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Two solid state anaerobic digesters (SSADs), 15 L each, were set up for co-digestion of switchgrass with primary digestate of a liquid anaerobic digester (LAD) and the recirculating leachate. Both the LAD and two SSADs were operated at 50°C. The results showed that the bioreactors were not started up stably until day 16 and day 47 for reactors A and B, respectively. The supplement of LAD digestate or injection of sodium hydroxide (NaOH) into the recirculating leachate readily reinitiated the biogas production to normal daily high rates of the two individual SSADs, one on day 16 and the other on day 47. In contrast to reactor A, there was a longer lag phase for bioreactor B, however, it showed 46.2% [77.9 vs 53.3 L kg−1 volatile solid (VS)] more cumulative biogas yields, and higher reduction rate of total solid, VS, cellulose and hemicellulose of 29.5, 31, 40.6 and 15%, respectively, which was likely due to optimized pH and NaOH pretreated switchgrass during start-up period. Methane contents of biogas increased gradually and stabilized at 50% for both reactors, indicating a normal operation of anaerobic digestion lasted for at least 100 days. The determined parameters of digested residues met China organic fertilizer standard (NY 525-2012) except for high moisture and low total nutrient contents. Therefore, the product of SSAD has the potential value of organic fertilizer. It is concluded that the LAD digestate can be reused as inoculums by co-digestion with agricultural residues for biogas and organic fertilizer production in SSAD.

The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host’s cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2–6.4 ID50 g−1 was reduced to a remaining infectivity of 104.6–5.7 ID50 g−1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5–800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

Ankyloglossia is a failure of the tongue to release from the oral floor with reported consequences that include breastfeeding difficulties and speech impediments. Frenotomy is a commonly performed procedure for the treatment of ankyloglossia. Laser (light amplification by stimulated emission of radiation) is one of several mediums used to perform frenotomies. Although most frenotomies are uncomplicated, there remains a small possibility of complication, such as infection, pain, ductal injury, and hemorrhage, even in expert hands. Because frenotomies are most often performed in infants, postoperative hemorrhage is an important complication to look for as even small amount of bleed may prove fatal, due to low blood volume reserve. We report a case of delayed hemorrhage after laser frenotomy in a 6-week old infant displaying shock symptoms and required fluid resuscitation.

The growth of two-dimensional materials into three-dimensional geometries holds the promise for high performance hybrid materials and novel architectures. The synthesis of such structures, however, proceeds in fundamentally different flow regimes compared to conventional CVD where pressure differences and wall collisions are neglected. We here demonstrate the remarkable stability of graphene growth under varying fluid dynamic flow regimes. We investigate the growth process across different flow conditions using confined growth in refractory pores. Analysis of the growth rate reveals a transport-limited process which allows experimental determination of the gas diffusion coefficient. The diffusion coefficient was found to be constant for large pore dimension but scales with pore dimension as the pore size decreases below the mean free path providing clear evidence for previously predicted Knudsen molecular-flow conditions for atomic confinement. Surprisingly, changes to the flow conditions by two orders of magnitude do not cause qualitative changes of the graphene growth process. This unique behavior was attributed to rarefied flow conditions by scaling analysis and an analytical relation between growth rate and constriction could be extracted that proves accurate throughout the investigated conditions. Our results demonstrate a fundamentally different growth process compared to traditional CVD processes that is akin to atomic layer deposition and highlight the feasibility of high-quality 2D-material growth on 3D morphologies with ultra-high aspect ratios.

Current anti-epidermal growth factor receptor (EGFR) therapy for oral cancer does not provide satisfactory efficacy due to drug resistance or reduced EGFR level. As an alternative candidate target for therapy, here we identified an oncogene, ROS1, as an important driver for oral squamous cell carcinoma (OSCC) metastasis. Among tumors from 188 oral cancer patients, upregulated ROS1 expression strongly correlated with metastasis to lung and lymph nodes. Mechanistic studies uncover that the activated ROS1 results from highly expressed ROS1 gene instead of gene rearrangement, a phenomenon distinct from other cancers. Our data further reveal a novel mechanism that reduced histone methyltransferase EZH2 leads to a lower trimethylation of histone H3 lysine 27 suppressive modification, relaxes chromatin, and promotes the accessibility of the transcription factor STAT1 to the enhancer and the intron regions of ROS1 target genes, CXCL1 and GLI1, for upregulating their expressions. Down-regulation of ROS1 in highly invasive OSCC cells, nevertheless, reduces cell proliferation and inhibits metastasis to lung in the tail-vein injection and the oral cavity xenograft models. Our findings highlight ROS1 as a candidate biomarker and therapeutic target for OSCC. Finally, we demonstrate that co-targeting of ROS1 and EGFR could potentially offer an effective oral cancer therapy.

Agriculture in the 21st Century is facing new challenges and transformation according to five general trends: commercialisation, internationalisation, science and technology, environment and energy production. In this laboratory, a series of research, development and commercialisation of biogas and enzyme technologies reflect these trends. Anaerobic digestion is a microbial process that converts organic waste into biogas, containing 60-70% methane. A simple thermophilic anaerobic digestion (TAnD) system for poultry manure was developed from the lab to the farm. TAnD at 50 to 60°C produces biogas at high rates and, as a result, the process is efficient and the digester is a compact unit. A pilot plug-flow TAnD was first constructed and operated at NCSU research farm. Multiple benefits of the system were demonstrated. Supported by UNDP, a TAnD to process five tons of manure daily (from 50,000 hens) was constructed in China and has been operating for 20 years. Large scale digesters processing hundreds of tons of manure daily are increasingly popular in China and Europe. Biogas from large digesters can be a significant source of energy to generate electricity or to fuel transportation. Beyond energy production, the digester is a rich source of biomaterials. A feather-degrading bacterium was discovered and isolated from TAnD, and its keratinase enzyme and the gene encoding for the enzyme were subsequently isolated. Genetic manipulation for over-expression and up-scale fermentation have now made the industrial production of the enzyme possible. As a heat-stable protease, the keratinase was found to be useful in processing feather meal, in enhancing feed digestibility and was able to degrade prion proteins (implicated as the disease vector in bovine spongiform encephalopathy).

A sliding mode controller u( t) is designed to achieve the following three objectives simultaneously: eigenvalue placement, H∞ norm constraint, and individual state variance constraint for bi-linear stochastic systems. By using the invariance property of sliding mode control, the matched bi-linear term of the system disappears on the sliding mode. With the aid of the upper bound covariance control theory, the controller u( t) is derived in which the control feedback gain matrix G is synthesized for achieving the above multiple objectives.

SUMMARY Versazyme (VZ) is an approved keratinase-based feed additive produced naturally as a fermentation product of Bacillus licheniformis PWD-1. The experiment was designed to test the recovery and efficacy of VZ using typical feed industry pelleting parameters. The pelleted diets were conditioned to 85°C for 30 s and pelleted using a 4 × 32 mm die, cooled, and crumbled. A total of 192 male broiler chicks were fed a corn-soybean meal starter diet formulated at 18.5% CP to 21 d of age. The 4 treatments used in the feeding study were as follows: VZ 0.0%-mash, VZ 0.0%-pellet, VZ 0.1%-mash, and VZ 0.1%-pellet. Body weight and feed consumption were determined at 21 d of age. The recovery of VZ in feed was 93% in the mash diet and 66% in the pelleted diet. There was a 55-g increase in BW gain and 0.17 improvement of FCR when birds were supplemented with 0.1% VZ. Pelleting improved BW gain (P < 0.05) but did not significantly improve the FCR. Growth performance results indicate that pelleting did not affect the efficacy of VZ.

All-trans retinoic acid (ATRA) can induce acute respiratory distress syndrome in patients with acute promyelocytic leukaemia (APL). The current study investigated the role of monocyte chemotactic protein (MCP)-1 in the chemotactic transmigration of ATRA-treated NB4 (ATRA-NB4) APL cells toward A549 alveolar epithelial cells. NB4 and A549 cells were separately cultured with ATRA and/or dexamethasone (DEX). ATRA-NB4 cells were then placed in an upper insert and co-incubated with A549 cells or their conditioned medium (CM) located in a lower plate to test their transmigration activity. ATRA stimulated NB4 cells to transmigrate toward the A549 cells. The secretion of MCP-1 was enhanced by ATRA treatment in both A549 and NB4 cells. The binding assay demonstrated that ATRA-NB4 cells bound MCP-1. Pre-treatment of both CM-A549 cells with antibodies against MCP-1 and of ATRA-NB4 cells with antibodies against MCP-1 receptors reduced ATRA-NB4 cell transmigration. DEX did not suppress MCP-1 secretion and transmigration in ATRA-NB4 cells, although when applied to A549 cells, MCP-1 secretion was suppressed and ATRA-NB4 cell transmigration was attenuated. Monocyte chemotactic protein-1 secreted from alveolar epithelial cells plays an important role in the cell-cell interaction involved in the chemotactic transmigration of all-trans retinoic acid-treated acute promyelocytic leukaemia cells toward alveolar epithelial cells.

A preparation of keratinase (KE, 400,000 enzyme units/g) was evaluated as a feed additive for its effects on growth performance, nutrient digestibility and intestinal development in broiler chickens. Four experimental diets were arranged as a 2 × 2 factorial with two sources of protein, either soybean meal (SBM) alone or a combination of SBM and cottonseed meal (1:1 on crude protein basis) and with or without 1.0 g KE/kg diet. Dietary supplementation of KE increased body weight gain from 1 to 21 days of age and improved feed conversion ratio from 1 to 42 days of age. Also, KE supplementation enhanced nitrogen retention (P

This study evaluated the antioxidant ability of Taisung No. 3 mulberry leaf extract (MLE) as well as the potential of mulberry leaf (ML)-based dietary supplementation for modulating the antioxidative status of laying hens. The results showed that the MLE had a total phenolic compound content of 7.4 ± 0.15 mg of gallic acid equivalent/g dry weight (DW) and a total flavonoid content of 4.4 ± 0.19 mg of quercetin equivalent/g DW. The 2, 2-diphenyl-1-picrylhydrazyl free-radical-scavenging ability was 45.9% when 0.1 mg/mL MLE was added. The lipid oxidation inhibition ability was 43.9% when 50 mg/mL MLE was added. We subjected 96 laying hens (Hendrix Genetics) to 4 treatments, namely diets supplemented with dry ML at 0 (control), 0.5, 1, or 2% for 12 weeks. Each treatment involved 8 replicates with 3 hens each. The results indicated that the 0.5% ML-supplemented group exhibited significantly higher mRNA levels of antioxidant-regulated genes, such as Nrf2, HO-1, and GST, and significantly lower ROMO1 gene expression levels at wk 12. The serum malondialdehyde level was lower and the catalase activity and superoxide dismutase activity were higher in all the ML-supplemented groups than in the control group. The egg mass and feed conversion rate significantly improved in the ML-supplemented groups compared with the control group, and, overall, 1% ML supplementation had the most favorable effects at one to 12 weeks. The egg yolk weight, shell weight, shell strength, shell thickness, yolk color, and Haugh unit were increased among all ML-supplemented groups at one to 12 weeks. On the basis of these observations, we conclude that 0.5% ML can be used as a new feed additive to potentially modulate the antioxidative status of laying hens and improve their production performance and egg quality.

A prion-like protein, Sup35NM-His6, which is safe, easy to produce and able to aggregate into stable amyloid, was developed. This study was to test the feasibility of using Sup35NM-His6 amyloid as a marker for standard sterilization methods known to be effective in inactivating infectious prions. Also Sup35NM-His6 aggregates were spiked into cow brain tissue homogenates to mimic prions in tissues to determine if the aggregate protein could be specifically detected by Western blot at the nanogram level. Like mammalian prions, the proteinase K (PK) resistant fractions of Sup35NM-His6 remain intact after autoclaving. Treatments with 0.1N NaOH or 1.0% NaOCl also resulted in PK undigestible residues. Exposure to the strong denaturant guanidine thiocyanate (>3 M) destabilized and made the protein PK-digestible. Under strong alkaline conditions of 1.0N NaOH, 2.5% NaOCl, or a combination of NaOH (0.1 and 1.0N) with autoclaving, Sup35NM-His6 was completely hydrolyzed such that it was no longer detectable by Western blot. Overall, these tests suggest that Sup35NM-His6 could be a useful tool to assess the effectiveness of prion degradation for the prevention of TSE.

Versazyme (VZ) is a keratinase-based feed additive. Broiler diets were formulated with low (95%), medium (100%), or high (105%) CP and amino acid levels relative to US Poultry Industry Reporting Service averages (2003). In each diet, amino acid minimums were maintained relative to the percentage of CP. The protein levels were as follows: starter—21, 22, 23%; grower—19, 20, 21%; finisher—17, 18, 19%; and withdrawal—16, 17, 18%. Mixed sex broilers 0 to 48 d of age were fed the 3 protein levels with and without VZ (0.10%) in a 3 × 2 factorial experiment. Statistical main effects indicated 48-d BW was greater on the high protein diets (2.42 kg) compared with low protein (2.36 kg), with the medium protein diet being intermediate (2.40 kg). Overall, VZ significantly (P

Recent studies indicate that enzymatic treatment of the infectious PrP Sc prion under defined conditions could be an effective method to inactivate infectious prions. However, field studies on prion inactivation are hampered by restricted access to the dangerous and expensive infectious prion material. Hence, a surrogate marker for infectious prions would facilitate more practical prion inactivation research. Protein Sup35p, a non-pathogenic prion-like protein produced in yeast, has physical and chemical properties very similar to the BSE prion. Sup35NM-His6, a derivative of Sup35p, was produced from Escherichia coli by gene cloning, protein expression and purification. Monomeric Sup35NM-His6 is soluble. When aggregated, it forms prion-like amyloid, insoluble and resistant to proteases. Similar to BSE prion, a pre-heating step renders this protein digestible by proteinase K, subtilisin and keratinase but not collagenase and elastase. These results indicated that Sup35NM-His6, being simple and inexpensive to produce and non-pathogenic, can be a potential ideal candidate of prion surrogate protein in the study of prion inactivation and prevention of prion diseases.

Aims: Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A sporulation-deficient mutant of B. licheniformis PWD-1, named B. licheniformis WBG, was developed and characterized. Methods and Results: The mutation was generated using the splicing by overlap extension PCR method (Gene SOEing) to create 256 bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise the nucleotide fragment 256 bp from the B. licheniformis chromosome. PCR analysis and DNA sequencing confirmed the spoIIAC gene deletion. Heat-treatment assays and electron microscopy verified the absence of spores. Conclusions: This asporogenic strain is able to express normal levels of keratinase when compared with its wild-type host. Significance and Impact of the Study: In this study, a method of constructing a stable sporulation-defective strain was developed. It can be potentially useful as a tool to generate asporogenic strains of Bacillus that retain their industrial capabilities for production of exoproteases and other exozymes.

A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.

Extracellular production of keratinase–streptavidin fusion protein (KER–STP) was accomplished by the cloning of Bacillus subtilis with a transforming plasmid carrying the kerA - stp fusion gene. The fusion protein was readily immobilized onto a biotinylated solid matrix by mixing in the culture medium. The properties and reaction kinetics of free and immobilized keratinase (KE) were characterized and compared. Heat stability and pH tolerance were greatly improved by immobilization, but the catalytic efficiency ( k cat / K m ) was reduced by eightfold. The yield of bio-immobilization using bioselective adsorption of the fusion protein was approximately 20%, as estimated from the activity of free KE. HPLC analysis of reaction products demonstrated the hydrolysis of feather keratin, casein, and bovine serum albumin (BSA) by the immobilized KE. The rates of reactions are lower than those of the free enzyme. On the other hand, the stability of the enzyme was greatly improved.

The objective of this study was to determine the efficacy of a broad-spectrum protease enzyme, PWD-1 keratinase, upon supplementation to corn-soy starter diets on growth performance of broiler chickens. Three experiments were conducted. In each experiment, 1-d-old broiler chicks were randomly assigned to 24 cage pens of eight birds per pen in a completely randomized design of five experimental treatments and grown to 21 or 26 d of age. Treatments in experiments 1 and 2 were control (C, 21.39% CP), low protein (LP, 18% CP), and LP supplemented with 0.05, 0.1, or 0.15% enzyme preparation (wt/wt). Treatments in experiment 3 were control (C), C+ 0.1% enzyme preparation (C+E) fed starting at either 1 or 5 d of age, LP and LP+ 0.1% enzyme preparation (LP+E). Feeding the LP+E diet produced numerically higher BW at 21 d of age (experiments 1 and 3) and a significantly higher BW at 26 d of age (experiment 2; 1,025 and 1,032 g vs. 965 g for 0.1 and 0.15% vs. LP, respectively, P < 0.05). Feed conversion ratio (FCR) was also improved when chicks were fed the LP+E diet both at 21 (experiment 3) and 26 d of age (experiment 2). In experiment 3, supplementing the C diets with 0.10% enzyme resulted in improvements (P < 0.05) in BW whether the enzyme was supplemented starting at 1 d (767 vs. 695 g for C+E vs. C, respectively) or 5 d of age (764 vs. 695 g for C+E vs. C, respectively). FCR was numerically improved. Furthermore, diets supplemented with the enzyme at any level resulted in reduction of jejunal viscosity at 22 and 27 d of age (P < 0.05). Results of these experiments indicate that the growth of broiler chickens can be significantly improved by dietary supplementation with PWD-1 keratinase.

Immobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties. Conventional purification followed by chemical immobilization is a laborious and costly process. A new genetic construct was developed to produce the keratinase-streptavidin fusion protein. Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step. The method was tested in both the Bacillus subtilis and Escherichia coli systems. In B. subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium. In E. coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract. The overall efficiencies were approximately the same, 24-28%, using both systems.

Fermentation scale-up was studied for the production of keratinase by Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6-10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37 degrees C for both strains, though B. licheniformis is thermophilic and grows best at 50 degrees C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37 degrees C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50 degrees C for growth followed by a shift to 37 degrees C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29.

Since Chinese Taipei joined the World Trade Organization (WTO) membership in year 2001, the global economy and trading activities have become more competitive in Taiwan, while the food safety and supply chain management have also developed into important management issues in the world. Therefore, with the intention of developing its cattle industry, the Taiwanese government has invested considerably in Information and Communication Technology (ICT) to enhance farm management efficiency and support the internal growth of its Radio Frequency Identification (RFID) industry. Recently, RFID has been attracted widespread hope, hype and optimism, and hailed in the mainstream media as a revolutionary technology. This case study introduces a cattle management pilot project using Ultra-high frequency (UHF) RFID technology to assist farmer’s cattle management and identification, and enhance process and efficiency in the milking rooms. The deployment experiences and solutions can be utilized as a reference for further study and the cattle management automation industry.

The intensive and large-scale production of food animals and animal products has generated an enormous waste disposal problem for the animal industry. These wastes, which include animal excreta, mortalities, hair, feathers, and processing wastes, are largely organic materials and are convertible to useful resources. Making the conversion processes efficient and economical presents a great challenge to modern biotechnology. An efficient thermophilic anaerobic digester system has been developed that converts animal manure to methane for an energy source, solid residues for feed supplements, and liquid nutrients for aquaculture. This digester system also destroys pathogens and thus protects environmental health. During the development of this system, a feather-degrading bacterium was discovered and identified as a thermophilic Bacillus licheniformis, Strain PWD-1. The bacterium can ferment and convert feathers to feather-lysate, a digestible protein source for feed use. An enzyme, keratinase, secreted by this bacterium was purified and characterized. This keratinase is a potent protease that hydrolyzes all proteins tested, including collagen, elastin, and feather keratin. When the enzyme was mixed as an additive in feed, it significantly enhanced the digestibility of feather meal in chickens. In addition to feed technology, the bacterium and the enzyme are believed to have many other industrial and environmental applications.

The major cellular components of atherosclerotic lesions in several species have been shown to be smooth muscle cells (SMC) and macrophages. Many studies suggest the composition of a lesion varies depending on the stage of lesion development. For example, macrophages are believed to be involved in the initial events of fatty streak formation in many animals. This communication presents the first cellular study of quail atherosclerosis and demonstrates the alteration of cellular structure during the process of the disease in quail fed a cholesterol diet. Monoclonal antibodies to alpha-actin and chicken macrophages effectively identified the presence of SMC and macrophages, respectively, as constituents of the atherosclerotic lesions. Macrophage presence, as well as SMC proliferation, was observed in early lesions. Although the first cell type to be involved in the initial stages of atherogenesis cannot be defined, the results suggest early intervention of macrophages and SMC. Scanning electron microscopic examination of the aortic arch demonstrates the obvious differences in appearance of the endothelial surface of normal and diseased quail. The accumulation of subendothelial foam cells causes the lumen surface to bulge irregularly into the lumen. The results of the present study are important to the evaluation of the key cellular events of atherogenesis.

Feeding trials were designed to evaluate the nutritive value of feathers treated by a feather-degrading bacterium, Bacillus licheniformis (Strain PWD-1). An initial experiment compared feathers that had been treated under aerobic or anaerobic conditions with untreated feathers as 25% of the feed protein in the diet. The test diets were fed to growing broiler chicks from Day 6 through Day 21 posthatch. The anaerobically fermented product, feather-lysate, resulted in improved growth responses of 6.9 and 19.3% over the aerobic- and nonbacterial-treated feather feed products, respectively, but 6.4% less than a standard com-soybean-based diet In a subsequent experiment, 3 or 6% of feather-lysate, untreated feathers, and commercial feather meal were fed to chicks on a basal diet containing only 10% protein. The linear growth response slopes were compared with the slope resulting when chicks were fed diets containing soybean protein in graded levels from 10 to 19% of the total feed. Feather-lysate supplemented with lysine, methionine, and histidine produced a growth curve identical to that of soybean meal. Commercial feather meal supported an improved growth rate over the untreated feathers but significantly less than feather-lysate. These results indicate that the anaerobic fermentation of feathers offers a potential new process for feather waste treatment to provide a nutritious feed protein.

The microstructure of as-received and post-annealed SiC whiskers were investigated by transmission electron microscopy. In the as-received SiC whiskers, the thinner beta-SiC parts were jointed with the one-dimensional disordered parts by {111}, twin boundaries. After annealing, beta-SiC parts became coarser than the one-dimensional disordered parts at 1900-degrees-C and then disappeared at 2000-degrees-C.

Background: The Janus kinase-2 (JAK-2) V617F mutation has been recently reported in patients with myeloproliferative disorders (MPD), which is believed to underlie growth factor hypersensitivity displayed by haematopoietic progenitors in these disorders. However, its frequency has been rarely determined in Taiwanese patients. Methods: The frequency of JAK2-V617F mutation in patients with polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis (IMF) was determined in the DNA from the peripheral blood leucocytes of 108 patients by genomic polymerase chain reaction and restriction enzyme-based assay. Results: The JAK2-V617F mutation could be detected in 28 of 33 polycythaemia vera patients (85%), 29 of 49 essential thrombocythaemia patients (59%) and 2 of 6 IMF patients (33%), but was not detected in 11 patients with myelodysplastic syndrome or another 10 with other haematological diseases. The presence of the JAK2 mutation was associated with specific MPD disease subtypes (P = 0.007), longer disease duration (P = 0.005), splenomegaly (P = 0.019), a higher white blood cell count (P = 0.002) and a higher haemoglobin level (P = 0.036). However, the overall risk of thrombosis or bleeding was not affected by the presence of the JAK2 mutation (32 vs 17%; P = 0.22). Conclusion: The JAK2-V617F mutation can be frequently detected in the Taiwanese patients with MPD disorders and therefore should be incorporated into the initial evaluation of patients suspected of MPD.

The low temperature (77 K) and room temperature (296 K) hardening-softening behaviour is studied of the primary system B4 and secondary systems A3 and C1 of Al-single crystals with axis, which are previously deformed under cyclic loading for certain number of cycles. Yield stresses are measured, as a result, the latent hardening ratios (LHRs) of the secondary systems are calculated. It is likely shown that: when the primary system hardens at most, the LHR may be the lowest and perhaps lower than one; when primary system softens at most, the LHR may be highest and the higher than one.

Extracts of snake venom have been widely used for the treatment of vascular thrombotic diseases, yet the therapeutic mechanism is not clear. The effect of snake venom fractions on atherosclerosis in Japanese quail was studied. The venom of Agkistrodon halys was fractionated by DEAE-cellulose chromatography and the pooled protein fractions that resulted were injected intravenously into the quail with aortic atherosclerosis induced by dietary cholesterol. After 7 weeks of injections on every other day, the quail were killed, blood clotting times and serum cholesterol levels were determined, and aortic atherosclerosis and fatty liver were scored. The results showed that while no regression of atherosclerosis was observed, the lowering of serum cholesterol, prolonged blood clotting time and reduced fatty liver were significantly affected by the injection of one of the pooled protein fractions. This venom fraction contained two major protein components, one of which had arginine esterase activity. From this study we conclude that snake venom has little effect on the regression of atherosclerosis, but it prolongs blood clotting and lowers serum cholesterol.

A bio-process called thermophilic anaerobic digestion that converts poultry waste into biogas energy was developed in our laboratory. During operation, feathers in the waste were completely degraded. This observation prompted a series of studies that have led to new technology and commercial developments. A feather-degrading bacterium was discovered and isolated. An enzyme called keratinase capable of hydrolysing the feather protein keratin was purified. The gene encoding this enzyme was isolated and sequenced. Genetic modification enabled the over expression of the gene in the parent strain as well as in other Bacillus strains. Scale-up fermentation, first in 150 l and then in 50 kl fermentors, made the mass production of the enzyme possible. Subsequently, in application research, keratinase was shown to partially hydrolyse feathers and convert them into a digestible feed protein. When supplemented as a feed additive on standard diet, the enzyme promoted chicken growth by improving dietary protein digestibility. Ifcommercially developed, keratinase can lower feed cost, increase poultry meat yield, and upgrade the utilization of less digestible protein feedstuffs. From an environmental point of view, keratinase has the potential to reduce waste nitrogen excretion by farm animals. More recently, keratinase was found to be able to degrade prion protein that is believed to cause transmissible spongiform encephalopathies (TSE) including ‘mad cow disease’, sheep scrapie, deer chronic wasting disease and human Creutzfeldt–Jakob disease. This discovery indicated the potential use of keratinase in a new enzymatic process to destroy prion protein and possibly prevent TSE, in a cost-effective and environmentally friendly manner. To test and to develop the enzymatic process, a prion-like surrogate protein (PSP) has been developed as a safe bio-marker. The combination of keratinase and PSP can provide a useful toolbox for TSE prevention. Based on the keratinase platform technology and its applications, BioResource International (BRI), a biotechnology company, was founded to serve the poultry and animal industries.

Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease-causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion-like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.

Day-old male broiler chicks were randomly assigned to 32 floor pens in a completely randomized block design and grown to 6 wk of age. Birds in experiment 1 were fed 1 of 2 basal diets supplemented with or without a protease containing feed additive, Versazyme (VZ). The 4 treatments were 1) control (C), a corn-soybean meal diet that contained 95% of amino acids recommended by NRC except for threonine and isoleucine; 2) C + 0.1% VZ (wt/wt) (C+) in the starter diet only; 3) high (HP) amino acid diet, a corn-soybean meal diet with 100 to 105% of amino acid recommended by NRC except for threonine and isoleucine; and 4) HP + 0.1% VZ (wt/wt) (HP+) in starter diet only. Supplementing both diets with VZ improved BW and feed conversion ratio (FCR) at 21 d of age and BW at 42 d of age. Cumulative 42-d FCR was only improved in birds fed the HP+ diet. Birds in experiment 2 received the following treatments: 1) HP, 2) HP + 0.1% VZ batch A (wt/wt) (A) in starter diet only, and 3) HP + 0.1% VZ batch B (wt/wt) (B) in starter diet only. Enzyme supplementation improved 22-d BW and FCR. There was no significant difference in BW at 43 d of age. Both A and B improved overall FCR (1.758 and 1.751 vs. 1.79 for A and B vs. HP, respectively). In conclusion, supplementation of starter broiler diets with VZ resulted in improved market growth performance.

We began to use cementless MicroStructured Omnifit acetabular components in November 1988. Liner dislodging and excessive polyethylene wear later occurred in some of our patients. This finding prompted us to review our experience with the use of such modular acetabular components.A total of 887 hips in 672 patients, which had been fitted with cementless MicroStructured Omnifit acetabular components, were evaluated for locking system complications using radiographic examinations. Of the cases, 240 hips which had received a 26-mm femoral head (group 1) and 110 hips which had received a 32-mm femoral head (group 2) were compared using a digitiser to determine the influence of femoral head size on polyethylene wear.There were two types of radiographic signs of locking system complications in 7 hips, at a mean time of 2.9 years after implantation. The incidence of liner locking system complications using this modular acetabular component was 0.8%. The mean liner wear rate per year was 0.15 mm in group 1 and 0.20 mm in group 2 (p0.005). The initial polyethylene thickness ranged from 5.9 to 13.6 mm in group 1, compared with 3.9 to 10.6 mm in group 2 (p0.005).A patient who has received a total hip arthroplasty that includes a MicroStructured Omnifit acetabular component should be monitored frequently for radiographic signs of locking system complications. In this study, the mean liner wear rate was significantly higher in group 2. Inadequate polyethylene thickness in this group was implicated as the major cause of excessive polyethylene wear.

Keratinase isolated from Bacillus licheniformis PWD-1 was immobilized on controlled-pore glass beads. The immobilized keratinase demonstrated proteolytic activities against both insoluble feather keratin and soluble casein. It also displayed a higher level of heat stability and an increased tolerance toward acidic pHs compared with the free keratinase. During a continuous reaction at 50(deg)C, the immobilized keratinase retained 40% of the original enzyme activity after 7 days. The immobilized keratinase exhibits improved stability, thereby increasing its potential for use in numerous applications.

To increase the production of keratinase, stable strains of Bacillus licheniformis carrying multiple keratinase gene copies in the chromosome were developed. Integrative vectors carrying kerA with or without P43-promoter were constructed and subcloned into B. licheniformis T399D and Bacillus subtilis DB104. In T399D, multiple copies of kerA integration into the chromosome were identified and determined by Southern blot. The optimal integration of kerA was found in the range of 3-5 copies. Higher integration of gene copies (>5) caused reduced processing and secretion of the extracellular keratinase. In DB104, kerA was cloned in the plasmid, not integrated into the chromosome. The strong constitutive promoter P43 not only increased the keratinase production in plasmid-based expression in DB104 but also improved the enzyme yield of the integrants of T399D. New strains were able to enhance cell growth and enzyme yield at higher concentrations of medium substrate. When they were grown in either soy or feather medium, the keratinase activity was stable and improved by about 4-6 times.

We retrospectively reviewed 45 hip arthroplasties which were performed over a period of 20 years in 38 patients with cirrhosis of the liver. There was a high perioperative 30-day complication rate (26.7%). Advanced cirrhosis was associated with a higher risk of complications (p = 0.004) as also was increased age, a high level of creatinine, a low level of albumin, a low platelet count, ascites, encephalopathy and an increased operative blood loss. The survival of the prosthesis at five years was 77.8% and infection was a major cause of failure. In view of the high rate of early complications and the limited longevity of the prosthesis, surgeons who perform hip arthroplasty on such patients should counsel them appropriately preoperatively.

This paper examines the current status of research work on mobile ATM and presents the authors' view on pending technical challenges that require attention and investigation. Mobile ATM is not restricted to the local area and it need not be ground based. This paper examines: (a) location management, (b) connection management, (c) handoff management, (d) routing, and (e) media access for two evolving architectures, namely: (a) wireless ad-hoc ATM networks, and (b) LEO-based satellite ATM networks.

Prions-infectious agents involved in transmissible spongiform encephalopathies-normally survive proteolytic and mild protein-destructive processes. Using bacterial keratinase produced by Bacillus licheniformis strain PWD-1, we tested conditions to accomplish the full degradation of prion protein (PrP) in brain-stem tissue from animals with bovine spongiform encephalopathy and scrapie. The detection of PrPSc, the disease-associated isoform of PrP, in homogenates was done by Western blotting and various antibodies. The results indicated that only in the presence of detergents did heat pretreatment at >100 degrees C allow the extensive enzymatic breakdown of PrPSc to a state where it is immunochemically undetectable. Proteinase K and 2 other subtilisin proteases, but not trypsin and pepsin, were also effective. This enzymatic process could lead to the development of a method for the decontamination of medical and laboratory equipment. The ultimate effectiveness of this method of prion inactivation has to be tested in mouse bioassays.

The multiple access with reduced handshake (MARCH) protocol utilizes the broadcast characteristics of an omnidirectional antenna to reduce the number of control messages required to transmit a data packet in multihop ad hoc networks. In MARCH, the RTS-CTS handshake is used only by the first hop of a route to forward data packets while for the rest it utilizes a new CTS-only handshake. Since fewer control packets are transmitted, the probability of packet collision is reduced and therefore the channel throughput is increased. Simulation results demonstrate that MARCH outperforms MAC protocols that employ only the RTS-CTS handshake.

Study objectives: Social epidemiologists have hypothesised that neighbourhood quality may exert an important contextual influence on mental and physical health. However, validated instruments do not exist for measuring neighbourhood quality in Taiwan. A self reported instrument to measure perceived neighbourhood quality in Taiwan was developed and tested. Design: Community survey. Setting: Southern Taiwan, including the metropolitan Kaohsiung area and eight surrounding communities, representing urban, suburban, and rural districts. Participants: A total of 1084 residents, aged 18 to 75, were surveyed during 1999 to 2000. Main results: Using factor analysis with varimax rotation, three subscales explained 54.8% of the variance in our 15 item Neighbourhood Quality Index: perceived social capital (Cronbach α=0.84), perceived security (α=0.78), and adequacy of services and facilities (α=0.67). Lower perceived neighbourhood social capital (odds ratio, OR, 1.26; 95% CI: 1.21 to 1.32), lower neighbourhood security (OR 1.37; 95% CI: 1.26 to 1.48), and inadequate neighbourhood services and facilities (OR 1.17; 95% CI: 1.06 to 1.28) were all related to higher residential dissatisfaction. Conclusions: A Neighbourhood Quality Index was developed for use in Taiwan with good internal consistency and test-retest reliability, as well as convergent validity. Future studies will examine the association between this index and measures of individual mental and physical health.

The purpose of this study was to retrospectively review the cases of pathologic long bone fractures caused by multiple myeloma treated in our hospital, to analyze the surgical method, complications, radiation therapy, survival time, and influence on quality of life.In a retrospective study, 22 patients with the pathological long bone fractures due to multiple myeloma who were surgically treated between 1987 and 1997 were analyzed. All patients received open reduction and internal fixation either with plates or intra-medullary nailing. Cement augmentations were performed in the majority of cases (91%). A detailed retrospective analysis was done to correlate the surgical methods, radiation therapy, functional results, and complications post-surgically.The most common site of fracture was the femur. The mean postoperative survival time was around 19 months. Post-operative pain relief was satisfactory, and only two patients required narcotics. No major complications were observed. However the union rate was only 30%, which might have been due to the inhibitory effect of radiation therapy on bone healing, or insufficient osteogentic ability of the myeloma-involved bone.Satisfactory pain relief and low implant failure rate was achieved and no definite evidence of tumor dissemination was found in this study. The authors suggest that open reduction and internal fixation with cement augmentation is a favorable treatment option for those patients suitable for surgery. However, postoperative radiation therapy may be associated with a low rate of union.

The lateral collateral ligament is the primary stabilizer against varus stress and is also an important contributor in maintaining posterolateral knee stability. Quadriceps tendon-patellar bone autograft has been used for anterior or posterior cruciate ligament reconstruction. We introduce a reconstructive procedure to restore the lateral collateral ligament using a quadriceps tendon-patellar bone autograft. The procedure is designed for unstable knees with concomitant cruciate ligament tear and posterolateral complex injury. This is a reasonable choice especially when allograft tissue is not available or in patients who are not suited for the use of bone-patellar tendon-bone autograft.

Vascular endothelial growth factor (VEGF) expression and tumor microvessel density (MVD) were examined by immunohistochemical staining in 117 cases of thoracic esophageal squamous cell carcinoma. Thirty-six (31%) of the 117 cases were evaluated as VEGF-positive. The average number of metastatic lymph nodes at surgery was 5.6 in the VEGF-positive cases and 3.0 in the VEGF-negative cases and was significantly higher in those with VEGF-positive cases (P = 0.04). The incidence of pathological tumor (PT)2-4 cases among the high-MVD cases was significantly higher than among the low-MVD cases (P = 0.01). MVD was 59.4 +/- 4.7 (mean +/- SE)/mm2 in the VEGF-positive cases and 47.9 +/- 3.8/mm2 in the VEGF-negative cases. The MVD of the VEGF-positive tumors was higher than that of VEGF-negative tumors, but the difference was not significant (P = 0.08). The survival rate of the patients with high-MVD tumors was significantly poorer than those with low-MVD tumors, and the survival rate of those patients with VEGF-positive tumors was significantly poorer than in those with VEGF-negative tumors (P = 0.009 and P = 0.04, respectively). The cumulative survival rates in the VEGF-positive groups were found to be significantly poorer in the pT3 and pathological node (pN)1 groups when stratified according to pT factor (pathological T category) and pN factor (pathological N category) in the tumor-node-metastasis (TNM) classification. VEGF expression had the second highest hazard ratio in the multivariate analysis, after pN factor. These results indicate that VEGF is a useful marker for predicting the outcome in patients with more advanced esophageal squamous cell carcinoma. It seems that TNM factors and VEGF expression are important factors in the selection of appropriate treatments.

This case report illustrates a rare case of motor weakness caused by a free fat graft herniation. A 40-year-old woman who had undergone surgery for a herniated lumbar intervertebral disc experienced right lower leg weakness. On magnetic resonance image (MRI) a herniated free fat graft was noted. An emergent operation was performed and the herniated fat graft was removed. Postoperatively, the patient recovered well with improvement of the motor weakness. MRI is a good method for diagnosis of fat graft herniation. The mechanisms of this complication have been documented, and the size of the fat graft plays an important role. The methods for prevention of this herniation are also discussed. Although the transplantation of adipose tissue has many advantages, including the prevention of postoperative epidural fibrosis, great care is needed when applying a fat graft intra-operatively. When a postoperative neurologic deficit develops, herniation of the fat graft must be considered. An emergent operation is the treatment of choice for this particular complication.