Predicting mechanical properties of silk from its amino acid sequences via machine learning.

The silk fiber is increasingly being sought for its superior mechanical properties, biocompatibility, and eco-friendliness, making it promising as a base material for various applications. One of the characteristics of protein fibers, such as silk, is that their mechanical properties are significantly dependent on the amino acid sequence. Numerous studies have been conducted to determine the specific relationship between the amino acid sequence of silk and its mechanical properties. Still, the relationship between the amino acid sequence of silk and its mechanical properties is yet to be clarified. Other fields have adopted machine learning (ML) to establish a relationship between the inputs, such as the ratio of different input material compositions and the resulting mechanical properties. We have proposed a method to convert the amino acid sequence into numerical values for input and succeeded in predicting the mechanical properties of silk from its amino acid sequences. Our study sheds light on predicting mechanical properties of silk fiber from respective amino acid sequences.

Periodontal Pathogens Modulate Lipid Flux via Fatty Acid Binding Protein 4.

A strong correlation between chronic periodontitis and systemic diseases (e.g., cardiovascular disease, metabolic disorders) has been suggested for several decades. However, the evidence supporting this correlation is restricted primarily to epidemiologic studies, with only a few experimental outcomes confirming such a correlation and providing information about the underlying molecular mechanisms. To reveal a correlation between periodontitis and systemic diseases as well as a relevant molecular pathway, we investigated the effects of Porphyromonas gingivalis and Fusobacterium nucleatum, which play roles in chronic periodontitis progression, on Raw264.7 and THP-1 macrophages. Infection with P. gingivalis or F. nucleatum significantly induced the expression of fatty acid binding protein 4 (FABP4), one of the most important adipokines that play a role in the progression of systemic diseases such as atherosclerosis and type 2 diabetes. Periodontal pathogen-induced FABP4 expression in macrophages promoted lipid uptake by these cells, as demonstrated by the diminished lipid accumulation in cells treated with an FABP4 inhibitor, BMS309403, or with knockdown of FABP4 expression. This periodontal pathogen-induced FABP4 expression was dependent on the JNK pathway, and JNK inhibition reduced lipid uptake by reducing FABP4 expression. Serum levels of antibodies against P. gingivalis correlated with serum FABP4 levels in humans, whereas no association occurred between F. nucleatum antibody titers and FABP4 levels. To our knowledge, this report is the first to experimentally demonstrate that periodontal pathogens stimulate lipid uptake in macrophages by modulating FABP4 expression. These findings strongly support the hypothesis that periodontitis may affect the progression of various systemic diseases.

Emotional and behavioural problems in Singaporean children based on parent, teacher and child reports.

INTRODUCTION: This study aims to determine the prevalence of emotional and behavioural problems in a community sample of Singaporean children aged 6-12 years, and its agreement according to parent, teacher and child reports. METHODS: The Child Behaviour Checklist (CBCL), Teacher Rating Form (TRF) and child report questionnaires for depression and anxiety were administered to a community sample of primary school children. 60 percent of the children sampled (n = 2,139) agreed to participate. Parents of a sub-sample of 203 children underwent a structured clinical interview. RESULTS: Higher prevalence of emotional and behavioural problems was identified by CBCL (12.5 percent) than by TRF (2.5 percent). According to parent reports, higher rates of internalising problems (12.2 percent) compared to externalising problems (4.9 percent), were found. Parent-teacher agreement was higher for externalising problems than for internalising problems. Correlations between child-reported depression and anxiety, and parent and teacher reports were low to moderate, but were better for parent reports than for teacher reports. CONCLUSION: The prevalence rates of emotional and behavioural problems in Singaporean children based on CBCL ratings are comparable to those in the West, but the low response rate and exclusion of children with special needs limit the generalisability of our findings. Singaporean children have higher rates of internalising problems compared to externalising problems, while Western children have higher rates of externalising problems compared to internalising problems.

Floor of mouth haemorrhage and life-threatening airway obstruction during immediate implant placement in the anterior mandible.

A majority of the procedures performed in the dental office setting are considered safe and minimally invasive. Despite this fact, as healthcare providers it is our responsibility to be able to anticipate, recognize and manage life-threatening emergencies that may occur. In the following report, the authors will describe a life-threatening complication that resulted from the placement of mandibular implants.

In vitro calibration and validation of a digital subtraction radiography system using scanned images.

OBJECTIVES: To calibrate and validate a digital subtraction radiography system using scanned images for quantification of alveolar bone changes by means of computer-assisted densitometric image analysis (CADIA) in vitro. MATERIALS AND METHODS: Noise levels were determined using 10 standardized periapical radiographs of the same lower molar region in a human dry skull. For validation of the system, radiographs were taken before and after bovine bone particles in measures with increments of 2 mg weighing from 2 to 20 mg were added into each socket of three dry skulls. Radiographs were developed and scanned into a computer with a flatbed scanner. After digitization, the images were subjected to alignment, normalization and subtraction. Appropriate regions of interest (ROIs) were selected and their CADIA values were calculated for the determination of noise levels, and correlations between the CADIA values and the actual bone mass were performed. RESULTS: When the threshold value was 7, the percentage of pixels deviating from the set threshold value was small (0-11.3%). There were statistically significant correlations between the actual bone mass and the CADIA value for anterior sockets (p<0.001, r2=0.89) and posterior sockets (p<0.001, r2=0.9). For pooled data of both anterior and posterior sockets, the correlation was also statistically significant (p<0.001, r2=0.88). CONCLUSIONS: A high and statistically significant correlation between the actual bone mass and CADIA value was obtained, which suggests that the system could be suitable for the detection of small alveolar bone changes.

Birth prevalence of Down syndrome in Singapore from 1993 to 1998.

OBJECTIVE: To examine the livebirth prevalence rate of Down Syndrome in Singapore from 1993 to 1998. DESIGN: Index cases for the National Birth Defects Register were obtained from all neonatal nurseries in Singapore, all hospital discharge summaries, cytogenetic and pathology reports from all pathology laboratories in Singapore and from the compulsory reporting of all termination of pregnancy cases and stillbirths delivered. SETTING: Information for the Register was obtained from case notes retrieved from the medical record offices, antenatal clinics, cytogenetic laboratories, pathology departments and the Registry of Births and Deaths. SUBJECTS: All foetuses with Trisomy 21 diagnosed prenatally together with livebirths and stillbirths with Down Syndrome diagnosed at or after birth were identified from the Registry database. MAIN OUTCOME MEASURES: Prevalence of Down Syndrome RESULTS: From 1993 to 1998, there were 295 Down Syndrome livebirths, four stillbirths and 197 Down Syndrome foetuses aborted. There has been an increasing number of Down Syndrome foetuses diagnosed antenatally ending in termination and this is accompanied by a falling trend in the Down Syndrome livebirth rate in the same years from 1.17 to 0.89 per 1000 total live births. This is despite an expected increase in Down Syndrome livebirth rate obtained by modelling maternal Down Syndrome age-related risks on the maternal age distribution over the years. CONCLUSIONS: The livebirth prevalence of Down Syndrome in Singapore has fallen over the years from 1.17/1000 livebirths in 1993 to 0.89/1000 livebirths in 1998 due to antenatal diagnosis and selective termination.

Is air pollution a risk factor for low birth weight in Seoul?

Environmental factors contributing to reduced birth weight are of great concern because of the well-known relation of birth weight to infant mortality and adverse effects in later life. We examined the associations between air pollution exposures during pregnancy and low birth weight among all full-term births (gestational age 37-44 weeks) for a 2-year period (January 1996 through December 1997) in Seoul, South Korea. We evaluated these associations with a generalized additive logistic regression adjusting for gestational age, maternal age, parental educational level, parity, and infant sex. We used smoothing plots with generalized additive models to analyze the exposure-response relation for each air pollutant. The adjusted relative risk of low birth weight was 1.08 [95% confidence interval (CI) = 1.04-1.12] for each interquartile increase for carbon monoxide concentrations during the first trimester of pregnancy. The relative risks were 1.07 (95% CI = 1.03-1.11) for nitrogen dioxide, 1.06 (95% CI = 1.02-1.10) for sulfur dioxide, and 1.04 (95% CI = 1.00-1.08) for total suspended particles also for interquartile increase in exposure. Carbon monoxide, nitrogen dioxide, sulfur dioxide, and total suspended particle concentrations in the first trimester of pregnancy period are risk factors for low birth weight.

A comparison of male and female theft offenders remanded to a state psychiatric hospital.

We report a retrospective study examining the prevalence, demographic profile and pattern of psychiatric morbidity of theft offenders remanded for psychiatric assessment by the Singapore courts. Case records of 100 male and 100 female consecutively remanded theft offenders were reviewed. There was a high rate of psychopathology, with schizophrenia as the commonest disorder. Sex differences were identified in demographic profiles, psychiatric diagnoses, types of offences committed and likely psychiatric determinants of the offence. Depression was more common in females while substance abuse and antisocial personality disorder were diagnosed more frequently in males.

Preparation, characterization and in vivo evaluation of 120-day poly(D,L-lactide) leuprolide microspheres.

A 120-day poly(D,L-lactide) (PLA) microsphere delivery system for a luteinizing hormone-releasing hormone (LHRH) analogue, leuprolide, was prepared and evaluated. Leuprolide microspheres were prepared with PLA (m.w. 11000 Da) by a dispersion/solvent extraction-evaporation method and characterized for drug load by HPLC, particle size by laser diffractometry and surface morphology by scanning electron microscopy. In vitro peptide release and polymer degradation were studied using a modified dialysis method. Serum peptide and testosterone levels were analyzed after subcutaneous administration using a rat model. Spherical microspheres with a mean diameter of 52 microm containing 13.4% peptide released 10% of the peptide within 24 h, followed by a linear release for 150 days. Serum leuprolide levels increased immediately after administration of the microspheres to 45.6 ng/ml, but then fell to 4.3 ng/ml at 15 days and approximately 2.0 ng/ml at 30 days where they remained for 120 days. The testosterone levels increased initially to 15 ng/ml and then decreased to below 0.5 ng/ml by day 4 where they remained for 120 days. In conclusion, a 120-day microsphere formulation of leuprolide was developed with excellent controlled peptide release characteristics and in vivo efficacy.

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 mRNA expression in ectopic and eutopic endometrium in women with endometriosis: a rationale for endometriotic invasiveness.

OBJECTIVE: To investigate mRNA expression of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in ectopic endometriosis tissue and uterine endometrium from women with and without endometriosis throughout the menstrual cycle. DESIGN: Molecular studies in human tissue. SETTING: Department of Gynecology and Obstetrics, Reproductive Immunology Laboratory, Stanford University Medical Center. PATIENT(S): Fifty-three premenopausal woman (23 women with endometriosis and 30 women without endometriosis undergoing laparoscopic surgery). Endometrium and ectopic endometriosis tissue were obtained at the time of surgery. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): mRNA expression from eutopic and ectopic endometrium was analyzed by quantitative, competitive PCR. RESULT(S): Both uterine endometrium and ectopic endometriotic tissue from women with endometriosis expressed significantly (P<.05) lower levels of TIMP-3 than endometrium from normal women. Also, ectopic endometrium expressed higher levels of MMP-9 and a higher ratio of MMP-9/TIMP-3 than eutopic endometrium from normal and endometriosis patients. CONCLUSION(S): These results suggest that ectopic and eutopic endometrium from endometriosis patients may be more invasive and prone to peritoneal implantation because of greater MMP and less TIMP-3 mRNA expression than endometrium from women without endometriosis. Thus, increased proteolytic activity may be one of the reasons for the invasive properties of the endometrium, resulting in the development of endometriosis.

Sepharose-unbinding ricin E as a source for ricin A chain immunotoxin.

To evaluate the Sepharose-unbinding ricin E as a preference source material for ricin A chain (RTA) in immunotoxin studies, RTA of ricin E (RTA(E)) was characterized and compared with RTA of the Sepharose-binding ricin D (RTA(D)). RTA(E) and RTA(D) were separated into two subunits of A(1) and A(2) by capillary electrophoresis. The isoelectric points of A(1) and A(2) subunits were determined to be 7.6 and 7.4, respectively, for RTA(E), while they were 7.4 and 7.3, respectively, for RTA(D). The molecular masses of A(1) and A(2) isomers determined by the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry were 31059 and 32266 Da, respectively, for RTA(E), while they were 30892 and 32179 Da, respectively, for RTA(D). There were no significant differences in the cell surface affinity and cytotoxicity between RTA(E) and RTA(D). Anti-CD4-RTA(E) immunotoxin was prepared by conjugating RTA(E) with anti-CD4 monoclonal antibody using a heterobifunctional crosslinker, 4-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio) toluene. Anti-CD4-RTA(E) immunotoxin showed comparable cytotoxic effects to anti-CD4-RTA(D) immunotoxin to antigen-positive CEM cells in vitro. It is concluded that RTA(E) from ricin E is one of different variants of RTA(D) and may be used as a preference source material of RTA in immunotoxin studies.

Enhancement of bone growth by sustained delivery of recombinant human bone morphogenetic protein-2 in a polymeric matrix.

PURPOSE: The purpose of this study was to develop a polymeric sustained delivery system for recombinant human bone morphogenetic protein-2 (BMP-2) and to evaluate local bone growth induced by the sustained release of BMP-2 in an animal model. METHODS: BMP-2 was incorporated in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres to obtain different release rates. Two sustained and an immediate release implants were produced by suspending the BMP-2 loaded PLGA microspheres in aqueous sodium carboxymethylcellulose (CMC), lyophilizing, and cutting the dried materials to the size of the animal bone defects. The local in vivo release at the implantation site in rat calvarial defects was determined by gamma scintigraphy using radiolabeled BMP-2. The local bone induction in the critical size of rabbit calvarial defects was evaluated six weeks post implantation. RESULTS: The immediate release implant showed about 65% initial drug release within 24 h and the remaining BMP-2 quickly exhausted from the implantation site within 7 days. The sustained release implants, showing 45-55% initial release followed by a prolonged release for 21 days, released a greater amount of BMP-2 at the implantation site and maintained higher serum BMP-2 for the longer period of time compared to the immediate release implant. Significant bone growth was observed in all BMP-2 treated defects while the defects without treatment or with BMP-2-free implant showed minimal bone healing. 75-79% of rabbit calvarial defect area was healed with newly induced bone matrix by the sustained release implants in 6 weeks as compared to 45% recovery from the immediate release implant. CONCLUSION: The sustained delivery of BMP-2 based on the biodegradable PLGA microsphere system resulted in faster and more complete bone healing in the animal model.

Disposable amperometric glucose sensor electrode with enzyme-immobilized nitrocellulose strip.

Electrochemical properties of screen-printed carbon paste electrodes (CPEs) with a glucose oxidase-immobilized and hexamineruthenium (III) chloride ([Ru(NH(3))(6)](3+)) containing nitrocellulose (NC) strip were examined. The NC strip (2x8 mm) placed on the CPEs printed on polyester (PE) film is tightly sealed using another PE film on the top with open edges on both sides. Samples containing macromolecules and particles (e.g. proteins and blood cells) are applied at one edge of the NC strip and reach the detection area, chromatographically separating small molecules (e.g. glucose, ascorbate, acetaminophen, and uric acid) of analytical interests. Since sample volumes and the amount of catalytic reagents (mediator and glucose oxidase) are precisely predefined by the dimension and pore size (8 mum) of the NC strip, the sensor-to-sensor reproducibility and accuracy of analysis are greatly improved. The use of [Ru(NH(3))(6)](3+) mediator, which exhibits characteristic substantially lowers the applied potential (0.0 V vs Ag/AgCl) for glucose determination and eliminates the interference from other oxidizable species, providing improved analytical results.

Preparation and characterization of a composite PLGA and poly(acryloyl hydroxyethyl starch) microsphere system for protein delivery.

PURPOSE: To prepare and characterize a novel composite microsphere system based on poly(D,L-lactide-co-glycolide) (PLGA) and poly(acryloyl hydroxyethyl starch) (acHES) hydrogel for controlled protein delivery. METHODS: Model proteins, bovine serum albumin, and horseradish peroxidase were encapsulated in the acHES hydrogel, and then the protein-containing acHES hydrogel particles were fabricated in the PLGA matrix by a solvent extraction or evaporation method. The protein-loaded PLGA-acHES composite microspheres were characterized for protein loading efficiency, particle size, and in vitro protein release. Protein stability was examined by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and monitoring the enzymatic activity. RESULTS: Scanning electron microscopy showed discrete PLGA microspheres containing many acHES particles. The composite microspheres were spherical and smooth in size range of 39-93 microm. The drug loading efficiency ranged from 51 to 101%. The composite microspheres showed more favorable in vitro release than conventional PLGA microspheres. The composite microspheres showed 20% less initial with a gradual sustained release compared to high burst (approximately 60%) followed by a very slow release with the conventional PLGA microspheres. The composite microspheres also stabilized encapsulated proteins from the loss of activity during the microsphere preparation and release. Proteins extracted from the composite microspheres showed good stability without protein degradation products and structural integrity changes in the size-exclusion chromatography and SDS-PAGE analyses. Horseradish peroxidase extracted from microspheres retained more than 81% enzymatic activity. CONCLUSION: The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.

Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes.

Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of approximately 25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.

Formulation and in vitro transfection efficiency of poly (D, L-lactide-co-glycolide) microspheres containing plasmid DNA for gene delivery.

The stability, in vitro release, and in vitro cell transfection efficiency of plasmid DNA (pDNA) poly (D,L-lactide-co-glycolide) (PLGA) microsphere formulations were investigated. PLGA microspheres containing free and polylysine (PLL)-complexed pDNA were prepared by a water-oil-water solvent extraction/evaporation technique. Encapsulation enhanced the retention of the supercoiled structure of pDNA as determined by gel electrophoresis. PLL complexation of pDNA prior to encapsulation increased both the stability of the supercoiled form and the encapsulation efficiency. Free pDNA was completely degraded after exposure to DNase, while encapsulation protected the pDNA from enzymatic degradation. Rapid initial in vitro release of pDNA was obtained from microspheres containing free pDNA, while the release from microspheres containing PLL-complexed pDNA was sustained for more than 42 days. Bioactivity of encapsulated pDNA determined by in vitro cell transfection using Chinese hamster ovary cells (CHO) showed that the bioactivity of encapsulated pDNA was retained in both formulations but to a greater extent with PLL-complexed pDNA microspheres. These results demonstrated that PLGA microspheres could be used to formulate a controlled-release delivery system for pDNA that can protect the pDNA from DNase degradation without loss of functional activity.