Embedded Tissues Research Articles

CARDIAC AMYLOIDOSIS IN ELDERLY WITH POSITIVE SCINTIGRAPHY AND MONOCLONALITY: A DIAGNOSTIC DILEMMA

Abstract Background and Aims Clinical identification of elderly patients with both wild–type transthyretin amyloidosis (ATTR) and monoclonal gammopathy of unknown significance (MGUS) is increasing. For this reason, accurate amyloid histologic subtyping is crucial in case of concomitant positivity of bisphosphonate scintigraphy and monoclonal protein testing. We report the diagnostic work–up of cardiac amyloidosis in this specific patient subtype in our tertiary referral center. Methods Among 133 patients who received the diagnosis of cardiac amyloidosis from March 2021 to March 2023, 21 (16%) presented with an abnormality of both bisphosphonate scintigraphy and at least one of the serum monoclonal protein tests. Patients underwent abdominal fat biopsy/needle aspiration as the first diagnostic procedure. In case of negative results, an endomyocardial biopsy (EMB) was planned. Amyloid was identified at histology by Congo Red staining. Amyloid typing was obtained by immunohistochemistry using a recently developed amyloid antibody panel AmYkit (Amimed) applicable on fixed paraffin–embedded tissues in an automated platform. The results were compared with those obtained with standard immunohistochemistry. Results Sixteen of the 21 patients were older than 75 years (76%, 81±10 ys) with a grade 3 (62%) or 2 (33%) scintigraphy. A grade 1 positive scintigraphy was detected in one patient. Serum immunofixation revealed a previously unknown monoclonal protein peak in 78%, while 22% had a history of MGUS. None of the patients had hematologic disorders. TTR pathogenic mutation was detected in one patient. On pathologic examination, abdominal fat was negative for amyloid in all cases, while Congo Red staining was positive in all examined EMBs. Immunohistochemistry performed with standard antibodies was not able to discriminate between AL and ATTR, due to signal overlapping of variable intensity. Conversely, our preliminary results with AmYkit panel of antibodies demonstrate absence of signal overlapping in over 90% of cases, allowing accurate histologic discrimination between AL and TTR. Conclusion Based on our clinical and histologic observations, in elderly patients with concomitant positivity of both bisphosphonate scintigraphy and monoclonal protein testing, TTR is the most frequent type of cardiac amyloid. EMB is a useful diagnostic tool to confirm the clinical suspicion of amyloidosis. Accurate amyloid subtyping is granted by a more sensitive panel of antibodies.

Monte Carlo simulation of handheld probes to detect non-invasive ductal carcinoma from diffuse optical reflectance signals

To identify the malignancy in human breast, hand-held optical probes are simulated using the Monte Carlo method. These devices were used to scan different tissue equivalent numerical breast phantoms to detect the embedded cancerous tissues in them. The phantoms were simulated with glandular tissues embedded with ductal carcinoma of size 5 mm and 2 mm diameter at various depths. Two optical probes namely dual side detector (DSD) probe and a single side detector (SSD) probe are designed through which one million light photons of wavelengths 600 nm and 800 nm respectively were passed into the numerical phantoms. The photons that were backscattered from the phantoms were received by the detectors in both probes. They were measured as signals and both probes were able to detect the presence of the embedded cancerous tissues at different depths. The results indicate that the probes with a light source at 800 nm could detect deep-seated inhomogeneities. The absolute value of Peak Intensity (|PI|) and Full Width at Half Maximum (FWHM) would be indicative of their location and approximate size of the embedded tissues. The |PI| decreases with the size and depth of the embedded tissue, and the FWHM increases with depth. The results show that the designed probes are capable of detecting small variations in tissues.

Processing Neonatal Mouse Brains for Immunohistochemical Analysis: As Required for Including Spindle Orientation Analysis and Analysis of DNA Damage and Apoptosis.

Analyzing sections of neonatal mouse brain using immunohistochemistry can inform microcephaly pathogenesis, but obtaining and staining high-quality sections can be challenging. The neonatal brain shows less structural integrity than the adult brain. As a result, embedding technique must be optimized to allow sections without cracks or other anatomic disruptions. Moreover, paraffin embedding, which maximized tissue preservation, can reduce antigenicity of proteins in the embedded tissues. We describe an optimized embedding technique and antigen recovery technique that allows successful sectioning and immunohistochemical staining.

Cochlear implant imaging in the mouse and guinea pig using light-sheet microscopy

Postmortem examination of the cochlea with a cochlear implant in the scala tympani presents several challenges. It is technologically difficult to section a cochlea with an implant due to the presence of its wires and metal components that are adjacent to the membranous and bony tissues of the cochlea. These metal components damage traditional steel blades of a microtome in celloidin, paraffin or frozen embedded tissues. However, plastic embedded implanted cochleas have been successfully sectioned using specialized methods (Irving et al., 2013). An alternative non-destructive method is to optically section a chemically cleared cochlea using light-sheet microscopy, which we will describe in this publication. However, since this method uses a light-sheet to section the cochlea the opaque and reflective metal components of the implant results in some artifacts in the 2D optical sections. The best image quality using light-sheet fluorescent microscopy is when the implant is removed prior to imaging.

Agarose-paraffin double embedding assisted with 3D printing for high throughput rice root sectioning

Anatomical analysis of the plant tissues is an important technique used in plant research for studying the process of plant growth, development, adaptation to environmental stress as well as for characterizing gene localization. In Vietnam, most anatomical analysis for plant material was manually conducted which is time-consuming and labor-intensive. In this report, we present an innovative method that can deliver high-quality rice root sectioning. To optimize the efficiency of the conventional paraffin sectioning technique, the agarose-paraffin double embedding was assisted with 3D-printing technique for designing the specific mold which allow to fix from 3 to 12 rice roots in agarose. Molds consist of a lower section with grooves for firmly holding the rice roots in the right place, the additional middle sections allowing up to 3 layers of root samples to be inserted and an upper section to hold the samples before pouring the embedding. The agarose blocks obtained from the molds were then fixed in the formalin solution, then dehydrated through a gradient concentration of ethanol before being impregnated through a series of mixtures of paraffin/xylene and finally embedded in the paraffin. Anatomical sections of 10 μm of thickness were cut with a microtome, spread on glass slides, and then used for staining and collecting the images. As a result, the agarose-paraffin double embedded tissue blocks were more easily orientated and better solidified for rapid and high-quality sectioning. The sections also showed no interference of agarose with staining, as result, the tissues were much clear. In conclusion, this method represents an innovative technique for high throughput plant sectioning which could be applied to different types of plant tissues.

Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 1: Data acquisition, anatomical feature segmentation, tracking global volume and density changes

BackgroundMicrometer-resolution neuroimaging with gold-standard conventional histology requires tissue fixation and embedding. The exchange of solvents for the creation of sectionable paraffin blocks modifies tissue density and generates non-uniform brain shrinkage. New methodWe employed synchrotron radiation-based X-ray microtomography for slicing- and label-free virtual histology of the mouse brain at different stages of the standard preparation protocol from formalin fixation via ascending ethanol solutions and xylene to paraffin embedding. Segmentation of anatomical regions allowed us to quantify non-uniform tissue shrinkage. Global and local changes in X-ray absorption gave insight into contrast enhancement for virtual histology. ResultsThe volume of the entire mouse brain was 60%, 56%, and 40% of that in formalin for, respectively, 100% ethanol, xylene, and paraffin. The volume changes of anatomical regions such as the hippocampus, anterior commissure, and ventricles differ from the global volume change. X-ray absorption of the full brain decreased, while local absorption differences increased, resulting in enhanced contrast for virtual histology. These trends were also observed with laboratory microtomography measurements. Comparison with existing methodsMicrotomography provided sub-10 μm spatial resolution with sufficient density resolution to resolve anatomical structures at each step of the embedding protocol. The spatial resolution of conventional computed tomography and magnetic resonance microscopy is an order of magnitude lower and both do not match the contrast of microtomography over the entire embedding protocol. Unlike feature-to-feature or total volume measurements, our approach allows for calculation of volume change based on segmentation. ConclusionWe present isotropic micrometer-resolution imaging to quantify morphology and composition changes in a mouse brain during the standard histological preparation. The proposed method can be employed to identify the most appropriate embedding medium for anatomical feature visualization, to reveal the basis for the dramatic X-ray contrast enhancement observed in numerous embedded tissues, and to quantify morphological changes during tissue fixation and embedding.

Abstract 319: MALDI-IMS of N-glycan profiles of molecular subclasses of human hepatocellular carcinoma

Abstract Introduction: Hepatocellular Carcinoma (HCC) is the second leading cause of cancer deaths globally, and the incidence rate in the US is increasing. Recent work has identified changes in N-linked glycosylation directly in HCC tissue by MALDI-IMS glycan imaging. However, there was significant inter-tumor heterogeneity, suggesting a potential correlation between the glycan changes and molecular tumor heterogeneity. Therefore, this work will analyze HCC tissues, categorized by subtype, in an effort to fit glycosylation patterns within different classes of HCC. Methods: To analyze the glycosylation of HCC tissue samples, MALDI-IMS was utilized, which uniquely allows for the analysis of spatially mapped N-glycans to paraffin embedded tissues. The sample set of HCC tissues consisted of Hoshida HCC subtypes 1, 2, and 3 tissues. Tissues were prepared through a previously published protocol that involved antigen retrieval, spraying of PNGase F Prime with a TM-Sprayer, then spraying of MALDI matrix CHCA. Each tissue is then imaged on a Bruker MALDI solariX FT-ICR, and data is analyzed using flexImaging and SCiLS software. Results: The primary goal of this work was to determine if differences within N-glycan profiles of HCC tissues are related to the differences between Hoshida genetic subtypes. Preliminary data showed that HCC tumors contain increased fucosylation of glycoproteins, yet there was significant heterogeneity within analyzed HCC tissues regarding the expressed glycan structures. With HCC tissues that were categorized into different Hoshida subtypes, glycan MALDI-IMS data showed trends that could approximately separate the subtypes. Subtype 1, which is less differentiated and is associated with poor survival, showed the most extreme tumor-associated glycan expression when compared to the surrounding normal and cirrhotic tissue. Subtype 1 tissues showed a higher number of unique glycan structures that associated with the tumor tissue. Subtype 1 showed an average of 18.00 ± 8.48 tumor-associated glycans, subtype 2 with 4.25 ± 2.21, and subtype 3 with 2.25 ± 1.71. While the identity of these glycan structures varied from tissue to tissue, trends emerged, particularly regarding how branched and fucosylated glycans associate to tumor tissue with each subtype, including decreased fucosylation in subtype 2. Overall, this data shows that some of the heterogeneity in glycan expression of HCC tumors may be attributable to differences between genetic subtypes of HCC. Conclusion: HCC tissues exhibit glycan expression that is noticeably different from normal or cirrhotic tissue, yet there is significant variation between HCC tissues regarding both the number and identity of glycan structures that are observed on the tissue. Using the Hoshida classification system, some of that variation can be explained, which could have profound impact on the utilization of N-linked glycosylation for biomarker applications. Citation Format: Andrew DelaCourt, Anand Mehta, Alyson Black, Richard Drake, Peggi Angel, Yujin Hoshida. MALDI-IMS of N-glycan profiles of molecular subclasses of human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 319.

Comprehensive Sequencing Analysis in Subcutaneous Panniculitis-like T-Cell Lymphoma

Subcutaneous panniculitis-like T-cell lymphoma (SPTL) is a rare subtype of peripheral T-cell lymphomas predominantly affecting younger individuals. Typically, SPTL presents with multifocal subcutaneous nodules, and nearly 20% of the patients have autoimmune disorders, including systemic lupus erythematosus, juvenile rheumatoid arthritis and Sjögren disease. Unlike other lymphomas, which usually require chemotherapy for long-term survival, immunosuppressive therapy alone yields favorable outcomes in SPTL. A previous study showed allelic NAV3 gene aberrations (LOH or deletion by FISH) in 44% of the SPTL samples and comparative genomic hybridization (CGH) revealed numerous DNA copy number changes. In another study, recurrent mutations were identified in various epigenetic modifiers (72%) and components of the PI3K/AKT/mTOR signaling pathway (44%). However, molecular pathogenesis of this disease has not been clearly understood.To elucidate the causative genetic lesions of SPTL, we collected 13 patients from Thailand (n=12) and Japan (n=1). Histopathology was reviewed by hematopathologists showing karyorrhexis of tumor cells infiltrating fat lobules. In all cases, lymphoma cells expressed CD3, CD5, CD8 and TIA1 but no CD56 suggesting the cytotoxic T-cells in origin. Tumor cells expressed T-cell receptor alpha/beta. The monoclonality was confirmed by southern blot (n=12). The mean percentage of SPTL cells in tissue blocks was 38% (range 20-80%). The majority of cases (75%) showed a high proliferative index (Ki-67 expression > 70%).Regarding the clinical data, 10 of 13 patients (80%) were female. The median age at diagnosis was 30 years old. Five cases (38.5%) showed systemic symptoms and 2 cases (20%) were associated with hemophagocytic syndromes. Seven cases received cyclosporine and prednisolone. All showed complete response but 2 cases relapsed. Six cases received anthracycline-based chemotherapy but 3 cases relapsed. All relapses responded to immunosuppressive therapy.We collected paired samples from each of cases (n=13). Tumor samples were collected from Formaldehyde Fixed-Paraffin Embedded (FFPE) tissues whereas germline samples were collected from either bone marrow FFPE or blood which showed no tumor involvement. We performed whole-exome and targeted-capture sequencing in those paired tumor/germline DNA samples by SureSelect v5 kit and custom kit, respectively. To identify somatic mutations and structural variations, sequencing data was analyzed using Genomon (ver. 2.3.0) and Genomon-SV. Copy number was analyzed based on sequencing data as described previously (ASH 2016 abstract by Saeki, et al.).By whole exome sequencing, 149 mutation calls were identified in 8 samples. The somatic mutations and copy number variations were grouped into 4 biological pathways which are commonly affected in cancer.1. Apoptotic associated pathway (FADD , ACIN1 , and PPM1L) : Interestingly, a nonsense mutation of FADD was found and validated. FADD protein is critical for promoting apoptosis of T-cell progenitors at the pre-TCR checkpoint.2. Epigenetic modifier pathway (GFIB , SAB130 and CHD9) : Notably, GFI1B mutation was found to be frameshift deletion causing protein truncation. Gfi1b repressed the Gata3, a transcription factor required for survival of T-lymphocyte progenitors.3. Translation initiation pathway (DHX29 and CTIF) : Missense mutation of DHX29 was identified whereas copy number analysis revealed one case possessed focal amplification of CTIF , a component of the CBP80/CBP20 translation initiation complex and associated with nonsense-mediated decay of mRNA.4. Transcription factors (MEF2B and MGA ): Targeted sequence detected mutations in MEF2B, MGA and PMS2 , which are associated with regulation of BCL6, MYC-MAX pathway and DNA mismatch repair, respectively.At least one mutation involved in either of such 4 pathways was identified in 5/8cases (62.5%). In addition, Sequence based T-cell receptor repertoire analysis proved monoclonal expansion in 5 cases.In conclusion, the identifications of novel genetic alterations provide an insight into the lymphomagenesis of SPTL. Heterogeneous mutations in different candidate pathways suggest complexpathogenesis of this rare tumor. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

A miniature mechanical testing device for testing hydrogel-based biomaterials in a confocal microscope.

Cardiac muscle cells are the fundamental building blocks of the heart, yet little is known about their mechanical properties in either healthy or diseased states. While many have explored unloaded myocyte behavior under a variety of interventions, methods for force measurements are limited due to cell fragility. Here, we present a custom device for manipulation and mechanical testing of hydrogels embedded with delicate cardiac muscle cells. Consisting of a custom disposable flexure, which is easily interchangeable, the device has the potential for high throughput testing of cell-gel constructs. Additionally, the mechanical testing device is the size of a microscope slide - appropriate for use in most microscopes, for simultaneous imaging of the sample. The mechanical properties of a gelatin-methacryloyl hydrogel sample were assessed, and 3D volumes of gel imaged using a confocal microscope. The Young's modulus of the gel was found to be 33kPa.Clinical Relevance- High-throughput testing provides the potential to gain insight into cardiac cell mechanics. Experimentation under the influence of a variety of pharmacological interventions could improve the rate at which treatments for cardiac disease are developed. Furthermore, methods may be extended to other embedded biological tissues.

Bioresorbable, Miniaturized Porous Silicon Needles on a Flexible Water-Soluble Backing for Unobtrusive, Sustained Delivery of Chemotherapy.

Conventional melanoma therapies suffer from the toxicity and side effects of repeated treatments due to the aggressive and recurrent nature of melanoma cells. Less-invasive topical chemotherapies by utilizing polymeric microneedles have emerged as an alternative, but the sustained, long-lasting release of drug cargos remains challenging. In addition, the size of the microneedles is relatively bulky for the small, curvilinear, and exceptionally sensitive cornea for the treatment of ocular melanoma. Here, we report a design of bioresorbable, miniaturized porous-silicon (p-Si) needles with covalently linked drug cargos at doses comparable to those of conventional polymeric microneedles. The p-Si needles are built on a water-soluble film as a temporary flexible holder that can be intimately interfaced with the irregular surface of living tissues, followed by complete dissolution with saline solution within 1 min. Consequently, the p-Si needles remain embedded inside tissues and then undergo gradual degradation, allowing for sustained release of the drug cargos. Its utility in unobtrusive topical delivery of chemotherapy with minimal side effects is demonstrated in a murine melanoma model.

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy.

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 µm x 1,000 µm) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of ≤5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Immunofluorescence Tomography: High-resolution 3-D reconstruction by serial-sectioning of methacrylate embedded tissues and alignment of 2-D immunofluorescence images

Immunofluorescence tomography is a high-resolution 3-D reconstruction method based on methacrylate embedding and serial-sectioning, where 2-D images of immuno-stained serial-sections are computationally aligned into image stacks, and the 3-D volume rendered. Butyl-Methyl Methacrylate (BMMA) plastic was adopted as it preserves excellent tissue morphology and can be de-plasticized easily using an organic solvent, which enables immuno-staining of serial-sections without antibody penetration issues over millimeters of 3-D reconstructed tissue (Z-depth). High axial Z-resolution over a large volume was achieved by cutting serial-sections at 2 µm thickness. Stained sections were imaged by multiple modalities, including immunofluorescence, electron microscopy and second harmonic generation (SHG), and there are advantages over confocal microscopy as the tissue does not need to be cleared, while antibody penetration or light scattering issues are minimized. The plastic serial-sections can be re-probed, without a loss in tissue structure, using low pH glycine hydrochloride antibody elution. It is a cost-effective approach as the microscopes needed are significantly cheaper than confocal microscopes and sections can be kept indefinitely. Therefore, immunofluorescence tomography is a powerful new tool to quantify sub-populations of cells in high-resolution 3-D using antibody fluorescence. This article describes the immunofluorescence tomography method for 3-D reconstruction of epithelial tissues such as mammary gland, cornea and the hair follicle.

Evaluation of the potential clinical prognostic value of lncRNA-BANCR gene in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the seventh most common cause of cancer death in worldwide. LncRNA-BANCR is a long non-coding RNA (lncRNA), which has made new windows in cancer investigations. The aim of this survey was to determine the lncRNA-BANCR gene expression changes in patients with ESCC. In case-control investigation was performed on 150 formalin fixed-paraffin embedded tissues (75 cancerous and 75 non-cancerous tissues) of ESCC patients. The lncRNA-BANCR gene expression alteration was assessed by Real-Time PCR technique. Our findings revealed that lncRNA-BANCR gene expression was increased significantly in tumor tissues compared with the non-cancerous tissues (p = 0.0025). In addition, lncRNA-BANCR gene expression changes was positively associated with the lymph node metastasis (p = 0.013), tumor differentiation (p = 0.019) and tumor stage (p = 0.017). Our results suggest a possible role of lncRNA-BANCR in proliferation of esophageal tissues and may be considered as a potential prognostic value for ESCC metastasis.

Dynamic expression of 11 miRNAs in 83 consecutive primary and corresponding recurrent glioblastoma: correlation to treatment, time to recurrence, overall survival and MGMT methylation status.

BackgroundGlioblastoma (GBM) is the most common and the most malignant glioma subtype. Among numerous genetic alterations, miRNAs contribute to pathogenesis of GBM and it is suggested that also to GBM recurrence and resistance to therapy. Based on publications, we have selected 11 miRNAs and analyzed their expression in GBM. We hypothesized that selected miRNAs are differentially expressed and involved in primary as well as in recurrent GBM, that show significant expressional differences when different treatment options are in question, and that are related to certain patients and tumor characteristics.Patients and methodsParaffin embedded tissues, obtained from primary and corresponding recurrent tumor from 83 patients with primary GBM were used. Eleven miRNAs (miR-7, miR-9, miR-15b, miR-21, miR-26b, miR-124a, miR-199a, let-7a, let-7b, let-7d, and let-7f) were selected for qPCR expression analysis. For patients who received temozolamide (TMZ) as chemotherapeutic drug, O6-methylguanine-DNA methyltransferase (MGMT) methylation status was defined using the methyl-specific PCR.ResultsThere was a significant change in expression of miR-7, miR-9, miR-21, miR-26b, mirR-124a, miR-199a and let-7f in recurrent tumor compared to the primary. In recurrent tumor, miR-15b, let-7d and let-7f significantly changed comparing both treatment options. We also observed difference in progression free survival between patients that received radiotherapy and patients that received radiotherapy and chemotherapy, and longer survival for patients who received chemotherapy after second surgery compared to not treated patients. miR-26b showed correlation to progression free survival and let-7f to overall survival. We did not find any expression difference between the tumors with and without methylated MGMT.ConclusionsOur data suggest that analyzed miRNAs may not only contribute to pathogenesis of primary GBM, but also to tumor progression and its recurrence. Moreover, expression of certain miRNAs appears to be therapy-dependent and as such they might serve as additional biomarker for recurrence prediction and potentially predict a therapy-resistance.

Association between Toll-like receptor and tumor necrosis factor immunological pathways in uterine cervical neoplasms.

The immune system plays a critical role in the defense against human papillomavirus (HPV) infection and its persistence. Toll-like receptors (TLRs) are membrane receptors responsible for activation of the innate immune response, and an association between TLR expression and uterine cervical cancer has been shown. Tumor necrosis factors (TNFs) are among the main mediators of skin and mucosa inflammation. The aim of this study was to demonstrate the association between TLR and TNF immune expression and cervical cancer and premalignant cervical lesions. A total of 64 embedded tissues were obtained from gynecological procedures, including 35 specimens with cervical intraepithelial neoplasia (CIN) and 10 specimens with cervical squamous cell carcinoma (CSCC) as well as 19 normal cervical samples. The expression of TLR2, TLR3, TLR4, TNF-α and TNF-β was measured by immunohistochemistry and graded into low and high levels of expression. There was an association between the expression levels of TLR2 and those of TNF-α and TNF-β (p = 0.01 and p = 0.021, respectively) in the cervical cancer and CIN groups. TLR4 expression was associated with TNF-α and TNF-β expression (p = 0.016 and p = 0.025, respectively) in these 2 groups. By contrast, TLR3 was not statistically associated with TNF-α or TNF-β in any of the groups. There might be an association of the TLR2 and TLR4 pathways with the immunological response of TNF-α and TNF-β in cervical cancer. These markers are also expressed at higher levels in cervical cancer and premalignant lesions compared to normal controls.

Predictive value of APAF-1 and COX-2 expression in pathologic complete response to neoadjuvant chemoradiotherapy for patients with locally advanced rectal adenocarcinoma.

PurposeTo investigate predictive value of APAF-1 and COX-2 expression in pathologic complete response (pCR) for patients with rectal adenocarcinoma (RAC) who were treated with neoadjuvant chemoradiotherapy (neo-CRT) followed by total mesorectal excision (TME).Materials and MethodsImmunohistochemistry assay was used to detect expression of APAF-1 and COX-2 in paraffin-wax embedded tissues obtained before neo-CRT for patients with RAC. A 5-point tumor-regression grade (TRG) based on the ratio of residual tumor to fibrosis according to Dworak's scoring system was used to assess neo-CRT response. The relationship between expression of APAF-1 and COX-2 genes and pCR was explored.ResultspCR (TRG4) was observed in 23 patients (28.0%). pCR were more likely to be achieved for those with APAF-1 over-expression or lower expression of COX-2. pCR rate in patients with combination of high APAF-1 and low COX-2 expression was 56.0%, significantly higher than those with other combination of APAF1 and COX-2 expression. Multivariate analysis showed that over-expression of APAF-1 and suppressed expression of COX-2 were independent predictive factors for pCR.ConclusionImmunohistochemical evaluation of APAF-1 and COX-2 expression on pretreatment specimen may be used to predict pCR to neo-CRT in patients with RAC. The potential of the markers in monitoring pCR patient merits further investigation.

Evidencia genética revela oportunidades de mejora para preparación de tejidos en análisis forenses

Introducción: los tejidos embebidos en parafina constituyen una excelente alternativa para obtener adn, especialmente cuando no es posible contar con muestras frescas o el almacenamiento y conservación del tejido no es viable, de manera que esta muestra es el único elemento disponible para realizar un cotejo. El éxito en cualquier análisis genético es contar con métodos de fijación de tejidos y de extracción de adn adecuados que permitan obtener moléculas de buena calidad y cantidad, libres de contaminantes biológicos, químicos y microbiológicos que pueden actuar como inhibidores en los procesos de amplificación por pcr. Métodos y materiales: en el laboratorio de genética del Instituto Nacional de Medicina Legal y Ciencias Forenses, Regional Suroccidente, Colombia, se realizó una prueba preliminar para la validación de la extracción de adn en tejidos embebidos en parafina, utilizando el método orgánico validado y empleado de rutina por ellaboratorio de genética de Bogotá, Colombia. Resultados y discusión: se corroboró la eficacia del método a partir del análisis de reproducibilidad, respetabilidad y concordancia en cuanto a concentración de adn, y obtención de perfiles genéticos a partir de los extractos de adn de cuatro réplicas de tres diferentes tipos de tejidos obtenidos a partir de un feto, con respecto a una muestra de sangre en soporte fta. Conclusiones: con los resultados preliminares obtenidos, se encontraron algunas oportunidades de mejora para los laboratorios que intervienen en el proceso.

An automatically contamination-avoiding technique for intracorporeal esophagojejunostomy using a transorally inserted anvil during laparoscopic total gastrectomy for gastric cancer.

BackgroundIntracorporeal Roux-en-Y esophagojejunostomy during laparoscopic total gastrectomy for gastric cancer remains a challenging manipulation due to the uncontrolled direction of the jejunal side or unintended embedded tissues, although several methods have been introduced. In this study, we simplified the procedure based on a surgical string fixing technique using a transorally inserted anvil (OrVil™; Covidien Ltd., Mansfield, MA, USA).MethodsFrom March 2012 to September 2013, 14 consecutive patients underwent simplified intracorporeal Roux-en-Y esophagojejunostomy using OrVil™ during laparoscopic total gastrectomy for gastric cancer at our hospital. Clinicopathologic characteristics and surgical outcomes of these patients were retrospectively analyzed.ResultsAll of the procedures were successful completed with no complication or conversion to open surgery. The mean overall operative time was 193.8 ± 41.8 min, whereas the mean reconstruction time was 32.6 ± 4.6 min. The mean estimated blood loss was 105.7 ± 65.4 ml. The mean diameter of anastomosis measured by upper gastrointestinal contrast X-ray test at 1 month after operation was 2.3 cm. During a median follow-up period of 12 months, neither local recurrence nor anastomosis-related morbidity was observed.ConclusionsOur preliminary results suggested that this automatically contamination-avoiding technique based on a surgical-string-fixing strategy using OrVil™ during laparoscopic total gastrectomy for gastric cancer might be feasible and safe and provide a simple solution for intracorporeal Roux-en-Y esophagojejunostomy.

Ultrastructurally smooth thick partitioning and volume stitching for large-scale connectomics.

FIB-SEM has become an essential tool for studying neural tissue at resolutions below 10×10×10 nm, producing datasets superior for automatic connectome tracing. We present a technical advance, ultrathick sectioning, which reliably subdivides embedded tissue samples into chunks (20 µm thick) optimally sized and mounted for efficient, parallel FIB-SEM imaging. These chunks are imaged separately and then ‘volume stitched’ back together, producing a final 3D dataset suitable for connectome tracing.

Developing 3D SEM in a broad biological context

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.Lay DescriptionLife happens in three dimensions. For many years, first light, and then EM struggled to image the smallest parts of cells in 3D. With recent advances in technology and corresponding improvements in computing, scientists can now see the 3D world of the cell at the nanoscale. In this paper we present the results of high resolution 3D imaging in a number of diverse cells and tissues from multiple species. 3D reconstructions of cell structures often revealed them to be significantly more complex when compared to extrapolations made from 2D studies. Correlating functional 3D LM studies with 3D EM results opens up the possibility of making new strides in our understanding of how cell structure is connected to cell function.