Field Microscopy Research Articles

Abstract 4675: Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2

Abstract Lung cancer will account for 21% of US cancer deaths in 2023 with 238,340 cases and 127,070 deaths. Treatment with EGFR-TKI (epidermal growth factor receptor tyrosine kinase) inhibitors initially decrease tumor growth, but often leads to TKI resistance. According to studies, non-small cell lung cancer (NSCLC) tumors express more SphK2 (Sphingosine kinase 2) than normal lung tissue and patients with overexpression have worse survival chances. However, SphK2's role in TKI resistance is unclear. We hypothesize that inhibiting/downregulating SphK2 could reduce tumorigenicity and overcome ER (Erlotinib) and OR (Osimertinib) resistance. We studied the expression of SphK2 in both drug-resistant (OR/ER) and drug sensitive (parental) H358, H2170, H3255, and PC9 NSCLC cell lines using Western blotting, qRT-PCR, and immunofluorescence. H3255OR cells were also bioprinted with GelMA hydrogel using an R-Gen 200 bioprinter to mimic the physiological tumor environment and treated with SphK2 siRNA or ABC294640 (SphK2i). Immunoblotting was done to analyze changes in the expression of SphK2 in H358 (WT-EGFR) and H3255 (mutant EGFR- L858R) parental and OR-resistant cell lines. Results indicated a 1.5-1.8-fold upregulation of SphK2. Increased SphK2 gene expression was observed using qPCR in TKI-resistant H3255OR (4.3-fold), PC9ER/OR with mutant EGFR (E746-A750del) (1.8-3.3-fold), and H2170ER/OR with WT EGFR (1.3-1.5-fold) compared to parental cells. Immunofluorescence studies showed increased fluorescence of SphK2 in H3255OR (2.6-fold), H358 ER (2.4-fold) and OR cells (2.3-fold) compared to parental cells. Spheroids formed from single cancer cells can "re-create" the growth of a tumor. The spheroid count of H3255OR was reduced by 73.98% and 56%, respectively, when SphK2i was added to the 3D-cell culture with Matrigel, compared to the diluent and OR alone (p≤0.01). We used 3D bioprinting to precisely stack cells on biological scaffolds to recreate lung tumors from patients and boost the therapeutic efficacy of these findings. When bioprinted spheroids of H3255OR were treated with SphK2i in combination with OR, the number of spheroids per microscopic field were reduced by 87.3% compared to OR alone. Bioprinted H3255OR spheroids treated with SphK2 siRNA and OR revealed a 75.8% and 84% reduction of spheroid count, respectively, compared to treatments with siRNA alone or OR alone (p≤0.05). This data supports the hypothesis that SphK2 suppression or downregulation can assist in mitigating oncogenesis. We also used H3255OR transwell migration assays to examine how SphK2 inhibition affects cell migration. Combining OR with SphK2i resulted in a 94.7% and 91.4% decrease in cell migration compared to diluent and OR alone (p≤0.01). In H358OR-resistant cells, combining OR with SphK2i resulted in a 94.97% inhibition compared to diluent (p≤0.01). These results suggest that targeting SphK2 could be a valuable approach for treating lung cancer. Citation Format: Ayesha Khan, Bamby Dieng, Namrata Dube, Mohammad Fazle Alam, Neelu Puri. Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4675.

Abstract 221: Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies

Abstract Organotypic, three-dimensional (3D) cancer tissue models (OCTM) have enabled investigators to develop predictive markers that can be used in preclinical cancer drug development. However, a drawback of the existing models is that they are mostly based on cell lines based are not physiological. In vitro development of 3D tumoroids using patient derived primary tumors (PDPT) in tumor microenvironment (TME) will have clinical significant and can predict human responses to cancer therapeutics. Here, we describe the generation of OCTM from patient-derived metastatic colorectal cancer, the second most deadly cancer. PDPT were grown in Matrigel for 5 passages and for biobanking purposes tumoroids were further expanded in faCellitate 384 well plates or Sun Bioscience plates to harvest large amounts of well-defined tumoroids cultured for over 21 days. Expanded tumoroids were collected and cryopreserved or gently mixed several times using a pipet tip to break up tumoroids into smaller fragments. These tumor fragments were mixed with endothelial cells, dendritic cells, fibroblasts and embedded in a collagen matrix to simulate TME. The collagen-cell mixture was then added to Transwells and induced to gel to form a collagen gel matrix that simulates TME. The tumoroids were cultured with a specialized medium that supports the growth of complex cell types. The tumor development was monitored daily using bright field microscopy and image analysis was used to monitor tumor size. To evaluate the utility of the OCTM, treatment was initiated at day 10 of the culture period by exposing tissues with the chemotherapeutic drug Cisplatin on days 0, 2, 4, 7, 9, and 11. After each treatment, OCTM tissues (N=2) were fixed for H&E and live/dead staining. Overall, the results of the study showed that: 1) PDPT can be expanded for 5 passages (this low passage number is not expected to change the phenotype of the tumor) and further expanded in high throughput format, 384 well plates, 2) the PDPT could be cultured with multiple cell types in a collagen-gel matrix to recreate the TME, 3) histological evaluation of the OCTM showed a glandular-like tumoroid, 4) immunohistochemical staining revealed that the PDPT in the in vivo-like microenvironment were positive for CK20, and 5) the PDPT shrank following 3 treatment cycles with Cisplatin (day 4). Conclusion: The data shows metastatic colorectal PDPT can be expanded in vitro and cultured in a complex tumor microenvironment on tissue culture inserts. These OCTMs can be used to screen drug safety and efficacy. This study opens new avenues for high-throughput cancer drug screening format using PDPT embedded in a in vivo like TME. This model can be used as a tool for personalized oncology drug testing and for translational assays tailored to other cancer types from our patient derived primary tumor pool. Citation Format: Seyoum Ayehunie, Megan Groves, Bryda Bryda, Alex Armento, Anthony Tolcher. Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 221.

Abstract 2103: Small extracellular vesicles derived from macrophages show anti-tumor effects in MDA-MB-231 cells

Abstract Triple-negative breast cancer is the primary cause of death in women globally due to the absence of targeted receptors on the tumor, making it hard to treat. In addition, the rapid spread of triple-negative breast cancer makes it difficult to manage using current treatment options such as lumpectomies, mastectomies, radiation, and chemotherapy. The survival rate for patients with triple-negative breast cancer is only 8-16%. Therefore, there is a critical need to develop new and effective treatment options. The objective of this study was to investigate the potential of M1 macrophage-derived exosomes to induce cancer cell apoptosis. To test this, we isolated M1 macrophage-derived exosomes to see their effects on triple-negative breast cancer MDA-MB-231 cells. We induce polarization in RAW 264.7 macrophage cells via lipopolysaccharide (LPS). Macrophage polarization was characterized using an enzyme-linked immunosorbent assay (ELISA), real-time PCR, nitric oxide production, and bright field microscopy. We characterized exosomes using nanoparticle tracking analysis (NTA) and scanning electron microscopy (SEM). The apoptosis marker, caspase 3/7 activation, was assessed using confocal microscopy in MDA-MB-231 cells. M1 macrophage-derived exosomes were isolated, characterized, and incubated with breast cancer cells to see their anticancer effects. Data showed the induction of apoptosis in 48 hours in triple-negative breast cancer cells. We conclude that exosomes derived from M1 macrophages may have the capability to induce apoptosis in triple-negative breast cancer. Citation Format: Parth Desai, Anjali Kumari, Saqer Al Abdullah, Kristen Dellinger. Small extracellular vesicles derived from macrophages show anti-tumor effects in MDA-MB-231 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2103.

Abstract 6185: Generative AI for virtual HE-staining of whole slide images of unstained breast cancer tissue

Abstract Introduction Hematoxylin and eosin (HE) is the standard stain used in histology to make tissue visible to the human eye by highlighting certain cellular and tissue structures, and it is a technique that is widely used in the diagnosis of cancer and other pathologies. Chemical staining, however, is irreversible, making the tissue unusable for subsequent measurements, such as spatial transcriptomics. Here we utilize generative AI method based on pix2pix image-to-image translation to generate virtual HE-staining for whole slide images (WSIs) acquired of unstained tissue with brightfield microscopy and perform a thorough histological evaluation for the feasibility in breast cancer diagnostics. Materials and Methods We optimized sample preparation and imaging setup for virtual staining purposes, and developed a custom generative adversarial network architecture for learning the virtual staining from paires samples of unstained and H&E stained tissue. Here, we focus on the utility of virtual staining in breast cancer diagnostics, using a set of breast cancer samples for acquiring whole slide images from unstained tissue before H&E staining and from reference H&E stained tissue after chemical staining. Hold-out set of sample pairs are left for validation, allowing us to evaluate the virtual staining performance for a vast array of tissue components and to examine the potential shortcomings in staining reproduction. We use a comprehensive set of quantitative metrics both on pixel and object level to evaluate virtual staining quality. In addition, we perform thorough visual evaluation of histological feasibility by histology experts to examine the computational staining. Results We demonstrate that by careful optimization of both sample preparation and imaging workflow, as well as the computational methods, generative adversarial networks can be used for virtual staining of whole slide images of breast cancer tissue acquired from unstained tissue using regular bright field microscopy. We analyzed the virtual staining performance quantitatively and visually, and highlight the potential of the method through successful cases and demonstrate the applicability in breast cancer diagnostics, as well as discuss the challenges and shortcomings of virtual staining for clinical samples. Conclusions We demonstrate the potential of virtual HE staining for clinical histopathology of breast cancer tissue. Notably, our virtual staining based on generative AI shows promise towards more sustainable and streamlined sample processing and staining process in digital pathology. Citation Format: Leena Latonen, Umair Khan, Sonja Koivukoski, Johan Hartman, Pekka Ruusuvuori. Generative AI for virtual HE-staining of whole slide images of unstained breast cancer tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6185.

Regulating loading strain rates under shockless quasi-isentropic compression using a resin-based areal density gradient flyer

Areal density gradient flyers (ADGFs) have important applications in quasi-isentropic compression and high strain rate loading. The wave impedance gradient distribution of the reported ADGFs is single, and the effects of different wave impedance gradient distributions and spikes number density on the loading results are unknown. In this study, the resin-based ADGFs with different spike areal density distribution and spikes number density were designed and prepared using projection micro stereolithography technology (PμSL). The printing accuracy of ADGFs was evaluated using three-dimensional optical profiler, optical microscope, Scanning Electron Microscopy (SEM) and ultra depth of field microscope. The loading process of the ADGF on the target was studied by experiment and simulation. The simulation result was consistent with the experimental result. Augmenting the wave impedance distribution index (P) of spikes and spikes number density increases the loading strain rate. Due to the high printing accuracy of PμSL, the fine ADGF structures with large spikes number density can be prepared, thereby promoting the formation of quasi-isentropic compression plane waves. This work realizes the regulation of loading strain rate under shockless quasi-isentropic compression, which is significant for understanding the mechanical response of materials under high strain rate loads.

A P2/P3 Biphasic Layered Oxide Composite as a High-Energy and Long-Cycle-Life Cathode for Potassium-Ion Batteries.

Layered transition metal oxides are extensively considered as appealing cathode candidates for potassium-ion batteries (PIBs) due to their abundant raw materials and low cost, but their further implementations are limited by slow dynamics and impoverished structural stability. Herein, a layered composite having a P2 and P3 symbiotic structure is designed and synthesized to realize PIBs with large energy density and long-term cycling stability. The unique intergrowth of P2 and P3 phases in the obtained layered oxide is plainly characterized by X-ray diffraction refinement, high-angle annular dark field and annular bright field-scanning transmission electron microscopy at atomic resolution, and Fourier transformation images. The synergistic effect of the two phases of this layered P2/P3 composite is well demonstrated in K+ intercalation/extraction process. The as-prepared layered composite can present a large discharge capacity with the remarkable energy density of 321 Wh kg-1 and also manifest excellent capacity preservation after 600 cycles of K+ uptake/removal.

A non-perturbative mixed anomaly and fractional hydrodynamic transport

We present a new non-perturbative ’t Hooft anomaly afflicting a quantum field theory with symmetry group G = U(1) × ℤ2 in four dimensions. We use the Adams spectral sequence to compute that the bordism group \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\Omega }_{5}^{{\ ext{Spin}}}$$\\end{document}(BG), which classifies anomalies that remain when perturbative anomalies cancel, is ℤ4. By constructing a mapping torus and evaluating the Atiyah-Patodi-Singer η-invariant, we show that the mod 4 anomaly is generated by a pair of Weyl fermions that are vector-like under U(1), but with only one component charged under ℤ2. We construct a simple microscopic field theory that realises the anomaly, before investigating its impact in the hydrodynamic limit. We find that the anomaly dictates transport phenomena in the U(1) current and energy-momentum tensor akin to the chiral vortical and magnetic effects (even though the perturbative anomalies here vanish), but with the conductivities being fractionally quantised in units of a quarter, reflecting the mod 4 nature of the bordism group. Along the way, we compute the (relevant) bordism groups \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\Omega }_{d}^{{\ ext{Spin}}}$$\\end{document}(Bℤ2 × BU(1)) and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\Omega }_{d}^{{{\ ext{Pin}}}^{-}}$$\\end{document} (BU(1)) in all degrees d = 0 through 5.

High-speed and wide-field 3D Raman imaging using flower-like gap-enhanced Raman tags

Raman imaging technology plays a pivotal role in biomedical research, with the pursuit of higher resolution, faster imaging speed, and a larger field of view consistently driving the development of Raman imaging systems. In this work, we propose a high-speed and wide-field three-dimensional (3D) Raman imaging method based on light field microscopy and flower-like gap-enhanced Raman tags (F-GERTs). F-GERTs with enhanced Raman signals and high stability are used to label microbial and cell samples, avoiding the problem of photobleaching. The micro-lens array and scientific camera are incorporated to capture light field Raman images containing spatial information of the sample in a single snapshot acquisition process. The 3D reconstruction algorithm can complete the processing of the light field image within 1 min, reconstructing the 3D Raman image of the sample and the precisely slice structure at different depths. This high-speed and wide-field Raman imaging technology has great potential in the field of real-time 3D Raman imaging of microorganisms and cells.

Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman’s correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70–23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05–39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, −49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.

Arterial supply and morphological characteristics of sympathetic neurons in the human superior cervical ganglion.

The aim of this study was the micromorphological analysis of the distribution of microvessels, mast cells and ganglionic neurons in two parts, proximal and distal of the human superior cervical sympathetic ganglions (SCSGs). Statistical analyses were applied to detect the possible metric regional differences in their densities. Five injected human SCSGs with colored India ink and gelatin were microdissected and examined. Second group of five human SCSGs was prepared and serially sliced for CD34 and mast cell tryptase immunostaining. The microscopic fields of two parts of the SCSGs were analyzed for the following quantifications: microvessel density (MVD), mast cell density (MCD), and ganglionic cell count and measurements. The mean number of CD34-positive microvessels in microscopic fields, the MVD, had a value of 83 for the upper parts, and 82.7 for the lower parts of SCSGs. The mean number of tryptase-positive mast cells in microscopic fields, the MCD, was 4.5 in the proximal parts, and 4.7 in the distal parts of SCSGs. The mean number of ganglionic neurons in microscopic fields was 19.5 in the proximal parts, and 19.8 in the distal parts of SCSGs. The density of CD34-positive microvessels, the density of tryptase-positive mast cells, and the density, mean diameters and mean areas of ganglionic neurons were not significantly different in two observed parts, upper and lower of the SCSGs. In conclusion, the distributions of microvessels, mast cells, and neurons in two parts of the SCSGs were uniform with no specific micromorphological variations, there is a homogenous vascular and cellular pattern within the SCSGs.

Chemical Mechanical Polishing for Monocrystal SiC Assisted by the Heterogeneous Sono-Fenton Oxidation

The efficient chemical mechanical polishing (CMP) ability of monocrystal SiC determines the development of advanced semiconductor device. In order to improve the ultra-precision polishing efficiency for SiC, a CMP method based on the heterogeneous sono-Fenton oxidation was proposed. The effects of heterogeneous sono-Fenton on SiC surface softening and polishing based on hydroxyl radical oxidation were studied by means of the corrosion and CMP experiments. Utilizing the indentation tester, the ultra depth of field microscope and optical profilometer, the synergistic effects of heterogeneous sono-Fenton method on the improvement of SiC surface softening and polishing performance were revealed. The research results showed that the change rates of SiC hardness and elastic modulus after heterogeneous sono-Fenton corrosion were the highest, which were 0.12 and 0.6, respectively. The depth variation of indentation on SiC after heterogeneous sono-Fenton corrosion was the highest, which was 1.741 μm. Correspondingly, the improvement degree of SiC surface quality after polishing based on the heterogeneous sono-Fenton corrosion was the greatest and the material removal rate was the highest, which were Sa2.94 nm and 0.469 μm h−1, respectively.

High-density volumetric super-resolution microscopy

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs μm−2) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs μm−2) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs μm−2) in dense cytosolic tubulin datasets.

Microsurgical Practice with Use of Smartphone Camera as the Microscopic Field.

Accessibility of microsurgical equipment is a major barrier to proper training of surgeons before live patient free flap surgery. A technique is presented that uses a smartphone camera as the microscopic field, eliminating the need for an expensive operative microscope for surgical practice. A convenient and cost-effective simulation protocol could reduce the time frame of the microsurgery learning curve. Furthermore, the use of the smartphone video function may allow improved feedback by mentors, improving access and communication between microsurgical teachers and learners. The PocketSuture smartphone stand is a commercially available device that allows the smartphone camera to be used as magnification. The proposed education protocol included suture practice, vessel dissection, and free tissue transfer in nonliving animal models, with vessel anastomosis and patency confirmation performed with a smartphone camera for field magnification. Video of the suturing technique allowed feedback from the mentor. A progressive suturing protocol leading to the ability to perform microsurgical anastomosis on nonliving animal models was developed. The basic costs for the stand, instrument set and suture were less than $500. The PocketSuture smartphone stand can be used for microsurgical training with real-time video for plastic surgery learners with limited access to microscopes and local mentors.

Comparative study of total thoracoscopic mitral valve surgery and traditional open mitral valve surgery :propensity score matching study

Background: Conventional median open mitral valve replacement surgery usually requires a 20-30cm size incision to ensure adequate visual exposure. So, while we complete the valve replacement, it also brings about a series of problems. The appearance of TV thoracoscope changes the exposure of the surgical field. Because the microscopic field is enlarged, it not only makes the surgical vision clearer, but also greatly shortens the incision, reduces bleeding, and conceals the incision, minimizing the trauma and shortening the postoperative recovery time, which is in line with the concept of modern health. Aim: All technological innovation is controversial in the early stage, and it is such debate that can promote the progress and development of technology.This study was conducted in the context of this debate to summarize the experience of Total thoracoscopic mitral valve surgery in our center.Total thoracoscopic mitral valve replacement surgery and traditional median incision mitral valve replacement surgery were compared by the propensity score matching method.Evaluate its safety and merits. Methods: From January 2018 to December 2020, a total of 139 patients in the thoracoscopic group were eligible in the cardiothoracic surgery department of our hospital, and 1066 cases of traditional median sternotomy mitral valve replacement. After propensity score matching, 125 patients were selected in the thoracoscopic group, among 1066 patients with median incision MVR, 1:1 matched to 125, as the control group in the thoracoscopic group. Results: The results showed that the mean operative time in the thoracoscopic MVR group was significantly longer than that in the control group (185.73 ± 43.04 vs. 164.58 ± 40.28, p=0.0016). Similarly, both the mean CPB time and aortic block time were significantly longer than those in the control group (108.37±30.57min vs. 79.18±17.65min, p <0.0001; 66.59±29.63min vs. 45.09±19.68min, p <0.0001. Moreover, the proportion of patients with CPB greater than 120min and aortic block greater than 90min was significantly higher than the control group (24.1% vs. 3.2%, p <0.0001,14.4% vs. 2.4%, p=0.0237). In addition, there was no significant difference in the mean ICU observation time between the two groups (49.35±34.51h vs. 48.72±25.89h, p=0. 8792). Differently, the mean endotracheal intubation time was significantly lower in the thoracoscopic MVR group compared to the control group (18.52±6.07h vs. 25.45±14.18h, p=0.0032). Similarly, the length of stay in the thoracoscopic group was 9.13 ± 4.58 days, which was significantly lower than 11.12 ± 5.76 days in the control group (p=0.0004). There was no significant difference in the postoperative hemoglobin concentration or platelet count between the two groups of MVR patients. Mean intraoperative bleeding volume in the thoracoscopic MVR group was significantly less than that in the control group (158.37±100.91ml vs.250.74 ± 81.34, P <0.0001). Similarly, the mean postoperative discharge in the thoracoscopic MVR group was significantly than in the control group (428.39±361.38ml vs. 514.07 ± 347.92ml, p=0.0383). For patients with postoperative blood products, there were 37 (29.6%) in the thoracoscopic group and 33 (26.4%) in the control group. There was no significant difference between the two groups. Similarly, there was no significant difference in the mean total hospital costs between the two groups. Conclusion: This study shows that it has feasibility and safety for total thoracoscopic mitral valve replacement for mitral valve lesions, which can reduce the time and stay of the hospital compared with conventional direct surgery, and obtain good minimally invasive and cosmetic effect, and lay a theoretical and clinical foundation for the application of thoracoscopic technology in cardiac surgery, which has important scientific significance.

Alterations in histopathology and stress-associated gene expression induced by infection with Prohemistomum vivax encysted metacercariae in Nile tilapia

Due to limited data on the pathogenicity of Prohemistomum vivax (P. vivax) and its impacts on fish health, this study aimed to determine the morphological, molecular characteristics, pathogenicity, and histopathological alterations in fish infected with P. vivax. Eight hundred (800) Nile tilapia (Oreochromis niloticus) were collected from various farms in Kafr El Sheikh Governorate. The fish were examined for encysted metacercariae (EMC) in different organs. Tissue specimens were collected and underwent histopathological analysis, expression of stress-related genes, and genetic characterization by sequencing of the internal transcribed spacer 2 (ITS2). P. vivax metacercariae were oval to round in shape and were collected from various organs including the muscle, skin, eyes, intestine, liver, kidney, and gills of infected O. niloticus. Sequencing and phylogenetic analysis of the ITS2 region revealed a 507-bp fragment, confirming parasite identity and matching within the same clade as other P. vivax isolates. Infected fish displayed abdominal hydropsy, skin darkening, and emaciation. P. vivax encysted metacercariae were detected during the study period in 620/800 fish, with an overall prevalence of 77.5%. The seasonal prevalence was 95% in summer, 85% in spring, 55% in autumn, and 75% in winter. The intensity of infection was 1–40 cysts per microscopic field. Histopathological examination of muscles revealed parasitic cysts embedded within muscle fibers, causing severe degeneration and necrosis. Upregulation of cytochrome P450 (cpy1a1), heat shock protein 70 (hsp-70), and tumor suppressor p53 (p53) was recorded in both liver and muscle samples of infected tilapia compared to controls. This indicates activation of detoxification, cellular stress, and apoptotic pathways in response to P. vivax infection. There is limited data available on the pathogenicity of P. vivax and its impacts on fish health; thus, this study provides key insights into the morphology, pathogenicity, and histopathological impacts of P. vivax in Nile tilapia.

Detection of estradiol with a digital immunoassay using an anti-immunocomplex antibody and single-molecule observation of gold nanoparticles.

Gold nanoparticles (AuNPs) have been widely applied to molecular sensors due to their optical properties. We previously reported a molecular detection by observing the scattered light of AuNPs at a single nanoparticle level using dark field microscopy (DFM). Recently, a molecular detection method using digital immunoassay has been reported, taking advantage of the characteristics of DFM. However, the digital immunoassays reported so far have been performed by a conventional sandwich immunoassay, which is difficult to apply to the detection of small molecules. In this study, with the aim of small molecule detection, we developed a digital immunoassay method using an anti-immunocomplex antibody that specifically recognizes immunocomplexes of small molecules with antibodies. The number of AuNPs modified with anti-immunocomplex antibody bound to immunocomplex of estradiol and anti-estradiol antibody was counted at a single nanoparticle level using DFM. We demonstrated for the first time that estradiol molecule can be detected by digital immunoassay using DFM and an anti-immunocomplex antibody with a detection sensitivity of 1pg/mL.

Cortisol as a cerebral cortex neurons apoptosis regulator in acute phase of ischemic stroke (clinical and pathological study)

In response to ischemic stroke (IS) a natural activation of the stress-realizing system occurs. The features of this activation influence the outcome of the acute period and the prognosis of recovery and can be adjusted. At the same time, the role of the stress-realizing system in the pathogenesis of IS is still unexplored.Objective: to investigate the effect of peripheral blood cortisol concentration on the regulation of apoptosis of neurons of the cerebral cortex in the acute phase of IS.Material and methods. A prospective clinical and pathological study was performed. It included 9 patients with IS in the left middle cerebral artery territory who were admitted to hospital and died in the hyperacute phase of IS and had no infectious complications, allergic reactions or oncological diseases and who did not undergo thrombolysis. The cerebral cortex was examined. Neuron-specific enolase (NSE), protein 53 (p53), caspase 3, caspase 8, Fas receptor (CD95), and Fas apoptotic inhibitory molecule 2 (FAIM2) were determined on the slices using an indirect immunoperoxidase immunohistochemical staining method. A total of 567 microscopic fields were analysed for the group of patients with IS and 63 fields for the control group (three people). Before death, the blood concentrations of sFas, sFasL, cortisol, adrenocorticotropic hormone, adrenaline and norepinephrine were determined by enzyme immunoassay (the control group consisted of 28 people).Results. Significant correlation was found between the proportion of casp3-positive neurons and the concentration of cortisol in peripheral blood in zones 2 (r=0.263; p<0.01) and 3 (r=0.383; p<0.01). In the 2nd zone, significant negative correlation was found with the concentrations of sFas (r=-0.177; p<0.05) and sFasL (r=-0.164; p<0.05); in the 3rd zone, significant positive correlation was found with the ratio of the concentrations of sFasL and sFas (r=0.240; p<0.01). The proportion of Fas-positive neurons in the cerebral cortex correlated significantly with the concentration of the soluble form of this molecule (for the 1st zone – r=0.222, for the 2nd zone – r=0.438, for the 3rd zone – r=0.289; p<0.01) and the ratio of the concentrations of sFasL and sFas (respectively: r=0.231, r=0.266 and r=0.281; p<0.01) in the peripheral blood.Conclusion. Peripheral blood cortisol concentration is a factor that determines the regulation of apoptosis of neurons in the cerebral cortex in the acute phase of IS.

Intranasal vaccine for Lyme disease provides protection against tick transmitted Borrelia burgdorferi beyond one year

Strategies for disease control are necessary to reduce incidence of Lyme Disease (LD) including development of safe vaccines for human use. Parainfluenza virus 5 (PIV5) vector has an excellent safety record in animals and PIV5-vectored vaccines are currently under clinical development. We constructed PIV5-vectored LD vaccine candidates expressing OspA from B. burgdorferi (OspAB31) and a chimeric protein containing sequences from B. burgdorferi and B. afzelii (OspABPBPk). Immunogenicity and vaccine efficacy were analyzed in C3H-HeN mice after prime-boost intranasal vaccination with live PIV5-OspAB31 or PIV5-OspABPBPk, subcutaneous (s.c.) vaccination with rOspAB31+Alum, and the respective controls. Mice vaccinated intranasally with live PIV5-AB31 or PIV5-ABPBPk had higher endpoint titers of serum antibody against OspAB31 at 6- and 12- months post vaccination, compared to mice vaccinated s.c. with rOspAB31. Neutralization activity of antibody was maintained up to 18-months post-immunization, with the response greater in live PIV5-delivered OspA vaccines, than that induced by s.c. rOspAB31. Challenge with infected ticks carrying 10-19 strains of B. burgdorferi performed at 4-, 9- or 15-months post-immunization showed increased breakthrough infections in mice vaccinated with s.c. rOspAB31 compared to intranasal PIV5-AB31 or PIV5-ABPBPk at 9- and 15-months, as determined by quantification of serologic antibodies to B. burgdorferi proteins as well as flaB DNA in tissues, and by visualization of motile B. burgdorferi in culture of tissues under dark field microscope. These findings indicate that immunization of mice with PIV5 delivered OspA generates immune responses that produce longer-lasting protection ( > 1 year) against tick-transmitted B. burgdorferi than a parenteral recombinant OspA vaccine.

Experimental evidences of the direct influence of external magnetic fields on the mechanism of the electrocatalytic oxygen evolution reaction

The use of magnetic fields as external stimuli to improve the kinetics of electrochemical reactions is attracting substantial attention, given their potential to reduce energy losses. Despite recent reports showing a positive effect on catalytic performance upon applying a magnetic field to a working electrode, there are still many uncertainties and a lack of experimental evidence correlating the presence of the magnetic field to the electrocatalytic performance. Here, we present a combination of electrochemical and spectroscopic tools that demonstrate how the presence of an external magnetic field alters the reaction mechanism of the electrocatalytic oxygen evolution reaction (OER), accelerating the overall performance of a Ni4FeOx electrode. Complementary experimental evidence has been gathered supporting the participation of this microscopic magnetic field effect. Electrochemical impedance spectroscopy (EIS) points to a speed-up of the intrinsic reaction kinetics, independent of other indirect effects. In the same direction, the spectro-electrochemical fingerprint of the intermediate species that appear during the electrocatalytic cycle, as detected under operando conditions, indicates a change in the order of the reaction as a function of hole accumulation. All these experimental data confirm the direct influence of an external magnetic field on the reaction mechanism at the origin of the magnetically enhanced electrocatalytic OER.

Multi-scale FE[formula omitted] investigation of non linear rate dependent 3D composite structures accounting for fiber–matrix damage

In the present paper, a two-scale FE2 technique based on periodic homogenization theory is investigated to predict the macroscopic non-linear behavior of polymer matrix composite structures.The computational technique accounts for the fiber/matrix interfacial damage, the matrix ductile damage and the effect of 3D periodic microstructure. The developed approach integrates the geometric description and the non-linear time-dependent local behavior of the different constituents (fibers, matrix and interface). For numerical calculations, advanced User Defined Material and User Element subroutines are developed at the two scales, simultaneously activated to solve macroscopic and microscopic problems through an incremental scheme in the finite element commercial code Abaqus/Implicit The computational efficiency of the developed multi-scale approach is demonstrated by predicting the overall response of 3D composite structures under complex loading paths. The composite structures consist of thermoplastic polymer matrix with elasto-viscoplastic behavior and ductile damage, reinforced by elastic aligned short fibers that are coated by a cohesive zone, which obeys the general unified potential. The numerical results obtained by FE2 simulation with and without accounting for interface effect are analyzed and compared for two examples: Meuwissen-like and 3D corner shape structures. The main benefits of the developed approach lie in accessing the microscopic strain fields, the distributions of the internal variables and the damage evolution in both polymer matrix and interface, as well as identifying their repercussions on the macroscopic response.