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The accumulation of the microtubule-associated tau protein in and around blood vessels contributes to brain microvascular dysfunction through mechanisms that are incompletely understood. Delivery of nutrients to active neurons in the brain relies on capillary calcium (Ca 2+ ) signals to direct blood flow. The initiation and amplification of endothelial cell Ca 2+ signals require an intact microtubule cytoskeleton. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced microtubule destabilization would impair endothelial Ca 2+ signaling. We tested the hypothesis that tau disrupts the regulation of local cerebral blood flow by reducing endothelial cell Ca 2+ signals and endothelial-dependent vasodilation. We used a pathogenic soluble tau peptide (T-peptide) model of tau aggregation and mice with genetically encoded endothelial Ca 2+ sensors to measure cerebrovascular endothelial responses to tau exposure. T-peptide significantly attenuated endothelial Ca 2+ activity and cortical capillary blood flow in vivo . Further, T-peptide application constricted pressurized cerebral arteries and inhibited endothelium-dependent vasodilation. This study demonstrates that pathogenic tau alters cerebrovascular function through direct attenuation of endothelial Ca 2+ signaling and endothelium-dependent vasodilation., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Optimal bladder compliance is essential to urinary bladder storage and voiding functions. Calculated as the change in filling volume per change in pressure, bladder compliance is used clinically to characterize changes in bladder wall biomechanical properties that associate with lower urinary tract dysfunction. But because this method calculates compliance without regard to wall structure or wall volume, it gives little insight into the mechanical properties of the bladder wall during filling. Thus, we developed Pentaplanar Reflected Image Macroscopy (PRIM): a novel ex vivo imaging method to accurately calculate bladder wall stress and stretch in real time during bladder filling. The PRIM system simultaneously records intravesical pressure, infused volume, and an image of the bladder in five distinct visual planes. Wall thickness and volume were then measured and used to calculate stress and stretch during filling. As predicted, wall stress was nonlinear; only when intravesical pressure exceeded ~ 15 mmHg did bladder wall stress rapidly increase with respect to stretch. This method of calculating compliance as stress vs stretch also showed that the mechanical properties of the bladder wall remain similar in bladders of varying capacity. This study demonstrates how wall tension, stress and stretch can be measured, quantified, and used to accurately define bladder wall biomechanics in terms of actual material properties and not pressure/volume changes. This method is especially useful for determining how changes in bladder biomechanics are altered in pathologies where profound bladder wall remodeling occurs, such as diabetes and spinal cord injury., (© 2023. The Author(s).)

Background: Frequent blood donors are at high risk of developing iron deficiency. Currently, there is no potent screening during blood donation to detect iron deficient erythropoiesis (IDE) before anemia develops and deferral from donation is inevitable., Study Design and Methods: In addition to capillary and venous hemoglobin, the iron status of 99 frequent blood donors was assessed by various venous blood parameters and zinc protoporphyrin IX (ZnPP). ZnPP was determined by high-performance liquid chromatography (HPLC) and a new prototype fiber-optic device was employed for non-invasive measurements of ZnPP through the blood collection tubing (NI-tubing) and on lip tissue (NI-lip). We aimed to evaluate the feasibility and diagnostic value of the NI-tubing measurement for early detection of severe iron deficiency in blood donors., Results: NI-tubing and HPLC reference measurements of ZnPP showed narrow limits of agreement of 12.2 μmol ZnPP/mol heme and very high correlation (Spearman's Rho = 0.938). Using a cutoff of 65 μmol ZnPP/mol heme, NI-tubing measurements ( n = 93) identified 100% of donors with iron deficiency anemia (IDA) and an additional 38% of donors with IDE. Accordingly, NI-tubing measurements would allow detection and selective protection of particularly vulnerable donors., Conclusion: NI-tubing measurements are an accurate and simple method to implement ZnPP determination into the routine blood donation process. ZnPP was able to identify the majority of subjects with IDE and IDA and might therefore be a valuable tool to provide qualified information to donors about dietary measures and adjustments of the donation interval and thereby help to prevent IDA and hemoglobin deferral in the future., Competing Interests: A.S., A.L., L.H., M.V., and R.S. have disclosed no conflicts of interest. CH is shareholder and managing director of the start-up company FerroSens GmbH (Munich, Germany), which aims at commercializing the technology for NI detection of ZnPP. C.H., G.H., and H.S. are co-inventors of the patent family with application number PCT/EP2016/053389 “Apparatus and method for fluorescence measurements on tissue for the determination of blood fluorophores.” F.W. and E.Q. are affiliated to the company “Blutspendedienst des Bayerischen Roten Kreuzes gemeinnützige GmbH” (Munich, Germany)., (Copyright © 2023 by The Author(s). Published by S. Karger AG, Basel.)

Background: Trauma patients with abnormal fibrinolysis have increased morbidity and mortality. Knowledge of mechanisms differentiating fibrinolytic phenotypes is important to optimize treatment. We hypothesized that subjects with abnormal fibrinolysis identified by whole blood viscoelastometry can also be distinguished by plasma thrombin generation, clot structure, fibrin formation, and plasmin generation measurements., Methods: Platelet-poor plasma (PPP) from an observational cross-sectional trauma cohort with fibrinolysis shutdown (% lysis at 30 minutes [LY30] < 0.9, n = 11) or hyperfibrinolysis (LY30 > 3%, n = 9) defined by whole blood thromboelastography were studied. Noninjured control subjects provided comparative samples. Thrombin generation, fibrin structure and formation, and plasmin generation were measured by fluorescence, confocal microscopy, turbidity, and a fluorescence-calibrated plasmin assay, respectively, in the absence/presence of tissue factor or tissue plasminogen activator (tPA)., Results: Whereas spontaneous thrombin generation was not detected in PPP from control subjects, PPP from hyperfibrinolysis or shutdown patients demonstrated spontaneous thrombin generation, and the lag time was shorter in hyperfibrinolysis versus shutdown. Addition of tissue factor masked this difference but revealed increased thrombin generation in hyperfibrinolysis samples. Compared with shutdown, hyperfibrinolysis PPP formed denser fibrin networks. In the absence of tPA, the fibrin formation rate was faster in shutdown than hyperfibrinolysis, but hyperfibrinolysis clots lysed spontaneously; these differences were masked by addition of tPA. Tissue plasminogen activator-stimulated plasmin generation was similar in hyperfibrinolysis and shutdown samples. Differences in LY30, fibrin structure, and lysis correlated with pH., Conclusion: This exploratory study using PPP-based assays identified differences in thrombin generation, fibrin formation and structure, and lysis in hyperfibrinolysis and shutdown subgroups. These groups did not differ in their ability to promote tPA-triggered plasmin generation. The ability to characterize these activities in PPP facilitates studies to identify mechanisms that promote adverse outcomes in trauma., Level of Evidence: Prognostic/Epidemiological; Level III., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Aneurysms of the ascending aorta, unrelated to xenobiotic administration, are described in 5 rats and 2 mice in nonclinical safety studies conducted at Charles River Laboratories (CRL) sites over the past 10 years. The most prominent microscopic finding was focal dilation with disruption of the wall of the ascending aorta with chronic adventitial inflammation or fibroplasia. The pathogenesis of this finding is unknown. There were no associated macroscopic findings, clinical abnormalities, or vascular lesions elsewhere. The results of a search of historical control data from toxicology studies of 1 day to 72 weeks' duration performed at CRL for aortic findings from 5900 mice and 23,662 rats are also reported. Aortic lesions are uncommon in mice and rats used in nonclinical safety studies, but toxicologic pathologists should be aware that aneurysms of the ascending aorta with fibroplasia and inflammation in the aortic wall and adventitia may occur spontaneously or iatrogenically, as they have the potential to impact interpretation in toxicology studies.

Monitoring of transcranial electrical motor evoked potentials (tcMEP) during carotid endarterectomy (CEA) has been shown to effectively detect intraoperative cerebral ischemia. The unique purpose of this study was to evaluate changes of MEP amplitude (AMP), area under the curve (AUC) and signal morphology (MOR) as additional MEP warning criteria for clamping-associated ischemia during CEA. Therefore, the primary outcome was the number of MEP alerts (AMP, AUC and MOR) in the patients without postoperative motor deficit (false positives). We retrospectively reviewed data from 571 patients who received CEA under general anesthesia. Monitoring of somatosensory evoked potentials (SSEP) and tcMEP was performed in all cases (all-or-none MEP warning criteria). The percentages of false positives (primary parameter) of AMP, AUC and MOR were evaluated according to the postoperative motor outcome. In the cohort of 562 patients, we found significant SSEP/MEP changes in 56 patients (9.96%). In 44 cases (7.83%) a shunt was inserted. Nine patients (1.57%) were excluded due to MEP recording failure. False positives were registered for AMP, AUC and MOR changes in 121 (24.01%), 148 (29.36%) and 165 (32.74%) patients, respectively. In combination of AMP/AUC and AMP/AUC/MOR false positives were found in 9.52% and 9.33% of the patients. This study is the first to evaluate the correctness of the MEP warning criteria AMP, AUC and MOR with regard to false positive monitoring results in the context of CEA. All additional MEP warning criteria investigated produced an unacceptably high number of false positives and therefore may not be useful in carotid surgery for adequate detection of clamping-associated ischemia.

Methanol is a sustainable substrate for biotechnology. In addition to natural methylotrophs, metabolic engineering has gained attention for transfer of methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cut-offs, i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes., Competing Interests: The authors declare no conflict of interest.

Background: Both hyperfibrinolysis and fibrinolysis shutdown can occur after severe trauma. The subgroup of trauma patients with fibrinolysis shutdown resistant to tissue plasminogen activator (t-PA)-mediated fibrinolysis have increased mortality. Fibrin polymerization and structure may influence fibrinolysis subgroups in trauma, but fibrin architecture has not been characterized in acutely injured subjects. We hypothesized that fibrin polymerization measured in situ will correlate with fibrinolysis subgroups., Methods: Blood samples were collected from trauma patients and noninjured controls. We selected samples across a range of fibrinolysis phenotypes (shutdown, physiologic, hyperfibrinolysis) and t-PA sensitivities (sensitive, physiologic, resistant) determined by thrombelastography. Plasma clots were created in situ with fluorescent fibrinogen and imaged using confocal microscopy for analysis of clot architecture in three dimensions. For each clot, we quantified the fiber resolvability, a metric of fiber distinctness or clarity, by mapping the variance of fluorescence intensity relative to background fluorescence. We also determined clot porosity by measuring the size and distribution of the gaps between fibrin fibers in three-dimensional space. We compared these measures across fibrinolysis subgroups., Results: Fiber resolvability was significantly lower in all trauma subgroups compared with controls (n = 35 and 5, respectively; p < 0.05). We observed markedly different patterns of fibrin architecture among trauma patients stratified by fibrinolysis subgroup. Subjects with t-PA-resistant fibrinolysis shutdown exhibited abnormal, densely packed fibrin clots nearly devoid of pores. Individuals with t-PA-hypersensitive fibrinolysis shutdown had highly irregular clots with pores as large as 2500 μm to 20,000 μm, versus 78 μm to 1250 μm in noninjured controls., Conclusion: Fiber resolvability was significantly lower in trauma patients than controls, and subgroups of fibrinolysis differ in the porosity of the fibrin clot structure. The dense fibrin network in the t-PA-resistant group may prevent access to plasmin, suggesting a mechanism for thrombotic morbidity after injury.

Background: Approximately every third surgical patient is anemic. The most common form, iron deficiency anemia, results from persisting iron-deficient erythropoiesis (IDE). Zinc protoporphyrin (ZnPP) is a promising parameter for diagnosing IDE, hitherto requiring blood drawing and laboratory workup., Study Design and Methods: Noninvasive ZnPP (ZnPP-NI) measurements are compared to ZnPP reference determination of the ZnPP/heme ratio by high-performance liquid chromatography (ZnPP-HPLC) and the analytical performance in detecting IDE is evaluated against traditional iron status parameters (ferritin, transferrin saturation [TSAT], soluble transferrin receptor-ferritin index [sTfR-F], soluble transferrin receptor [sTfR]), likewise measured in blood. The study was conducted at the University Hospitals of Frankfurt and Zurich., Results: Limits of agreement between ZnPP-NI and ZnPP-HPLC measurements for 584 cardiac and noncardiac surgical patients equaled 19.7 μmol/mol heme (95% confidence interval, 18.0-21.3; acceptance criteria, 23.2 μmol/mol heme; absolute bias, 0 μmol/mol heme). Analytical performance for detecting IDE (inferred from area under the curve receiver operating characteristics) of parameters measured in blood was: ZnPP-HPLC (0.95), sTfR (0.92), sTfR-F (0.89), TSAT (0.87), and ferritin (0.67). Noninvasively measured ZnPP-NI yielded results of 0.90., Conclusion: ZnPP-NI appears well suited for an initial IDE screening, informing on the state of erythropoiesis at the point of care without blood drawing and laboratory analysis. Comparison with a multiparameter IDE test revealed that ZnPP-NI values of 40 μmol/mol heme or less allows exclusion of IDE, whereas for 65 μmol/mol heme or greater, IDE is very likely if other causes of increased values are excluded. In these cases (77% of our patients) ZnPP-NI may suffice for a diagnosis, while values in between require analyses of additional iron status parameters., (© 2019 The Authors. Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.)

Background: Reliable screening for iron deficiency (ID) has required a blood sample and cost-intensive laboratory measurements. A novel method to non-invasively measure erythrocyte zinc protoporphyrin (ZnPP), an established marker for ID, is evaluated in children., Methods: ZnPP was determined non-invasively by fluorescence measurements on the wet vermillion of the lower lip in 99 hospitalized children aged 9 months to 5 years. For comparison, conventional ID parameters and ZnPP were determined from blood samples., Results: The non-invasively measured ZnPP values had limits of agreement (LoA) of 14 µmol ZnPP/mol heme (95% confidence interval: 9-20) compared to fluorescence measurements directly in blood. Repeated high-performance liquid chromatography reference determinations had comparable LoA of 14 µmol ZnPP/mol heme (9-17). Non-invasive ZnPP measurements had sensitivity and specificity of 67% (39-88%) and 97% (91-99%), and negative and positive predictive value of 94% (90-97%) and 80% (55-93%), for detecting ID as defined by the soluble transferrin receptor (sTfR). In groups with more severe ID as defined by serum ferritin and sTfR, higher ZnPP values were found, with the highest ZnPP values for the group with ID anemia., Conclusion: Non-invasive ZnPP measurements are reliably feasible in children. The simple and fast method has the potential to enable wide-spread screening for ID.

Background: Assessment of hemolysis in vivo is becoming increasingly relevant in critical care. Current methods (Harboe, 1959) for quantifying the free hemoglobin (fHb) content produce unsatisfactory results in case of hyperbilirubinemia, a frequent condition in patients at risk for intravascular hemolysis., Methods: A novel evaluation method based on second-derivative fitting to quantify fHb content was developed. The method uses spectrophotometric data from 350 to 650 nm recorded with standard instruments as input. To evaluate the power of the new method, plasma of patients and non-icteric plasma of healthy volunteers were spiked with fHb concentrations up to 2000 mg/L and compared to methods described in the literature by Harboe, Noe and Fairbanks. All measurements were done in compliance with the bioanalytical method validation protocol from the European Medicines Agency., Results: Both the second-derivative fitting algorithm as well as the methods of Harboe, Noe and Fairbanks quantified fHb accurately in non-icteric samples, with inaccuracy and imprecision below 10%. For icteric specimen, false high results were obtained with the established formulas for fHb concentrations below 700 mg/L. In contrast, no interference was found with the second-derivate fitting method for bilirubin concentrations up to 465 μmol/L. The lower limits of quantifications for the second-derivative fitting algorithm were specified in agreement with the EMA guideline with 25 mg/L fHb for both non-icteric and icteric specimens., Conclusions: A user-friendly, computer-based algorithm is reported that allows the accurate quantification of fHb concentrations in the presence of high bilirubin concentrations. The new method allows for uniform sample preparation with only a single dilution step and can be readily implemented in any laboratory on standard spectrophotometers using the provided supplementary Microsoft Excel macro., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Autosomal-dominant acute porphyria, a group of rare diseases, can lead to life-threatening neurovisceral attacks. No efficient screening test is available today. Elevated urinary porphobilinogen in addition to elevated porphyrins is highly specific for an attack of acute porphyria. This study proposes and evaluates a custom-made device, algorithm, and methods for a two-step quantification of urinary porphyrins and porphobilinogen. The first step is oxidation of the nonfluorescent porphyrinogens and subsequent fluorescence-spectroscopic determination of total urinary porphyrins (TUP) using second derivative spectral fitting. Photo-oxidation is compared with chemical oxidation methods. The second step is the quantification of porphobilinogen in case of elevated TUP. Heat-induced conversion products of porphobilinogen, namely uroporphyrin and porphobilin, are quantified by fluorescence and absorption spectroscopy. Results show that the preferred method combination is TUP quantification (lower limit of quantification: 0.2  μmol  /  L) after photo-oxidation with subsequent absorption-spectroscopic determination of porphobilin after heating for indirect quantification of porphobilinogen (quantification range: 0 to 20  mg  /  L). Urinary porphobilinogen and porphyrins of one acute porphyria patient were quantified with <10  %   deviation from an external reference determination. The spectrophotometric approach requires only minimal sample processing and yields a result within 15 min, thus closing the screening gap for acute porphyria., ((2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Background: Baroreceptor-activating therapy (BAT) has been shown to control resistant hypertension in one sham-controlled and further observational studies. Incremental but significant reincrease of blood pressure (BP) have been described after open-label temporary withdrawal of such therapy., Method: Our study in 16 randomized patients investigated the course of automated office, ambulatory, and home BP in a randomized, controlled cross-over design., Results: After 4 weeks of blinded and randomized withdrawal in hypertension-controlled long-term carriers of BAT (2.67 ± 1.3 years, 145/104 mmHg), the primary end point of 35 mmHg difference, similar to initial BP drop after BAT initiation, was not reached in any patient. Ambulatory BP rose significantly during BAT off by 10/8 ± 4/3 mmHg (3.13/2.10, P = 0.007/0.002) and automated office BP by 10/4 ± 2/1 (4.17/0.58, P = 0.005/0.03) at 4 weeks after BAT on while mean home BP did not change significantly by 2/2 ± 3/2 mmHg (-5.9/-3.5, P = 0.6/0.5)., Conclusion: Our data in a limited study population show, that BP rise after temporary BAT withdrawal is significant but does not reach a magnitude comparable with the initial drop after de novo implantation. Such results points to preserved hypertension control after electrical BAT withdrawal and deserves further pathophysiological and clinical clarification.

An 8-year-old boy suffering from progressive glioblastoma was scheduled for neurosurgery. Prior to induction of anaesthesia pulse oximetry measured 64 % saturation of oxygen (SpO 2 ). Arterial blood gas analysis revealed normal oxygen saturation and normal oxygen partial pressure. After having ruled out technical problems of pulse oximetry the neurosurgical procedure was halted. Meticulous examination of the child's history and medication did not explain a possible interaction of drugs with pulse oximetry. A Chinese herb tea had been given to the child, but was then stopped on the day of admission. The surgical procedure took place the next day without any complications. During the subsequent inpatient stay, repeated blood gas analyses showed normal oxygenation, but pulse oximetry measured initially SpO 2 values of 64 %, gradually increasing over 7 days up to 91 % by the time of discharge from hospital. Blood samples were taken and analysed. Absorption spectroscopy from the patient's blood showed an uncommon absorption maximum at 684 nm besides the normal maxima. The normalisation of SpO 2 values after stopping Chinese herb tea administration leads to the conclusion that one of its ingredients caused the distorted pulse oximetry measurement.

Most biotechnological processes are based on glucose that is either present in molasses or generated from starch by enzymatic hydrolysis. At the very high, million-ton scale production volumes, for instance for fermentative production of the biofuel ethanol or of commodity chemicals such as organic acids and amino acids, competing uses of carbon sources e.g. in human and animal nutrition have to be taken into account. Thus, the biotechnological production hosts E. coli, C. glutamicum, pseudomonads, bacilli and Baker's yeast used in these large scale processes have been engineered for efficient utilization of alternative carbon sources. This flexible feedstock concept is central to the use of non-glucose second and third generation feedstocks in the emerging bioeconomy. The metabolic engineering efforts to broaden the substrate scope of E. coli, C. glutamicum, pseudomonads, B. subtilis and yeasts to include non-native carbon sources will be reviewed. Strategies to enable simultaneous consumption of mixtures of native and non-native carbon sources present in biomass hydrolysates will be summarized and a perspective on how to further increase feedstock flexibility for the realization of biorefinery processes will be given., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Worldwide, more individuals have iron deficiency than any other health problem. Most of those affected are unaware of their lack of iron, in part because detection of iron deficiency has required a blood sample. Here we report a non-invasive method to optically measure an established indicator of iron status, red blood cell zinc protoporphyrin, in the microcirculation of the lower lip. An optical fibre probe is used to illuminate the lip and acquire fluorescence emission spectra in ∼1 min. Dual-wavelength excitation with spectral fitting is used to distinguish the faint zinc protoporphyrin fluorescence from the much greater tissue background fluorescence, providing immediate results. In 56 women, 35 of whom were iron-deficient, the sensitivity and specificity of optical non-invasive detection of iron deficiency were 97% and 90%, respectively. This fluorescence method potentially provides a rapid, easy to use means for point-of-care screening for iron deficiency in resource-limited settings lacking laboratory infrastructure.

Background: Resistant arterial hypertension and chronic kidney disease (CKD) are interlinked via sympathetic overactivity. Baroreflex activation therapy (BAT) is a well tolerated therapy, which has been shown to reduce BP in patients with resistant hypertension. The effects of BAT in patients with resistant hypertension and end stage renal disease have not been reported., Method and Results: We retrospectively analyzed procedural effectiveness and safety in seven CKD stage 5D patients with resistant hypertension who underwent BAT. One year after activation, office SBP decreased significantly from 194 ± 28 to 137 ± 16 mmHg (P < 0.01). Ambulatory SBP showed a trend to be decreased from 167 ± 30 to 137 ± 24 mmHg (P = 0.17), whereas the median number of prescribed antihypertensive classes decreased from 5 (4-9) to 3 (1-4) (P = 0.01). Intraoperative drop of SBP was -34.3 ± 34.4 mmHg (P = 0.04). With respect to adverse events there were minor side-effects (mainly paresthesia and dysphagia) reported in our patients, which occurred according to treatment intensity and modality., Conclusion: BAT is an effective and well tolerated intervention to reduce BP in patients suffering from end-stage renal disease and resistant hypertension. Therefore, BAT might contribute to a reduction of cardiovascular events in those high-risk patients.

Background: A controversy exists regarding which monitoring technique is superior in cases in which general anesthesia (GA) is necessary for carotid endarterectomy (CEA). Multimodal evoked potential (mEP) monitoring was investigated under GA during CEA and compared with a historical control group undergoing neurological evaluations awake under loco-regional anesthesia (LA)., Methods: We retrospectively studied 651 patients undergoing elective CEA. In groupHISTORY (N.=349; 1997-1999) LA was provided using superficial or deep/superficial cervical plexus blocks. In groupmEP, (N.=302; 2009-2013) GA was performed by administering remifentanil/propofol infusion. The multimodal EPs included the median-nerve-somatosensory and motor evoked potentials. The primary outcome was the rate of technical failure. The arterio-arterial shunt rate and immediate postoperative motor outcomes were also compared., Results: GroupmEP showed a significantly lower rate of technical failure (OR 0.17; CI 0.03-0.6; P=0.002). Because the groups differed systematically, logistic regression analysis was used to compare shunt rates and motor outcomes. Since shunt rates were 8.3% (groupmEP) versus 8.2% (groupHISTORY), but logistic regression model showed significant differences (OR 3.77; CI 1.67-8.95; P=0.001) correct comparison was impossible. Immediate postoperative deficits were 4.3% (groupmEP) and 4.9% (groupHISTORY); logistic regression analysis: transient OR 0.77, CI 0.28 to 0.22, P=0.61 and permanent OR 0.37, CI 0.02-7.74, P=0.49., Conclusion: Monitoring mEPs was associated with less technical failure than awake evaluation and showed similar motor outcomes. Because the groups differed systematically, the interpretation of shunt rates was impossible. Monitoring mEP should be considered to detect intraoperative ischemia in cases in which patients undergo CEA under GA.

Intraoperative smoke-generation limits the quality of vision during laparoscopic/endoscopic laser-assisted surgeries. The current study aimed at the evaluation of factors affecting this phenomenon. As a first step, a suitable experimental setup and a test tissue model were established for this investigation. The experimental setup is composed of a specific sample container, a laser therapy component suitable for the ablation of model tissue at different treatment wavelengths (λ = 980 nm, 1350 nm, 1470 nm), a suction unit providing continuous smoke extraction, and a detection unit for smoke quantification via detection of light (λ = 633 nm) scattered from smoke particles. The ablation rate (AR) was calculated by dividing the ablated volume by the ablation time (60 sec). The laser-induced scattering signal intensity of the smoke (SI) was determined from time-charts of the signal intensity as a measure for vision, in addition a delay-time tdelay could be derived defining the onset of SI after the laser was switched on. The ratio SI/AR is used as a measure for smoke generation in relation to the ablation rate. Additionally the light transmission of the tissue samples was used to estimate their optical properties. In this set-up, smoke generation using λ = 980 nm as ablation laser wavelength was detected after a delay-time tdelay = (121.6 ± 24.8) sec which is significantly longer compared to the wavelengths λ = 1350 nm with tdelay = (89.8 ± 19.3) sec and λ = 1470 nm with tdelay = (24.7 ± 5.4) sec. Thus, the delay Experimental set-up consisting of sample container, laser therapy component, suction unit and scattered-light detection compartment. time is wavelength-dependent. The SI/AR ratio was significantly different (p < 0.001) for 1470 nm irradiation compared to 980 nm irradiation [SI/AR(1470) = (11.8 ± 2.6) · 10(3) vs. SI/AR(980) = (8.6 ± 2.0) · 10(3) ]. The ablation crater for 980 nm irradiation was comparable with 1470 nm irradiation, but the coagulation rim was thicker in the 980 nm case. In conclusion, it could be shown experimentally that smoke-generation depends on the wavelength used for laser ablation., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Medical laser applications based on widespread research and development is a very dynamic and increasingly popular field from an ecological as well as an economic point of view. Conferences and personal communication are necessary to identify specific requests and potential unmet needs in this multi- and interdisciplinary discipline. Precise gathering of all information on innovative, new, or renewed techniques is necessary to design medical devices for introduction into clinical applications and finally to become established for routine treatment or diagnosis. Five examples of successfully addressed clinical requests are described to show the long-term endurance in developing light-based innovative clinical concepts and devices. Starting from laboratory medicine, a noninvasive approach to detect signals related to iron deficiency is shown. Based upon photosensitization, fluorescence-guided resection had been discovered, opening the door for photodynamic approaches for the treatment of brain cancer. Thermal laser application in the nasal cavity obtained clinical acceptance by the introduction of new laser wavelengths in clinical consciousness. Varicose veins can be treated by innovative endoluminal treatment methods, thus reducing side effects and saving time. Techniques and developments are presented with potential for diagnosis and treatment to improve the clinical situation for the benefit of the patient.

Background: Methods for detecting intraoperative cerebral ischemia arising from internal carotid artery (ICA) cross-clamping during carotid endarterectomy (CEA) should be sensitive, specific, and rapid to prevent intraoperative stroke. We had 3 objectives pertaining to this: (1) investigation of the rates of success of multimodal evoked potential (mEP) monitoring using a combination of median nerve (m) somatosensory (SS) EPs, tibial nerve SSEPs (tSSEPs), and transcranial electrical stimulated motor EPs (tcMEPs); (2) evaluation of the rates of false-negative mEP results; and (3) analysis of the relationship between different time periods associated with ICA cross-clamping and the postoperative outcome of motor function in patients with significant changes in mEP monitoring., Methods: Two hundred sixty-four patients undergoing CEA using general anesthesia with monitoring of bilateral mSSEPs, tSSEPs, and tcMEPs were retrospectively reviewed between 2009 and 2012. The rates of successful assessment of mEPs were investigated, and the rate of false-negative mEP results was analyzed. Different time periods (T1--time of clamping, T2--clamping to significant mEP changes, T3--significant mEP change to intervention, and T4--intervention to recovery of EP) were tested using Welch t test for significant association with postoperative motor deficit., Results: (1) Multimodal EP monitoring was achieved in 241 patients (91.3%, point estimate [PE] 0.91, confidence interval [CI] 0.87 to 0.94), whereas none of the modalities were recordable in one case (PE 0.0038, CI 0.0002 to 0.019). Additionally, tSSEP was not recordable in 21 patients (PE 0.08, CI 0.05 to 0.12), and we found one case of isolated failure of tcMEP recording (PE 0.0038, CI 0.0002 to 0.019). (2) False-negative mEP results were found in 1 patient (0.4%; PE 0.0038, CI 0.0002 to 0.019). Significant mEP changes occurred in 32 patients (12.1%), and thus, arterioarterial shunt was performed in 17 (6.4%) patients. Eleven patients (4.2%) showed transient and 1 showed permanent postoperative motor deficit. (3) There was no significant difference regarding any of the time periods associated with ICA cross-clamping and postoperative alteration of motor function (T1: P = 0.19, CI -30.1 to 6.8 minutes; T2: P = 0.38, CI -23 to 9.5 minutes; T3: P = 0.25, -9.7 to 2.8 minutes; T4: P = 0.42, CI to -15.5 to 7.0 minutes)., Conclusions: Multimodal EP monitoring is applicable during CEA. The 0.4% false-negative rate suggests an advantage of mEP monitoring when compared with isolated mSSEP monitoring. Our data suggest that periods of time during cross-clamping were not significantly associated with postoperative motor deficit. However, the small number of patients limits the conclusiveness of these findings. mEP monitoring could not prevent a postoperative motor deficit in all patients, but our results suggest that it is a useful adjunct to mSSEP monitoring.

Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology.

Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133 + 1,083 blocks), were determined by quantitative reverse transcription polymerase chain reaction for all samples, as well as by microarray for 133 validation samples. In the training set, agreement between high vs. low mRNA expression from whole slide and tumor-enriched sections was absolute for ESR1 and ERBB2, and 83 % for PGR. Overall agreements, when comparing mRNA expression to immunohistochemistry, were 100 % (ERBB2), 89 % (ESR1) and 83 % (PGR), which was confirmed in the validation sets. Percentage of tumor in the sections did not influence the results. In conclusion, reliable quantification of ESR1, PGR and ERBB2 mRNA expression can be obtained from a whole slide section, and correlates well with immunohistochemistry. Prior removal of surrounding tissue was found to be unnecessary even with minimal tumor content in the section.

Interstitial cells of Cajal (ICC) provide a pacemaker signal for coordinated motility patterns in the mammalian gastrointestinal (GI) tract. Kit signaling is required for development and maintenance of ICC, and these cells can be identified by Kit-like immunoreactivity. The zebrafish GI tract has two distinct ICC networks similar to mammals, suggesting a similar role in the generation of GI motility; however, a functional role for Kit-positive cells in zebrafish has not been determined. Analysis of GI motility in intact zebrafish larvae was performed during development and after disruption of Kit signaling. Development of coordinated motility patterns occurred after 5 days post-fertilization (dpf) and correlated with appearance of Kit-positive cells. Disruptions of Kit signaling using the Kit antagonist imatinib mesylate, and in Sparse, a null kita mutant, also disrupted development of coordinated motility patterns. These data suggest that Kit signaling is necessary for development of coordinated motility patterns and that Kit-positive cells in zebrafish are necessary for coordinated motility patterns.

Objective: This study was to investigate the utility of motor evoked potential monitoring elicited by transcranial electrical stimulation (tcMEP) during CEA in addition to the established median nerve somatosensory evoked potentials (mSSEPs)., Methods: We retrospectively reviewed data from 600 patients undergoing CEA under general anesthesia with monitoring of mSSEPs and tcMEPs in a multicenter study. MSSEP and tcMEP parameters were recorded during internal carotid artery (ICA) cross clamping and compared with the postoperative motor outcome, demographic and patient history data., Results: The intraoperative monitoring of tcMEPs was successful in 594 of the patients (99%) and selective shunt was performed in 29 of them (4.83%). Nine of the patients showed a transient contralateral loss of tcMEPs, without changes in mSSEPs and required intervention (1.5% "false-negative"). Three of them showed postoperative motor deficits. The time period from tcMEP loss to intervention was significantly longer (p = 0.01) in this group compared to the patients without postoperative motor deficit., Conclusion: TcMEPs during CEA may be an adjunct to mSSEP monitoring to avoid "false-negative" mSSEP results, as mSSEPs seem to lack specificity for detecting isolated ischemia of corticospinal pathway., Significance: TcMEPs seem to improve postoperative outcome, especially in case of a timely correction of cerebral ischemia., (Copyright © 2012 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.)

An optical filter unit is demonstrated, which uses two successively arranged tunable thin-film optical band-pass filters and allows for simultaneous adjustment of the central wavelength in the spectral range 522-555 nm and of the spectral bandwidth in the range 3-16 nm with a wavelength switching time of 8 ms∕nm. Different spectral filter combinations can cover the complete visible spectral range. The transmitted intensity was found to decrease only linearly with the spectral bandwidth for bandwidths >6 nm, allowing a high maximum transmission efficiency of >75%. The image of a fiber bundle was spectrally filtered and analyzed in terms of position-dependency of the transmitted bandwidth and central wavelength.

Monte Carlo (MC) simulations are frequently used to simulate the radial distribution of remitted fluorescence light from tissue surfaces upon pencil beam excitation to gather information about influences of different tissue parameters. Here, the "weighted direct emission method" (WDEM) is proposed, which uses a weighted MC simulation approach for both excitation and fluorescence photons, and is compared to four other methods in terms of accuracy and speed, and using a broad range of tissue-relevant optical parameters. The WDEM is 5.2× faster on average than a fixed weight MC approach while still preserving its accuracy. Additional gain of speed can be achieved by implementing it on graphics processing units.

Due to the approval of various new targeted therapies for the treatment of cancer, molecular pathology laboratories with a diagnostic focus have to meet new challenges: simultaneous handling of a large number of samples, small amounts of input material, and fragmentation of nucleic acids because of formalin fixation. As a consequence, fully automated systems for a fast and standardized extraction of high-quality DNA from formalin-fixed paraffin-embedded (FFPE) tissues are urgently needed. In this study, we tested the performance of a fully automated, high-throughput method for the extraction of nucleic acids from FFPE tissues. We investigated the extraction performance in sections of 5 different tissue types often analyzed in routine pathology laboratories (cervix, colon, liver, lymph node, and lung; n=340). Furthermore, we compared the quality, labor input, and applicability of the method for diagnostic purposes with those of a laboratory-validated manual method in a clinical setting by screening a set of 45 colorectal adenocarcinoma for the KRAS mutation. Automated extraction of both DNA and RNA was successful in 339 of 340 FFPE samples representing 5 different tissue types. In comparison with a conventional manual extraction protocol, the method showed an overall agreement of 97.7% (95% confidence interval, 88.2%-99.9%) for the subsequent mutational analysis of the KRAS gene in colorectal cancer samples. The fully automated system is a promising tool for a simple, robust, and rapid extraction of DNA and RNA from formalin-fixed tissue. It ensures a standardization of sample processing and can be applied to clinical FFPE samples in routine pathology.

Laparoscopic/robotic partial nephrectomy (LPN) is increasingly considered for small renal tumors (RT). This demands new compatible surgical tools for RT-resection, such as lasers, to optimize cutting and coagulation. This work aims to characterize ex vivo handling requirements for six medically approved laser devices emitting different light wavelengths (940, 1064, 1318, 1470, 1940, and 2010 nm) amenable for LPN. Incisions were made by laser fibers driven by a computer-controlled stepping motor allowing precise linear movement with a preset velocity at a fixed fiber-tip distance to tissue. Optical parameters were measured on 200 μm tissue slices. Cutting quality depended on power output, fiber velocity and fiber-tip distance to tissue. Contact manner is suitable for cutting while a noncontact manner (5 mm distance) induces coagulation. Ablation threshold differs for each wavelength. Ablation depth is proportional to power output (within limit) while axial and superficial coagulation remains mostly constant. Increased fiber velocity compromises the coagulation quality. Optical parameters of porcine kidney tissue demonstrate that renal absorption coefficient follows water absorption in the 2 μm region while for other spectral regions (900 to 1500 and 1 μm) the tissue effects are influenced by other chromophores and scattering. Tissue color changes demonstrate dependencies on irradiance, scan velocity, and wavelength. Current results clearly demonstrate that surgeons considering laser-assisted RT excisions should be aware of the mentioned technical parameters (power output, fiber velocity and fiber-tip tissue-distance) rather than wavelength only.

Purpose: According to current guidelines, molecular tests predicting the outcome of breast cancer patients can be used to assist in making treatment decisions after consideration of conventional markers. We developed and validated a gene expression signature predicting the likelihood of distant recurrence in patients with estrogen receptor (ER)-positive, HER2-negative breast cancer treated with adjuvant endocrine therapy., Experimental Design: RNA levels assessed by quantitative reverse transcriptase PCR in formalin-fixed, paraffin-embedded tumor tissue were used to calculate a risk score (Endopredict, EP) consisting of eight cancer-related and three reference genes. EP was combined with nodal status and tumor size into a comprehensive risk score, EPclin. Both prespecified risk scores including cutoff values to determine a risk group for each patient (low and high) were validated independently in patients from two large randomized phase III trials [Austrian Breast and Colorectal Cancer Study Group (ABCSG)-6: n = 378, ABCSG-8: n = 1,324]., Results: In both validation cohorts, continuous EP was an independent predictor of distant recurrence in multivariate analysis (ABCSG-6: P = 0.010, ABCSG-8: P < 0.001). Combining Adjuvant!Online, quantitative ER, Ki67, and treatment with EP yielded a prognostic power significantly superior to the clinicopathologic factors alone [c-indices: 0.764 vs. 0.750, P = 0.024 (ABCSG-6) and 0.726 vs. 0.701, P = 0.003 (ABCSG-8)]. EPclin had c-indices of 0.788 and 0.732 and resulted in 10-year distant recurrence rates of 4% and 4% in EPclin low-risk and 28% and 22% in EPclin high-risk patients in ABCSG-6 (P < 0.001) and ABCSG-8 (P < 0.001), respectively., Conclusions: The multigene EP risk score provided additional prognostic information to the risk of distant recurrence of breast cancer patients, independent from clinicopathologic parameters. The EPclin score outperformed all conventional clinicopathologic risk factors., (©2011 AACR.)

Warm ischemia (WI) and bleeding during laparoscopic partial nephrectomy (LPN) place technical constraints on surgeons. Our aim was to develop a safe and effective laser-assisted LPN-technique without the need for WI. In this study, a diode laser-emitting light at a wavelength of 1,318 nm at output powers between 45 and 70 W in continuous-wave mode was used. Light was coupled into a flexible 600-μm bare fiber to be transported to the tissues. After dry lab experience, 13 patients (six males, seven females) underwent five open and eight laparoscopic/retroperitoneoscopic partial nephrectomies. Postoperative renal function and serum C-reactive protein (CRP) were monitored and coagulation depth and effects on resection margins (RR) were evaluated. Demographic, clinical, and follow-up data are presented. Mean operative time was 116.5 min (range 60-175 min) with mean blood loss of 238 ml (range 50-600 ml). Laser light application took a maximum of 17 min. All patients had a favorable outcome. The locations of the treated tumors (eight left and five right) were central (two), upper pole (two), lower pole (three) and middle kidney parenchyma (six anterior, two posterior, and five peripheral). Mean tumor size was 3.3 cm (range 1.8-5 cm). Two WI (19 and 24 min) were needed. Immediate postoperative serum creatinine and CRP were elevated within 0.1 to 0.6 mg/dl (mean 0.18) and 2.1-10 mg/dl (mean 6.24), respectively. Coagulation depth ranged from <1 to 2 mm without an effect on histopathological evaluation of tumors or RR. One patient had positive RR. During follow-up (2-6 months), one patient developed an A-V fistula that needed embolization. This prospective in-vivo feasibility study showed that the diode laser is a safe and promising device for LPN. Its advantages are minimal gas formation, good hemostasis, and minimal parenchymal damage. Oncological safety appears to be warranted by the use of a diode laser.

Interstitial photodynamic therapy (iPDT) is being investigated for the treatment of high-grade human brain malignancies. In recent clinical studies, fluorescence monitoring during iPDT of glioblastoma multiforme has revealed patient-specific accumulation of photosensitizer (aminolevulinic acid (ALA) induced protoporphyrin IX, PpIX) and its photobleaching kinetics. As photosensitizer degradation, also referred to as photobleaching, and tissue damage are caused by the same underlying processes, the photobleaching kinetics might provide a tool for real-time treatment supervision. Here, we show with computer simulations that varying optical properties have a strong influence on the irradiation time required to fully bleach the photosensitizer. We propose a method to potentially determine the time point during iPDT, when the photosensitizer within the target volume has been largely photobleached. Simulations show that it is possible to determine this time point by continuously monitoring the ratio of the fluorescence intensities at two time points during irradiation. We show that this method works for a large range of optical properties, different photobleaching rates and varying inter-fibre distances. In conclusion, the relative fluorescence method offers the potential to individualize irradiation times to consume the photosensitizer within the target tissue during iPDT., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: The reliability of intra-operative recordings of trigeminal scalp-induced somatosensory-evoked potentials (T-SSEP) is controversial. This investigation aimed to provide evidence that T-SSEP recordings are stable using standardised neurophysiological methodology and anaesthesiological regime., Methods: We investigated 99 patients undergoing carotid endarterectomy under total intravenous anaesthesia (propofol/remifentanil infusion). Long-latency T-SSEPs were recorded from the scalp after simultaneously stimulating 2nd and 3rd branches of the trigeminal nerve. The analysis included visual assessments of traces and measurements of latencies and amplitudes of the N13 and P19 peaks of T-SSEP. Furthermore, additional groups of patients were investigated to identify changes in the parameters of T-SSEP that might correspond to different states of anaesthesia and artificial muscle activity., Results: We reproducibly recorded T-SSEP responses in 99 patients with a mean latency of 12.4 ms (SD=0.93) and amplitude of 5.7 μV (SD=4.7). Collateral investigations concerning changes of T-SSEP caused by neuromuscular blockade improved independence of T-SSEP recordings to muscle relaxation in contrast to facial and cervical muscle activity., Conclusions: We demonstrated the reliability of recording stable intra-operative T-SSEP responses with standardised electrophysiological and anaesthesiological regimes., Significance: We provided evidence of the non-muscular origin of T-SSEPs recorded from the scalp., (Copyright © 2010 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.)

Intraluminal manometry is a tool commonly used to record motility in the human digestive tract. The recorded signal results from a combination of factors, including the hydrodynamic pressure transmitted through the intestinal contents due to contraction of the gut wall and the force of the gut wall acting on the sensors in regions of a luminal occlusion. However, the actual relationships between small bowel wall contraction, the measured intraluminal pressure, and the resultant flow have not been directly addressed. Video recording and high-resolution fiber-optic manometry were used to create spatiotemporal video maps of diameter and intraluminal pressure from isolated segments of rabbit small intestine. In the unstimulated gut, longitudinal muscle contractions were the only detectable motor pattern; circular muscle contractions were elicited by distension or erythromycin (1 μM). Longitudinal muscle contractions were not lumen-occlusive, although they caused measurable low-amplitude changes in pressure. Localized nonpropagating circular muscle contractions caused small localized, nonpropagating peaks of intraluminal pressure. Propagating contractions of circular muscle evoked larger, propagating pressure changes that were associated with outflow. Propagating circular muscle contractions often caused dilation of aboral receiving segments, corresponding to "common cavities"; these were propulsive, despite their low intraluminal pressure. The highest-amplitude pressure events were caused by lumen-occlusive circular muscle contractions that squeezed directly against the catheter. These data allow us to define the complex relationships between wall motion, intraluminal pressure, and flow. A strong correlation between circular and longitudinal muscle contraction and intraluminal pressure was demonstrated. Common-cavity pressure events, caused by propulsion of content by circular muscle contractions into a receptive segment, were often of low amplitude but were highly propulsive. Studies of wall motion in isolated preparations, combined with manometry, can assist in interpretation of pressure recordings in vivo.

The development of optimized therapy strategies against malignant tumors is critically dependent on the assessment of tissue-based biomarkers in routine diagnostic tissue samples. We investigated a novel, fully automated, and xylene-free method for RNA isolation and biomarker determination using formalin-fixed paraffin-embedded (FFPE) tissue. The aim was to show that this approach is feasible and gives results that are comparable to the current gold standards. Expression of the breast cancer biomarkers ESR1, PGR, and HER2 was measured in a total of 501 FFPE tissue samples from 167 breast carcinomas, which had been stored for up to 21 years. Total RNA was extracted from tissue sections and biomarker expression was measured by kinetic RT-PCR (RT-kPCR). The results of the new method were compared with immunohistochemistry as the current gold standard.RNA was successfully isolated from all samples, with a mean yield of 1.4 μg/sample and fragment lengths of at least 150 bp in 99% of samples. RT-kPCR analysis of ESR1, PGR, and HER2 was possible in all samples. Comparing RT-kPCR results with standard IHC, we found a good concordance for ESR1 (agreement: 98.4%), PGR (84.4%), and HER2 (89.8%). We observed a low section-to-section variability of kPCR results for all 3 biomarkers (root of mean squared errors: 0.2 to 0.5 Ct values). The new approach is a reliable high-throughput instrument for standardized testing of biomarkers in clinical routine and for research studies on archived FFPE material up to 21 years old. For the assessment of ESR1, PGR, and HER2 the results are comparable to the current gold-standard.

Background: There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer., Methods: We copurified both RNA and DNA from a single 10-μm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1-25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry., Results: In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%-100% of tumor tissues and 21%-100% of normal tissues. The 7 SNPs were successfully genotyped in 91%-97% of tumor and 94%-97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%-99.5%., Conclusions: This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples.

A 1.5-year-old, male blue and gold macaw (Ara ararauna) was anesthetized for a health examination and blood collection. The following day it was returned for episodes of coughing. The bird was presented again 13 days after the initial presentation with severe dyspnea. A tracheal stenosis was diagnosed by endoscopy and treated by surgical resection of 5 tracheal rings and tracheal anastomosis. The bird was discharged but returned 2 days later with a recurrent stenosis. Bougienage and balloon dilation of the stenotic area were performed separately; each resulted in less than 48 hours' improvement in clinical signs after treatment. A second tracheal resection and anastomosis was performed, during which an additional 10 tracheal rings were removed. This second anastomosis was significantly more difficult to complete given the marked variation in diameter of the proximal and distal tracheal segments. The macaw recovered without complication and has had no recurrence of respiratory abnormalities 2 years after the second surgery. This report describes the longest total tracheal segment to be resected, followed by tracheal anastomosis, in a psittacine bird. The positive outcome in this case suggests that, when surgical therapy is elected, an aggressive approach is necessary to prevent recurrence of tracheal stenosis. In addition, macaws can recover well even after significant lengths of the trachea are resected.

Interstitial cells of Cajal (ICC) generate pacemaker activity (slow waves) in gastrointestinal (GI) smooth muscles, but the mechanism(s) of pacemaker activity are controversial. Several conductances, such as Ca(2+)-activated Cl() channels (CaCC) and non-selective cation channels (NSCC) have been suggested to be involved in slow wave depolarization. We investigated the expression and function of a new class of CaCC, anoctamin 1 (ANO1), encoded by Tmem16a, which was discovered to be highly expressed in ICC in a microarray screen. GI muscles express splice variants of the Tmem16a transcript in addition to other paralogues of the Tmem16a family. ANO1 protein is expressed abundantly and specifically in ICC in all regions of the murine, non-human primate (Macaca fascicularis) and human GI tracts. CaCC blocking drugs, niflumic acid and 4,4-diisothiocyano-2,2-stillbene-disulfonic acid (DIDS) reduced the frequency and blocked slow waves in murine, primate, human small intestine and stomach in a concentration-dependent manner. Unitary potentials, small stochastic membrane depolarizations thought to underlie slow waves, were insensitive to CaCC blockers. Slow waves failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16a(tm1Bdh)(/tm1Bdh)) and did not develop subsequent to birth in organ culture, as in wildtype and heterozygous muscles. Loss of function of ANO1 did not inhibit the development of ICC networks that appeared structurally normal as indicated by Kit antibodies. These data demonstrate the fundamental role of ANO1 in the generation of slow waves in GI ICC.