In-process monitoring of a tissue-engineered oral mucosa fabricated on a micropatterned collagen scaffold: use of optical coherence tomography for quality control.

Background: We previously reported a novel technique for fabricating dermo-epidermal junction (DEJ)-like micropatterned collagen scaffolds to manufacture an ex vivo produced oral mucosa equivalent (EVPOME) for clinical translation; however, more biomimetic micropatterns are required to promote oral keratinocyte-based tissue engineering/regenerative medicine. In addition, in-process monitoring for quality control of tissue-engineered products is key to successful clinical outcomes. However, evaluating three-dimensional tissue-engineered constructs such as EVPOME is challenging. This study aimed to update our technique to fabricate a more biomimetic DEJ structure of oral mucosa and to investigate the efficacy of optical coherence tomography (OCT) in combination with deep learning for non-invasive EVPOME monitoring. Methods: A picosecond laser-textured microstructure mimicking DEJ on stainless steel was used as a negative mould to fabricate the micropatterned collagen scaffold. During EVPOME manufacturing, OCT was applied twice to monitor the EVPOME and evaluate its epithelial thickness. Findings: Our moulding system resulted in successful micropattern replication on the curved collagen scaffold. OCT imaging visualised the epithelial layer and the underlying micropatterned scaffold in EVPOME, enabling to non-invasively detect specific defects not found before the histological examination. Additionally, a gradual increase in epithelial thickness was observed over time. Conclusion: These findings demonstrate the feasibility of using a stainless-steel negative mould to create a more biomimetic micropattern on collagen scaffolds and the potential of OCT imaging for quality control in oral keratinocyte-based tissue engineering/regenerative medicine.

Genetic analyses of GII.17 norovirus strains in diarrheal disease outbreaks from December 2014 to March 2015 in Japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region.

A novel GII.P17-GII.17 variant norovirus emerged as a major cause of norovirus outbreaks from December 2014 to March 2015 in Japan. Named Hu/GII/JP/2014/GII.P17-GII.17, this variant has a newly identified GII.P17 type RNA-dependent RNA polymerase, while the capsid sequence displays amino acid substitutions around histo-blood group antigen (HBGA) binding sites. Several variants caused by mutations in the capsid region have previously been observed in the GII.4 genotype. Monitoring the GII.17 variant's geographical spread and evolution is important.

Amygdala volume in a population with special educational needs at high risk of schizophrenia.

BACKGROUND: The mildly learning disabled population has a three-fold elevated risk for schizophrenia. It has been proposed that in some individuals this cognitive limitation is a pre-psychotic manifestation of early onset schizophrenia. We examined clinical and neuroanatomical measures of a putative extended phenotype of schizophrenia in an adolescent population receiving special educational assistance. We predicted that people with intellectual impairment and schizotypal features would exhibit amygdala volume reduction as one of the neuroanatomical abnormalities associated with schizophrenia. METHOD: Assessment by clinical interview, neuropsychological assessment and magnetic resonance imaging scanning was carried out in 28 intellectually impaired individuals identified as being at elevated risk of schizophrenia due to the presence of schizotypal traits, 39 intellectually impaired controls and 29 non-intellectually impaired controls. Amygdala volume was compared in these three groups and the relationship between symptomatology and amygdala volume investigated. RESULTS: Right amygdala volume was significantly increased in the elevated risk group compared with the intellectually impaired controls (p=0.05). A significant negative correlation was seen between left amygdala volume and severity of negative symptoms within this group (p<0.05), but not in either control group. CONCLUSIONS: Intellectually impaired subjects judged to be at elevated risk of schizophrenia on the basis of clinical assessment exhibit structural imaging findings which distinguish them from the generality of learning disabled subjects. Within this population reduced amygdala volume may be associated with negative-type symptoms and be part of an extended phenotype that reflects particularly elevated risk and/or early manifestations of the development of psychosis.

Bacterial community dynamics during reduction of odorous compounds in aerated pig manure slurry.

AIMS: To study the microbial community responsible for the reduction of the polluting load during aerobic digestion of pig slurry. METHODS AND RESULTS: We analysed bacterial succession by nonculture-based methods and determined the physicochemical parameters and polluting substances during 6 days of aerobic digestion. The bacterial subpopulations evolved by aeration, predominantly Bacillus spp., degraded organic matter and vigorously consumed oxygen, as indicated by low oxidation-reduction potential (ORP). In this phase, the volatile fatty acid (VFA) levels drastically decreased, and VFAs were almost depleted on day 4. Simultaneously, the ammonia concentration decreased to its lowest level on day 4; thereafter, it increased until the end of the process. After the decrease in the total organic carbon content in the supernatant of the decomposed slurry, the ORP increased (approximately 0 mV), and the microbial community showed an abundance of lineages belonging to the phylum Proteobacteria. CONCLUSIONS: Bacillus was the predominant member of the bacterial community driving the VFA-removal process. Their predominance was related to the presence of available carbon, including VFAs and changes in ORP. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the relationships among the involved microbes, polluting materials and physicochemical parameters will aid process design and retrofitting of the process.

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

Pilocarpine-induced salivation and thirst in conscious rats.

The muscarinic receptor agonist pilocarpine is widely used as a sialogogue. It has been well-established that it also induces water intake in animals. However, the mechanisms underlying the relationships between these events are unknown. To address this problem, we examined water intake and parotid salivary secretion in conscious rats. Intraperitoneally injected pilocarpine increased both water intake and salivary secretion. Intracerebroventricularly injected pilocarpine also induced water intake, but not salivary secretion. Intracerebroventricularly applied atropine, a muscarinic receptor antagonist, suppressed the water intake produced by pilocarpine applied intraperitoneally and intracerebroventricularly. However, it did not affect the salivary secretion induced by pilocarpine applied peripherally. We conclude that peripherally applied pilocarpine affects the parotid glands and the thirst center in the central nervous system, while it may induce salivary secretion mainly via peripheral responses, but water intake mainly via the central nervous system.

Personality change after stroke: some preliminary observations.

OBJECTIVES: To describe changes in personality after stroke and effects on carers. METHODS: A consecutive series of patients was recruited from hospital admissions with stroke. A novel questionnaire was administered to the patients' main carer at nine months after the stroke to determine their perception of the patients' pre-stroke and post-stroke personality. Personality change was identified by changes in these ratings, and associations between personality change and the following variables explored: emotional disorder in patients and carers (measured using the hospital anxiety and depression scale and a structured psychiatric interview), stroke classification (Oxford community stroke classification), residual disability (Barthel index and Nottingham extended activities of daily living scale), and lesion characteristics on computed tomography (CT). RESULTS: Carers of 35 patients with stroke took part. Reported changes in personality after stroke included: reduced patience and increased frustration (both p<0.0001, t test of difference), reduced confidence, more dissatisfaction, and a less easy going nature (all p<0.005). Occasionally, aspects of personality change were seen as positive by carers. There were relations between greater personality change and interviewer rated patient depression or anxiety (p<0.001) but not when this was self rated; and between personality change and both emotional disorder in carers (p<0.005) and greater disability (p<0.01) but not CT lesion characteristics. CONCLUSIONS: Carers commonly perceive personality change in stroke patients. This is associated with self rated emotional distress in the carer. More research is needed to understand what carers mean by "personality change" and what factors contribute to the perceived change.

Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

[Mitral valve stenosis associated with a floating ball thrombus in the left atrium; report of a case].

A floating ball thrombus in the left atrium is relatively a rare case. A 70 year-old female patient who showed a symptom of heart failure was admitted to our hospital. By echocardiography, the mass in the dilated left atrium was appeared to be a floating ball thrombus, and the patient was diagnosed to be mitral valve stenosis. Following the treatment of heart failure, the patient underwent mitral valve replacement and the ball thrombus was successfully removed. Because of the high risk of sudden death with strangulated ball thrombus and systemic embolization, surgical removal of the ball thrombus should be done immediately after the diagnosis was established.

[Health status and lifestyle issues of homeless people in Sapporo city, 2000].

OBJECTIVE: To clarify the health status and lifestyle issues of homeless people in Sapporo city, voluntary "health consultations and medical examinations" were carried out near an emergency kitchen. METHODS: The voluntary activities were held in a park near the shelter tents of homeless people seven times from December 1999 to December 2000. The homeless people who consulted us, medical doctors, were asked detailed questions about past history, present illness, subjective symptoms, lifestyles and so on, and were examined for their blood pressure and urinary parameter. RESULTS: A total of 60 homeless people, including 59 men and 1 woman, were consulted and examined, Fifty-seven percent of them were 50 years old of older, and 30% had been homeless for less than half a year. Forty percent had some dental problems, 28% suffered neck stiffness, and 27% back pain. The medical examination found 53% of them to be hypertensive and 26% to be diabetic. Twenty-five percent had meals only once a day, 55% had meat or fish in their diet not more than twice a week and 57% had vegetables in their diet not more than twice a week. Forty-two percent slept not more than 5 hours a day, 13% often drank alcohol in the daytime, and 83% were smokers. CONCLUSION: The present results suggest that lifestyle-related chronic diseases are more significant problems among homeless people in Sapporo city than common infectious diseases such as tuberculosis or dysentery, probably because it is colder and therefore there are fewer homeless people in Sapporo city than in other major cities in Japan such as Tokyo and Osaka. Further studies of the homeless people living in such a cold environment are warranted to develop better health policies for them in the context of their social and economical determinants. In addition, it is important to establish a more reliable registration system for these people in order to plan and provide a comprehensive social and health support network as needed.

Enhancement of the thermophilic stage in cattle waste composting by addition of tofu residue.

The microbial degradation and temperature rise during the composting of a cattle waste and rice straw mixture blended with tofu (soybean curd) residue was investigated using an insulated and unheated in-vessel composter (effective volume, 12 1) and a static pile with passive aeration. The addition of 11% (dry weight basis) of tofu residue shortened the time required for temperature to reach the thermophilic phase and increased the duration of the temperatures above 55 degrees C significantly, but the maximum temperature was not affected by the additive level. As shown by the change in BOD, most of the easily biodegradable matter in the tofu residue was consumed during 12 days of composting. The same results were observed in the temperature profile of the static pile with passive aeration. Tofu residue addition yielded a higher maximum temperature and a nearly two times longer duration of temperatures above 55 degrees C in almost all locations of the pile. The use of tofu residue as a co-composting material would promote thermophilic degradation throughout the entire composting mass.

Crystal structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin at 2.0 A resolution.

1-Deoxynojirimycin, a pseudo-monosaccharide, is a strong inhibitor of glucoamylase but a relatively weak inhibitor of cyclodextrin glucanotransferase (CGTase). To elucidate this difference, the crystal structure of the CGTase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin was determined at 2.0 A resolution with the crystallographic R value of 0.154 (R(free) = 0.214). The asymmetric unit of the crystal contains two CGTase molecules and each molecule binds two 1-deoxynojirimycins. One 1-deoxynojirimycin molecule is bound to the active center by hydrogen bonds with catalytic residues and water molecules, but its binding mode differs from that expected in the substrate binding. Another 1-deoxynojirimycin found at the maltose-binding site 1 is bound to Asn-667 with a hydrogen bond and by stacking interaction with the indole moiety of Trp-662 of molecule 1 or Trp-616 of molecule 2. Comparison of this structure with that of the acarbose-CGTase complex suggested that the lack of stacking interaction with the aromatic side chain of Tyr-100 is responsible for the weak inhibition by 1-deoxynojirimycin of the enzymatic action of CGTase.

X-ray telescope onboard Astro-E: optical design and fabrication of thin foil mirrors.

X-ray telescopes (XRT's) of nested thin foil mirrors are developed for Astro-E, the fifth Japanese x-ray astronomy satellite. Although the launch was not successful, the design concept, fabrication, and alignment procedure are summarized. The main purpose of the Astro-E XRT is to collect hard x rays up to 10 keV with high efficiency and to provide medium spatial resolution in limited weight and volume. Compared with the previous mission, Advanced Satellite for Cosmology and Astrophysics (ASCA), a slightly longer focal length of 4.5-4.75 m and a larger diameter of 40 cm yields an effective area of 1750 cm2 at 8 keV with five telescopes. The image quality is also improved to 2-arc min half-power diameter by introduction of a replication process. Platinum is used instead of gold for the reflectors of one of the five telescopes to enhance the high-energy response. The fabrication and alignment procedure is also summarized. Several methods for improvement are suggested for the reflight Astro-E II mission and for other future missions. Preflight calibration results will be described in a forthcoming second paper, and a detailed study of images will be presented in a third paper.

Blue-light-dependent osmoregulation in protoplasts of Phaseolus vulgaris Pulvini.

Blue light was found to induce shrinkage of the protoplasts isolated from first-leaf lamina pulvini of 18-day-old Phaseolus vulgaris. The response was transient following pulse stimulation, while it was sustainable during continuous stimulation. No apparent difference was found between flexor and extensor protoplasts. Protoplasts of the petiolar segment located close to the pulvinus showed no detectable response. In the plants used, the pulvinus was fully matured and the petiole was ceasing its elongation growth. When younger, 12-day-old, plants were used, however, the petiolar protoplasts did respond to blue light. The pulse-induced response was similar to that in pulvinar protoplasts, although the response to continuous stimulation was transient and differed from that in pulvinar protoplasts. No shrinkage was induced in pulvinar protoplasts when the far-red-light-absorbing form of phytochrome was absent for a period before blue-light stimulation, indicating that the blue-light responsiveness is strictly controlled by phytochrome. Inhibitors of anion channels and H(+)-ATPase abolished the shrinking response, supporting the view that protoplasts shrink by extruding ions. The response of pulvinar protoplasts is probably involved in the blue-light-induced, turgor-based movement of pulvini. The blue-light responding system in pulvini is suggested to have evolved from that functioning in other growing organs.

Differential expression and regulation of K(+) channels in the maize coleoptile: molecular and biophysical analysis of cells isolated from cortex and vasculature.

UNLABELLED: Recently, two K(+) channel genes, ZMK1 and ZMK2, were isolated from maize coleoptiles. They are expressed in the cortex and vasculature, respectively. Expression in Xenopus oocytes characterized ZMK1 as an inwardly rectifying K(+) channel activated by external acidification, while ZMK2 mediates voltage-independent and proton-inhibited K(+) currents. In search of the related gene products in planta, we applied the patch-clamp technique to protoplasts isolated from the cortex and vasculature of Zea mays coleoptiles and mesocotyls. In the cortex, a 6-8 pS K(+) channel gave rise to inwardly rectifying K(+) currents. Like ZMK1, this channel was activated by apoplastic acidification. In contrast, protoplasts from vascular tissue expressing the sucrose transporter ZmSUT1 were dominated by largely voltage-independent K(+) currents with a single-channel conductance of 22 pS. The pronounced sensitivity to the extracellular protons Ca(2+), Cs(+) and Ba(2+) is reminiscent of ZMK2 properties in oocytes. Thus, the dominant K(+) channels in cortex and vasculature most likely represent the gene products of ZMK1 and ZMK2. Our studies on the ZMK2-like channels represent the first in planta analysis of a K+ channel that shares properties with the AKT3 K(+) channel family. KEYWORDS: K(+) channel, voltage-independent, proton block, maize coleoptile.

Crystal structure of alkalophilic asparagine 233-replaced cyclodextrin glucanotransferase complexed with an inhibitor, acarbose, at 2.0 A resolution.

The product specificity of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 is improved to near-uniformity by mutation of histidine-233 to asparagine. Asparagine 233-replaced CGTase (H233N-CGTase) no longer produces alpha-cyclodextrin, while the wild-type CGTase from the same bacterium produces a mixture of predominantly alpha-, beta-, and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains. In order to better understand the protein engineering of H233N-CGTase, the crystal structure of the mutant enzyme complexed with a maltotetraose analog, acarbose, was determined at 2.0 A resolution with a final crystallographic R value of 0.163 for all data. Taking a close look at the active site cleft in which the acarbose molecule is bound, the most probable reason for the improved specificity of H233N-CGTase is the removal of interactions needed to form a compact ring like a-cyclodextrin.

Crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 determined at 1.9 A resolution.

The crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 was determined at 1.9 A resolution. While the wild-type CGTase from the same bacterium produces a mixture of mainly alpha-, beta- and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains, site-directed mutation of histidine-233 to asparagine changed the nature of the enzyme such that it no longer produced alpha-cyclodextrin. This is a promising step towards an industrial requirement, i.e. unification of the products from the enzyme. Two independent molecules were found in an asymmetric unit, related by pseudo two-fold symmetry. The backbone structure of the mutant enzyme was very similar to that of the wild-type CGTase except that the position of the side chain of residue 233 was such that it is not likely to participate in the catalytic function. The active site cleft was filled with several water molecules, forming a hydrogen bond network with various polar side chains of the enzyme, but not with asparagine-233. The differences in hydrogen bonds in the neighborhood of asparagine-233, maintaining the architecture of the active site cleft, seem to be responsible for the change in molecular recognition of both substrate and product of the mutant CGTase.

[Delayed emesis induced by the chemotherapeutic agent doxorubicin hydrochloride in dogs].

The occurrence of delayed emesis induced 24 h after the administration of a non-platina chemotherapeutic agent, doxorubicin hydrochloride (doxorubicin), as well as behaviors such as feeding, drinking and defecation were examined in dogs. A single intravenous administration of 2 mg/kg doxorubicin induced emesis within 24 h of administration in some dogs, while delayed emesis was observed 24 h after administration in all dogs. This delayed emesis emerged strongly at day 3 or 4 and decreased at day 5. Hypophagia, the decreased frequency of drinking and the increased frequency of defecation were induced shortly after delayed emesis. Twenty-four hours after the administration of doxorubicin, a daily dose of 0.3 and 1 mg/kg/day, p.o. azasetron, a 5-HT3 antagonist, was administered for 4 days. Doxorubicin-induced delayed emesis was observed to decrease by about 30 and 50%, respectively. This result suggests that 5-HT3 receptors play a role in the mechanism of delayed emesis. Azasetron was found to improve the increased frequency of defecation, but exerted no obvious effect on hypophagia or on the decreased frequency of drinking. Taken together, we suggest that doxorubicin-induced emesis in dogs is a useful method to study further the mechanisms of delayed emesis and to investigate novel therapeutic agents against delayed emesis.

A mutation in the 3-phosphoglycerate kinase gene allows anaerobic growth of Bacillus subtilis in the absence of ResE kinase.

The Bacillus subtilis ResD-ResE two-component signal transduction system is essential for aerobic and anaerobic respiration. A spontaneous suppressor mutant that expresses ResD-controlled genes and grows anaerobically in the absence of the ResE histidine kinase was isolated. In addition, aerobic expression of ResD-controlled genes in the suppressed strain was constitutive and occurred at a much higher level than that observed in the wild-type strain. The suppressing mutation, which mapped to pgk, the gene encoding 3-phosphoglycerate kinase, failed to suppress a resD mutation, suggesting that the suppressing mutation creates a pathway for phosphorylation of the response regulator, ResD, which is independent of the cognate sensor kinase, ResE. The pgk-1 mutant exhibited very low but measurable 3-phosphoglycerate kinase activity compared to the wild-type strain. The results suggest that accumulation of a glycolytic intermediate, probably 1, 3-diphosphoglycerate, is responsible for the observed effect of the pgk-1 mutation on anaerobiosis of resE mutant cells.