Quantitative Proteomics for the Development and Manufacturing of Human-Induced Pluripotent Stem Cell-Derived Neural Stem Cells Using Data-Independent Acquisition Mass Spectrometry.

Human-induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) have several potential applications in regenerative medicine. A deep understanding of stem cell characteristics is critical for developing appropriate products for use in the clinic. This study aimed to develop approaches for characterizing iPSC-derived NSCs. Data-independent acquisition mass spectrometry (DIA-MS) was used to obtain temporal proteomic profiles of differentiating cells. Principal component analysis of the proteome profiles allowed for the discrimination of cells cultured for different periods. Cells were characterized by Gene Ontology analysis to annotate the upregulated proteins based on their functions. We found that trophoblast glycoprotein (TPBG), a membrane glycoprotein that inhibits the Wnt/ß-catenin pathway, was elevated in NSC and that silencing TPBG promoted proliferation rather than neuronal differentiation. Treatment with Wnt/ß-catenin pathway activators and inhibitors showed that modulating the Wnt/ß-catenin pathway is crucial for differentiation into NSC. These results suggest that the level of TPBG is critical for differentiation into NSC, and TPBG is a potentially critical quality attribute of differentiating cells. In summary, DIA-MS-based proteomics is a promising multi-attribute method for characterizing stem cell-derived products.

Structural determination of the cell wall polysaccharide LCPS-1 in Lacticaseibacillus paracasei strain Shirota YIT 9029.

The neutral polysaccharides LCPS-1 and LCPS-2 play functional roles in the cell wall of the lactic acid bacterium Lacticaseibacillus paracasei strain Shirota YIT 9029 (LcS; formerly Lactobacillus casei strain Shirota YIT 9029), which has long been used as a probiotic food product. Studies have shown that LCPS-1 is associated with the immunomodulatory functions of LcS. We hypothesized that the structure of LCPS-1 is crucial for elucidating the mechanism of action of LcS on host immune responses and aimed to solve the undetermined primary structure of LCPS-1. Our results showed that LCPS-1 has a molecular weight of >400 kDa and is composed of Glc, Rha, Gal, and GlcNAc, with a repeating structure. Using limited degradation reactions, including controlled Smith and deamination degradations, we obtained key fragments with low molecular weight. Subsequently, their structures were analyzed using NMR spectra and other analytical techniques. Further, we integrated the results for each key fragment to derive the complete structure of LCPS-1. Our results indicated that the most probable structure of LCPS-1 is composed of two types of units (X, Y), each with a basic structure of seven sugars in which the C2-position of Rha is substituted with an acetyl group. The structure of X is {6[Glcß1-2] Galα1-3[2-OAc] Rhaß1-4Glcß1-4[Rhaα1-3] [Glcα1-6] Glcß1-} and that of Y is {6[Glcß1-2] Galα1-3[2-OAc] Rhaß1-4Glcß1-4[Rhaα1-3] [Glcα1-6)] GlcNAcß1-}, which can be expressed as (X6Y12)n. In this study, we identified the primary structure of LCPS-1, and our results may enable an improved understanding of the immunomodulatory abilities of LcS.

Glycoproteomic analysis of the changes in protein N-glycosylation during neuronal differentiation in human-induced pluripotent stem cells and derived neuronal cells.

N-glycosylation of glycoproteins, a major post-translational modification, plays a crucial role in various biological phenomena. In central nervous systems, N-glycosylation is thought to be associated with differentiation and regeneration; however, the state and role of N-glycosylation in neuronal differentiation remain unclear. Here, we conducted sequential LC/MS/MS analyses of tryptic digest, enriched glycopeptides, and deglycosylated peptides of proteins derived from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells, which were used as a model of neuronal differentiation. We demonstrate that the production profiles of many glycoproteins and their glycoforms were altered during neuronal differentiation. Particularly, the levels of glycoproteins modified with an N-glycan, consisting of five N-acetylhexosamines, three hexoses, and a fucose (HN5H3F), increased in dopaminergic neuron-rich cells (DAs). The N-glycan was deduced to be a fucosylated and bisected biantennary glycan based on product ion spectra. Interestingly, the HN5H3F-modified proteins were predicted to be functionally involved in neural cell adhesion, axon guidance, and the semaphorin-plexin signaling pathway, and protein modifications were site-selective and DA-selective regardless of protein production levels. Our integrated method for glycoproteome analysis and resultant profiles of glycoproteins and their glycoforms provide valuable information for further understanding the role of N-glycosylation in neuronal differentiation and neural regeneration.

Reproducibility and reliability of performance indicators to evaluate the therapeutic effectiveness of biofeedback therapy after elbow surgery: An observational case series.

Electromyographic biofeedback (EMG-BF) therapy provides information on the state of contraction of the targeted muscles and relaxation of their antagonists, which can facilitate early active range of motion (RoM) after elbow surgery. Our aim in this study was to calculate the minimum detectable change (MDC) during EMG-BF therapy, initiated in the early postoperative period after elbow surgery.This study is an observational case series. EMG-BF of muscle contraction and relaxation was provided during active elbow flexion and extension exercises. Patients completed 3 sets of 10 trials each of flexion and extension over 4 weeks. The total range of flexion-extension motion and scores on the Japanese Society for Surgery of the Hand version of the disability of the arm, shoulder, and hand questionnaire and the Japanese version of the Patient-Rated Elbow Evaluation were obtained at baseline and weekly during the 4-week intervention period. A prediction formula was developed from the time-series data obtained during the intervention period, using the least-squares method. The estimated value was calculated by removing the slope from the prediction formula and adding the initial scores to residuals between the measured scores and predicted scores individually. Systematic error, MDC at the 95th percentile cutoff (MDC95), repeatability of the measures, and the change from the baseline to each time-point of intervention were assessed.The MDC95 was obtained for all 3 outcome measures and the range of values was as follows: RoM, 8.3° to 22.5°; Japanese version of the Patient-Rated Elbow Evaluation score, 17.6 to 30.6 points; and disability of the arm, shoulder, and hand questionnaire subscale: disability and symptoms score, 14.2 to 22.9 points.The efficacy of EMG-BF after elbow surgery was reflected in earlier initiation of elbow RoM after surgery and improvement in patient-reported upper limb function scores. The calculated MDC95 cut-offs could be used as reference values to assess the therapeutic effects of EMG-BF in individuals.

Vascularized Iliac Bone Lining in Downgraded Treatment of Campanacci Grade III Giant Cell Tumor of the Distal Radius.

It is commonly accepted that wide en bloc resection followed by reconstruction is essential in progressive lesions (Campanacci grade III) for local control of possible recurrence. However, specific grade III can be downgraded and treated with intralesional curettage to preserve better wrist function, without increasing the recurrency rates. In this report, Grade III giant cell tumor of the distal radius was successfully treated using vascularized osseous graft from the inner lip of the iliac bone in addition to downgrading strategy.

Diverse galactooligosaccharides consumption by bifidobacteria: implications of ß-galactosidase--LacS operon.

Galactooligosaccharides (GOS) possess prebiotic properties that specifically increase the number of bifidobacteria in the human intestine, thus giving health benefits to the host. Although the bifidogenic effect of GOS has been demonstrated in numerous studies, the utilization of GOS by specific bifidobacteria remains unclear. The goal of our study was to elucidate GOS consumption by specific bifidobacteria and gain insights into the mechanism. First, we examined GOS consumption by 14 bifidobacterial strains belonging to seven different species by comparing growth rate, carbohydrate consumption, and acid production. We then performed a transcription analysis in the case of one strong GOS consumer, Bifidobacterium adolescentis YIT 4011(T), to predict the operon contributing to GOS use. The study indicated the contribution of an operon consisted of LacS symporter and ß-galactosidase to bifidobacterial GOS consumption.

Development of hypoallergenic galacto-oligosaccharides on the basis of allergen analysis.

Galacto-oligosaccharides (GOSs) are recognized as prebiotics beneficial to human health through their abilities to modulate gut microbiota. On the other hand, it has been reported that immediate allergic reactions are caused by a GOS product (Bc-GOS) produced by treating lactose with ß-galactosidase derived from Bacillus circulans. The objective of this study was to create a safer GOS product that is less likely to cause GOS-induced allergy (GOS-AL). First, we identified two derivatives of tetrasaccharide sugar chains in Bc-GOS as the factors responsible for GOS-AL by histamine release test (HRT) using blood samples obtained from two GOS-AL patients. Through our search for non-allergic GOS, we developed a new GOS product, SK-GOS, which was produced by catalyzing lactose with ß-galactosidase derived from Sporobolomyces singularis and Kluyveromyces lactis. We regard it as a hypoallergic and safe GOS product that does not cause GOS-AL.

Articular synovial chondromatosis of the finger.

A 40-year-old woman presented with a six-month history of synovial chondromatosis of the metacarpophalangeal joint of the right ring finger, which was resected through both dorsal and volar incisions. To our knowledge there have been only 17 reported cases of articular synovial chondromatosis of the digital joint so far. We present a case affecting the metacarpophalangeal joint with a review of scattered information found in other 17 reports.

Biosynthesis of the carbohydrate moieties of arabinogalactan proteins by membrane-bound ß-glucuronosyltransferases from radish primary roots.

A membrane fraction from etiolated 6-day-old primary radish roots (Raphanus sativus L. var hortensis) contained ß-glucuronosyltransferases (GlcATs) involved in the synthesis of the carbohydrate moieties of arabinogalactan proteins (AGPs). The GlcATs transferred [(14)C]GlcA from UDP-[(14)C]GlcA on to ß-(1 → 3)-galactan as an exogenous acceptor substrate, giving a specific activity of 50-150 pmol min(-1) (mg protein)(-1). The enzyme specimen also catalyzed the transfer of [(14)C]GlcA on to an enzymatically modified AGP from mature radish root. Analysis of the transfer products revealed that the transfer of [(14)C]GlcA occurred preferentially on to consecutive (1 → 3)-linked ß-Gal chains as well as single branched ß-(1 → 6)-Gal residues through ß-(1 → 6) linkages, producing branched acidic side chains. The enzymes also transferred [(14)C]GlcA residues on to several oligosaccharides, such as ß-(1 → 6)- and ß-(1 → 3)-galactotrioses. A trisaccharide, α-L-Araf-(1 → 3)-ß-Gal-(1 → 6)-Gal, was a good acceptor, yielding a branched tetrasaccharide, α-L-Araf-(1 → 3)[ß-GlcA-(1 → 6)]-ß-Gal-(1 → 6)-Gal. We report the first in vitro assay system for ß-GlcATs involved in the AG synthesis as a step toward full characterization and cloning.

Two-stage tendon sheath reconstruction using sublimis tendon and silicone Penrose drain after severe purulent flexor tenosynovitis: a case report.

Two-stage tendon sheath reconstruction, a method of pulley reconstruction using the sublimis tendon and a pseudosheath formed with a silicone Penrose drain wrapped around the profundus tendon, is a new technique for improving a poor functional prognosis after purulent flexor tenosynovitis.

Percutaneous flexible double pinning (Py-Desmanet's procedure) for pediatric distal radius fractures.

PURPOSE: The purpose of this study is to report the results of percutaneous flexible double pinning for pediatric distal radius fractures. METHODS: Thirteen unstable fractures (three physeal and ten metaphyseal) of the distal radius in which the physeal plate could be still identified were treated with percutaneous flexible double pinning between 2008 and 2011. The average age of these cases was 9.8 years (range, 2-16 years). According as Py-Demanet's original technique, the fracture was fixed with two percutaneous transepiphyseal intramedullary wires. Kirschner wires or c-wires of 1.4-1.6 mm in diameter were used in each case depending on age. Operative and short follow-up outcomes were assessed. RESULTS: The average operative duration was 23 min (range, 5-45 min). Comorbid distal ulnar fractures were further stabilized by intramedullary pinning. Additional external splintage was administered in all cases for 6 weeks postoperatively. Wires were removed after an average of 7.2 weeks (range, 4-10 weeks). Bone union was achieved in all cases. Neither malunion nor early epiphyseal closure of the distal radius was identified at mean follow-up of 12 months (range, 3-51 months). DISCUSSION: Flexible double pinning has been successfully used for distal radius fractures in adults. Since this technique is minimally invasive, quick, and technically easy, it is also a good treatment option for unstable distal radius fractures in children. LEVEL OF CLINICAL EVIDENCE: Therapeutic Level IV.

Regional first web flap used to cover the amputated stump of the thumb.

The regional first web flap is a distally-based flap that is raised from the radiodorsal aspect of the metacarpophalangeal joint of the index finger towards the dorsal first web, and used to cover a thumb amputation. The advantage of this flap over the conventional cross-finger flap is that is gives a thicker and hairless flap with no skin graft to the donor site. Mobilisation of the joints, including the metacarpophalangeal joint of the index finger, can also be maintained during the immobilisation period between two stages.

Vascularized genicular osseous-periosteal flap for pseudarthrosis of the proximal phalanx of the little finger: a case report.

This study describes pseudoarthrosis of the proximal phalanx of the little finger and its successful treatment using a free vascularized genicular osseous-periosteal flap. Since this thin and pliable flap can be harvested as a small vascularized unit, it is ideal for the treatment of phalangeal pseudoarthrosis.

Chemical characterization of oligosaccharides in the milk of six species of New and Old World monkeys.

Human and great ape milks contain a diverse array of milk oligosaccharides, but little is known about the milk oligosaccharides of other primates, and how they differ among taxa. Neutral and acidic oligosaccharides were isolated from the milk of three species of Old World or catarrhine monkeys (Cercopithecidae: rhesus macaque (Macaca mulatta), toque macaque (Macaca sinica) and Hamadryas baboon (Papio hamadryas)) and three of New World or platyrrhine monkeys (Cebidae: tufted capuchin (Cebus apella) and Bolivian squirrel monkey (Saimiri boliviensis); Atelidae: mantled howler (Alouatta palliata)). The milks of these species contained 6-8% total sugar, most of which was lactose: the estimated ratio of oligosaccharides to lactose in Old World monkeys (1:4 to 1:6) was greater than in New World monkeys (1:12 to 1:23). The chemical structures of the oligosaccharides were determined mainly by (1)H-NMR spectroscopy. Oligosaccharides containing the type II unit (Gal(ß1-4)GlcNAc) were found in the milk of the rhesus macaque, toque macaque, Hamadryas baboon and tufted capuchin, but oligosaccharides containing the type I unit (Gal(ß1-3)GlcNAc), which have been found in human and many great ape milks, were absent from the milk of all species studied. Oligosaccharides containing Lewis x (Gal(ß1-4)[Fuc(α1-3)]GlcNAc) and 3-fucosyl lactose (3-FL, Gal(ß1-4)[Fuc(α1-3)]Glc) were found in the milk of the three cercopithecid monkey species, while 2-fucosyl lactose (5'-FL, Fuc(α1-2)Gal(ß1-4)Glc) was absent from all species studied. All of these milks contained acidic oligosaccharides that had N-acetylneuraminic acid as part of their structures, but did not contain oligosaccharides that had N-glycolylneuraminic acid, in contrast to the milk or colostrum of great apes which contain both types of acidic oligosaccharides. Two GalNAc-containing oligosaccharides, lactose 3'-O-sulfate and lacto-N-novopentaose I (Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc) were found only in the milk of rhesus macaque, hamadryas baboon and tufted capuchin, respectively. Further research is needed to determine the extent to which the milk oligosaccharide patterns observed among these taxa represent wider phylogenetic trends among primates and how much variation occurs among individuals or species.

Side-chain structure of cell surface polysaccharide, mannan, affects hypocholesterolemic activity of yeast.

We previously reported that Kluyveromyces marxianus YIT 8292 exhibited more potent hypocholesterolemic activity than other yeasts containing Saccharomyces cerevisiae . To clarify the reason for the higher hypocholesterolemic activity, we examined the side-chain structure of cell surface polysaccharide, mannan, of K. marxianus YIT 8292. The result shows that K. marxianus YIT 8292 had shorter alpha-(1,2)-linked oligomannosyl side chains and lower phosphate content in mannan than S. cerevisiae. The association between its structural features and hypocholesterolemic activity was investigated by comparing the hypocholesterolemic activities of S. cerevisiae mannan mutants in rats fed a high-cholesterol diet. S. cerevisiae mnn5 mutant with deficiencies in the phosphorylation and elongation of mannan side chains showed higher hypocholesterolemic activity than the wild-type strain. These results show that the side-chain length and phosphate contents of mannan affect hypocholesterolemic activity.

A rare nodular fasciitis involving the finger: a case report.

This is a report of a 43-year-old man with nodular fasciitis of the little finger. Nodular fasciitis with its feature of rapid growth and aggressive histological appearance is often mistaken for a malignant neoplasm and rarely involves fingers.

Streptococcus thermophilus produces exopolysaccharides including hyaluronic acid.

Hyaluronic acid (HA) is an important material for medical, cosmetic and food applications. HA is obtained commercially from rooster combs and the fermentation of streptococci. However, the safety problems such as hyaluronidase or exotoxin contamination remain controversial. To reduce the risk of hyaluronidase or exotoxin contamination, we attempted to isolate strains of Streptococcus thermophilus with high productivity of useful exopolysaccharides (EPSs) including HA from traditional dairy food products. Forty-six S. thermophilus strains were isolated from dairy food products, and examined of their HA production using a HA binding protein method. According to the results, six S. thermophilus strains produced EPSs including HA. S. thermophilus YIT 2084 had a markedly high HA productivity (approximately 8 mg/l). We focused on the high-molecular-mass fraction of EPS (2000 kDa) from S. thermophilus YIT 2084. By using high-performance liquid chromatography, it was found that a high-molecular-mass fraction of EPS included N-acetylglucosamine (54.4%) and glucuronic acid (45.6%), which are components of HA. Furthermore, 13C-nuclear magnetic resonance spectroscopy showed that the spectra of the high-molecular-mass fraction corresponded well to those of commercial HA. Here, we described for the first time that S. thermophilus, which is a generally recognized safe bacterium, produces HA. The novel HA-producing bacterium S thermophilus YIT 2084 has great potential for applications in the medical, cosmetic and food fields, although its culture conditions remain to be improved.