Rapid Detection of high-level oncogene amplifications in ultrasonic surgical aspirations of brain tumors

ABSTRACT: BACKGROUND: Genomic tumor information, such as identification of amplified oncogenes, can be used to plan treatment. The two sources of a brain tumor that are commonly available include formalin-fixed, paraffin-embedded (FFPE) sections from the small diagnostic biopsy and the ultrasonic surgical aspiration that contains the bulk of the tumor. In research centers, frozen tissue of a brain tumor may also be available. This study compared ultrasonic surgical aspiration and FFPE specimens from the same brain tumors for retrieval of DNA and molecular assessment of amplified oncogenes. METHODS: Surgical aspirations were centrifuged to separate erythrocytes from the tumor cells that predominantly formed large, overlying buffy coats. These were sampled to harvest nuclear pellets for DNA purification. Four glioblastomas, 2 lung carcinoma metastases, and an ependymoma were tested. An inexpensive PCR technique, multiplex ligation-dependent probe amplification (MLPA), quantified 79 oncogenes using 3 kits. Copy number (CN) results were normalized to DNA from non-neoplastic brain (NB) in calculated ratios, [tumor DNA]/[NB DNA]. Bland-Altman and Spearman rank correlative comparisons were determined. Regression analysis identified outliers. RESULTS: Purification of DNA from ultrasonic surgical aspirations was rapid (<3 days) versus FFPE (weeks) and yielded greater amounts in 6 of 7 tumors. Gene amplifications up to 15-fold corresponded closely between ultrasonic aspiration and FFPE assays in Bland-Altman analysis. Correlation coefficients ranged from 0.71 to 0.99 using 3 kit assays per tumor. Although normalized CN ratios greater than 2.0 were more numerous in FFPE specimens, some were found only in the ultrasonic surgical aspirations, consistent with tumor heterogeneity. Additionally, CN ratios revealed 9 high-level ([greater than or equal to] 6.0) gene amplifications in FFPE of which 8 were also detected in the ultrasonic aspirations at increased levels. The ultrasonic aspiration levels of these amplified genes were also greater than 6.0 CN ratio, except in one case (3.53 CN ratio). Ten of 17 mid-level ([greater than or equal to]3.0 & <6.0 CN ratio) amplifications detected in FFPE were also detected as being increased ([greater than or equal to] 2.0 CN ratio) in the aspirations. CONCLUSIONS: Buffy coats of centrifuged ultrasonic aspirations contained abundant tumor cells whose DNA permitted rapid, multiplex detection of high-level oncogene amplifications that were confirmed in FFPE. Virtual slides http://www.diagnosticpathology.diagnomx.eu/vs/1883718801686466.     

Microbial production host selection for converting second-generation feedstocks into bioproducts

Although many secondary metabolites with diverse biological activities have been isolated from myxobacteria, most strains of these biotechnologically important gliding prokaryotes remain difficult to handle genetically. In this study we describe the new fast growing myxobacterial thermophilic isolate GT-2 as a heterologous host for the expression of natural product biosynthetic pathways isolated from other myxobacteria. According to the results of sequence analysis of the 16S rDNA, this moderately thermophilic isolate is closely related to Corallococcus macrosporus and was therefore named C. macrosporus GT-2. Fast growth of moderately thermophilic strains results in shorter fermentation and generation times, aspects which are of significant interest for molecular biological work as well as production of secondary metabolites. Development of a genetic manipulation system allowed the introduction of the complete myxochromide biosynthetic gene cluster, located on a transposable fragment, into the chromosome of GT-2. Genetic engineering of the biosynthetic gene cluster by promoter exchange leads to much higher production of myxochromides in the heterologous host C. macrosporus GT-2 in comparison to the original producer Stigmatella aurantiaca and to the previously described heterologous host Pseudomonas putida (600 mg/L versus 8 mg/L and 40 mg/L, respectively).     

Environmental epidemiological study and estimation of benchmark dose for renal dysfunction in a cadmium-polluted area in China

We have performed a study aimed at investigating the critical concentration of urinary cadmium (UCd) required for the development of renal dysfunction. We studied population groups (totally 790 persons) living in two cadmium exposed areas and one control area in China. UCd, was determined as an indicator of cadmium exposure and accumulation, while the concentrations of N-acetyl-beta-D-glucosaminidase (NAG), its iso-form B (NAG-B), beta2-microglobulin (B2M), retinol binding protein (RBP), and albumin (ALB) in urine were measured as indicators of the renal effects caused by cadmium. There was a significantly increased prevalence of hyperNAGuria, hyperNAG-Buria, hyperB2Muria, hyperRBPuria and hyperALBuria with increasing levels of Cd excretion in urine. We used the benchmark dose (BMD) procedure to estimate the critical concentration of urinary cadmium in this general population. The lower confidence limit of the BMD (LBMD-05) of urinary cadmium for a 5% level of risk above the background level was estimated for each of the renal effect indicators. The BMD-05/LBMD-05 were estimated to be 4.46/3.99, 6.70/5.87, 8.36/7.31, 7.98/6.98 and 15.06/12.18 microg/g creatinine for urinary NAG-B, NAG, B2M, RBP and ALB, respectively. Our findings suggest, based on the present study, that the Lower Confidence Limit of the Population Critical Concentration of UCd (LPCCUCd-05) of tubular dysfunction for 5% excess risk level above the background may be ca. 3-4 microg/g creatinine, and that cadmium concentration in urine should be kept below this level to prevent renal tubular damage. This report is the first to use the BMD method in this field and to define the concept of critical concentration in urine.     

Effects on the prostate of environmental cadmium exposure – A cross-sectional population study in China

To explore possible effects of environmental cadmium exposure on prostate in humans, and the possible relationship of serum sex hormones to occurrence of clinic signs of tissue changes in the prostate, a case-control study was undertaken in the southeast part of China in 1998. A total of 297 male volunteers from a control area and two cadmium-polluted areas were included as subjects in this study. All the subjects were required to answer a questionnaire and to undergo a complete physical examination including digital-rectal examination (DRE). Blood and urine samples were collected. Serum total prostate specific antigen (PSA), total serum testosterone (T), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured by radioimmunoassay and enzymeimmunoassay method, respectively. The data of urinary cadmium (U-Cd) and blood cadmium (B-Cd) were obtained by atomic absorption spectrometry (AAS) as an indicator of cadmium body burden. Statistical analysis was applied to investigate a possible relation between cadmium exposure and prostate pathological changes. The results show that there is a clear dose-response relationship between cadmium exposure and the prevalence of cases with abnormal PSA. The blood cadmium content in cases with positive DRE was significantly higher than that of subjects with negative DRE (P<0.05). Significant differences in the level of FSH between cases with positive DRE and the normal subjects were also noted (P<0.05). These results indicate that chronic environmental cadmium exposure is associated with injuries to human prostate. A possible relationship to changes in circulating sex hormones needs further investigation.     

Relationship between mercury concentration in blood, cognitive performance, and blood pressure, in an elderly urban population

Concentration of mercury (Hg) in whole blood in an elderly urban population with a mean age of 87 years was studied in relation to cognitive function, arterial blood pressure (arterial BP), age, gender, body mass index (BMI) and smoking habits. This population-based study consisted of 106 subjects both males and females. Clinical assessment of the subjects included medical and social history, physical and neurologic examination and assessment of cognitive functions with Mini-Mental State Examination (MMSE). Information on use of all potentially anti-hypertensive drugs was collected. Whole blood from 106 subjects were collected and analysed for mercury by Cold Vapour Atomic Absorption Spectrometry (Milton Ray ASS-CV) with Seronorm Trace Element as matrix matched quality control. Males and females did not differ in blood-mercury (B-Hg) concentrations or in any of the other studied variables. B-Hg concentrations did not differ between smokers and non-smokers. Females were treated more often than males with antihypertensive drugs. There was no relation found between B-Hg concentration and cognitive function, arterial BP, age, gender or BMI. In conclusion, no relations were found between B-Hg concentrations and the studied variables.     

Splinting or surgery for carpal tunnel syndrome? Design of a randomized controlled trial [ISRCTN18853827]

Background  

Carpal tunnel syndrome is a common disorder, which can be treated with surgery or conservative options. However, there is insufficient evidence and no consensus among physicians with regard to the preferred treatment for carpal tunnel syndrome. Therefore, a randomized controlled trial is conducted to compare the short- and long-term efficacy of surgery and splinting in patients with carpal tunnel syndrome. An attempt is also made to avoid the (methodological) limitations encountered in earlier trials on the efficacy of various treatment options for carpal tunnel syndrome.     

A comparison in product‐value potential in four treatment strategies for food waste and faeces – assessing composting,fly larvae composting and anaerobic digestion

Municipalities are expected to provide solid waste management, which is funded by tax revenue or/and waste treatment fees. In many low‐ and middle‐income countries, municipalities struggle to provide an adequate level of service, and in these places, the informal sector plays a major role in the collection and treatment of solid waste. In contrast to the plastic and metal fraction, the organic fraction is not managed by the informal sector, primarily because it has low or no financial value and treatment would cost more than the possible revenue. If the organic fraction could be converted to valuable products, the treatment could bear its own cost and this could act as an incentive to collect and treat this fraction. In this study, the potential product value generated through four treatment strategies treating food waste and faeces was compared in a Swedish context: (i) thermophilic composting; (ii) black soldier fly treatment (BSF treatment); (iii) anaerobic digestion (AD); and (iv) BSF treatment followed by AD (BSF + AD). In order to assess the AD strategies, the biomethane potentials of the substrates were assessed. Food waste had the highest biomethane potential, while BSF‐treated faeces had the lowest (417 and 188 NmL g VS^?1, respectively). Thermophilic composting yielded the lowest value product (organic fertilizer; 26 € t^?1 treated food waste) and BSF treatment + AD the highest total value of products (animal feed, vehicle gas and organic fertilizer; 215 € t^?1 treated food waste). The treatment costs were not taken into account here; the total value gives an indication of the cost margin for the different strategies studied. In places with an existing AD plant, BSF treatment + AD strategy is the most economically viable. In places where no such plant exists, BSF treatment is likely to be the most economically favourable treatment.     

AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple‐drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β‐fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn‐type or DAP‐type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature T _m 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.