A novel DNAJB6 mutation causes dominantly inherited distal-onset myopathy and compromises DNAJB6 function.

BACKGROUND: Mutations in the DNAJB6 gene have been identified as a rare cause of dominantly inherited limb-girdle muscular dystrophy or distal-onset myopathy. MATERIALS AND METHODS: Exome sequencing was performed to investigate a Taiwanese family with a dominantly inherited distal-onset myopathy. Functional effects of the causal mutation were investigated in vitro. RESULTS: Exome sequencing of the two affected individuals in this family identified a heterozygous mutation, c.287C>T (p.Pro96Leu) in the DNAJB6 gene, which co-segregated with the myopathy within all 12 family members. Notably, this mutation is novel and localizes within the glycine and phenylalanine-rich (G/F) domain and alters an amino acid residue previously reported with a different mutation. Furthermore, immunofluorescence analyses and filter trap assay demonstrated that the c.287C>T (p.Pro96Leu) mutation possessed a dominant negative effect on the anti-aggregation function of DNAJB6 protein. CONCLUSION: This study expands the molecular spectrum of DNAJB6 mutations and also emphasizes the pathogenic role of DNAJB6 dysfunction in distal-onset myopathy.

Designing a stronger interface through graded structures in amorphous/nanocrystalline ZrCu/Cu multilayered films.

Many multilayered nano-structures appear to fail due to brittle matter along the interfaces. In order to toughen them, in this study, the microstructure and interface strength of multilayered thin films consisting of amorphous ZrCu and nanocrystalline Cu (with sharp or graded interfaces) are examined and analyzed. The interface possesses a gradient nature in terms of composition, nanocrystalline phase size and volume fraction. The bending results extracted from the nano-scaled cantilever bending samples demonstrate that multilayered films with graded interfaces would have a much higher interface bending strength/strain/modulus, and an overall improvement upgrade of more than 50%. The simple graded interface design of multilayered thin films with improved mechanical properties can offer much more promising performance in structural and functional applications for MEMS or optical coating.

Vertical habitat shift of viviparous and oviparous deep-sea cusk eels revealed by otolith microstructure and stable-isotope composition.

Otolith stable-oxygen-isotope composition and microstructure were analysed in order to investigate the vertical habitat shift of deep-sea cusk eels (Ophidiiformes). Otolith δ18 O profiles suggested that both viviparous blind cusk eels and oviparous cusk eels experienced a pelagic larval stage and then settled to the deep-sea floor over a vertical distance that ranged among individuals from 200 to >1000 m. This result shows that the larvae of viviparous Barathronus maculatus undertake an ontogenetic vertical migration after a period of larval drift that may facilitate their wide distribution on the sea floor.

Exploring the significance of sex hormone-binding globulin examination in the treament of women with polycystic ovarian syndrome (PCOS).

OBJECTIVES: To explore whether sex hormone-binding globulin (SHBG) and free androgen index (FAI) can be seen as therapeutic effect indexes of women with polycystic ovarian syndrome (PCOS). MATERIALS AND METHODS: The body mass index (BMI), basal sexual hormones, SHBG, fasting blood glucose (FBG), and fasting insulin (FINS) were collected from 579 women with PCOS, were divided into two groups according to BMI: obese group (n = 145) and non-obese group (n = 434), according to homeostasis model assessment of insulin status (HOMA-IR). Patients were then divided into four groups: A: non-obese without insulin resistance (n = 174), B: non-obese with insulin resistance (n = 260), C: obese without insulin resistance (n = 34), D: obese with insulin resistance (n = 111). A and B groups received Diane-35 alone, C and D groups received Diane-35 plus metformin for three months. Then clomiphene citrate and HMAG were used to induce ovulation then compared ovulation rate and pregnancy outcome. RESULTS: FAI decreased significantly and SHBG increased significantly in all groups. In A group FINS and HOMA-IR increased significantly (p < 0.05), but in B and D groups FINS and HOMA-IR decreased significantly (p < 0.05). After treatment the ovulation rate in non-obese group was higher than obese group (p < 0.01). Compared with non-ovulation patients, SHBG increased significantly and FAI decreased significantly in the patient with ovulation. Regarding the pregnancy outcome, FAI decreased significantly in delivery patients than spontaneous abortion patients. Furthermore, SHBG increased significantly. CONCLUSION: It was important to check SHBG and FAI during the treatment of PCOS patient. They could be used to assess whether the treatment was effective and as a guidance of clinical medication.

First Report of Fusarium Maize Ear Rot Caused by Fusarium kyushuense in China.

From September 2009 to October 2012, surveys to determine population structure of Fusarium species on maize were conducted in 22 provinces in China, where the disease incidence ranged from 5 to 20% in individual fields. Maize ears with clear symptoms of Fusarium ear rot (with a white to pink- or salmon-colored mold at the ear tip) were collected from fields. Symptomatic kernels were surface-sterilized (1 min in 0.1% HgCl2, and 30 s in 70% ethanol, followed by three rinses with sterile distilled water), dried, and placed on PDA. After incubation for 3 to 5 days at 28°C in the dark, fungal colonies displaying morphological characteristics of Fusarium spp. (2) were purified by transferring single spores and identified to species level by morphological characteristics (2), and DNA sequence analysis of translation elongation factor-1α (TEF) and ß-tubulin genes. A large number of Fusarium species (mainly F. graminearum species complex, F. verticillioides, and F. proliferatum) were identified. These Fusarium species are the main causal agents of maize ear rot (2). Morphological characteristics of six strains from Anhui, Hubei, and Yunnan provinces were found to be identical to those of F. kyushuense (1), which was mixed with other Fusarium species in the natural infection in the field. Colonies grew fast on PDA with reddish-white and floccose mycelia. The average growth rate was 7 to 9 mm per day at 25°C in the dark. Reverse pigmentation was deep red. Microconidia were obovate, ellipsoidal to clavate, and 5.4 to 13.6 (average 8.8) µm in length. Macroconidia were straight or slightly curved, 3- to 5-septate, with a curved and acute apical cell, and 26.0 to 50.3 (average 38.7) µm in length. No chlamydospores were observed. Identity of the fungus was further investigated by sequence comparison of the partial TEF gene (primers EF1/2) and ß-tubulin gene (primers T1/22) of one isolate (3). BLASTn analysis of the TEF amplicon (KC964133) and ß-tubulin gene (KC964152) obtained with cognate sequences available in GenBank database revealed 99.3 and 99.8% sequence identity, respectively, to F. kyushuense. Pathogenicity tests were conducted twice by injecting 2 ml of a prepared spore suspension (5 × 105 spores/ml) into maize ears (10 per isolate of cv. Zhengdan958) through silk channel 4 days post-silk emergence under field conditions in Wuhan, China. Control plants were inoculated with sterile distilled water. The ears were harvested and evaluated 30 days post-inoculation. Reddish-white mold was observed on inoculated ears and the infected kernels were brown. No symptoms were observed on water controls. Koch's postulates were fulfilled by re-isolating the pathogen from infected kernels. F. kyushuense, first described on wheat in Japan (1), has also been isolated from rice seeds in China (4). It was reported to produce both Type A and Type B trichothecene mycotoxins (1), which cause toxicosis in animals. To our knowledge, this is the first report of F. kyushuense causing maize ear rot in China and this disease could represent a serious risk of yield losses and mycotoxin contamination in maize and other crops. The disease must be considered in existing disease management practices. References: (1) T. Aoki and K. O'Donnell. Mycoscience 39:1, 1998. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) F. Van Hove et al. Mycologia 103:570, 2011. (4) Z. H. Zhao and G. Z. Lu. Mycotaxon 102:119, 2007.

Resistance to Fusarium head blight and seedling blight in wheat is associated with activation of a cytochrome p450 gene.

ABSTRACT One plant genotype displays a resistance phenotype at one development stage but a susceptible reaction to the same pathogen at another stage, which is referred to here as resistance inversion. In wheat, Fusarium head blight (FHB)-resistant cv. Sumai3 showed a Fusarium seedling blight (FSB)-susceptible reaction whereas FHB-susceptible cv. Annong8455 exhibited FSB resistance when challenged with a Fusarium asiaticum strain that produces deoxynivalenol (DON). The resistance to FHB and FSB in wheat was closely associated with expression of a plant cytochrome P450 gene in response to FHB pathogens and mycotoxins. Quantitative real-time polymerase chain reaction analyses showed that expression of nine defense-related genes in spikes and seedlings was induced by the fungal infection, in which a massive accumulation of a plant cytochrome P450 gene, CYP709C1, was clearly associated with the resistance reaction in both seedling and spike. The FHB-resistant Sumai3 accumulated 7-fold more P450 transcripts than did the FHB-susceptible Annong8455, while 84-fold more P450 transcripts were accumulated in the FSB-resistant Annong8455 than the FSB-susceptible Sumai3. A Fusarium strain with a disrupted Tri5 gene, which is not able to produce the first enzyme essential for trichothecene mycotoxin biosynthesis, also induced more P450 transcripts in FHB- and FSB-resistant cultivars. The fungal activation of the P450 gene was more profound in the FSB-resistant reaction than the FHB-resistant reaction relative to their susceptible counterparts. DON triggered a differential expression of the P450 gene with comparable patterns in spikes and seedlings in a resistance-dependent manner. These results may provide a basis for dissecting mechanisms underlying FHB and FSB resistance reactions in wheat and revealing functions of the cytochrome P450 in plant detoxification and defense.

Human prion protein cDNA: molecular cloning, chromosomal mapping, and biological implications.

A human complementary DNA whose protein product is considered to be the major component of scrapie-associated fibrils in Creutzfeldt-Jakob disease, kuru, and Gerstmann-Straussler syndrome has been identified and characterized. The extensive homology of this gene sequence to the hamster PrP 27- to 30-kilodalton prion protein complementary DNA clone, and its existence as a single copy in the human genome, leads to the conclusion that this is the human prion gene. This human prion gene has been mapped to human chromosome 20, negating a direct link between the prion protein and Down's syndrome or the amyloid of Alzheimer's disease.

Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene.

A novel potential cell surface receptor of the tyrosine kinase gene family has been identified and characterized by molecular cloning. Its primary sequence is very similar to that of the human epidermal growth factor receptor and the v-erbB oncogene product; the chromosomal location of the gene for this protein is coincident with the neu oncogene, which suggests that the two genes may be identical.

Rhabdomyolysis and brain ischemic stroke in a heroin-dependent male under methadone maintenance therapy.

OBJECTIVE: There are several complications associated with heroin abuse, some of which are life-threatening. Methadone may aggravate this problem. METHOD: A clinical case description. RESULTS: A 33-year-old man presented with rhabdomyolysis and cerebral ischemic stroke after intravenous heroin. He had used heroin since age 20, and had used 150 mg methadone daily for 6 months. He was found unconsciousness at home and was sent to our hospital. In the ER, his opiate level was 4497 ng/ml. In the ICU, we found rhabdomyolysis, acute renal failure and acute respiratory failure. After transfer to an internal ward, we noted aphasia and weakness of his left limbs. After MRI, we found cerebral ischemic infarction. CONCLUSION: Those using methadone and heroin simultaneously may increase risk of rhabdomyolysis and ischemic stroke. Patients under methadone maintenance therapy should be warned regarding these serious adverse events. Hypotheses of heroin-related rhabdomyolysis and stroke in heroin abusers are discussed.

Structure of the rat alpha 1-acid glycoprotein gene.

The complete nucleotide sequence of the rat alpha 1-acid glycoprotein gene has been determined from an isolated lambda recombinant bacteriophage. Southern blot analysis and DNA sequencing indicate that there is only one gene per genome; it contains six exons and is located within a 3,200-base-pair fragment starting from a TATA box and extending to the polyadenylation signal AATAAA. Transcription starts 37 base pairs upstream from the beginning of the translation codon ATG. The TATA box (TATAAA) lies 26 base pairs upstream from this site. The gene contains several potential glucocorticoid receptor-binding sites, both inside and outside the structural gene.

Inhibitory effect of CDA-II, a urinary preparation, on aflatoxin B(1)-induced oxidative stress and DNA damage in primary cultured rat hepatocytes.

The effects of CDA-II (cell differentiation agent II; a urinary preparation) on both aflatoxin B(1) (AFB(1))-induced cell injury and DNA damage were investigated using cultured rat hepatocytes. CDA-II was able to suppress both the lipid peroxidation and lactate dehydrogenase leakage induced by AFB(1). Glutathione (GSH) depletion by AFB(1) was replenished by CDA-II treatment. Under these experimental conditions, CDA-II enhanced the activity of GSH peroxidase, but not GSH S-transferase. By evaluation of unscheduled DNA synthesis, CDA-II reduced AFB(1)-induced DNA damage in hepatocyte cultures. These findings suggest that CDA-II can inhibit cytotoxicity of AFB(1) through enhancing the activity of GSH peroxidase and preventing GSH depletion.

Effect of transpiration on Pb uptake by lettuce and on water soluble low molecular weight organic acids in rhizosphere.

The effect of transpiration (high and low) on Pb uptake by leaf lettuce and on water soluble low molecular weight organic acids (LMWOAs) in rhizosphere has been studied. After two weeks of growth the plants were cultured in greenhouse for more four weeks and two days. Pb(NO(3))(2) solutions of different concentrations (100, 200, and 300 mg l(-1) of Pb) were then added to the quartz sand pots of different plants and studies were initiated. Blank experiments (without treating the quartz sand pots with Pb(NO(3))(2) solutions) were also run in parallel. No significant differences in the growth of the plants with the concentrations of added Pb(NO(3))(2) solutions were observed by both low and high transpirations at the end of the 0, 3rd, and 10th days of studies. The total evaporation of the volatiles during 10 days did not depend on the concentration of Pb(2+) but with high transpiration the rate of evaporation was significantly higher than with low transpiration. Uptake of Pb by shoots and roots of the plants was found to be proportional to the concentration of various Pb(NO(3))(2) solutions added and more accumulation was observed in roots than in shoots at the end of 3rd and 10th days. High transpiration created more Pb uptake than low transpiration did. One volatile acid, propionic acid and nine non-volatile acids, lactic, glycolic, oxalic, succinic, fumaric, oxalacetic, D-tartaric, trans-aconitic, and citric acids in rhizosphere quartz sands were identified and quantified by gas chromatography (GC) analysis. D-Tartaric and citric acids were major among the non-volatile acids. The amount of LMWOAs in rhizosphere quartz sands increased with the higher amount of Pb uptake and also with the duration of studies. The total quantities of the LMWOAs in the rhizosphere quartz sands were significantly higher under high transpiration with 300 mg l(-1) Pb solution addition at the end of 10th day. The present study shows prominent correlation between transpiration and uptake of heavy metal and interesting correlation between Pb contaminated level and quantity of water soluble LMWOAs in rhizosphere quartz sands. The latter thus deserves of further studies.

First Report of the Causal Agent of Huanglongbing ("Candidatus Liberibacter asiaticus") Infecting Kumquat in Taiwan.

Huanglongbing (greening) disease caused by a nonculturable, phloem-limited bacterium is a severe disease of citrus. On the basis of the influence of temperature on host symptoms and the causal agent, this disease can be categorized as Asian caused by "Candidatus Liberibacter asiaticus", African caused by "Ca. L. africanus", and American caused by "Ca. L. americanus". Kumquat (Fortunella margarita (Lour.) Swingle), a member of the Rutaceae is an economically important crop for export and local consumption in Taiwan. Recently, a Huanglongbing-like disease was found on kumquat in Yilan County, the largest kumquat-producing area in northeastern Taiwan. Even though the disease has been reported on Citrus spp. from Taiwan, it has never been reported on kumquat. Symptoms of infected kumquat were mottling, yellowing, hardening, and curling of leaves followed by premature defoliation, twig dieback, decay of feeder rootlets and lateral roots, and ultimately the death of the entire plant. Typical sieve-tube-restricted bacteria were observed in kumquat cells by electron microscopy (1). In addition, psyllid-transmission tests demonstrated that the Asian psyllid (Diaphorina citri) could transmit this bacterium to healthy kumquats. Positive bud graft transmissions were obtained to F. margarita, F. japonica (Thunb.) Swingle, F. obovata Hort. ex Tanaka, Luchen sweet orange (Citrus sinensis (L.) Osb.), and Wentan pummelo (C. maxima f. sp. butan Hay.). These inoculated plants showed symptoms in 3 to 8 months, and bacteria could be detected by polymerase chain reaction (PCR) using a common primer pair that amplified a 226-bp specific DNA fragment (2). For further molecular identification, the bacterial DNA was extracted from the inoculated plants and PCR was performed by using two sets of primers selected from the 16S rRNA region (GenBank Accession No. L22532) and 16S/23S intergenic spacer region (GenBank Accession No. AB019793). The expected DNA fragments of 1,389 bp and 862 bp were, respectively, amplified from symptomatic plants but not from healthy plants. The PCR products were cloned and sequenced (GenBank Accession Nos. DQ302750 and DQ207841). The 16S rRNA has 98 to 99% identity and 16S/23S intergenic spacer region has 99% identity to the corresponding region of "Ca. L. asiaticus" in GenBank. These molecular analyses confirm the presence of "Ca. L. asiaticus" in kumquat. Since Huanglongbing has been rarely reported naturally on kumquat, further analysis of this bacterium as a special strain of "Ca. L. asiaticus" is needed. References: (1) M. Garnier et al. Ann. Microbiol. 135A:169, 1984. (2) T. H. Hung et al. J. Phytopathol. 147:599, 1999.

First Report of Bougainvillea spectabilis chlorotic vein-banding virus Infecting Bougainvillea Plants in Taiwan.

Bougainvillea (Bougainvillea spectabilis), native to Amazonian rainforests in South America, is an important ornamental and landscaping plant that is widely distributed in tropical and subtropical areas. A new virus disease, Bougainvillea chlorotic vein-banding, caused by a Badnavirus, Bougainvillea spectabilis chlorotic vein-banding virus (BsCVBV), was first reported in Brazil in 2001 (1) and recently discovered in Taiwan. Infected bougainvillea developed symptoms such as mottling, chlorosis, vein-banding, and stunting. Severe leaf-distortion symptoms were observed in the susceptible hybrid Taipei Red, the most popular bougainvillea cultivar in Taiwan. In electron microscopic observations, typical bacilliform virions measuring 28 × 130 to 150 nm were observed in infected bougainvillea cells. In addition, our transmission tests demonstrated that the virus could be easily transmitted among different bougainvillea cultivars by bud grafting but not by mechanical inoculation. Bougainvillea plants showed apparent symptoms 1 month after grafting. For molecular identification, viral DNA was extracted from the test plants (2), and polymerase chain reaction (PCR) was performed using the primers selected from the DNA sequences of ORF III of Sugarcane bacilliform virus (GenBank Accession No. M89923). The sequence of the forward primer was 5'-TCA AAG TTT GAT TTG AAG AGC GGG-3' and the sequence of the reverse primer was 5'-CTT GCA TAC TGC TCC CCA TCC-3' The primers amplified a 676-bp PCR product (GenBank Accession No. DQ103759). Nucleotide and deduced amino acid sequences were 82 and 90% identical, respectively, to the corresponding region of the Brazilian strain of BsCVBV (GenBank Accession No. AY532653). These data indicate that the bougainvillea disease occurring in Taiwan is caused by a strain of BsCVBV. Reference: (1) C. M. Chagas et al. Virus Rev. Res. 6:153, 2001. (2) H.-J. Su et al. J. Phytopathol. 151:290, 2003.

Programmable and on-demand drug release using electrical stimulation.

Recent advancement in microfabrication has enabled the implementation of implantable drug delivery devices with precise drug administration and fast release rates at specific locations. This article presents a membrane-based drug delivery device, which can be electrically stimulated to release drugs on demand with a fast release rate. Hydrogels with ionic model drugs are sealed in a cylindrical reservoir with a separation membrane. Electrokinetic forces are then utilized to drive ionic drug molecules from the hydrogels into surrounding bulk solutions. The drug release profiles of a model drug show that release rates from the device can be electrically controlled by adjusting the stimulated voltage. When a square voltage wave is applied, the device can be quickly switched between on and off to achieve pulsatile release. The drug dose released is then determined by the duration and amplitude of the applied voltages. In addition, successive on/off cycles can be programmed in the voltage waveforms to generate consistent and repeatable drug release pulses for on-demand drug delivery.

Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture.

A TMV-specific full-size murine IgG-2b/K antibody (mAb24) was expressed in a Nicotiana tabacum cv. Petite Havana SR1 suspension culture (P9s), which was derived from a stably transformed transgenic plant (P9). The integration of an N-terminal murine leader peptide directed the assembled immunoglobulin for secretion. However, in suspension culture, the full-size recombinant antibody, rAb24, was retained by the plant cell wall and was not present in the culture medium. rAb24 expression reached a basal level of 15 microg per gram wet cell weight, corresponding to 0.3% of the total soluble plant cell protein. The level of rAb24 could be increased three-fold by amino acid supplementation of the culture medium. For purification of the recombinant antibody from batch-cultured tobacco suspension cells, the primary plant cell wall was partially digested by enzymatic treatment. This resulted in a total release of recombinant full-size rAb24 into the extraction buffer. A three-step procedure was used to purify the immunoglobulins, starting with cross-flow filtration (step 1) followed by protein A affinity chromatography (step 2) and gel filtration as a final purification step (step 3). This procedure gave a recovery of more than 80% of the expressed rAb24 from plant cell extracts. SDS-PAGE, IEF and immunoblot analyses demonstrated a high degree of homogeneity for the affinity-purified rAb24. An ELISA procedure demonstrated that the specificity and affinity of the protein A affinity purified antibody was indistinguishable from its murine counterpart, indicating the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies.

GST fusion proteins cause false positives during selection of viral movement protein specific single chain antibodies.

Glutathione S-transferase (GST) fusion proteins are used frequently for investigating protein-protein and protein-DNA interactions. The present study demonstrates that the use of GST fusion proteins caused false positives during selection of phage-displayed single-chain antibody fragments (scFvs) specific for three domains of the movement protein (NS(M)) of tomato spotted wilt virus (TSWV). To identify and exclude the false positives when using GST as a fusion partner linked to the antigen of interest, indirect phage enzyme-linked immunosorbent assay (ELISA) was compared with capture phage ELISA. Of 210 enriched phage clones, indirect phage ELISA identified 106 clones specific for binding to GST-domain fusions but not to GST. In contrast, using capture phage ELISA, all 106 selected clones were identified as false positives, reacting with the GST fusion proteins and GST. This was confirmed by characterization of soluble scFv antibodies. The data indicate that GST fusion proteins seem unsuitable for screening of phage-displayed antibody fragments and it is essential to use capture phage ELISA, instead of the indirect phage ELISA used commonly to exclude false positives in characterization of selected clones with GST fusion proteins.

Slow release from a coated sphere with slight deformations of coating film and drug matrix.

This paper, a continuation of our previous work, is a presentation of the effects of slight deformations of both the coating film and the drug matrix on the concentration distributions and the slow release characteristics of a coated drug particle. The coated particle shape, in practice, is not perfectly spherical. With restriction of the deformation shape functions considered, it is noted that if the deformation is slight or if only the average release rate is of interest, the effects of shape deformation are secondary. Otherwise, the shape deformation can have a significant effect on the concentration profiles and/or the release rate from different parts of the particle surface. The deformations of both coating film and the drug matrix could introduce a greater deviation from the spherical case than those considering only coating film or drug matrix shape deformation.

Slow release from a coated sphere with a slightly deformed coating.

Effects of a slight deformation of the coating film on the concentration distribution and the slow release characteristics of a coated drug particle is theoretically investigated in this work. A perturbation technique is employed for transformation of the deformed spherical problem into a series of concentric spherical problems with the degree of deformation considered as the perturbation parameter. In addition, the governing equations are solved analytically by further restricting the deformation shape function. Results in this study indicate that if the deformation is slight or only the average release rate is of interest, the effects of coating shape deformation are secondary. However, if the deformation is not so small and if the drug diffusivity in the film is low, the shape deformation could have a significant effect on the concentration profiles and/or the release rate from different parts of the particle surface.