Fresh Sections Research Articles

Characterization of the Native Oxide Shell of Copper Metal Powder Spherical Particles.

The native oxide layer that forms on copper (Cu) metal spherical particle surfaces under ambient handling conditions has been shown to have a significant effect on sintering behavior during microwave heating in a previous study, where an abnormal expansion was observed and characterized during sintering of Cu compacts using reducing gases. Because microwave (MW) heating is selective and depends greatly on the dielectric properties of the materials, this thin oxide layer will absorb MW energy easily and can consequently be heated drastically starting from room temperature until the reduction process occurs. In the current study, this oxide ceramic layer was qualitatively and quantitatively characterized using the carrier gas hot extraction (CGHE) method, Auger electron spectroscopy (AES), and a dual-beam focused ion beam (FIB)/scanning electron microscope (SEM) system that combines both FIB and SEM in one single instrument. Two different commercial gas-atomized spherical Cu metal powders with different particle sizes were investigated, where the average oxygen content of the powders was found to be around 0.575 wt% using the CGHE technique. Furthermore, AES spectra along with depth profile measurements were used to qualitatively characterize this oxide layer, with only a rough quantitative thickness approximation due to method limitations and the electron beam reduction effect. For the dual-beam FIB-SEM system, a platinum (Pt) coating was first deposited on the Cu particle surfaces prior to any characterization in order to protect and to preserve the oxide layer from any possible beam-induced reduction. Subsequently, the Pt-coated Cu particles were then cross-sectioned in the middle in situ using an FIB beam, where SEM micrographs of the resulted fresh sections were characterized at a 36° angle stage tilt with four different detector modes. Quantitative thickness characterization of this native oxide layer was successfully achieved using the adapted dual-beam FIB-SEM setup with more accuracy. Overall, the native Cu oxide layer was found to be inhomogeneous over the particles, and its thickness was strongly dependent on particle size. The thickness ranged from around 22–67 nm for Cu powder with a 10 µm average particle size (APS) and around 850–1050 nm for one with less than 149 µm.

An ultrasonically actuated fine-needle creates cavitation in bovine liver.

Ultrasonic cavitation is being used in medical applications as a way to influence matter, such as tissue or drug vehicles, on a micro-scale. Oscillating or collapsing cavitation bubbles provide transient mechanical force fields, which can, e.g., fractionate soft tissue or even disintegrate solid objects, such as calculi. Our recent study demonstrates that an ultrasonically actuated medical needle can create cavitation phenomena inside water. However, the presence and behavior of cavitation and related bioeffects in diagnostic and therapeutic applications with ultrasonically actuated needles are not known. Using simulations, we demonstrate numerically and experimentally the cavitation phenomena near ultrasonically actuated needles. We define the cavitation onset within a liver tissue model with different total acoustic power levels. We directly visualize and quantitatively characterize cavitation events generated by the ultrasonic needle in thin fresh bovine liver sections enabled by high-speed imaging. On a qualitative basis, the numerical and experimental results show a close resemblance in threshold and spatial distribution of cavitation. These findings are crucial for developing new methods and technologies employing ultrasonically actuated fine needles, such as ultrasound-enhanced fine-needle biopsy, drug delivery, and histotripsy.

Relationship between the initial number of carried Bursaphelenchus xylophilus and its transmission by Monochamus carolinensis with reference to virulence

Summary The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the causative agent of pine wilt disease and is transmitted by cerambycid adults of the genus Monochamus. It is believed to have been introduced into Japan from North America in the 1900s and since then it has been devastating the native pine forests. By contrast, the epidemics of the disease have never occurred in pine forests native to North America in the original habitats, indicating no expression of virulence. To determine the evolutionary change in the transmission traits of PWN, the temporal pattern of PWN transmission to fresh branch sections of Pinus sylvestris was studied using 38 Monochamus carolinensis adults in Illinois, USA, and compared with previous studies on the PWN-M. alternatus and B. mucronatus-M. saltuarius systems in Japan. A great difference was observed in the initial nematode load among vectors irrespective of the three systems. The maximal numbers of PWN transmitted (5 days)−1 by individual vectors were greater and the efficiencies of nematode transmission and invasion were higher for the Japanese PWN-M. alternatus system than for the North American PWN-M. carolinensis system in two heavy classes of initial load of over 1000 nematodes. The proportion of PWN transmitted to healthy pine trees by reproductively immature female vectors was small (2.5-11.5%) in the three nematode-vector systems in most cases. The evolutionary changes in the transmission traits and the control of the range expansion of the PWN were discussed in relation to the expression of virulence.

Cytobacts: Abundant and Diverse Vertically Seed-Transmitted Cultivation-Recalcitrant Intracellular Bacteria Ubiquitous to Vascular Plants

We have recently described ‘Cytobacts’ as abundant intracellular endophytic bacteria inhabiting live plant cells based on the observations with callus and cell suspension cultures of grapevine and other plant species with the origin ascribable to field explants. In this study, we investigated the prevalence of such cytoplasmic bacterial associations in field plants across different taxa, their cultivability, and the extent of taxonomic diversity and explored the possibility of their embryo-mediated vertical transmission. Over 100 genera of field plants were surveyed for ‘Cytobacts’ through bright-field live-cell imaging as per our previous experience using fresh tissue sections from surface-sterilized shoot-tissues with parallel cultivation-based assessments. This revealed widespread cellular bacterial associations visualized as copious motile micro-particles in the cytoplasm with no or sparse colony forming units (CFU) from the tissue-homogenates indicating their general non-cultivability. Based on the ease of detection and the abundance of ‘Cytobacts’ in fresh tissue sections, the surveyed plants were empirically classified into three groups: (i) motile bacteria detected instantly in most cells; (ii) motility not so widely observed, but seen in some cells; and (iii) only occasional motile units observed, but abundant non-motile bacterial cells present. Microscopy versus 16S-rRNA V3–V4 amplicon profiling on shoot-tip tissues of four representative plants—tomato, watermelon, periwinkle, and maize—showed high bacterial abundance and taxonomic diversity (11–15 phyla) with the dominance of Proteobacteria followed by Firmicutes/Actinobacteria, and several other phyla in minor shares. The low CFU/absence of bacterial CFU from the tissue homogenates on standard bacteriological media endorsed their cultivation-recalcitrance. Intracellular bacterial colonization implied that the associated organisms are able to transmit vertically to the next generation through the seed-embryos. Microscopy and 16S-rRNA V3–V4 amplicon/metagenome profiling of mature embryos excised from fresh watermelon seeds revealed heavy embryo colonization by diverse bacteria with sparse or no CFU. Observations with grapevine fresh fruit-derived seeds and seed-embryos endorsed the vertical transmission by diverse cultivation-recalcitrant endophytic bacteria (CREB). By and large, Proteobacteria formed the major phylum in fresh seed-embryos with varying shares of diverse phyla. Thus, we document ‘Cytobacts’ comprising diverse and vertically transmissible CREBs as a ubiquitous phenomenon in vascular plants.

Expression of GPR68, an Acid-Sensing Orphan G Protein-Coupled Receptor, in Breast Cancer.

BackgroundBreast cancer (BC) is the most diagnosed cancer and the leading cause of global cancer incidence in 2020. It is quite known that highly invasive cancers have disrupted metabolism that leads to the creation of an acidic tumor microenvironment. Among the proton-sensing G protein-coupled receptors is GPR68. In this study, we aimed to explore the expression pattern of GPR68 in tissues from BC patients as well as different BC cell lines. Methods: In-silico tools were used to assess the expression of GPR68 in BC patients. The expression pattern was validated in fresh and paraffin-embedded sections of BC patients using qPCR and immunohistochemistry (IHC), respectively. Also, in-silico tools investigated GPR68 expression in different BC cell lines. Validation of GPR68 expression was performed using qPCR and immunofluorescence techniques in four different BC cell lines (MCF-7, MDA-MB-231, BT-549 and SkBr3). Results: GPR68 expression was found to be significantly increased in BC patients using the in-silico tools and validation using qPCR and IHC. Upon classification according to the molecular subtypes, the luminal subtype showed the highest GPR68 expression followed by triple-negative and Her2-enriched cells. However, upon validation in the recruited cohort, the triple-negative molecular subtype of BC patients showed the highest GPR68 expression. Also, in-silico and validation data revealed that the triple-negative breast cancer cell line MDA-MB-231 showed the highest expression of GPR68. Conclusion: Therefore, this study highlights the potential utilization of GPR68 as a possible diagnostic and/or prognostic marker in BC.

Abstract 100: Meningeal Lymphatic System Modulates Intra-parenchymal Hematoma Clearance And Brain Recovery In Experimental Intracerebral Hemorrhage

Background: Intracerebral hemorrhage (ICH) brain injury is induced by toxic blood components and subsequent neurologic deficits can be alleviated by promoting hematoma resolution. The meningeal lymphatic system has been shown to mediate substances clearance from the central nervous system, including macromolecules and pathogenic metabolites. However, it remains unclear whether this system plays a role in hematoma clearance and ICH outcomes. In this study, we investigated the correlation between meningeal lymphatic function and ICH pathologies. Pharmacological promotion of meningeal lymphatic function was also used to determine whether targeting meningeal lymphatic system can facilitate ICH recovery. Methods: Whole-mount meninges were used to evaluate the complexity and coverage rate of meningeal lymphatic vessels. Meningeal lymphatic drainage was evaluated by injecting fluorescent microbeads and PKH26-labeled erythrocytes into the cisterna magna and brain parenchyma, respectively. Hematoma volume was measured in fresh coronal brain sections. Neurobehavioral performance was evaluated by cylinder test. Pathological outcomes including iron deposition, residual erythrocytes, neuronal loss, and astrogliosis were detected by immunohistochemistry or immunofluorescence. Results: Meningeal lymphangiogenesis and increased lymphatic drainage occur during the late phase of ICH. Depriving the function of meningeal lymphatic vessels impeded hematoma clearance, whereas boosting meningeal lymphatic drainage and lymphangiogenesis improved hematoma clearance rate and behavioral function as well as reduced iron deposition, neuronal loss, and astrogliosis in the ICH brain. Conclusion: Early enhancement of meningeal lymphatic function is beneficial for ICH recovery, implicating the meningeal lymphatic system as a potential therapeutic target for treating ICH.

Morphological, Phytochemical and Fouriertransform Infrared Spectroscopy (FTIR) Spectroscopic Analysis of Selected Species of Portulacaceae: A Study

Introduction: Morphological characters of plants to assess the differences or similarities in plant taxa and use these characters for plant identification. And also an attempt is made in the present study to analyze the functional group of active compounds present in the leaf and stem extract of selected species of Portulacaceae. Objective: To study morphological, phytochemical and Fourier-transform infrared spectroscopy (FTIR) spectroscopic analysis of selected species of Portulaceae. Methodology: Physical examination and measurement of physiognomic features of fresh floral and vegetative sections of the plants were used to conduct a morphological study on the genus Portulaca. Phytochemicals were screened and analyzed with standard methods. 2,2-diphenyl-1-picrylhydrazylquenching assay were selected to investigate antioxidant free radical scavenging activity respectively. Result: Morphologically they are dissimilar. Leaves are needle in P. grandiflora and obovate in P. umbraticola. Petals are numerous in P.grandiflora and 5 in P. umbraticola. The result of preliminary phytochemical screening indicated that leaf and stem of both species. Both plants were free from steroids. Moreover, quantitative estimation of phytochemicals also exhibited that leaf and stem of both species. Secondary metabolites, which are abundant in plants and have fascinating biological activities, are an important source with a variety of structural arrangements and properties. They have a rich source of protein and have high antioxidant scavenging activity. Conclusion: Both species of Portulacaceae carry rich protein and have high therapeutic values. Therefore further efficacy and safety studies are encouraged on this potential herb with the hope of replacing some less effective ones in clinical practice especially for antidiabetic and anticancer.

Effects of MCC950 on nerve injury in rats with intracerebral hemorrhage

Objective: To investigate the effects of the pyrin domain-containing protein 3 (NLRP3) inflammasome inhibitor MCC950 on nerve injury in rats with intracerebral hemorrhage(ICH). Methods: Seventy-two SD rats were randomly divided into three groups (n=24): Sham group, ICH group and MCC950 group. ICH group and MCC950 group rats were injected with autogenous non-anticoagulant blood to establish ICH model, and then the rats in MCC950 group were intraperitoneally injected with MCC950 at the dose of 10 mg/kg(2 mg/ml) for 3 days after ICH model was established. Seventy-two hours after the establishment of the model, the forelimb placement test, the corner test and mNSS score were performed to observe the neurological function of the rats with ICH. The volume of hematoma was observed in fresh brain tissue sections. HE staining was used to observe the pathological changes of brain tissue. The dry-wet weight ratio was calculated to evaluate the changes of brain tissue edema. The degeneration of neurons was observed by FJC staining. The neuronal apoptosis was observed by TUNEL staining. The protein expression and activation levels of NLRP3, ASC, caspase-1, IL-1β, IL-18 and GSDMD were determined by Western blot. Results: Compared with sham group, the percentage of successful placement of left forelimb and left turn was decreased significantly (P＜0.01, P＜0.05), mNSS score was increased significantly (P＜0.01) in ICH group. Hematoma volume was increased significantly, the number of microglial cells around the hematoma was increased, the number of neurons was decreased, nerve cell swelled, some cells showed pyknotic necrosis, and the staining was deepened. The water content of the right base was increased significantly (P＜0.05). The number of FJC positive and TUNEL positive cells around the hematoma was increased significantly (P＜0.05). The levels of NLRP3, ASC, caspase-1, pro-caspase-1, caspase-1/pro-caspase-1 ratio, GSDMD-N, GSDMD, GSDMD-N/GSDMD ratio, IL-1β and IL-18 were increased significantly (P＜0.01, P＜ 0.05). Compared with ICH group, the percentage of successful placement of left forelimb and left turn was increased significantly in MCC950 group (P＜0.05), while the mNSS score and the volume of hematoma were decreased significantly (P＜0.01), the swelling degree of nerve cells around the hematoma was reduced significantly, and the number of pyrotic necrotic cells was decreased. The water content of the right base was decreased significantly (P＜0.05), and the number of FJC positive and TUNEL positive cells around the hematoma was decreased significantly (P＜0.05). The levels of NLRP3, ASC, caspase-1, pro-caspase-1, caspase-1/pro-caspase-1 ratio, GSDMD-N, GSDMD, GSDMD-N/GSDMD ratio, IL-1β and IL-18 were decreased significantly (P＜0.05). Conclusion: MCC950 can ameliorate nerve injury after ICH by inhibiting NLRP3 inflammasome mediated inflammation and pyroptosis.

NOTTINGHAM HISTOLOGICAL GRADING OF CASES OF PRIMARY INVASIVE CARCINOMA OF THE BREAST AT LAUTECH TEACHING HOSPITALS, NIGERIA

Background Breast cancer has since been known to be a global public health problem being the most common malignancy in women worldwide. The incidence in Nigeria is yet to be determined but is said to range from three and a half to thirteen and a half percent in hospital-based reports. Objective Our study aimed at grading the cases of primary invasive carcinoma of the breast using the Nottingham Histological System of grading. Methods The study was a hospital-based retrospective study carried out at LAUTECH Teaching Hospitals in Osogbo and Ogbomoso with ethical approval being obtained from the research and Ethics Committee of LAUTECH Teaching Hospital Ogbomoso. The original request cards were retrieved, studied and essential clinical details which include age, and the side (right or left) of the breast tumor were extracted. Tissue blocks were also retrieved and fresh sections about 3-5µm were cut from formalin-fixed and paraffin-embedded blocks and stained with Hematoxylin and Eosin dyes for histological analyses. Data obtained was analyzed using both Microsoft Excel and EPI Info statistical software and statistical package for social sciences. The information obtained was reported using frequencies and percentages. Quantitative data were presented as mean ± SD [Standard Deviation]. A 95% confidence interval was used in this study and a P-value of ≤ 0.05 was considered statistically significant. Result Our study revealed the commonest histological variant of primary invasive carcinoma of the female breast to be infiltrating ductal carcinoma, not specified type with 305 cases (88.9%).

Deep-Learning-Based Segmentation of Fresh or Young Concrete Sections from Images of Construction Sites.

The objective of this study is to evaluate the feasibility of deep-learning-based segmentation of the area covered by fresh and young concrete in the images of construction sites. The RGB images of construction sites under various actual situations were used as an input into several types of convolutional neural network (CNN)–based segmentation models, which were trained using training image sets. Various ranges of threshold values were applied for the classification, and their accuracy and recall capacity were quantified. The trained models could segment the concrete area overall although they were not able to judge the difference between concrete of different ages as professionals can. By increasing the threshold values for the softmax classifier, the cases of incorrect prediction as concrete became almost zero, while some areas of concrete became segmented as not concrete.

Immunohistochemical characterization of KS cases seen in Nnewi Anambra state using HHV-8 LNA1 and HIV-1P24 antibodies

Introduction: Kaposi sarcoma (KS) is of public health significance in sub-Saharan Africa, including Nigeria, especially in the era of HIV/AIDS. Several works have been done on the prevalence and patterns of KS both in Nigeria and other parts of Africa, with a reported significant prevalence. We employed immunohistochemistry to characterise the morphologic KS cases seen in Nnewi. Materials and Methods: The Formalin Fixed, Paraffin Embedded (FFPE) tissue blocks of all haematoxylin and eosin (H&E) diagnosed cases of KS seen in the archives of histopathology facilities in Nnewi, Anambra State over 15 year period were retrieved. Fresh sections were made from the tissue blocks of the 82 cases that met the inclusion criteria for the study, and were subjected to immunohistochemistry using HHV-8 LNA1 (Human Herpes Virus-8 Latent nuclear antigen1) and HIV-1p24 antibodies and reviewed. Results: A total of 82 KS cases were studied, 69 of which were confirmed KS cases on immunohistochemistry. KS accounted for 1.20% and 14.47% of solid malignancies and sarcomas respectively. Nearly 80% of these were HIV/AIDS-associated, 59.3% of which occurred in females. KS occurred more in the third decade with an age range of 7-74years. Conclusion: KS is quite common in our environment and is largely HIV/AIDS associated. Reducing the burden of HIV/AIDS will invariably reduce KS burden.

Altering product placement to create a healthier layout in supermarkets: Outcomes on store sales, customer purchasing, and diet in a prospective matched controlled cluster study.

BackgroundPrevious product placement trials in supermarkets are limited in scope and outcome data collected. This study assessed the effects on store-level sales, household-level purchasing, and dietary behaviours of a healthier supermarket layout.Methods and findingsThis is a prospective matched controlled cluster trial with 2 intervention components: (i) new fresh fruit and vegetable sections near store entrances (replacing smaller displays at the back) and frozen vegetables repositioned to the entrance aisle, plus (ii) the removal of confectionery from checkouts and aisle ends opposite. In this pilot study, the intervention was implemented for 6 months in 3 discount supermarkets in England. Three control stores were matched on store sales and customer profiles and neighbourhood deprivation. Women customers aged 18 to 45 years, with loyalty cards, were assigned to the intervention (n = 62) or control group (n = 88) of their primary store. The trial registration number is NCT03518151. Interrupted time series analysis showed that increases in store-level sales of fruits and vegetables were greater in intervention stores than predicted at 3 (1.71 standard deviations (SDs) (95% CI 0.45, 2.96), P = 0.01) and 6 months follow-up (2.42 SDs (0.22, 4.62), P = 0.03), equivalent to approximately 6,170 and approximately 9,820 extra portions per store, per week, respectively. The proportion of purchasing fruits and vegetables per week rose among intervention participants at 3 and 6 months compared to control participants (0.2% versus −3.0%, P = 0.22; 1.7% versus −3.5%, P = 0.05, respectively). Store sales of confectionery were lower in intervention stores than predicted at 3 (−1.05 SDs (−1.98, −0.12), P = 0.03) and 6 months (−1.37 SDs (−2.95, 0.22), P = 0.09), equivalent to approximately 1,359 and approximately 1,575 fewer portions per store, per week, respectively; no differences were observed for confectionery purchasing. Changes in dietary variables were predominantly in the expected direction for health benefit. Intervention implementation was not within control of the research team, and stores could not be randomised. It is a pilot study, and, therefore, not powered to detect an effect.ConclusionsHealthier supermarket layouts can improve the nutrition profile of store sales and likely improve household purchasing and dietary quality. Placing fruits and vegetables near store entrances should be considered alongside policies to limit prominent placement of unhealthy foods.Trial registrationClinicalTrials.gov NCT03518151 (pre-results)

Abstract 455: Consistent multi-lab performance of highly multiplexed RNA fusion detection with low RNA input and limit of detection

Abstract Gene fusions, which are usually caused by chromosomal rearrangements, are frequently associated with various cancers. Detection of fusion mutations is an important part of cancer management, and are usually detected in RNA purified from biopsies of fresh tissue or FFPE sections. The amount of RNA from these samples, however, is usually limited. We developed a new, PCR-based fusion detection method, which even allows detection of unknown and novel fusion mutations. We demonstrate its consistent performance across various RNA inputs, and sequencing depths, in terms of technical performance and limit of detection. A multiplex PCR RNA fusion panel was designed to cover known RNA fusion targets and unknown fusion targets for target enrichment via single primer multiplex PCR. SeraSeq fusion Reference RNA was used to evaluate the limit of detection of the method. The amplicons were about 110 bp on average to allow for target amplification from challenging fragmented samples such as FFPE RNA. After reverse transcription, the panel was used in a multiplex PCR to amplify the targets, and a final PCR was used to add sequencing adapters and sample indexes. The resulting libraries were sequenced in Illumina sequencers. The library was analyzed for the detection of known fusion variants across different initial input of targets, as well as the mapping rate, onto the target, and the presence of human ribosomal RNA. We found that the lengths of the fragmented RNA had little effect on the performance of the libraries. With various amount of input RNA, the required numbers of PCR cycles were optimized. We observe a linear relationship between the library yields and input amounts of RNA from 10 to 50ng, which had R1 mapping rates above 95% and 98%, respectively. We report the detection of all twelve fusion variants, which are present in the panel and in the Seraseq Fusion RNA Mix v4 reference standard and covered by our panel on each NGS platform with an average sequencing depth of 0.2M reads, or 3600 reads/amplicon across all assays. Even at inputs as low as 3 ng, all 12 fusions were typically detected. In general, the results for individual fusions among the different replicates were concordant, with limited observed variance in reads across some fusion junctions between assays and replicates. Over 90% of reads supported the fusion call from all inputs (ranging from 1.6 to 50 ng). Over 97% of reads supported the fusion call between 12 and 50 ng. A multi-lab validation confirmed the above results, and were also comparable when using an additional distinct CleanPlex fusion panel. CleanPlex fusion technology allows for reliable and simultaneous detection of 12 designed clinically relevant RNA fusions even at low input amounts. The data from a multi-lab study support the use of this technology for targeted NGS RNA fusion assays. The CleanPlex fusion technology provides consistent and sensitive results for a wide variety of sample inputs. Citation Format: David N. Debruyne, Chenyu Li, Julia Spencer, Vidushi Kapoor, Lily Y. Liu, Lucie Lee, Rounak Feigelman, Grayson Burdon, Jeffrey Liu, Guoying Liu, Zhitong Liu. Consistent multi-lab performance of highly multiplexed RNA fusion detection with low RNA input and limit of detection [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 455.

Abstract 2705: Spatial transcriptomics - a valuable tool to visualize compound effect in precision cut cancer tissue slices (PCCTS)

Abstract The recent advances in immunotherapies, such as immune checkpoint modulators, bispecific antibodies, and adoptive T-cell transfer, opens new opportunities for the treatment of cancer. Having this broad spectrum of new therapeutic agents available, the demand for predictive and robust preclinical models to minimize translational failures in immuno-oncology is increasing. Indivumed has successfully implemented the model of Precision Cut Cancer Tissue Slices (PCCTS) derived from viable human tumor tissue to serve as a platform for testing different applications such as chemotherapeutic agents, small molecules and antibodies. In this study, we investigated the effects of OKT3® (Muromonab), a therapeutic antibody against CD3, on PCCTS from a patient diagnosed with non-small cell lung cancer (NSCLC) in respect of gene expression changes using Spatial Transcriptomics. Spatial Transcriptomics (10x Genomics) is a technique to spatially resolve whole RNA-seq information. Tissue sections were prepared using a Krumdieck Tissue slicer and fresh tissue sections were incubated for 24 h with 10 µg/ml OKT3 followed by performing the Visium Spatial Gene Expression Workflow from 10x Genomics. For this purpose, a glass slide containing spatially barcoded capture probes were used to bind mRNAs of a tissue section. Following cDNA synthesis and library preparation, the libraries were sequenced using standard RNA-seq technology. Specific sequence barcodes allowed to assign gene expression data to the histological positions in the tissue. For visualization of gene expression patterns, the software Space Ranger and Loupe Browser were used. This method enabled the identification of cellular subpopulations in the spatial context before and after treatment with OKT3, whereas molecular identities and positional information of diverse cells would have been lost upon transcriptome analysis of bulk samples. Our data showed significant differences between untreated and OKT3 treated tissue slices especially in the microenvironment that encompasses inflammatory cells, extracellular matrix, and stromal cells interacting with tumor cells for cancer growth and progression. The combination of the PCCTS platform with Spatial Transcriptomics has been shown to be most valuable for the understanding of compound effects. The location of cells and their changes in gene expression patterns are important to understand tissue functionality and corresponding pathological changes. Citation Format: Nicole Kerstedt, Nicole Grabinski, Kristina Bernoth, Monika Schoeppler, Bita Motamedi-Baniassad, Jana Krueger, Moiken Petersen, Hartmut Juhl, Kerstin A. David. Spatial transcriptomics - a valuable tool to visualize compound effect in precision cut cancer tissue slices (PCCTS) [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 2705.

A pilot study on coarse ware ceramic fabrics from the Ayios Vasileios Survey Project (Greece)

In this article, we present the results of our pilot study on coarse ware ceramic fabrics from the Ayios Vasileios Survey Project (Laconia, Greece). The aim of this pilot was to explore the potential of optical fabric analysis on coarse wares on the basis of (mineral) inclusions detectable by eye or under modest magnification. We aimed to answer the following question: can we discern Bronze Age coarse wares from Byzantine/Early Modern coarse wares by means of this technique? We studied 177 ceramic fragments by eye and by means of a stereo microscope. This resulted in the description of 51 different provisional fabrics. Only a few of these fabrics could be assigned to a specific time period with certainty, based on a consistent dating of the sherds by the ceramic specialists, who looked at shape, decoration and fabric. Most of the fabrics seem to consist of sherds stemming from various time periods. A comparison between our provisional fabric groups and those published by other researchers in Laconia shows that possible connections or matches between fabrics made by us should be considered either as tentative or as unreliable beyond the level of argued assumptions. To arrive at more reliable ceramic fabric connections, or the identification of similar fabrics, it will be necessary to publish not only textual descriptions and images of thin sections—as seems to be the common approach—but also series of high-resolution pictures of sherds and their fresh sections, as has been done in this article, together with more detailed descriptions of these sherds.

Geochemistry, origin and anatexis temperature of monzogranite formation in Mount Khalaj (Mashhad, Iran)

Geochemistry, origin and anatexis temperature of monzogranite formation in Mount Khalaj (Mashhad, Iran)

Consistent performance of highly multiplexed RNA fusion detection with low RNA input and limit of detection.

e15029 Background: Gene fusions are usually caused by chromosomal rearrangements and frequently associated with various cancers. Detection of fusion mutations is an important part of cancer management, and are usually detected in RNA purified from biopsies of fresh tissue or FFPE sections. However, the amount of RNA from these samples is usually limited. We developed a new, PCR-based fusion detection method, which allows detection of unknown and novel fusion mutations. We demonstrate its consistent performance across various RNA inputs, and sequencing depths, in terms of technical performance and limit of detection. Methods: A multiplex PCR RNA fusion panel was designed to cover known RNA fusion targets and unknown fusion targets for target enrichment via single primer multiplex PCR. SeraSeq fusion Reference RNA was used to evaluate the limit of detection of the method. The amplicons were about 110 bp on average to allow for target amplification from challenging fragmented samples such as FFPE RNA. After reverse transcription, the panel was used in a multiplex PCR to amplify the targets, and a final PCR was used to add sequencing adapters and sample indexes. The resulting libraries were sequenced on Illumina sequencers. The data was analyzed for the detection of known fusion variants across varying initial inputs of targets, as well as the performance in terms of mapping and on-target rates, as well as the presence of human ribosomal RNA. Results: We found that the fragmented RNA length had little effect on the performance of the libraries. The required numbers of PCR cycles were optimized based on testing with various amounts of starting RNA input material. We observe a linear relationship between the library yields and RNA input amounts from 10 to 50ng, which had R1 mapping rates above 95% and 98%, respectively. We report the detection of all twelve fusion variants, which are targeted by our panel and present in the Seraseq Fusion RNA Mix v4 reference standard, and which are covered with an average sequencing depth of 0.2M reads, or 3600 reads/amplicon, across all assays. Even at inputs as low as 3 ng, all 12 fusions were typically detected. In general, the results for individual fusions among the different replicates were concordant, with limited observed variance in reads across some fusion junctions between assays and replicates. Over 90% of the reads supported the fusion calls from all inputs (ranging from 1.6 to 50 ng). Over 97% of the reads supported the fusion calls between 12 and 50 ng. Conclusions: A multi-lab validation confirmed the above results, and were also comparable when using an additional distinct CleanPlex fusion panel. To conclude, our CleanPlex fusion technology allows for reliable and simultaneous detection of 12 designed clinically relevant RNA fusions even at low input amounts.

Lung immune tone via gut-lung axis: gut-derived LPS and short-chain fatty acids' immunometabolic regulation of lung IL-1β, FFAR2, and FFAR3 expression.

Microbial metabolites produced by the gut microbiome, e.g. short-chain fatty acids (SCFA), have been found to influence lung physiology and injury responses. However, how lung immune activity is regulated by SCFA is unknown. We examined fresh human lung tissue and observed the presence of SCFA with interindividual variability. In vitro, SCFA were capable of modifying the metabolic programming in LPS-exposed alveolar macrophages (AM). We hypothesized that lung immune tone could be defined by baseline detection of lung intracellular IL-1β. Therefore, we interrogated naïve mouse lungs with intact gut microbiota for IL-1β mRNA expression and localized its presence within alveolar spaces, specifically within AM subsets. We established that metabolically active gut microbiota, which produce SCFA, can transmit LPS and SCFA to the lung and thereby could create primed lung immunometabolic tone. To understand how murine lung cells sensed and upregulated IL-1β in response to gut microbiome-derived factors, we determined that, in vitro, AM and alveolar type II (AT2) cells expressed SCFA receptors, free fatty acid receptor 2 (FFAR2), free fatty acid receptor 3 (FFAR3), and IL-1β but with distinct expression patterns and different responses to LPS. Finally, we observed that IL-1β, FFAR2, and FFAR3 were expressed in isolated human AM and AT2 cells ex vivo, but in fresh human lung sections in situ, only AM expressed IL-1β at rest and after LPS challenge. Together, this translational study using mouse and human lung tissue and cells point to an important role for the gut microbiome and their SCFA in establishing and regulating lung immune tone.

Spiny Dogfish (Squalus Acanthias): Changes in Structural and Mechanical Properties Under Storage

Fresh histological sections of the Black Sea spiny dogfish have been examined using a 500xSDM electron microscope. Experimental histological sections of the Black Sea spiny dogfish have been made along and across muscle fibers. Sections of skin and adipose tissue between the skin and the muscle have also been examined. PH value in the spiny dogfish muscle layers has been measured using a Kelilong Electron pH-150MI device. Thus, there are three zones determined: the first one is within 24-42 hours of storage with a pH ranging from 6,77; the second one – 46-78 hours with a pH in the range of 6,55; the third one – from 85 to 97 hours of storage with a pH of 6,41. The study of the elastic properties of fish is a study of the deformation properties. Regularities of change of rheological properties of fish depending on term and temperature mode of storage have been established. Graphs of values of deforming force of samples of different shelf life have been obtained, comparison of deformation time and size of deforming values have been made. Results of physical properties are placed in the process of optimizing the parameters of shelf life of the spiny dogfish. Mathematical models describing changes in the basic physicochemical properties of spiny dogfish in different storage parameters are.

Chloroplast Distribution in the Stems of 23 Eucalypt Species.

Small diameter branchlets and smooth barked stems and branches of most woody plants have chloroplasts. While the stems of several eucalypt species have been shown to photosynthesise, the distribution of chloroplasts has not been investigated in detail. The distribution of chloroplasts in branchlets (23 species) and larger diameter stems and branches with smooth bark (14 species) was investigated in a wide range of eucalypts (species of Angophora, Corymbia and Eucalyptus) using fresh hand sections and a combination of bright field and fluorescence microscopy. All species had abundant stem chloroplasts. In both small and large diameter stems, the greatest concentration of chloroplasts was in a narrow band (usually 100–300 μm thick) immediately beneath the epidermis or phellem. Deeper chloroplasts were present but at a lower density due to abundant fibres and sclereids. In general, chloroplasts were found at greater depths in small diameter stems, often being present in the secondary xylem rays and the pith. The cells of the chlorenchyma band were small, rounded and densely packed, and unlike leaf mesophyll. A high density of chloroplasts was found just beneath the phellem of large diameter stems. These trees gave no external indication that green tissues were present just below the phellem. In these species, a thick phellem was not present to protect the inner living bark. Along with the chlorenchyma, the outer bark also had a high density of fibres and sclereids. These sclerenchyma cells probably disrupted a greater abundance and a more organised arrangement of the cells containing chloroplasts. This shows a possible trade-off between photosynthesis and the typical bark functions of protection and mechanical strength.