Optimized methods for extracting circulating small RNAs from long-term stored equine samples.

Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Furthermore, we showed that miRNA of NGS-sufficient quality can be recovered from blood samples after >10 years of storage at -80 °C. This allows retrospective analyses of miRNAs from archived samples.

Jatropha mahafalensis oil from Madagascar: properties and suitability as liquid biofuel

Access to affordable and renewable sources of energy is crucial to reducing poverty and enhancing rural development in countries of the global South. Straight vegetable oil was recently identified as a possible alternative to conventional biomass for rural energy supply. In this context, the Jatropha curcas Linn. species has been extensively investigated with regard to its potential as a biofuel feedstock. In contrast, only little is known about Jatropha mahafalensis Jum. & H. Perrier, which is an indigenous and endemic representative of the Jatropha genus in Madagascar. This paper explores the potential and suitability of J. mahafalensis as a biofuel feedstock. Seed samples were collected in the area of Soalara in south-western Madagascar in February and September 2011. Two agro-ecological zones (coastal area and calcareous plateau) and two plant age groups (below and above 10 years) were considered. These four sample groups were analyzed with regard to oil properties, element contents, and fatty acid profiles. Measured values differed greatly between the two harvests, probably owing to different climatic or storage conditions. No direct relation between age of trees or location and oil quality could be established. The analyses indicate that J. mahafalensis oil can be used in oil lamps, cooking stoves and stationary combustion engines for electrification or for biodiesel production. However, modifications in storage and extraction methods, as well as further processing steps are necessary to enable its utilization as a straight vegetable oil and feedstock for biodiesel production. If these technical requirements can be met, and if it turns out that J. mahafalensis oil is economically competitive in comparison with firewood, charcoal, paraffin and petroleum, it can be considered as a promising feedstock for rural energy supply.

Weblog extraction with fuzzy classification methods

This paper uses folksonomies and fuzzy clustering algorithms to establish term-relevant related results. This paper will propose a Meta search engine with the ability to search for vaguely associated terms and aggregate them into several meaningful cluster categories. The potential of the fuzzy weblog extraction is illustrated using a simple example and added value and possible future studies are discussed in the conclusion.

Multicentre validation study of nucleic acids extraction from FFPE tissues

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Percutaneous treatment of biliary cast syndrome after orthotopic liver transplantation: comparison of mechanical versus hydraulic rheolytic cast extraction

Biliary cast syndrome (BCS) is the presence of casts within the intrahepatic or extrahepatic biliary system after orthotopic liver transplantation. Our work compares two percutaneous methods for BCS treatment: the mechanical cast-extraction technique (MCE) versus the hydraulic cast-extraction (HCE) technique using a rheolytic system.

Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine.

Re-evaluating adjuvant breast cancer trials: assessing hormone receptor status by immunohistochemical versus extraction assays

BACKGROUND: Tumor levels of steroid hormone receptors, a factor used to select adjuvant treatment for early-stage breast cancer, are currently determined with immunohistochemical assays. These assays have a discordance of 10%-30% with previously used extraction assays. We assessed the concordance and predictive value of hormone receptor status as determined by immunohistochemical and extraction assays on specimens from International Breast Cancer Study Group Trials VIII and IX. These trials predominantly used extraction assays and compared adjuvant chemoendocrine therapy with endocrine therapy alone among pre- and postmenopausal patients with lymph node-negative breast cancer. Trial conclusions were that combination therapy provided a benefit to pre- and postmenopausal patients with estrogen receptor (ER)-negative tumors but not to ER-positive postmenopausal patients. ER-positive premenopausal patients required further study. METHODS: Tumor specimens from 571 premenopausal and 976 postmenopausal patients on which extraction assays had determined ER and progesterone receptor (PgR) levels before randomization from October 1, 1988, through October 1, 1999, were re-evaluated with an immunohistochemical assay in a central pathology laboratory. The endpoint was disease-free survival. Hazard ratios of recurrence or death for treatment comparisons were estimated with Cox proportional hazards regression models, and discriminatory ability was evaluated with the c index. All statistical tests were two-sided. RESULTS: Concordance of hormone receptor status determined by both assays ranged from 74% (kappa = 0.48) for PgR among postmenopausal patients to 88% (kappa = 0.66) for ER in postmenopausal patients. Hazard ratio estimates were similar for the association between disease-free survival and ER status (among all patients) or PgR status (among postmenopausal patients) as determined by the two methods. However, among premenopausal patients treated with endocrine therapy alone, the discriminatory ability of PgR status as determined by immunohistochemical assay was statistically significantly better (c index = 0.60 versus 0.51; P = .003) than that determined by extraction assay, and so immunohistochemically determined PgR status could predict disease-free survival. CONCLUSIONS: Trial conclusions in which ER status (for all patients) or PgR status (for postmenopausal patients) was determined by immunohistochemical assay supported those determined by extraction assays. However, among premenopausal patients, trial conclusions drawn from PgR status differed--immunohistochemically determined PgR status could predict response to endocrine therapy, unlike that determined by the extraction assay.

[Tooth extraction under medication with acetylsalicylic acid.]

PURPOSE: The purpose of this prospective study was to show and analyze the bleeding complications after teeth extraction under therapy with 100 mg acetylsalicylic acid (ASA) and to compare them to bleeding complications after teeth extraction in patients with a healthy blood profile.PATIENTS AND METHODS: In 65 patients under medication with 100 mg ASA and in 252 healthy patients, 151/ 543 teeth were extracted and the bleeding complications monitored.RESULTS: The postoperative bleeding frequency was 1.54% in the ASA 100 group and 1.59% in the healthy control group without any medication. No serious or uncontrollable postoperative bleedings arose in either group. All bleedings could be easily handled. No obvious difference concerning the bleeding frequency between the two groups was observed. The small number of bleeding events and the complexity of affecting parameters did not permit statistical tests.CONCLUSION: It is not necessary to interrupt the medication of 100 mg acetylsalicylic acid given to prevent thromboembolism before tooth extractions.

Early implant placement with simultaneous guided bone regeneration following single-tooth extraction in the esthetic zone: a cross-sectional, retrospective study in 45 subjects with a 2- to 4-year follow-up

BACKGROUND: The concept of early implant placement is a treatment option in postextraction sites of single teeth in the anterior maxilla. Implant placement is performed after a soft tissue healing period of 4 to 8 weeks. Implant placement in a correct three-dimensional position is combined with a simultaneous guided bone regeneration procedure to rebuild esthetic facial hard and soft tissue contours. METHODS: In this retrospective, cross-sectional study, 45 patients with an implant-borne single crown in function for 2 to 4 years were recalled for examination. Clinical and radiologic parameters, routinely used in implant studies, were assessed. RESULTS: All 45 implants were clinically successful according to strict success criteria. The implants demonstrated ankylotic stability without signs of a peri-implant infection. The peri-implant soft tissues were clinically healthy as indicated by low mean plaque (0.42) and sulcus bleeding index (0.51) values. None of the implants revealed a mucosal recession on the facial aspect as confirmed by a clearly submucosal position of all implant shoulders. The mean distance from the mucosal margin to the implant shoulder was -1.93 mm on the facial aspect. The periapical radiographs showed stable peri-implant bone levels, with a mean distance between the implant shoulder and the first bone-implant contact of 2.18 mm. CONCLUSIONS: This retrospective study demonstrated successful treatment outcomes for all 45 implants examined. The mid-term follow-up of 2 to 4 years also showed that the risk for mucosal recession was low with this treatment concept. Prospective clinical studies are required to confirm these encouraging results.

Immediate transmucosal implant placement in molar extraction sites: a 12-month prospective multicenter cohort study

AIM: To assess the clinical and radiographic outcomes of immediate transmucosal placement of implants into molar extraction sockets. STUDY DESIGN: Twelve-month multicenter prospective cohort study. MATERIAL AND METHODS: Following molar extraction, tapered implants with an endosseous diameter of 4.8 mm and a shoulder diameter of 6.5 mm were immediately placed into the sockets. Molars with evidence of acute periapical pathology were excluded. After implant placement and achievement of primary stability, flaps were repositioned and sutured allowing a non-submerged, transmucosal healing. Peri-implant marginal defects were treated according to the principles of guided bone regeneration (GBR) by means of deproteinized bovine bone mineral particles in conjunction with a bioresrobable collagen membrane. Standardized radiographs were obtained at baseline and 12 months thereafter. Changes in depth and width of the distance from the implant shoulder (IS) and from the alveolar crest (AC) to the bottom of the defect (BD) were assessed. RESULTS: Eighty-two patients (42 males and 40 females) were enrolled and followed for 12 months. They contributed with 82 tapered implants. Extraction sites displayed sufficient residual bone volume to allow primary stability of all implants. Sixty-four percent of the implants were placed in the areas of 36 and 46. GBR was used in conjunction with the placement of all implants. No post-surgical complications were observed. All implants healed uneventfully yielding a survival rate of 100% and healthy soft tissue conditions after 12 months. Radiographically, statistically significant changes (P<0.0001) in mesial and distal crestal bone levels were observed from baseline to the 12-month follow-up. CONCLUSIONS: The findings of this 12-month prospective cohort study showed that immediate transmucosal implant placement represented a predictable treatment option for the replacement of mandibular and maxillary molars lost due to reasons other than periodontitis including vertical root fractures, endodontic failures and caries.

Early implant placement with simultaneous guided bone regeneration following single-tooth extraction in the esthetic zone: 12-month results of a prospective study with 20 consecutive patients

BACKGROUND: Early implant placement is one of the treatment options in postextraction sites in the anterior maxilla. Implant placement is performed after a soft tissue healing period of 4 to 8 weeks. Implant placement is combined with a simultaneous guided bone regeneration (GBR) procedure to rebuild esthetic facial hard and soft tissue contours. METHODS: In this prospective case-series study, 20 consecutive patients treated with an implant-borne single crown were prospectively followed for 12 months. Clinical, radiologic, and esthetic parameters were recorded to assess treatment outcomes. RESULTS: At the 12-month examination, all 20 implants were successfully integrated, demonstrating ankylotic stability and healthy peri-implant soft tissues as documented by standard parameters. The esthetic outcomes assessed by a pink esthetic score (PES) and a white esthetic score (WES) demonstrated pleasing results overall. The WES values were slightly superior to the PES values. The periapical radiographs showed minimal crestal bone loss around the used bone level implants, with mean bone loss of 0.18 mm at 12 months. Only one implant showed >0.5 mm bone loss, combined with minor mucosal recession of 0.5 to 1.0 mm. CONCLUSIONS: This prospective case series study evaluating the concept of early implant placement demonstrated successful tissue integration for all 20 implants. The short-term follow-up of 12 months revealed pleasing esthetic outcomes overall, as assessed by objective parameters. The risk for mucosal recession was low; only one patient showed minor recession of the facial mucosa. These encouraging results need to be confirmed with 3- and 5-year follow-up examinations.

Dimensional ridge alterations following immediate implant placement in molar extraction sites: a six-month prospective cohort study with surgical re-entry

AIM: To assess dimensional ridge alterations following immediate implant placement in molar extraction sites. MATERIAL AND METHODS: Twelve subjects received 12 immediate transmucosal implants in molar extraction sites. Peri-implant defects were treated according to the principles of Guided Bone Regeneration by means of a deproteinized bone substitute and a bioresorbable collagen membrane. Changes in vertical (IS-BD, CREST-BD) and horizontal distances (EC-I, IC-I) of alveolar bony walls to the bottom of the defects (BD) and to the implant surfaces (I) were compared between implant placement and surgical re-entry at 6 months. RESULTS: The implant survival rate at 6 months was 100%. Statistically significant differences (P<0.01) were observed in the mean changes in vertical distances IS-BD and CREST-BD between baseline and re-entry. At re-entry, all peri-implant marginal defects assessed from the internal socket wall to the implant surface (IC-I) were healed. The residual combined thickness of the buccal wall with the newly formed peri-implant bone at sites with an initial thickness of 1 mm was statistically significantly smaller (P<0.05) compared with that of sites with an initial buccal thickness of 2 mm (2.50 +/- 0.76 vs. 4+/-0 mm). CONCLUSIONS: The marginal defects around immediate implants placed in molar extraction sites were completely filled after 6 months of healing through de novo bone formation. Bone resorption was observed from the external aspects of the buccal and oral socket walls. Dimensional changes of the external socket walls were mostly pronounced at the buccal aspects.

Soft tissues healing at immediate transmucosal implants placed into molar extraction sites with buccal self-contained dehiscences. A 12-month controlled clinical trial

AIM: To assess soft tissues healing at immediate transmucosal implants placed into molar extraction sites with buccal self-contained dehiscences. MATERIAL AND METHODS: For this 12-month controlled clinical trial, 15 subjects received immediate transmucosal tapered-effect (TE) implants placed in molar extraction sockets displaying a buccal bone dehiscence (test sites) with a height and a width of > or =3 mm, respectively. Peri-implant marginal defects were treated according to the principles of Guided Bone Regeneration (GBR) by means of deproteinized bovine bone mineral particles in conjunction with a bioresorbable collagen membrane. Fifteen subjects received implants in healed molar sites (control sites) with intact buccal alveolar walls following tooth extraction. In total, 30 TE implants with an endosseous diameter of 4.8 mm and a shoulder diameter of 6.5 mm were used. Flaps were repositioned and sutured, allowing non-submerged, transmucosal soft tissues healing. At the 12-month follow-up, pocket probing depths (PPD) and clinical attachment levels (CAL) were compared between implants placed in the test and the control sites, respectively. RESULTS: All subjects completed the 12-month follow-up period. All implants healed uneventfully, yielding a survival rate of 100%. After 12 months, statistically significantly higher (P<0.05) PPD and CAL values were recorded around implants placed in the test sites compared with those placed in the control sites. CONCLUSIONS: The findings of this controlled clinical trial showed that healing following immediate transmucosal implant installation in molar extraction sites with wide and shallow buccal dehiscences yielded less favorable outcomes compared with those of implants placed in healed sites, and resulted in lack of 'complete' osseointegration.

Parameter-free extraction of the functional network topology from intracranial EEG recordings: a time-resolved study of graph properties in focal onset seizures

Rationale: Focal onset epileptic seizures are due to abnormal interactions between distributed brain areas. By estimating the cross-correlation matrix of multi-site intra-cerebral EEG recordings (iEEG), one can quantify these interactions. To assess the topology of the underlying functional network, the binary connectivity matrix has to be derived from the cross-correlation matrix by use of a threshold. Classically, a unique threshold is used that constrains the topology [1]. Our method aims to set the threshold in a data-driven way by separating genuine from random cross-correlation. We compare our approach to the fixed threshold method and study the dynamics of the functional topology. Methods: We investigate the iEEG of patients suffering from focal onset seizures who underwent evaluation for the possibility of surgery. The equal-time cross-correlation matrices are evaluated using a sliding time window. We then compare 3 approaches assessing the corresponding binary networks. For each time window: * Our parameter-free method derives from the cross-correlation strength matrix (CCS)[2]. It aims at disentangling genuine from random correlations (due to finite length and varying frequency content of the signals). In practice, a threshold is evaluated for each pair of channels independently, in a data-driven way. * The fixed mean degree (FMD) uses a unique threshold on the whole connectivity matrix so as to ensure a user defined mean degree. * The varying mean degree (VMD) uses the mean degree of the CCS network to set a unique threshold for the entire connectivity matrix. * Finally, the connectivity (c), connectedness (given by k, the number of disconnected sub-networks), mean global and local efficiencies (Eg, El, resp.) are computed from FMD, CCS, VMD, and their corresponding random and lattice networks. Results: Compared to FMD and VMD, CCS networks present: *topologies that are different in terms of c, k, Eg and El. *from the pre-ictal to the ictal and then post-ictal period, topological features time courses that are more stable within a period, and more contrasted from one period to the next. For CCS, pre-ictal connectivity is low, increases to a high level during the seizure, then decreases at offset. k shows a ‘‘U-curve’’ underlining the synchronization of all electrodes during the seizure. Eg and El time courses fluctuate between the corresponding random and lattice networks values in a reproducible manner. Conclusions: The definition of a data-driven threshold provides new insights into the topology of the epileptic functional networks.

Long-term stability of contour augmentation with early implant placement following single tooth extraction in the esthetic zone: a prospective, cross-sectional study in 41 patients with a 5- to 9-year follow-up

BACKGROUND Early implant placement with simultaneous contour augmentation is documented with short- and medium-term studies. The long-term stability of contour augmentation is uncertain. METHODS In this prospective, cross-sectional study, 41 patients with an implant-borne single crown were examined twice, in 2006 and 2010. Clinical, radiologic, and esthetic parameters were assessed at both examinations. In addition, a cone beam computed tomographic (CBCT) image was obtained during the second examination to assess the dimensions of the facial bone wall. RESULTS All 41 implants demonstrated ankylotic stability without signs of peri-implant infection at both examinations. The clinical parameters remained stable over time. Satisfactory esthetic outcomes were noted, as assessed by the pink and white esthetic score (PES/WES) indices. Overall, the PES scores were slightly higher than the WES scores. None of the implants developed mucosal recession over time, as confirmed by values of the distance between implant shoulder and mucosal margin and cast measurements. The periapical radiographs yielded stable peri-implant bone levels, with a mean distance between implant shoulder and first visible bone-implant contact value of 2.18 mm. The CBCT analysis demonstrated a mean thickness of the facial bone wall ≈2.2 mm. In two implants (4.9%) no facial bone wall was detectable radiographically. CONCLUSIONS This prospective cross-sectional study demonstrates stable peri-implant hard and soft tissues for all 41 implants examined and satisfactory esthetic outcomes overall. The follow-up of 5 to 9 years confirmed again that the risk for mucosal recession is low with early implant placement. In addition, contour augmentation with guided bone regeneration was able to establish and maintain a facial bone wall in 95% of patients.