10 of vesicular stomatitis virus (VSV). Under conditions which inactivated VSV by 6.10 log10 (1.37 J/cm2 irradiation and 2 microM DMMB), there was little change in the methemoglobin (Met-Hb) formation, concentration of reduced glutathione (GSH), or superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPX) activities. However, the activity of glutathione reductase (GR) was decreased by 77%. Under conditions that inactivated VSV by 5.69 log10 (1.37 J/cm2 irradiation and 24 microM MB) there was little effect of MB phototreatment on SOD, CAT, GPX and GSH activities. However, GR activity was decreased by 74% and Met-Hb content reached 3.98%. Under conditions that inactivated VSV by more than 6.20 log10 (60 degrees C for 2 min), virucidal heat treatment resulted in 27% Met-Hb formation and decreased GPX activity by 43%. No significant decline in SOD, CAT or GR activities or GSH concentration was observed. These results suggest that, compared with heat treatment and MB phototreatment, virucidal DMMB treatment preserves not only the oxidative state of hemoglobin but also the antioxidant systems against superoxide and hydrogen peroxide, although the reduced GR activity may limit the quenching capacity of antioxidants in DMMB-treated SFH.

Genetic changes induced in human cells in Space Shuttle experiment (STS-95).

BACKGROUND: Results of past space experiments suggest that the biological effect of space radiation could be enhanced under microgravity. To assess the radiation risk for humans during long-term spaceflight, it is very important to clarify whether human cells exhibit a synergistic effect of radiation and microgravity. HYPOTHESIS: If significant synergism occurs in human cells, genetic changes induced during spaceflight may be detected by using human tumor HCT-116 cells which are hypermutable due to a defect in the DNA mismatch repair system. METHODS: Cultured HCT-116 cells were loaded on the Space Shuttle Discovery (STS-95) and grown during the 9-d mission. After landing, many single-cell clones were isolated, microsatellite repetitive sequences in each clone were amplified by PCR, and mutations in the microsatellite loci were detected as changes in the length of PCR fragments. Mutation frequencies of ouabain-resistant phenotype were also analyzed. RESULTS: The frequencies of microsatellite mutations as well as ouabain-resistant mutations in the flight sample were similar to those of the ground control samples. Some cells were treated in space with bleomycin which mimics the action of radiation, but the frequencies of microsatellite mutations were not significantly different between the flight and the ground control samples. CONCLUSION: Under the present flight conditions, neither space radiation (about 20 mSv during this mission) nor microgravity caused excess mutations in human cells.

Superoxide generation from human polymorphonuclear leukocytes by liposome-encapsulated hemoglobin.

We investigated the interactions between liposome-encapsulated hemoglobin (Neo Red Cells: NRC) and human polymorphonuclear leukocytes as assessed by superoxide generation. NRC triggered superoxide generation from neutrophils in a dose-dependent manner. Empty liposomes also induced superoxide production of neutrophils. Superoxide generation of neutrophils induced by phorbol myristate acetate (PMA) was delayed but intensified both by NRC and empty liposomes. The intensity of superoxide generation induced by NRC was smaller than that by the empty liposomes. As NRC contained superoxide dismutase (SOD) that was copurified with hemoglobin from red blood cells and its activity remained, SOD contained in NRC may partially eliminate superoxide.

Three patients with isolated adrenocorticotropin deficiency presenting with neuroleptic malignant syndrome-like symptoms.

We report 3 patients with isolated adrenocorticotropin (ACTH) deficiency presenting with neuroleptic malignant syndrome (NMS)-like symptoms. All patients were in their 60's or 70's and showed consciousness disturbance, a high-grade fever, extrapyramydal signs, and muscle enzyme elevations, which met the criteria for NMS. Also, they all showed hyponatremia induced by isolated ACTH deficiency. In addition to the standard therapy for NMS, corticosteroid supplement therapy was effective in all patients. There thus appear to be subjects with isolated ACTH deficiency among patients presenting with NMS-like symptoms, and adrenal and pituitary function should be checked in NMS patients with hyponatremia.

Involvement of reactive oxygen species in hemoglobin oxidation and virus inactivation by 1,9-dimethylmethylene blue phototreatment.

The participation of reactive oxygen species (ROS) in virus inactivation by 1,9-dimethylmethylene blue (DMMB) phototreatment in stroma-free hemoglobin (SFH) was investigated with the use of scavengers, quenchers and enhancer. Virus (R17 bacteriophage) photoinactivation by either activated monomer or dimer DMMB was suppressed by sodium azide (singlet oxygen quencher) and promoted by the substitution of H2O for deuterium oxide (D2O), which is known to prolong the lifespan of singlet oxygen. There was no or little effect of mannitol (hydroxyl radical scavenger) and superoxide dismutase (superoxide scavenger) on the photoinactivation. Similar experiments were conducted to investigate the mechanism of methemoglobin (Met-Hb) formation by the activated monomer of DMMB. There was little effect of the singlet oxygen quencher, histidine, or the enhancer, D2O, on Met-Hb formation. However, rutin, which inhibits not only singlet oxygen but also other ROS, and mannitol supressed the formation of Met-Hb by activated monomer. The addition of superoxide dismutase (SOD) did not inhibit the formation. In contrast to the activity of the DMMB monomer, that of the dimer was inhibited by histidine and enhanced by D2O. The addition of neither mannitol nor SOD affected Met-Hb formation by activated dimer. These results collectively suggest that virus photoinactivation by the activated monomer and dimer of DMMB as well as Met-Hb formation by the activated dimer proceed via a singlet oxygen mediated pathway. In contrast, singlet oxygen may play a less important role in Met-Hb formation by the activated monomer.