Determination of neurotransmitters in mouse brain using miniaturized and tableted QuEChERS for the sample preparation.

Neurotransmitters (NTs) are important endocrine molecules in the human body that have several characteristics and functions. Therefore, it is necessary to determine the accuracy and precision of the proposed method for the assessment of NTs functions. This study proposes a quick, easy, cheap, effective, rugged, and safe (QuEChERS) tablet for easy sample preparation and application to low-volume samples. We used response surface methodology to design and optimize the QuEChERS tablets. When we measured NTs in the brains of aged mice, whose samples were derivatized by DNS and extracted by QuEChERS, all analytes except VMA, NAS, 5-HTP, DOPA, and A, were detected in the mouse brain samples. Our approach may provide easy sample preparation and/or extraction techniques for small sample volumes, not only in animal samples but also in human samples to avoid invasive collection.

New parallel computing algorithm of molecular dynamics for extremely huge scale biological systems.

In this paper, we address high performance extreme-scale molecular dynamics (MD) algorithm in the GENESIS software to perform cellular-scale molecular dynamics (MD) simulations with more than 100,000 CPU cores. It includes (1) the new algorithm of real-space nonbonded interactions maximizing the performance on ARM CPU architecture, (2) reciprocal-space nonbonded interactions minimizing communicational cost, (3) accurate temperature/pressure evaluations that allows a large time step, and (4) effective parallel file inputs/outputs (I/O) for MD simulations of extremely huge systems. The largest system that contains 1.6 billion atoms was simulated using MD with a performance of 8.30 ns/day on Fugaku supercomputer. It extends the available size and time of MD simulations to answer unresolved questions of biomacromolecules in a living cell.

Complete Genome Sequence of Bifidobacterium longum Strain Jih1, Isolated from Human Feces.

We report the complete genome sequence of Bifidobacterium longum strain Jih1, isolated from human feces. The assembled genome comprised one circular chromosome of 2.37 Mb. The chromosome harbors 1,941 protein-coding genes.

Virtual Grid Engine: a simulated grid engine environment for large-scale supercomputers.

BACKGROUND: Supercomputers have become indispensable infrastructures in science and industries. In particular, most state-of-the-art scientific results utilize massively parallel supercomputers ranked in TOP500. However, their use is still limited in the bioinformatics field due to the fundamental fact that the asynchronous parallel processing service of Grid Engine is not provided on them. To encourage the use of massively parallel supercomputers in bioinformatics, we developed middleware called Virtual Grid Engine, which enables software pipelines to automatically perform their tasks as MPI programs. RESULT: We conducted basic tests to check the time required to assign jobs to workers by VGE. The results showed that the overhead of the employed algorithm was 246 microseconds and our software can manage thousands of jobs smoothly on the K computer. We also tried a practical test in the bioinformatics field. This test included two tasks, the split and BWA alignment of input FASTQ data. 25,055 nodes (2,000,440 cores) were used for this calculation and accomplished it in three hours. CONCLUSION: We considered that there were four important requirements for this kind of software, non-privilege server program, multiple job handling, dependency control, and usability. We carefully designed and checked all requirements. And this software fulfilled all the requirements and achieved good performance in a large scale analysis.

Chiral and Molecular Recognition through Protonation between Aromatic Amino Acids and Tripeptides Probed by Collision-Activated Dissociation in the Gas Phase.

Chiral and molecular recognition through protonation was investigated through the collision-activated dissociation (CAD) of protonated noncovalent complexes of aromatic amino acid enantiomers with l-alanine- and l-serine-containing tripeptides using a linear ion trap mass spectrometer. In the case of l-alanine-tripeptide (AAA), NH3 loss was observed in the CAD of heterochiral H⁺(d-Trp)AAA, while H2O loss was the main dissociation pathways for l-Trp, d-Phe, and l-Phe. The protonation site of heterochiral H⁺(d-Trp)AAA was the amino group of d-Trp, and the NH3 loss occurred from H⁺(d-Trp). The H2O loss indicated that the proton was attached to the l-alanine tripeptide in the noncovalent complexes. With the substitution of a central residue of l-alanine tripeptide to l-Ser, ASA recognized l-Phe by protonation to the amino group of l-Phe in homochiral H⁺(l-Phe)ASA. For the protonated noncovalent complexes of His enantiomers with tripeptides (AAA, SAA, ASA, and AAS), protonated His was observed in the spectra, except for those of heterochiral H⁺(d-His)SAA and H⁺(d-His)AAS, indicating that d-His did not accept protons from the SAA and AAS in the noncovalent complexes. The amino-acid sequences of the tripeptides required for the recognition of aromatic amino acids were determined by analyses of the CAD spectra.

Development of a novel calcium phosphate cement composed mainly of calcium sodium phosphate with high osteoconductivity.

Two novel calcium phosphate cements (CPC) have been developed using calcium sodium phosphate (CSP) as the main ingredient. The first of these cements, labeled CAC, contained CSP, α-tricalcium phosphate (TCP), and anhydrous citric acid, whereas the second, labeled CABC, contained CSP, α-TCP, ß-TCP, and anhydrous citric acid. Biopex(®)-R (PENTAX, Tokyo, Japan), which is a commercially available CPC (Com-CPC), and OSferion(®) (Olympus Terumo Biomaterials Corp., Tokyo, Japan), which is a commercially available porous ß-TCP, were used as reference controls for analysis. In vitro analysis showed that CABC set in 5.7 ± 0.3 min at 22 °C and had a compressive strength of 86.0 ± 9.7 MPa after 5 days. Furthermore, this material had a compressive strength of 26.7 ± 3.7 MPa after 2 h in physiologic saline. CAC showed a statistically significantly lower compressive strength in the presence of physiologic saline and statistically significantly longer setting times than those of CABC. CABC and CAC exhibited apatite-forming abilities in simulated body fluid that were faster than that of Com-CPC. Samples of the materials were implanted into the femoral condyles of rabbits for in vivo analysis, and subsequent histological examinations revealed that CABC exhibited superior osteoconductivity and equivalent bioresorbability compared with Com-CPC, as well as superior osteoconductivity and bioresorbability compared with CAC. CABC could therefore be used as an alternative bone substitute material.

[Comparison of usefulness of the dorsal branch of the radial artery with the radial artery for arterial cannulation].

BACKGROUND: The radial artery cannulation is often associated with damped arterial waveforms with the hand moving. We used cannulation of the dorsal branch of the radial artery (DRA) and compared the stability of measurement, safety and complications with those of the radial artery(RA). METHODS: The study was a prospective single-blinded comparative study. Seventy-six patients undergoing general anesthesia requiring arterial cannulation were included. Patients were divided randomly into two separate groups of 35 patients each according to cannulation site: the radial artery (RA group) or the dorsal branch of the radial artery (DRA group). After induction of general anesthesia, cannulation was performed. Three hours after the successful cannulation, the changes of waveforms were noted with the hand moving. We examined whether there were any complications around the cannulation site after cannula removal. RESULTS: With the wrist flexion at all angles (30, 60 and 90 degrees), the frequency of worsening of arterial waveforms was significantly higher in RA group compared with DRA group. Some difficulties in catheter placement were observed in DRA group. No concomitant complication was noticed. CONCLUSIONS: Arterial line monitoring from DRA had better waveforms compared with RA monitoring without any complications.

Risk factors for gastric distension in patients with acute appendicitis: a retrospective cohort study.

PURPOSE: There has been no report on risk factors for gastric distension (GD) when inducing general anesthesia in an emergency situation. The aim of this study was to clarify the risk factors for GD in patients with acute appendicitis at their hospital visit. METHODS: We reviewed medical records of patients from April 2007 to March 2010 who underwent open appendectomy for acute appendicitis and were diagnosed pathologically. GD was defined as a larger anteroposterior diameter and larger lateral diameter of the stomach than those of the left kidney in computed tomography (CT) imaging. The primary outcome was the presence of GD. Candidate variables such as patient characteristics, physical findings, and CT imaging findings associated with GD were assessed. Time after beginning of abdominal pain was categorized and compared. Determinants with significant univariate association (P < 0.20) with the primary outcome were used to construct multivariable logistic regression models. RESULTS: We enrolled 121 patients and divided this cohort into a GD group (44 cases, 36%) and a non-GD group (77 cases, 64%). Results of univariate analysis showed longer duration of time after beginning of abdominal pain (P = 0.016), younger age (P < 0.001), and more frequent distended small bowel (P < 0.001) in the GD group than in the non-GD group. In multivariate analysis, age [odds ratio (OR) = 0.939, P = 0.002] and time after beginning of abdominal pain (OR = 1.807, P = 0.031) were shown to be independent risk factors. CONCLUSION: Younger appendicitis patients with acute abdominal pain for 1 or more days should be treated as patients with high risk for GD.

Evaluation by bone scintigraphy of osteogenic activity of commercial bioceramics (porous beta-TCP and HAp particles) subcutaneously implanted in rats.

Osteogenic potential of biomaterials used in bone regenerative therapy has been mainly examined in an animal-implantation study. We have here evaluated the applicability of bone scintigraphy in imaging ectopic bone formation, especially its initial phase, by beta-tricalcium phosphate (beta-TCP) particles that were implanted in rat dorsal subcutaneous tissues. In implanted osteogenic osteosarcoma cells used as a positive control, osteoid formation was found by histological examination and bone scintigraphy using (99m)Tc- hydroxymethyl diphosphonate (HMDP) at 2 and 3 weeks post-implantation, respectively, while the microfocuscomputed tomography (muCT) system required further mineralization, which occurred at 4 weeks. Implantation of beta-TCP particles alone induced only faint biomineralization inside the particles, which could be microscopically detected by calcein chelation at 2 weeks post-implantation, but not by other histological examinations (e.g., HE staining) or muCT. However, the bone scintigraphy successfully detected this microscopic change at 1 week. Implanted hydroxyapatite (HAp) particles alone used as a negative control did not induce mineralization at microscopic levels, and therefore nothing was detected by either calcein chelation or bone scintigraphy. In conclusion, the bone scintigraphic methodology, although exhibiting less quantitation and resolution, would be applicable as a non-invasive, highly sensitive methodology in detecting the initial, microscopic changes associated with mineralization.

[The present situation and future prospects of bone substitute of bioinspired materials].

As for an artificial bone which is a substitute material of a bone which supports our body, a block/a granulation or paste properties are manufactured by HAP or beta-TCP. They are similar ingredient of the bone itself. In particular, beta-TCP shows excellent absorbency, and it is applied as a scaffold of tissue engineering of a bony structure. Because such an artificial bone does not obstruct bone metabolism such as the absorption by osteoblasts and bone formation by osteoblasts, such product has both interconnected pore and bioabsorbable properties. However, these artificial bones alone cannot meet demands of clinical practice side. Therefore it is necessary to have the material designs that can promote the work of three factors of cell/Growth factor/Scaffold to future artificial bones, a function more suitable for operation applications. And I would hope that the level of regenerative medicine would drastically improve by further development above mentioned artificial bone.

Accelerated repair of a bone defect with a synthetic biodegradable bone-inducing implant.

BACKGROUND: Nothing has ever had osteoinductive capacity and degradability equivalent to that of autogenous bone, although many types of biomaterials have been developed. To address this issue, we constructed a new bone graft substitute with osteogenic potential and degradability by using porous beta-tricalcium phosphate (beta-TCP) granules, bone morphogenetic protein (BMP), and a synthetic block copolymer composed of poly-D: ,L: -lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG). In this experimental study, the bone-inducing capacity and degradation properties of the composite implant during the bone healing process were examined in vivo in a cortical and cancellous bone defect model in rabbits. METHODS: The advantages of this type of implant have been examined in a cortical bone defect model created in the distal femur of rabbits. The defects (6.5 x 5 mm) were filled with 30 mg of various implants: BMP-H [rhBMP-2, 0.0025% (w/w)], BMP-L [rhBMP-2, 0.000625% (w/w)], control A (beta-TCP alone), and control B (no implant). The distal femurs were harvested at scheduled intervals after surgery and examined for the evaluation of the bony repair of the defects by three-dimensional computed tomography and histology. RESULTS: The repair of both cortical and cancellous bone occurred predominantly in the BMP-H group, and only minor cortical bone repair and cancellous bone formation were noted in the BMP-L and control A groups. Most of the beta-TCP was resorbed in the BMP-H group at 6 weeks after surgery, whereas a significant amount of beta-TCP remained in the BMP-L and control A groups. CONCLUSIONS: beta-TCP granules coated with a BMP-retaining synthetic polymer appear to be effective in enhancing the repair of both cancellous and cortical bone defects. The early disappearance of the implanted beta-TCP and restoration of the normal anatomy of bone tissue are two notable features of this approach.

Osteoinduction with highly purified beta-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation.

The aim of the study was to examine the chronological histology of osteoinduction of highly purified beta-tricalcium phosphate (beta-TCP) implanted in dog dorsal muscles. Specimens were harvested on days 14, 28, 42, 56, 112 and 168 after implantation, and were analyzed by hematoxylin and eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry, in situ hybridization, and silver impregnation. After day 28, abundant TRAP- and cathepsin K-positive multinucleated cells adhered to beta-TCP, suggesting that these cells are osteoclasts that can resorb beta-TCP. On day 56, new bone was formed and alpha1 chain of type I procollagen mRNA-positive osteoblasts lined the newly formed bone. Silver impregnation showed abundant collagen fibrils within the beta-TCP micropores. These results suggest that micropores function as a storage space for extracellular matrix components, including collagen. Newly formed bone never degenerated in the late stage, suggesting that beta-TCP has good biocompatibility and this material retains the conditions appropriate for osteointegration and bioresorption. In conclusion, beta-TCP has osteoinductivity after implantation in dog dorsal muscles without use of bone marrow cells or osteoinductive cytokines. The appearance of a large number of active osteoclasts precedes new bone formation.

Bone formation and resorption of highly purified beta-tricalcium phosphate in the rat femoral condyle.

The aim of this study was to examine the chronological histology associated with highly purified beta-tricalcium phosphate (beta-TCP) implanted in the rat femoral condyle. Specimens were harvested on days 4, 7, 14, 28 and 56 after implantation, and were analyzed by tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry of the ED1 protein as a marker of the phagocyte system, and in situ hybridization with digoxigenin-labeled alpha1 chain of type I procollagen (COL1A1), osteopontin and osteocalcin. beta-TCP was resorbed in a chronological manner. Although new bone was not observed on day 4, fibroblast-like cells around beta-TCP were positive for COL1A1 and osteopontin mRNA. New bone formation presented after day 7. In the double-staining for OPN and ED1 on day 7, most cells around beta-TCP were positive for either osteopontin mRNA or ED1 protein. However, there were some doubly positive multinucleated cells, suggesting that they belonged to the mononuclear phagocyte system. After day 28, the implanted region was replaced with bone marrow. Multinucleated TRAP-positive and ED1-positive cells which adhered to beta-TCP at all stages seemed to be osteoclasts and they continuously resorbed beta-TCP. beta-TCP has a good biocompatibility since both bioresorption and bone formation started at an early stage after implantation.

Repair of an intercalated long bone defect with a synthetic biodegradable bone-inducing implant.

Recombinant human bone morphogenetic protein (rhBMP)-2 in a block copolymer composed of poly-D,L-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) as a carrier and porous beta-tricalcium phosphate (beta-TCP) blocks were used to generate a new fully absorbable osteogenic biomaterial. The bone regenerability of the rhBMP-2/PLA-DX-PEG/beta-TCP composite was studied in a critical-sized rabbit bone defect model. In an initial study, a composite of PLA-DX-PEG (250 mg) and beta-TCP (300 mg) loaded with or without rhBMP2 (50 microg) was implanted into a 1.5 cm intercalated bone defect created in a rabbit femur. Defects were assessed by biweekly radiography until 8 weeks postoperatively. The bony union of the defect was recognized only in the BMP-loaded group. To obtain further data on biomechanical and remodeling properties, another BMP-loaded composites group was made and observed up to 24 weeks. All defects were completely repaired without residual traces of implants. Anatomical and mechanical properties of the repaired bone examined by histology, 3-dimensional CT (3D-CT) and mechanical testing were essentially equivalent to the nonoperated-on femur at 24 weeks. These experimental results indicate that fully absorbable rhBMP-2/PLA-DX-PEG/beta-TCP is a promising composite having osteogenicity efficient enough for repairing large bone defects.

A new bone-inducing biodegradable porous beta-tricalcium phosphate.

A new type of degradable biomaterial with bone-inducing capacity was made by combining porous beta-tricalcium phosphate (beta-TCP) with a delivery system for recombinant human bone morphogenetic protein-2 (rhBMP-2). The BMP delivery system consisted of a block copolymer composed of poly-D,L-lactic acid with random insertion of p-dioxanone and polyethylene glycol (PLA-DX-PEG), a known biocompatible and biodegradable material. The efficacy of this biomaterial in terms of its bone-inducing capacity was examined by ectopic bone formation in the dorsal muscles of the mouse. In the beta-TCP implants coated with the PLA-DX-PEG polymer containing more than 0.0025% (w/w) of rhBMP-2, new ectopic bone tissues with marrow were consistently found on the surface of implants. The radiographic density of beta-TCP was diminished in a time-dependent manner. On histological examination, numerous multinucleated osteoclasts with positive tartrate-resistant acid-phosphatase (TRAP) staining were noted on the surface of the beta-TCP. These experimental results indicate that beta-TCP implants coated with synthetic rhBMP-2 delivery system might provide effective artificial bone-graft substitutes with osteoinductive capacity and biodegradable properties. In addition, this type of biomaterial may require less rhBMP-2 to induce significant new bone mass.