Detection of Talaromyces macrosporus and Talaromyces trachyspermus by a PCR assay targeting the hydrophobin gene.

Talaromyces species are typical fungi capable of producing the heat-resistant ascospores responsible for the spoilage of processed food products. Hydrophobins, which are unique to fungi, are small secreted proteins that form amphipathic layers on the outer surface of fungal cell walls. In this study, species-specific primer sets for detecting and identifying Talaromyces macrosporus and Talaromyces trachyspermus were designed based on hydrophobin gene sequences. A conventional polymerase chain reaction (PCR) assay using these primer sets produced species-specific amplicons for T. macrosporus and T. trachyspermus. The detection limit for each primer set was 100 pg template DNA. This assay also detected fungal DNA extracted from blueberries inoculated with T. macrosporus. Other heat-resistant fungi, including Byssochlamys, Neosartorya and Talaromyces species, which cause food spoilage, were not detected in PCR amplifications with these primer sets. Furthermore, a conventional PCR assay using a crude DNA extract as the template also yielded amplicons specific to T. macrosporus and T. trachyspermus. The simple and rapid PCR assay described herein is highly species-specific and can reliably detect T. macrosporus and T. trachyspermus, suggesting it may be relevant for the food and beverage industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The heat-resistant ascospores of Talaromyces macrosporus and Talaromyces trachyspermus are responsible for food spoilage after pasteurization. Traditional methods for detecting fungal contamination based on morphological characteristics are time-consuming and exhibit low sensitivity and specificity. In this study, a conventional polymerase chain reaction (PCR) assay based on hydrophobin gene sequences was developed for the specific detection of T. macrosporus and T. trachyspermus. This detection method was simple, rapid and highly specific. These results suggest that the conventional PCR assay developed in this study may be useful for detecting T. macrosporus and T. trachyspermus in raw materials and processed food products.

Design of a species-specific PCR method for the detection of the heat-resistant fungi Talaromyces macrosporus and Talaromyces trachyspermus.

Heat-resistant fungi occur sporadically and are a continuing problem for the food and beverage industry. The genus Talaromyces, as a typical fungus, is capable of producing the heat-resistant ascospores responsible for the spoilage of processed food products. Isocitrate lyase, a signature enzyme of the glyoxylate cycle, is required for the metabolism of non-fermentable carbon compounds, like acetate and ethanol. Here, species-specific primer sets for detection and identification of DNA derived from Talaromyces macrosporus and Talaromyces trachyspermus were designed based on the nucleotide sequences of their isocitrate lyase genes. Polymerase chain reaction (PCR) using a species-specific primer set amplified products specific to T. macrosporus and T. trachyspermus. Other fungal species, such as Byssochlamys fulva and Hamigera striata, which cause food spoilage, were not detected using the Talaromyces-specific primer sets. The detection limit for each species-specific primer set was determined as being 50 pg of template DNA, without using a nested PCR method. The specificity of each species-specific primer set was maintained in the presence of 1,000-fold amounts of genomic DNA from other fungi. The method also detected fungal DNA extracted from blueberry inoculated with T. macrosporus. This PCR method provides a quick, simple, powerful and reliable way to detect T. macrosporus and T. trachyspermus. SIGNIFICANCE AND IMPACT OF THE STUDY: Polymerase chain reaction (PCR)-based detection is rapid, convenient and sensitive compared with traditional methods of detecting heat-resistant fungi. In this study, a PCR-based method was developed for the detection and identification of amplification products from Talaromyces macrosporus and Talaromyces trachyspermus using primer sets that target the isocitrate lyase gene. This method could be used for the on-site detection of T. macrosporus and T. trachyspermus in the near future, and will be helpful in the safety control of raw materials and in food and beverage production.

Hyperspectroscopic imager for baby fibers.

Hyperspectral imaging system for diagnosing digestive diseases was newly developed in order to obtain information on pathology beyond morphology of lesions. In order to guide light reflected from a lesion, a baby fiber, which can be inserted in a forceps channel of the electronic endoscope, was also developed. The performance of the system was evaluated by animal experiment. Obtained hyperspectral data were found to have sufficient quality endurable to practical use. Harmful phenomena to a living body were not observed within the experiment. It was considered from the animal experiment that the present system could be practically used for humans.

Hemodynamic assessment of celiaco-mesenteric anastomosis in patients with pancreaticoduodenal artery aneurysm concomitant with celiac artery occlusion using flow-sensitive four-dimensional magnetic resonance imaging.

OBJECTIVES: Many pancreaticoduodenal artery (PDA) aneurysms are associated with celiac artery (CA) stenosis. The pathogenesis of PDA aneurysm may be associated with hemodynamic changes due to CA stenosis/occlusion. The aim of this study was to assess the hemodynamic changes of celiaco-mesenteric anastomosis in patients with PDA aneurysms concomitant with CA occlusion using four-dimensional flow-sensitive magnetic resonance imaging (4D-Flow). METHODS: 4D-Flow was performed preoperatively on five patients. Seven age- and sex-matched individuals were used as controls. Hemodynamic parameters such as flow volume and maximum flow velocity in PDAs, gastroduodenal arteries, common hepatic arteries, and superior mesenteric arteries were compared between both groups. Wall shear stress (WSS) and oscillatory shear index (OSI) were mapped in both groups. RESULTS: In the patient group, 4D-Flow identified retrograde flow of both gastroduodenal arteries and common hepatic arteries. Heterogeneous distribution patterns of both WSS and OSI were identified across the entire PDA in the patient group. OSI mapping showed multiple regions with extremely high OSI values (OSI > 0.3) in all patients. All PDA aneurysms, which were surgically resected, were atherosclerotic. CONCLUSIONS: 4D-Flow identified hemodynamic changes in celiaco-mesenteric arteries in patients with PDA aneurysms with concomitant CA occlusion. These hemodynamic changes may be associated with PDA aneurysm formation.

[Aortic valve surgery for patients with severely calcified aorta].

Severe atherosclerosis of the ascending aorta frequently causes difficulties during heart operations, hindering surgical maneuvers and potentially leading to systemic embolism. There have been several methods to solve these problems but the best way to treat patients requiring aortic valve replacement (AVR) has not been established yet. Surgical techniques for AVR in these patients include AVR under deep hypothermic circulatory arrest with or without endarterectomy of the ascending aorta or replacement of the ascending aorta. Endovascular clamping using a balloon is another approach but requires manipulation of the heavily calcified aorta that may result in a certain risk for stroke. Another option to avoid the ascending aorta and cross-clamping is the apicoaortic conduit. Recently introduced trans-catheter AVR (TAVR), especially trans-apical AVR, has been shown to be feasible in such patients. Larger studies and longer follow-up will be required to scientifically prove the superiority of trans-apical AVR over conventional surgical strategies in patients with porcelain aorta requiring AVR.

EMMPRIN (CD147) expression and differentiation of papillary thyroid carcinoma: implications for immunocytochemistry in FNA cytology.

OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tumour progression, invasion and metastasis. EMMPRIN expression has been demonstrated in several tumours, but its expression profile in thyroid cancer remains unclear. METHODS: We evaluated the expression profile of EMMPRIN at various stages of differentiation of thyroid carcinoma, including 20 cases of well-differentiated papillary carcinoma (WDPC), 15 cases of papillary carcinoma with a poorly differentiated carcinoma component (PC/PDC) and four cases with an undifferentiated carcinoma (UDC) component, using paraffin-embedded sections for immunohistochemical stains. Also, we used 32 fine needle aspiration cytology and imprint smears from the same cases for immunocytochemical stains. The staining results were evaluated with a scoring system. RESULTS: Immunohistochemical staining showed that EMMPRIN expression was absent or weak in almost all WDPC specimens, whereas it was moderate or strong in PDC and UDC components. In tumours that showed a gradual morphological transformation from WDPC to PDC components, the expression of EMMPRIN was progressively stronger from the areas of WDPC to those of PDC. WDPC, PC/PDC and UDC had expression scores of 4.9, 45.0 and 245.7, respectively. Results of immunocytochemical staining showed almost the same staining profile as those of immunohistochemical staining. The cytological atypia of EMMPRIN-positive cells was greater than that of negative cells. CONCLUSION: These results indicated that EMMPRIN expression correlates significantly with the degree of dedifferentiation of thyroid carcinoma. This study demonstrates the feasibility of expression of EMMPRIN using fine needle aspiration samples. Therefore, immunocytochemical analysis of EMMPRIN may be a novel aid to evaluate the differentiation of thyroid carcinoma.

[Investigation of the accurate measurement of the basic imaging properties for the digital radiographic system based on flat panel detector].

PURPOSE/AIM OF THE EXHIBIT: The purpose of this exhibit is: 1. To explain "resampling", an image data processing, performed by the digital radiographic system based on flat panel detector (FPD). 2. To show the influence of "resampling" on the basic imaging properties. 3. To present accurate measurement methods of the basic imaging properties of the FPD system. CONTENT ORGANIZATION: 1. The relationship between the matrix sizes of the output image and the image data acquired on FPD that automatically changes depending on a selected image size (FOV). 2. The explanation of the image data processing of "resampling". 3. The evaluation results of the basic imaging properties of the FPD system using two types of DICOM image to which "resampling" was performed: characteristic curves, presampled MTFs, noise power spectra, detective quantum efficiencies. CONCLUSION/SUMMARY: The major points of the exhibit are as follows: 1. The influence of "resampling" should not be disregarded in the evaluation of the basic imaging properties of the flat panel detector system. 2. It is necessary for the basic imaging properties to be measured by using DICOM image to which no "resampling" is performed.

Babesia parasites develop and are transmitted by the non-vector soft tick Ornithodoros moubata (Acari: Argasidae).

Ornithodoros moubata ticks were fed on blood infected with Babesia equi. However, the parasites were quickly cleared as evidenced by the disappearance of B. equi-specific ribosomal RNA from the ticks. We hypothesized that if the Babesia parasite can escape midgut-associated barriers a non-vector tick can become infected with Babesia. To test this hypothesis, B. equi parasite-infected blood from in vitro culture was injected into the haemocoel of ticks. B. equi-specific rRNA was surprisingly detected 45 days after injection even in the eggs. Babesia-free dogs were infested with O. moubata ticks that were infected by inoculation with B. gibsoni-infected red blood cells. Parasitaemia and antibody production against Bg-TRAP of B. gibsoni increased gradually. These results indicate that O. moubata may be a useful vector model for Babesia parasites and also a very important tool for studies on tick immunity against Babesia parasites and tick-Babesia interactions.

Direct injection of calcitriol or its analog into hyperplastic parathyroid glands induces apoptosis of parathyroid cells.

Hyperplasia of the parathyroid gland (PTG) is associated not only with excessive secretion of parathyroid hormone (PTH) but also with changes in the parathyroid cell (PTC) characteristics (i.e. hyperproliferative activity, and low contents of vitamin D and calcium-sensing receptors). Control of PTG hyperplasia is most important in the management of secondary hyperparathyroidism, but the advanced stage of hyperplasia is considered irreversible. In the present study, dialysis patients with PTG hyperplasia underwent direct injection of calcitriol or maxacalcitol (OCT) into the PTG. Ultrasonography showed that this treatment had significantly reduced PTG volume and tissue analysis using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and DNA electrophoresis indicated that cellular apoptosis had been induced. The mechanism of apoptosis was evaluated in detail in uremic rats fed a high-phosphate diet. OCT or its vehicle was directly injected into the rats' PTGs. In the PTGs treated by OCT, there was a significantly increased number of TUNEL-positive PTCs and DNA electrophoresis revealed the characteristic ladder pattern of DNA fragmentation, both findings indicative of apoptosis. There was also a significant upregulation of both vitamin D and Ca-sensing receptors in the PTCs and a clear shift of the Ca-PTH response curve to the left and downward. None of these findings was observed in the PTGs treated by vehicle. This novel treatment is successful in causing regression of PTG hyperplasia. Thus, it is expected to significantly reduce the PTH level and ameliorate the abnormal bone turnover and mineral metabolism.

Direct maxacalcitol injection into hyperplastic parathyroids improves skeletal changes in secondary hyperparathyroidism.

Direct maxacalcitol (OCT) injection into a parathyroid gland (PTG) ameliorates several important etiologic factors of resistance to medical treatments for secondary hyperparathyroidism (s-HPT): the upregulations of vitamin D receptor (VDR) and Ca-sensing receptor (CaSR) in PTGs and the regression of PTG hyperplasia by the induction of apoptosis. In this study, we evaluated the bone histomorphology on the basis of maintaining these effects in advanced s-HPT. Five/six nephrectomized Sprague-Dawley rats were fed a high-phosphorus and low-calcium diet for 8 weeks. These rats were divided into four treatment groups: (1) basic uremic (at the baseline), (2) direct OCT single injection into PTGs (DI-OCT) followed by OCT intravenous administration for 4 weeks (IV-OCT), (3) direct vehicle injection and IV-OCT, and (4) no treatment for an additional 4 weeks. The effects of these treatments on serum intact-parathyroid hormone (PTH) level, PTG weight, VDR and CaSR expression levels in PTGs, and bone histomorphometric parameters were investigated. In the DI-OCT+IV-OCT group, the significant decrease in serum intact-PTH level was maintained by the following IV-OCT. A significant decrease in PTG weight and the upregulations of VDR and CaSR expression levels in PTGs were also observed. Bone histomorphometric analysis showed significant improvements in osteitis fibrosa in both cancellous and cortical bones. However, these findings were not observed in the other groups. These results suggest that osteitis fibrosa caused by advanced s-HPT can be successfully reversed by a control of PTH at an appropriate level through the improvement of PTG hyperplasia as induced by DI-OCT+IV-OCT.

Three novel polymorphisms in the beta globin gene.

We report on three novel polymorphisms in and around the beta globin gene. Two of them are intronic (IVS2) polymorphisms (IVS 2 nt 200-203 (-CTTT) and IVS2 82-83 (-AG)). The third is a novel G-->C substitution at nt +1707 related to the beta globin cap site. This +1707 G-->C polymorphism was detected in 23.5% of chromosomes among 140 samples from India. It seems to be a novel but common polymorphism among Indians. There was no linkage between these novel polymorphisms and any beta thalassemia mutation.

Isolated colonic hernia through the esophageal hiatus.

We report on a 75-year-old woman with an isolated colonic hernia through the esophageal hernia. The patient had suffered from cough, palpitation and dyspnea. A chest X-ray showed a colon loop gas in the mediastinum. Simultaneous barium swallow and enema showed the herniation of the only transverse colon into the mediastinum and displacement of the distal esophagus by the migrated colon. The patient underwent an open-mesh cruroplasty and a Hill's posterior gastropexy. The postoperative clinical course was uneventful. The patient has cessation of the symptoms. To our knowledge, there are only five reports presenting patients with isolated colonic hernia through the esophageal hiatus, including our case.

An interplay of alleviating mutations in the clinical phenotype of beta-thalassaemia intermedia.

Prediction of a beta-thalassaemia major phenotype from the beta-genotype is generally relatively straightforward. However, despite the ability to accurately define the beta-thalassaemia mutations, prediction of a beta-thalassaemia intermedia phenotype from the genotype sometimes remains problematic and this has important implications in genetic counselling and prenatal diagnosis. We report a 11-year-old Indian male child with a thalassaemia intermedia phenotype. beta-Globin gene analysis of the family showed that he was a compound heterozygote with the -88 (C-->T) beta+-mutation and the IVS1 nt 130 (G-->C) beta0-mutation. Both these mutations are rare among Indians. The propositus was also found to be heterozygous for the XmnI polymorphism and had a normal alpha-genotype. In this family interplay of two alleviating mutations (a milder promoter mutation along with a gene for raised HbF) might have synergistically compensated for lack of globin chains in the patient. Hence, the nature of the beta-genotype as well as the knowledge of the presence or absence of alleviating factors will help the clinician to decide whether early commencement of a regular transfusion regime is necessary.

T-shaped re-anastomosis of graft for outlet obstruction after free jejunal graft.

We experienced three patients with persistent outlet obstruction after free jejunal graft and performed T-shaped re-anastomosis for relief of this symptom. Two patients underwent a laryngopharyngectomy for hypopharyngeal cancer and the other patient underwent a laryngopharyngectomy and total esophagectomy for concurrent hypopharyngeal cancer and esophageal cancer. We reconstructed alimentary conduit by a free jejunal reconstruction without using surgical microscopes. In brief, a graft vein and the internal jugular vein were anastomosed and a graft artery and the carotid artery were anastomosed. Then, the anastomosis of pharyngojejunostomy was carried out in a side-to- end fashion, followed by an end-to- end jejunesophagostomy. In a T-shaped re-anastomosis, the flexure of the transplanted jejunum was separated by GIA (US Surgical Corporation, Norwalk, CT, USA). In cases where the efferent part was redundant, the proximal or distal site was resected and straightened in order to avoid outlet stasis. After this, the end-to-side anastomosis between the efferent part and the bottom of proximal horizontal portion of the graft was performed by CDH (Ethicon, Somerville, NJ, USA) or Olsen's one layer method. These three patients received this operation and were relieved from persistent dysphagia. This method is a safe and easy procedure for relief from dysphagia and for recovery of quality of life for patients with this complication. However, it is of utmost importance to perform a reconstruction followed by profluent passage at the first operation.

Large-scale isolation of ESTs from medaka embryos and its application to medaka developmental genetics.

The medaka is becoming an attractive model organism for the study of vertebrate early development and organogenesis and large-scale mutagenesis projects that are aimed at creating developmentally defective mutants are now being conducted by several groups in Japan. To strengthen the study of medaka developmental genetics, we have conducted a large-scale isolation of ESTs from medaka embryos and developed tools that facilitate mutant analysis. In this study, we have characterized a total of 132,082 sequences from both ends of cloned insert cDNAs from libraries generated at different stages of medaka embryo development. Clustering analysis with 3-prime sequences finally identified a total of 12,429 clusters. As a pilot analysis, 924 clusters were subjected to in situ hybridization to determine the spatial localization of their transcripts. Using EST sequence data generated in the present study, a 60-mer oligonucleotide microarray with 8,091 unigenes (Medaka Microarray 8K) was constructed and tested for its usefulness in expression profiling. Furthermore, we have developed a rapid and reliable mutant mapping system using a set of mapped EST markers (M-marker 2003) that covers the entire medaka genome. These resources will accelerate medaka mutant analyses and make an important contribution to the medaka genome project.

Safety and growth suppressive effect of intra-hepatic arterial injection of AdCMV-p53 combined with CDDP to rat liver metastatic tumors.

Surgical resection is thought to be the best treatment for liver carcinoma, including hepatocellular carcinoma and metastatic liver carcinoma if there are a small number of tumors. Liver carcinoma is one of the main causes of death from cancer worldwide. The prognosis of liver carcinoma is still poor. Mutation of p53, which is well known as a tumor suppressor gene, is observed in many cases of advanced liver carcinoma. Cancer gene therapy using p53, which transduces the wild-type p53 gene in the tumor, is a promising new strategy for treating liver carcinoma. Selective and less invasive gene delivery to the liver tumor is necessary for clinical liver tumor gene therapy. The first purpose of the current study was to determine the best way to deliver the gene of interest to the liver tumor selectively. The second purpose was to study the tumor suppressive effect of intrahepatic arterial injection of an adenovirus vector with the p53 gene (AdCMV-p53), followed by administration of CDDP and noting its side effects. We injected AdCMV-LacZ via hepatic arteries of rats bearing RCN-9 colon cancer metastasis in the liver. Injection via the hepatic artery resulted in more successful gene transduction to the liver tumor in a tumor-selective manner than did injection via the portal vein. At 48 hrs after arterial injection of AdCMV-p53, CDDP (3 mg/kg) was administered in the peritoneal cavity of each rat. The use of CDDP with arterial injection of AdCMV-p53 resulted in more extensive apoptosis in the rat liver tumors without any deterioration in liver function. In conclusion, hepatic arterial injection of an adenovirus vector is better than portal vein injection for gene transduction efficiency, and causes no liver function disorder even when the injection is combined with CDDP.

Adult case of squamous cell carcinoma arising on congenital esophageal stenosis due to fibromuscular hypertrophy.

This study relates to an adult case of squamous cell carcinoma arising on congenital esophageal stenosis. The patient was a 65-year-old man who had suffered from dysphagia and vomiting since birth and was diagnosed as having congenital esophageal stenosis. The patient had not received any treatment because his symptoms were mild. The patients suffered from severe dysphagia since he was 20 years old and had received balloon therapies several times; however, the effects were transient. After admission to our hospital, he underwent a transhiatal esophagectomy without thoracotomy. Histopathological examination of the resected specimen revealed a thick muscular mucosae associated with hypertrophic fibromuscular components and poorly to moderately differentiated squamous cell carcinoma in the region of stenosis. This case report is the first of a patient with squamous cell carcinoma arising on congenital esophageal stenosis.

Expression of p73 in colorectal carcinoma: clinicopathological relevance.

p73 gene expression and its correlation with clinicopathological variables were examined in 92 patients (53 male, 39 female; age range 38 - 89 years) with colorectal carcinoma. Gene expression was determined by immunohistochemical analysis of resected formalin-fixed and paraffin-embedded carcinoma specimens. When p73 staining of each specimen was divided into two modes (small expression, 0-50% and large expression, > 50%), small expression of p73 was obtained in most cases. Tumour, grade, stage, site and size did not correlate with the mode of expression, but tumour recurrence differed according to mode. Expression of p73 was also related to prognosis: large expression indicated a shorter survival period. Our findings suggest that p73 expression plays a role in inducing tumour recurrence, and that analysis of the expression is useful in evaluating the clinical course of colorectal carcinoma post-surgery.