Real‐time augmented reality for delineation of surgical margins during neurosurgery using autofluorescence lifetime contrast

Current clinical brain imaging techniques used for surgical planning of tumor resection lack intraoperative and real‐time feedback; hence surgeons ultimately rely on subjective evaluation to identify tumor areas and margins. We report a fluorescence lifetime imaging (FLIm) instrument (excitation: 355 nm; emission spectral bands: 390/40 nm, 470/28 nm, 542/50 nm and 629/53 nm) that integrates with surgical microscopes to provide real‐time intraoperative augmentation of the surgical field of view with fluorescent derived parameters encoding diagnostic information. We show the functionality and safety features of this instrument during neurosurgical procedures in patients undergoing craniotomy for the resection of brain tumors and/or tissue with radiation damage. We demonstrate in three case studies the ability of this instrument to resolve distinct tissue types and pathology including cortex, white matter, tumor and radiation‐induced necrosis. In particular, two patients with effects of radiation‐induced necrosis exhibited longer fluorescence lifetimes and increased optical redox ratio on the necrotic tissue with respect to non‐affected cortex, and an oligodendroglioma resected from a third patient reported shorter fluorescence lifetime and a decrease in optical redox ratio than the surrounding white matter. These results encourage the use of FLIm as a label‐free and non‐invasive intraoperative tool for neurosurgical guidance.     

Mesoscopic fluorescence lifetime imaging: Fundamental principles,clinical applications and future directions

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real-time assessments of tissue properties in clinical settings. Label-free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real-time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.     

Multiparametric Characterization of Grade 2 Glioma Subtypes Using Magnetic Resonance Spectroscopic,Perfusion, and Diffusion Imaging

BACKGROUND AND PURPOSE: The purpose of this study was to derive quantitative parameters from magnetic resonance (MR) spectroscopic, perfusion, and diffusion imaging of grade 2 gliomas according to the World Health Organization and to investigate how these multiple imaging modalities can contribute to evaluating their histologic subtypes and spatial characteristics. MATERIALS AND METHODS: MR spectroscopic, perfusion, and diffusion images from 56 patients with newly diagnosed grade 2 glioma (24 oligodendrogliomas, 18 astrocytomas, and 14 oligoastrocytomas) were retrospectively studied. Metabolite intensities, relative cerebral blood volume (rCBV), and apparent diffusion coefficient (ADC) were statistically evaluated. RESULTS: The 75th percentile rCBV and median ADC were significantly different between oligodendrogliomas and astrocytomas (P < .0001) and between oligodendrogliomas and oligoastrocytomas (P < .001). Logistic regression analysis identified both 75th percentile rCBV and median ADC as significant variables in the differentiation of oligodendrogliomas from astrocytomas and oligoastrocytomas. Group differences in metabolite intensities were not significant, but there was a much larger variation in the volumes and maximum values of metabolic abnormalities for patients with oligodendroglioma compared with the other tumor subtypes. CONCLUSIONS: Perfusion and diffusion imaging provide quantitative MR parameters that can help to differentiate grade 2 oligodendrogliomas from grade 2 astrocytomas and oligoastrocytomas. The large variations in the magnitude and spatial extent of the metabolic lesions between patients and the fact that their values are not correlated with the other imaging parameters indicate that MR spectroscopic imaging may provide complementary information that is helpful in targeting therapy, evaluating residual disease, and assessing response to therapy.     

Estimating occupancy of Chinese pangolin (Manis pentadactyla) in a protected and non‐protected area of Nepal

Chinese pangolin is the world's most heavily trafficked small mammal for luxury food and traditional medicine. Although their populations are declining worldwide, it is difficult to monitor their population status because of its rarity and nocturnal behavior. We used site occupancy (presence/absence) sampling of pangolin sign (i.e., active burrows) in a protected (Gaurishankar Conservation Area) and non‐protected area (Ramechhap District) of central Nepal with multiple environmental covariates to understand factors that may influence occupancy of Chinese pangolin. The average Chinese pangolin occupancy and detection probabilities were  ± SE = 0.77 ± 0.08;  ± SE = 0.27 ± 0.05, respectively. The detection probabilities of Chinese pangolin were higher in PA (  ± SE = 0.33 ± 0.03) than compared to non‐PA (  ± SE = 0.25 ± 0.04). The most important covariates for Chinese pangolin detectability were red soil (97%), food source (97.6%), distance to road (97.9%), and protected area (97%) and with respect to occupancy was elevation (97.9%). We recommended use of remote cameras and potentially GPS collar surveys to further investigate habitat use and site occupancy at regular intervals to provide more reliable conservation assessments.     

Hub gene identification and prognostic model construction for isocitrate dehydrogenase mutation in glioma

Our study attempted to identify hub genes related to isocitrate dehydrogenase (IDH) mutation in glioma and develop a prognostic model for IDH-mutant glioma patients. In a first step, ten hub genes significantly associated with the IDH status were identified by weighted gene coexpression analysis (WGCNA). The functional enrichment analysis demonstrated that the most enriched terms of these hub genes were cadherin binding and glutathione metabolism. Three of these hub genes were significantly linked with the survival of glioma patients. 328 samples of IDH-mutant glioma were separated into two datasets: a training set (N = 228) and a test set (N = 100). Based on the training set, we identified two IDH-mutant subtypes with significantly different pathological features by using consensus clustering. A 31 gene-signature was identified by the least absolute shrinkage and selection operator (LASSO) algorithm and used for establishing a differential prognostic model for IDH-mutant patients. In addition, the test set was employed for validating the prognostic model, and the model was proven to be of high value in classifying prognostic information of samples. The functional annotation revealed that the genes related to the model were mainly enriched in nuclear division, DNA replication, and cell cycle. Collectively, this study provided novel insights into the molecular mechanism of IDH mutation in glioma, and constructed a prognostic model which can be effective for predicting prognosis of glioma patients with IDH-mutation, which might promote the development of IDH target agents in glioma therapies and contribute to accurate prognostication and management in IDH-mutant glioma patients.     

Protein Tyrosine Kinases in Human Brain and Gliomas

Tyrosine kinase activity was determined in neonatal and adult human brain, oligodendrogliomas, and astrocytomas. The astrocytomas were divided into low- (grade I and grade II) and high-grade (grade III and grade IV) tumors. We measured the tyrosine kinase activity in the cytosolic and membrane fraction using poly(glutamic acid:tyrosine, 4:1) as an artificial substrate. The cytosolic activity in oligodendrogliomas (n = 7), low-grade astrocytomas (n = 7), and neonatal brain (n = 1) was increased, on average, two- to fourfold compared with that in normal adult brain (n = 14). The cytosolic activities of high-grade astrocytomas (n = 11) were in approximately the same range as found in normal adult brain. The absence of an increase in cytosolic activity in high-grade astrocytomas compared with adult brain is likely due to the occurrence of necrosis in these tumors. In contrast to the cytosolic activity, no differences were found in the membrane-bound activity. By fast protein liquid chromatography, at least three forms of cytosolic protein tyrosine kinase could be separated, which eluted at 0, 115, and 210 mM NaCl. In most cases the highest amount of activity eluted at 210 mM NaCl. However, in oligodendrogliomas, high-grade astrocytomas, and neonatal brain, more activity eluted at 115 mM NaCl than in normal adult brain (p = 0.043). Nevertheless, protein tyrosine kinases from all three peaks contributed to the elevated levels of total cytosolic activity of oligodendrogliomas and low-grade astrocytomas.     

Aurora A is differentially expressed in gliomas,is associated with patient survival in glioblastoma and is a potential chemotherapeutic target in gliomas

Aurora A is critical for mitosis and is overexpressed in several neoplasms. Its overexpression transforms cultured cells, and both its overexpression and knockdown cause genomic instability. In transgenic mice, Aurora A haploinsufficiency, not overexpression, leads to increased malignant tumor formation. Aurora A thus appears to have both tumor-promoting and tumor-suppressor functions. Here, we report that Aurora A protein, measured by quantitative protein gel blotting, is differentially expressed in major glioma types in lineage-specific patterns. Aurora A protein levels in WHO grade II oligodendrogliomas (n = 16) and grade III anaplastic oligodendrogliomas (n = 16) are generally low, similar to control epilepsy cerebral tissue (n = 11). In contrast, pilocytic astrocytomas (n = 6) and ependymomas (n = 12) express high Aurora A levels. Among grade II to grade III astrocytomas (n = 7, n = 14, respectively) and grade IV glioblastomas (n = 31), Aurora A protein increases with increasing tumor grade. We also found that Aurora A expression is induced by hypoxia in cultured glioblastoma cells and is overexpressed in hypoxic regions of glioblastoma tumors. Retrospective Kaplan-Meier analysis revealed that both lower Aurora A protein measured by quantitative protein gel blot (n = 31) and Aurora A mRNA levels measured by real-time quantitative RT-PCR (n = 58) are significantly associated with poorer patient survival in glioblastoma. Furthermore, we report that the selective Aurora A inhibitor MLN8237 is potently cytotoxic to glioblastoma cells, and that MLN8237 cytotoxicty is potentiated by ionizing radiation. MLN8237 also appeared to induce senescence and differentiation of glioblastoma cells. Thus, in addition to being significantly associated with survival in glioblastoma, Aurora A is a potential new drug target for the treatment of glioblastoma and possibly other glial neoplasms.     

Multispectral fluorescence lifetime imaging system for intravascular diagnostics with ultrasound guidance: in vivo validation in swine arteries

Fluorescence lifetime technique has demonstrated potential for analysis of atherosclerotic lesions and for complementing existing intravascular imaging modalities such as intravascular ultrasound (IVUS) in identifying lesions at high risk of rupture. This study presents a multimodal catheter system integrating a 40 MHz commercial IVUS and fluorescence lifetime imaging (FLIm) using fast helical motion scanning (400 rpm, 0.75 mm/s), able to acquire in vivo in pulsatile blood flow the autofluorescence emission of arterial vessels with high precision (5.08 ± 0.26 ns mean average lifetime over 13 scans). Co‐registered FLIm and IVUS data allowed 3D visualization of both biochemical and morphological vessel properties. Current study supports the development of clinically compatible intravascular diagnostic system integrating FLIm and demonstrates, to our knowledge, the first in vivo intravascular application of a fluorescence lifetime imaging technique. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect on simultaneous removal of ammonia,nitrate, and phosphorus via advanced stacked assembly biological filter for rural domestic sewage treatment

The discharge of ammonia–nitrogen (NH₃–N), total nitrogen (TN), chemical oxygen demand (COD), and total phosphorus (TP) in rural sewage usually exceeds the Pollutant Discharge Standard for Urban Sewage Treatment Plants (GB18918-2002). Efficient and cost-effective removal of these pollutants cannot be simultaneously realized using conventional rural sewage treatment methods. Thus, an assembled biological filter (D50?×?W50?×?H113 cm), including a phosphorus removal layer filled with solid polymeric ferric sulfate and alternating aerobic-anaerobic layers, is proposed herein. The aerobic (anerobic) layers were filled with zeolite (zeolite and composite soil) at different intervals. This system was used for the treatment of synthetic sewage having COD: 122.0–227.0 mg/L; NH₃–N: 29.1–47.0 mg/L; TN: 28.0–58.0 mg/L; and TP: 2.0–3.8 mg/L. Based on optimal operation conditions (40 L/h reflow rate, without artificial aeration, and 12-h operation cycle), the system showed NH₃–N, TN, COD, and TP removal efficiencies of 87.1? ± ?8.1, 83.4? ± ?7.9, 91.0? ± ?9.4, and 80.0? ± ?6.4%, respectively. Further, in the pilot-scale test, under the same optimal parameters, the removal efficiencies of NH₃–N, TN, COD, and TP were 78.9? ± ?8.1, 75.4? ± ?7.9, 82? ± ?9.4, and 76? ± ?6.4%, respectively. Furthermore, in the different functional units of the system, a large number of functional bacteria capable of efficiently facilitating the simultaneous removal of the different pollutants from sewage were identified. Therefore, this proposed system, which complies with current environmental discharge regulations, can be a more sustainable approach for the treatment of unattended rural sewage.

Graphic abstract

     

In‐vitro investigations on laser‐induced smoke generation mimicking the laparoscopic laser surgery purposes

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 t_delay 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 t_delay = (121.6 ± 24.8) sec which is significantly longer compared to the wavelengths λ = 1350 nm with t_delay = (89.8 ± 19.3) sec and λ = 1470 nm with t_delay = (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) · 10³ vs. SI/AR(980) = (8.6 ± 2.0) · 10³]. 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.     

Soybean Hull Insoluble Polysaccharides: Improvements of Its Physicochemical Properties Through High Pressure Homogenization

Soybean hull is an agroindustrial waste which has not been fully studied as a food ingredient. The aims of this work were to obtain insoluble fibers from soybean hull and to evaluate the effect of high pressure homogenization (HPH) on its physicochemical properties. Hull insoluble polysaccharides (HIPS) were obtained in a single step, as the insoluble residue after pectin removal. FTIR showed bands corresponding to cellulose and hemicellulose in HIPS, and thermogravimetric analysis showed two degradation events at 236.3 °C and 325.6 °C, corresponding to cellulose and hemicellulose, respectively. HIPS dispersions (pH 3.00) were subjected to HPH by three cycles at increasing pressures (up to 1000 bar), obtaining soybean hull nanofibers. SEM images show that HPH at 1000 bar reduced the dimensions of the fiber bundle from 30 to 90 μm in length and 9–15 μm in diameter to nanofibers of 10–30 μm in length and 100–400 nm in diameter. AFM further confirms a heterogeneous distribution of sizes in HIPS₈₀₀ and HIPS₁₀₀₀, evidencing the presence of individual nanofibers with diameters around 50 ± 10 nm and 40 ± 10 nm, respectively, with several μm in length. Furthermore, an increase in water holding capacity from 2.1 to 61 g_water/g_{dry matter} and viscosity from 0.39 to 34,945 Pa.s were achieved as HPH at 1000 bar treatment was applied. HPH increased the interfacial area and promoted the interconnection of fibers in a hydrated gel-like structure. This explains flow behavior, which was extensively studied in this work: three-region viscosity profile (shear-thinning, plateau or shear-thickening and shear-thinning) and a pronounced hysteresis loop. Oscillatory rheology was used to study the viscoelastic behavior of HIPS dispersions. HIPS are a source of nanofibers, easy to obtain through a single step of chemical treatment followed by the application of high pressures. It is remarkable that the use of few chemical solvents is favorable from an environmental point of view. This work also suggests a potential application of HIPS to improve physicochemical and structural properties in acidic foods.

Graphical Abstract

     

Decreased expression of microRNA-200b is an independent unfavorable prognostic factor for glioma patients

Background and aimAs a member of the microRNA (miR)-200 family, miR-200b has been recognized as one of the fundamental regulators of epithelial–mesenchymal transition, chemosensitivity, cell proliferation, and cell cycle. Especially in glioma, miR-200b targets the CREB1 gene and suppresses the tumor cell growth in vitro. However, its involvement in human glioma has not yet been determined. The aim of this study was to investigate the clinical significance of miR-200b expression in this disease.MethodsmiR-200b expression in 266 pairs of human gliomas and matched nonneoplastic brain tissues was measured by real-time quantitative RT-PCR assay.ResultsCompared with nonneoplastic brain tissues, the expression level of miR-200b was significantly decreased in glioma tissues (tumor vs. normal: 2.87 ± 2.05 vs. 8.78 ± 2.50, P < 0.001). Of 266 patients with gliomas, 166 (62.41%) were in low miR-200b expression group. In addition, we found that the glioma tissues from high-grade tumors (grade III and IV) had much lower miR-200b expression than glioma tissues from low grade tumors (grade I and II). Moreover, the expression level of miR-200b was positively correlated with Karnofsky performance status (KPS) scores of glioma tissues. The results of a 60-month follow-up in 266 glioma patients further demonstrated that lower miR-200b expression was correlated with worse progression-free survival and overall survival in the patients with grade III and IV gliomas. Both univariate and multivariate analyses revealed that miR-200b was an independent prognostic indicator for glioma.ConclusionThese findings prove that the decreased expression of miR-200b may be associated with malignant tumor progression and poor prognosis in patients with gliomas, suggesting the potential role of miR-200b in glioma management. miR-200b may be a novel and valuable signature for predicting the clinical outcome of patients with gliomas.     

Amplification of KIT, PDGFRA, VEGFR2, and EGFR in gliomas

Receptor tyrosine kinase aberrations are implicated in the genesis of gliomas. We investigated expression and amplification of KIT, PDGFRA, VEGFR2, and EGFR in 87 gliomas consisting of astrocytomas, anaplastic astrocytomas, oligodendrogliomas, or oligoastrocytomas in tumor samples collected at the time of the diagnosis and in samples of the same tumors at tumor recurrence. Gene amplifications were investigated using either chromogenic in situ hybridization or fluorescence in situ hybridization, and protein expression using immunohistochemistry. In samples collected at glioma diagnosis, KIT and PDGFRA amplifications were more frequent in anaplastic astrocytomas than in astrocytomas, oligodendrogliomas, and oligoastrocytomas [28% versus 5% (P = 0.012) and 33% versus 2% (P = 0.0008), respectively]. VEGFR2 amplifications occurred in 6% to 17% of the gliomas at diagnosis, and EGFR amplifications in 0% to 12%. Amplified KIT was more frequently present in recurrent gliomas than in newly diagnosed gliomas (P = 0.0066). KIT amplification was associated with KIT protein expression and with presence of PDGFRA and EGFR amplifications both at the time of the first glioma diagnosis and at tumor recurrence, and with VEGFR2 amplification at tumor recurrence. Three (4%) primary gliomas and 10 (14%) recurrent gliomas that were evaluable for coamplification of KIT, PDGFRA, and VEGFR2 showed amplification of at least two of these genes; the amplicon contained amplified KIT in all 13 cases. In conclusion, besides glioblastoma, amplified KIT, PDGFRA, and VEGFR may also occur in lower-grade gliomas and in their recurrent tumors. It is currently not known whether specific tyrosine kinase inhibitors are effective in the treatment of such gliomas.     

Ultrasound assisted rapid synthesis of mefenamic acid based indole derivatives under ligand free Cu-catalysis: Their pharmacological evaluation

An improved and rapid synthesis of mefenamic acid based indole derivatives has been achieved via the ligand free Cu-catalyzed coupling-cyclization method under ultrasound irradiation. This simple, straightforward and inexpensive one-pot method involved the reaction of a terminal alkyne derived from mefenamic acid with 2-iodosulfanilides in the presence of CuI and K₂CO₃ in PEG-400. The reaction proceeded via an initial CC bond formation (the coupling step) followed by CN bond formation (the intramolecular cyclization) to afford the mefenamic acid based indole derivatives in good to acceptable yields. Several of these compounds showed inhibition of PDE4 in vitro and the SAR (Structure Activity Relationship) within the series is discussed. The compound 3d has been identified as a promising and selective inhibitor of PDE4B (IC₅₀ = 1.34 ± 0.46 µM) that showed TNF-α inhibition in vitro (IC₅₀ = 5.81 ± 0.24 µM) and acceptable stability in the rat liver microsomes.     

Hsp27 (HSPB1): a possible surrogate molecular marker for loss of heterozygosity (LOH) of chromosome 1p in oligodendrogliomas but not in astrocytomas

In oligodendrogliomas, 1p loss of heterozygosity (LOH) is a predictor of good prognosis and treatment response. In contrast, in uveal melanomas, LOH of chromosome 3 has been linked to poor prognosis and downregulation of Hsp27. In the present study, we have analyzed the expression of heat-shock proteins (Hsps) to characterize subtypes of gliomas and their histopathologic features and to correlate with other molecular markers including LOH of 1p. Biopsies from patients with primary gliomas (n = 65) were analyzed by immunohistochemistry, chromogenic in situ hybridization and fluorescent in situ hybridization and methylation-specific PCR (MSP). Elevated Hsp27 and total Hsp70 expression levels were associated with high-grade astrocytomas (p = 0.0001 and p = 0.01, respectively). In grade III oligodendrogliomas, the Hsp27 levels were significantly higher (p = 0.03). Low O6-methylguanine-DNA methyltransferase (MGMT) expression was associated with grade II astrocytomas. Elevated β-catenin expression was associated with grade III/IV astrocytomas (p = 0.003); p53 (+) tumors were more frequently found in grade III/IV astrocytomas (p = 0,001). LOH on 1p was associated with oligodendroglial tumours. In addition, a higher Hsp27 expression correlated with LOH of 1p (p = 0.017); this was also tested in two glioma cell lines. MSP was successful in only six samples. No significant correlations were found for the other markers. In conclusion, in oligodendroglial tumors, Hsp27 appeared as a surrogate marker of LOH of 1p which could also help to predict the disease prognosis. In gliomas, p53, Hsp27, Hsp70, MGMT, and β-catenin correlated with histopathological characteristics, suggesting that these markers could predict the disease outcome and the response to treatments.     

Genetic analysis to complement histopathological diagnosis of brain tumors

Gliomas, the most frequent tumors originating in the human nervous system, are divided into various subtypes. Currently, microscopic examination alone is insufficient for classification and grading so that genetic profiles are increasingly being emphasized in recognition of the emerging role of molecular diagnostic approaches to glioma classification. Glioblastomas (WHO grade IV) may develop de novo (primary glioblastomas) or through progression from lower-grade astrocytomas (secondary glioblastomas), while both glioblastomas show similar histological features. In contrast, they do constitute distinct disease entities that evolve through different genetic pathways, and are likely to differ in prognosis and response to therapy. Oligodendrogliomas (WHO grade II) account for 2.7% of brain tumors and 5-18% of all gliomas. Since this tumor is recognized as a particular subtype of glioma that shows remarkable responses to chemotherapy, a correct diagnosis is of prime importance. The difficulty is that histological differentiation of oligodendrogliomas from diffuse astrocytomas is highly subjective in cases without typical morphological features and there is a lack of reliable immunohistochemical markers. While histological distinction of low-grade gliomas from reactive astrocytes is also often difficult, reactive astrocytes usually lack genetic alterations. More biological and molecular approaches to glioma classification thus appear warranted to provide improved means to achieve correct diagnoses.