Tissue Localization Research Articles

Molecular Characteristics of JAK2 and Its Effect on the Milk Fat and Casein Synthesis of Ovine Mammary Epithelial Cells.

In addition to its association with milk protein synthesis via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, JAK2 also affects milk fat synthesis. However, to date, there have been no reports on the effect of JAK2 on ovine mammary epithelial cells (OMECs), which directly determine milk yield and milk contents. In this study, the coding sequence (CDS) region of ovine JAK2 was cloned and identified and its tissue expression and localization in ovine mammary glands, as well as its effects on the viability, proliferation, and milk fat and casein levels of OMECs, were also investigated. The CDS region of ovine JAK2, 3399 bp in length, was cloned and its authenticity was validated by analyzing its sequence similarity with JAK2 sequences from other animal species using a phylogenetic tree. JAK2 was found to be expressed in six ovine tissues, with the highest expression being in the mammary gland. Over-expressed JAK2 and three groups of JAK2 interference sequences were successfully transfected into OMECs identified by immunofluorescence staining. When compared with the negative control (NC) group, the viability of OMECs was increased by 90.1% in the pcDNA3.1-JAK2 group. The over-expression of JAK2 also increased the number and ratio of EdU-labeled positive OMECs, as well as the expression levels of three cell proliferation marker genes. These findings show that JAK2 promotes the viability and proliferation of OMECs. Meanwhile, the triglyceride content in the over-expressed JAK2 group was 2.9-fold higher than the controls and the expression levels of four milk fat synthesis marker genes were also increased. These results indicate that JAK2 promotes milk fat synthesis. Over-expressed JAK2 significantly up-regulated the expression levels of casein alpha s2 (CSN1S2), casein beta (CSN2), and casein kappa (CSN3) but down-regulated casein alpha s1 (CSN1S1) expression. In contrast, small interfered JAK2 had the opposite effect to JAK2 over-expression on the viability, proliferation, and milk fat and milk protein synthesis of OMECs. In summary, these results demonstrate that JAK2 promotes the viability, proliferation, and milk fat synthesis of OMECs in addition to regulating casein expression in these cells. This study contributes to a better comprehension of the role of JAK2 in the lactation performance of sheep.

Expression of Collagen XIII in Tissues of the Thyroid and Orbit With Relevance to Thyroid-Associated Ophthalmopathy.

Antibodies against collagen XIII have previously been identified in patients with active thyroid-associated ophthalmopathy (TAO). Although collagen XIII expression has been described in extraocular muscles and orbital fat, its detailed localization in extraocular and thyroid tissues and the connection to autoimmunity for collagen XIII remain unclear. Our objective was to map the potential targets for these antibodies in the tissues of the orbit and thyroid. We evaluated the expression of collagen XIII in human patient and mouse orbital and thyroid tissues with immunostainings and RT-qPCR using Col13a1-/- mice as negative controls. COL13A1 expression in Graves' disease and goiter thyroid samples was compared with TGF-β1 and TNF, and these were also studied in human thyroid epithelial cells and fibroblasts. Collagen XIII expression was found in the neuromuscular and myotendinous junctions of extraocular muscles, blood vessels of orbital connective tissue and fat and the thyroid, and in the thyroid epithelium. Thyroid expression was also seen in germinal centers in Graves' disease and in neoplastic epithelium. The expression of COL13A1 in goiter samples correlated with levels of TGF-B1. Upregulation of COL13A1 was reproduced in thyroid epithelial cells treated with TGF-β1. We mapped the expression of collagen XIII to various locations in the orbit, demonstrated its expression in the pathologies of the Graves' disease thyroid and confirmed the relationship between collagen XIII and TGF-β1. Altogether, these data add to our understanding of the targets of anti-collagen XIII autoantibodies in TAO.

A novel long non-coding RNA SLNCR1 promotes proliferation, migration, and invasion of melanoma via transcriptionally regulating SOX5

Steroid receptor RNA activator (SRA)-like non-coding RNA (SLNCR1) has been implicated in various tumorigenic processes, but the precise regulatory role in melanoma progression remains uncertain. We performed a comprehensive analysis to investigate the prognostic value of SLNCR1 expression in patients with melanoma by TCGA database and melanoma tissue samples via the Kaplan–Meier method. Subsequently, we conducted qRT-PCR and Fluorescence in Situ Hybridization (FISH) assays to identify SLNCR1 expression levels and localization in tissues and cells, respectively. Loss-of-function assays utilizing shRNAs vectors were used to investigate the potential impact of SLNCR1. Our data showed that SLNCR1 is significantly up-regulated in human malignant melanoma tissues and cell lines and functions as an oncogene. Silencing of SLNCR1 suppressed melanoma cell proliferation, migration, invasion, and inhibited tumorigenesis in a mouse xenograft model. Additionally, we employed bioinformatic predictive analysis, combined with dual-luciferase reporter analysis and functional rescue assays, to elucidate the mechanistic target of the SLNCR1/SOX5 axis in melanoma. Mechanistically, we discovered that SLNCR1 promotes EMT of human melanoma by targeting SOX5, as downregulation of SLNCR1 expression leads to a decrease in SOX5 protein levels and inhibits melanoma tumorigenesis. Our research offers promising insights for more precise diagnosis and treatment of human melanoma.

Enhancing upper tract urothelial carcinoma diagnosis: Utility of cytokeratin 17 and CK20/CD44/p53 immunohistochemical panel

Upper tract urothelial carcinoma (UTUC) presents diagnostic challenges due to small biopsy specimen size, poor orientation, and technical obstacles that can yield equivocal diagnoses. This uncertainty often mandates repeated biopsies to evaluate the necessity of nephroureterectomy. Prior studies have suggested cytokeratin 17 (CK17) immunostain as an adjunctive tool for diagnosing bladder urothelial neoplasia in both urine cytology and tissue biopsy specimens. We evaluated the utility of CK17 in differentiating UTUC from benign urothelium and its ability to stratify low-grade from high-grade neoplasia. Our study involved a cohort of previously diagnosed cytology (n = 29) and tissue specimens from biopsies and resections (n = 85). We evaluated CK17 staining percentage in cytology and tissue samples and localization patterns in biopsy/resection samples. Our findings showed a statistically significant distinction (p < 0.05) between UTUC and benign tissue specimens based on full thickness localization pattern (odds ratio 8.8 [95% CI 1.53–67.4]). The percentage of CK17 staining failed to significantly differentiate neoplastic from non-neoplastic cases in cytology or tissue samples. Additionally, based on prior research showing the efficacy of CK20/CD44/p53 triple panel in bladder urothelial neoplasia, we utilized tissue microarrays to evaluate if these markers could distinguish UTUC from benign urothelium. We found that CK20/CD44/p53, individually or in combination, could not distinguish urothelial neoplasia from non-neoplasia. Full thickness CK17 urothelial localization by immunohistochemistry was highly reproducible with excellent interobserver agreement and may play a supplementary role in distinguishing upper tract urothelial neoplasia from benign urothelium.

Hepatocyte nuclear factor 4 located in different developmental stages in Schistosoma japonicum and involved in important metabolic pathways

Nuclear receptors (NRs) are vital for regulating gene expression un organisms. Hepatocyte nuclear factor 4 (HNF4), a class of NRs, participates in blood feeding and intestinal maintenance in schistosomes. However, there is limited research on the molecular and functional characterization of HNF4 in Schistosoma japonicum (S. japonicum). Highly specific polyclonal antibodies were generated to analyze the expression and tissue localization of S. japonicum HNF4 (SjHNF4). The potential biological functions of SjHNF4 were characterized by transcriptome and pull-down analysis. Subsequently, enrichment analysis was performed to identify the specific signaling pathways linked to SjHNF4. The SjHNF4 protein was expressed heterologously and purified successfully. High purity and high potency polyclonal antibodies were further prepared. The expression of SjHNF4 was higher in female compared to male worms at both transcriptional and protein levels. Female worms expressed SjHNF4 in their perithecium, reproductive system, and certain parts of the intestinal tissues. SjHNF4 was also detected in the perithecium of male worms, as well as in the head, body of cercaria, and eggs. Furthermore, our findings highlighted the potential role of SjHNF4 in blood feeding and its interaction with crucial pathways such as glucose metabolism, lipid metabolism, and nucleotide metabolism. This study shed light on the location of SjHNF4 in different life stages of S. japonicum, particularly associated with the female schistosomes. A strong correlation was observed between SjHNF4 and essential metabolic pathways. These findings laid a solid groundwork for the research on the relationship between NRs and schistosomes.

SAM-IE: SAM-based image enhancement for facilitating medical image diagnosis with segmentation foundation model

The Segment Anything Model (SAM) is a large-scale model developed for general segmentation tasks in computer vision. Trained on a substantial dataset, SAM can accurately segment various objects in natural scene images. However, due to significant semantic differences between medical and natural images, directly applying SAM to medical image segmentation does not yield optimal results. Therefore, effectively utilizing such a comprehensive foundation model for medical image analysis is an emerging research topic. Despite SAM’s current suboptimal performance in medical image segmentation, it shows preliminary recognition and localization of tissues and lesions that radiologists focus on in medical images. This implies that SAM’s generated masks, features, and stability scores hold potential value for medical image diagnosis. Therefore, based on the model output of SAM, this study introduces a SAM-based Image Enhancement (SAM-IE) method for disease diagnosis. Targeting popular medical image classification models (e.g., ResNet50 and Swin Transformer), SAM-IE is proposed to enhance image inputs by combining the binary mask and contour mask generated by SAM with the original image to create attention maps, thereby improving diagnostic performance. To validate the effectiveness of SAM-IE for diagnosis, experiments were conducted on four medical image datasets for eight classification tasks. The results demonstrate the effectiveness of our proposed SAM-IE model, showcasing SAM’s potential value in medical image classification. This study provides a feasible approach for integrating SAM into disease diagnosis.

Abstract 1158: Spatially resolved cell profiling unveils tumor metabolic states associated with immunotherapy response in NSCLC

Abstract Background. Only a subset of non-small cell lung cancer (NSCLC) patients benefit from immune checkpoint inhibitors (ICIs). Thus, there is a clinical need to develop predictive biomarkers for ICIs. Deeper insights from the tumor microenvironment (TME) can lead to identification of spatial features associated with clinical responses to immune checkpoint inhibitors (ICI). In this study, we investigated functional profiles within cell subtypes as a surrogate for ICI clinical endpoints. Methods. We profiled a retrospective cohort of 39 NSCLC tissue cores from 27 patients treated with ICI, using highly multiplexed immunofluorescence (mIF) stained by the Phenocycler Fusion platform (Akoya Biosciences) capturing 45 proteins. Utilizing a deep learning based multiplex imaging analysis pipeline, cells were classified to 15 cell types by known marker expression and were further subclassified by unsupervised clustering. Cells were assigned to the tumor area or TME and more than 1000 spatial features were calculated based on cell type, marker positivity, and area assignments, and were compared between responders and non-responders to ICI therapy using Fisher’s exact and logrank tests. Results. Unsupervised cell subtyping of the 15 cell types identified 43 cell subsets, which were mostly segregated by their metabolism and activation status. Interestingly, all lymphocytes showed a similar pattern of clustering resulting in two clusters of metabolically active and inactive cells. The most significantly differentially expressed proteins between these cell types were oxidative phosphorylation proteins such as CS, SDHA and ATPA5, and other metabolic enzymes such as HK1, GLUT1 and LDHA. We found a direct connection between metabolic state, effector functions and tissue localization as the metabolically active lymphocytes exhibited higher levels of PD-1, MHC class I and II and CD44 positivity, and were more abundant within tumor infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLS). Unsupervised clustering of tumor cells demonstrated segregation to three main metabolic states - OXPHOS+, OXPHOS- and a third cluster (PPP+) which was characterized by upregulation of ASCT2, a glutamine transporter, as well as pNRF2 and G6PD, regulators of the pentose phosphate pathway. These cells also exhibited higher proliferation rate and CD44, a tumor stemness marker, positivity. All tumors with high content of PPP+ tumor cells (&amp;gt;40%) were resistant to PD-1 blockade (0/8 vs. 10/18 response rate in other tumors, p=0.009) and showed reduced overall survival (OS) rates (median OS of 27.6 vs 90.3 months, p=0.02). Conclusions. Taken together, this study reveals connections between metabolic states, effector functions, and immunotherapy outcome and contributes to the evolving landscape of predictive biomarkers for ICI therapies. Citation Format: James Monkman, Rotem Czertok, Shai Bookstein, Becky Arbiv, Yuval Shachaf, Ron Elran, Kenneth Bloom, Oscar Puig, Ken O'Byrne, Ettai Markovits, Arutha Kulasinghe. Spatially resolved cell profiling unveils tumor metabolic states associated with immunotherapy response in NSCLC [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 1158.

Abstract 3647: Digital spatial transcriptomic reveals different epithelial and immune cells signatures across endometriosis-related diseases: From simple ovarian endometriosis to ovarian cancer

Abstract Endometriosis represents a pressing medical concern, imposing a relevant burden on patients including potential malignant transformation into endometriosis-associated ovarian cancer (EAOC). Despite the process of cancer development remains unclear, immune cells abnormalities arose as putative biomarkers in the neoplastic progression. One-third of ovarian endometriosis cases exhibit PD-1/PD-L1 expression levels comparable to those observed in cancer. To substantiate the biological basis of these findings, this study aimed to elucidate the spatial transcriptomic profile of epithelial and immune components from different endometriosis-related conditions, along with their genetic interplay. Formalin-fixed paraffine embedded (FFPE) specimens from 4 ovarian endometriosis (OE) cases, two of which displayed elevated levels of PD-1/PD-L1 expression and were referred to as cancer-like endometriosis (CLE), 1 Clear Cell Ovarian Cancer (CCOC) and 1 Endometrioid Ovarian Cancer (EOC) were cut in 5um section for spatial transcriptomic analysis using GeoMX Human Whole Transcriptome Atlas (WTA) probe-set (&amp;gt;18000 genes). For all sections, the regions of interest (ROIs) were identified using a set of morphology markers: anti-human CD3, CD68 and PanCK antibodies. Spatial transcriptomic analysis revealed condition-specific gene enrichment of epithelial and immune cells across the different conditions. In spite of similar H&amp;E findings, PanCKpos epithelial cells from CLE showed an increased expression of genes involved in stress response and defense regulation pathways (e.g. CXCL6, S100A9, and PECAM-1), when compared to OE, in which PanCKpos cells were enriched in DUSP1/2 and VIM. Interestingly, in CCOC, location-specific gene-enrichment was found in PanCKpos cells, with a significant up regulation of tumor suppressor genes (e.g. EGR-1, NR4A1) in normal-looking peri-tumoral epithelium compared to cancerous intra-tumoral PanCKpos cells. Similarly, in the immune compartment, spatial transcriptomic analysis revealed that CD3pos cells from CLE displayed an up-regulation of genes involved in IL-17 signaling, response to oxydative stress and complement activation (e.g. CXCL6, HMOX1, S100A9, and PECAM-1), when compared to OE. Moreover, in CCOC, CD68pos cells of the tumoral mass showed over-expression of tumor growth and progression markers (e.g. SPP1) compared to their counterpart in the outer area of the tumoral mass. In this first comprehensive spatial transcriptomic study of endometriosis-related diseases with GeoMx WTA, our findings uncovered remarkable differences in epithelial and immune cells among different stages and tissue locations. Importantly, CLE emerged as a transitional entity with a unique immune profile. Ongoing analysis of six additional samples will further refine our results. Citation Format: Valentina Iacobelli, Camilla Nero, Stefano Alivernini, Giulia Scaglione, Floriana Camarda, Francesca Sillano, Rita Trozzi, Alessia Piermattei, Barbara Tolusso, Giulia Mantini, Luciano Giacò, Gloria Anderson, Gian Franco Zannoni, Elisa Gremese, Anna Fagotti, Giovanni Scambia. Digital spatial transcriptomic reveals different epithelial and immune cells signatures across endometriosis-related diseases: From simple ovarian endometriosis to ovarian cancer [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 3647.

Abstract 5650: RNA regulatory disruption by 3’-dATP: A novel approach to inhibit ribosome biogenesis in cancer

Abstract Background: Ribosomes are a complex ensemble of ribosomal RNA and proteins. As the site where genetic information is translated to protein, they have a key role in cell survival, growth and proliferation. Ribosome biogenesis (RiBi), the process of creating ribosomes, is a complex function governed by precise checkpoints and surveillance mechanisms which may become dysregulated in cancer leading to tumor growth and therapeutic resistance. Ribosome-targeted therapy could provide a promising treatment for cancer. NUC-7738 (3'-deoxyadenosine [3’-dA] which is phosphorylated and protected with a phosphoramidate moiety attached at the 5’-position) generates sustained intracellular levels of 3’-dATP, a molecule that profoundly influences RNA regulatory processes, including poly(A) tail length, leading to alternative polyadenylation (APA) and splicing, resulting in impaired cellular responses. NUC-7738 is currently being investigated in combination with pembrolizumab in patients with advanced melanoma in the Phase 2 part of the clinical study (NCT03829254). Here, we investigate the impact of 3’-dATP on RNA regulation and RiBi utilizing a novel bioinformatic pipeline. Material and Methods: RNA was extracted from melanoma and renal carcinoma cell lines treated with NUC-7738 for 24h and paired biopsies (pre and post treatment) from patients treated with NUC-7738 + pembrolizumab. Sequencing libraries used for long-read PCR-cDNA sequencing were generated. Gene-level expression was quantified using Salmon DeSeq2. In addition to gene expression, poly(A) tail length, APA and isoform switch were analyzed. Protein expression was determined using JESS Western analysis. Multiplexed analysis of protein localization and expression in tissue was carried out using an in-house immunofluorescence protocol. Results: NUC-7738 caused a global reduction in poly(A) tail length in cell lines and patient biopsies that was most pronounced in the poly(A) tail exosome targeting (PAXT) long non-coding RNA (lncRNA) transcripts, including SNHG19, 5’TOP and YAE1, all of which play a role in RiBi. PAXT lncRNAs such as SNHG3 and SNHG19 increased in abundance. Ribosomal subunit proteins such as RPS3, RPS6, RPL17 decreased in expression. Expression of YAE1, a regulator of RiBi, also decreased. Distinct APA usage of various transcripts was observed in cell lines and patient biopsies. Conclusion: Through the development of a novel bioinformatic pipeline we have been able to demonstrate that NUC-7738 disrupts RNA regulation, leading to perturbed RiBi. Targeting RiBi provides a promising approach to treating cancers Citation Format: Mustafa Elshani, Ying Zhang, In Hwa Um, Ruth Plummer, Sarah P. Blagden, Stefan N. Symeonides, Natalie Cook, T.R. Jeffry Evans, Alison L. Dickson, David J. Harrison. RNA regulatory disruption by 3’-dATP: A novel approach to inhibit ribosome biogenesis in cancer [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 5650.

Abstract 3652: Spatial transcriptomic study of the tumor microenvironment in HNSCC

Abstract Background: The complex and dynamic nature of the tumor-immune microenvironment (TME) presents challenges for identification of robust and predictive biomarkers in immuno-oncology (IO). Standard multiplex immunohistochemistry (mIHC) or multiplex immuno-fluorescent assays (mIF) allow for the phenotyping on the cellular level but are limited by the number of markers available and the detection systems. Gene expression or next generation sequencing (NGS) platforms provide high-plex bulk data sets about the TME, but the spatial context of the tumor and immune cell interactions is lost. The NanoString GeoMx® Digital Spatial Profiler (DSP) combines spatial context, utilizing fluorescent morphological markers, with molecular profiling capabilities. This combination allows for the quantitative analysis of high-plex analyte abundance which can be traced back to a region of interest within a sample, providing biological inference in the region of interest. Methods: Formalin-fixed paraffin-embedded (FFPE) specimens from HNSCC patients were cut in 5µm sections for all analyses. Whole section tissue analysis was previously performed utilizing the NanoString nCounter® PanCancer IO 360™ Panel. Tumor inflammation signatures (TIS), an 18-gene signature measuring the adaptive immune response within tumors, was generated for each specimen and categorized each as immune-hot or immune-cold based on predefined parameters. Four specimens were selected based on their TIS score and two sets of cut sections were processed by either Labcorp lab in Morrisville, NC or Nanostring lab in Seattle, WA for precision evaluation, using the GeoMx Human Whole Transcriptome Atlas (WTA) panel (18,000 genes) with the NanoString GeoMx digital spatial profiling technology. The TME regions of interest (ROI) were identified using a set of morphology markers consisting of panCK, CD45 and Syto13; 12 ROIs were selected from each sectioned tumor material. The same ROIs were selected at the two testing sites with matching tissue location and morphology characters. Results: GeoMx WTA panel provided an unbiased view of 18,000 protein-coding genes at specific TME regions. These counts were successfully mapped to the 12 ROIs on the tissue. A pseudo-TIS score was generated using the same 18 gene signature from the WTA panel for each ROI. The pseudo-TIS scores from ROIs from the four specimens showed consistency with the specimen-wise TIS score, yet variations were observed across different TME/ROI within each specimen. Whole transcriptome expression results showed high correlation between the two testing sites, providing confidence in the reproducibility of the technology. High resolution fluorescent staining images were able to be fed to downstream quantitative digital analysis to generate complementary spatial data. Citation Format: Caitlin Schroyer, Shanshan Zhou. Spatial transcriptomic study of the tumor microenvironment in HNSCC [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 3652.

Gene-Expression Patterns of Tumor and Peritumor Tissues of Smoking and Non-Smoking HPV-Negative Patients with Head and Neck Squamous Cell Carcinoma.

We studied the gene-expression patterns in specimens of tumor and peritumor tissue biopsies of 26 patients with head and neck carcinomas depending on smoking status. Histological and immunohistochemical examinations verified that all tumors belonged to the "classical" subgroup of head and neck carcinomas, and the HPV-negative tumor status was confirmed. The expression of 28 tumor-associated genes determined by RT-PCR was independent of patients' sex or age, TNM status, degree of differentiation, or tissue localization. Moreover, in peritumor tissue, none of the 28 genes were differentially expressed between the groups of smoking and nonsmoking patients. During oncotransformation in both studied groups, there were similar processes typical for HNSCC progression: the expression levels of paired keratins 4 and 13 were reduced, while the expression levels of keratin 17 and CD44 were significantly increased. However, further investigation revealed some distinctive features: the expression of the genes EGFR and TP63 increased significantly only in the nonsmoking group, and the expression of IL6, CDKN2A, EGF, and PITX1 genes changed only in the smoking group. In addition, correlation analysis identified several clusters within which genes displayed correlations in their expression levels. The largest group included 10 genes: TIMP1, TIMP2, WEE1, YAP, HIF1A, PI3KCA, UTP14A, APIP, PTEN, and SLC26A6. The genetic signatures associated with smoking habits that we have found may serve as a prerequisite for the development of diagnostic panels/tests predicting responses to different therapeutic strategies for HNSCC.

Effect of angular variation and in vitro degradation on mechanical properties of PBAT 3D scaffolds

AbstractThe customization of polymeric prototypes is available to meet the specific needs of individual patients, considering tissue location and patient‐specific characteristics. Poly (butylene adipate‐co‐terephthalate) (PBAT) has shown great promise among various polymers in this scenario due to its biodegradability and processability properties. However, the effect of geometry changes on the mechanical properties of PBAT 3D scaffolds and their mechanical stability after in vitro degradation are crucial to determining the final application. This study focuses on the structural, morphological, and mechanical characterization of PBAT prototypes with different architectures and evaluating their mechanical properties before and after in vitro degradation. Three‐dimensional structures were 3D printed with rectilinear deposition angles of 15°, 30°, 45°, 60°, 75°, and 90°. Mechanical tests revealed that the prototype architecture influenced the stiffness, elongation, and maximum tension, where a greater resistance was observed for filaments containing the lowest angles. The thermal stability of PBAT remained unaffected either by reprocessing or hydrolytic degradation; however, in vitro degradation affected the mechanical behavior by increasing the stiffness and decreasing the elongation of the scaffold. Biological assays have demonstrated the nontoxic nature of PBAT and its potential to promote cell viability at a rate of 95%. These findings underscore the possibility of optimizing the PBAT design and structure to tune the mechanical properties and contribute to advancing tissue engineering strategies.

Nanoparticle-based optical interfaces for retinal neuromodulation: a review.

Degeneration of photoreceptors in the retina is a leading cause of blindness, but commonly leaves the retinal ganglion cells (RGCs) and/or bipolar cells extant. Consequently, these cells are an attractive target for the invasive electrical implants colloquially known as "bionic eyes." However, after more than two decades of concerted effort, interfaces based on conventional electrical stimulation approaches have delivered limited efficacy, primarily due to the current spread in retinal tissue, which precludes high-acuity vision. The ideal prosthetic solution would be less invasive, provide single-cell resolution and an ability to differentiate between different cell types. Nanoparticle-mediated approaches can address some of these requirements, with particular attention being directed at light-sensitive nanoparticles that can be accessed via the intrinsic optics of the eye. Here we survey the available known nanoparticle-based optical transduction mechanisms that can be exploited for neuromodulation. We review the rapid progress in the field, together with outstanding challenges that must be addressed to translate these techniques to clinical practice. In particular, successful translation will likely require efficient delivery of nanoparticles to stable and precisely defined locations in the retinal tissues. Therefore, we also emphasize the current literature relating to the pharmacokinetics of nanoparticles in the eye. While considerable challenges remain to be overcome, progress to date shows great potential for nanoparticle-based interfaces to revolutionize the field of visual prostheses.

Cre-LoxP and tamoxifen-induced deletion of ovarian quiescin sulfhydryl oxidase 2 showed disruption of ovulatory activity in mice

BackgroundQuiescin sulfhydryl oxidase 2 (QSOX2) is a flavin adenine dinucleotide-dependent sulfhydryl oxidase that is known to be involved in protein folding, cell growth regulation, and redox state modification through oxidative activities. Earlier studies demonstrated the tissue and cellular localization of QSOX2 in the male reproductive tract, as well as the highly-regulated mechanism of QSOX2 protein synthesis and expression through the coordinated action of testosterone and epididymal-enriched amino acid, glutamate. However, the presence and the functions of QSOX2 in female reproduction are unknown. In this study, we applied the Cre-loxP gene manipulation system to generate the heterozygous and homozygous Qsox2 knockout mice and examined its effects on ovarian function.ResultsWe demonstrated that QSOX2 was detected in the follicle-supporting cells (granulosa and cumulus cells) of ovarian follicles of all stages but was absent in the corpus luteum, suggesting its supportive role in folliculogenesis. In comparison with reproductive organogenesis in wild-type mice, there was no difference in testicular and epididymal structure in male Qsox2 knockout; however, Qsox2 knockout disrupted the regular ovulation process in female mice as a drastic decrease in the formation of the corpus luteum was detected, and no pregnancy was achieved when mating males with homozygous Qsox2 knockout females. RNAseq analyses further revealed that Qsox2 knockout altered critical signaling pathways and genes that are responsible for maintaining ovarian functions.ConclusionOur data demonstrated for the first time that Qsox2 is critical for ovarian function in mice.

The impact of multiple abiotic stresses on ns-LTP2.8 gene transcript and ns-LTP2.8 protein accumulation in germinating barley (Hordeum vulgare L.) embryos.

Abiotic stresses occur more often in combination than alone under regular field conditions limiting in more severe way crop production. Stress recognition in plants primarily occurs in the plasma membrane, modification of which is necessary to maintain homeostasis in response to it. It is known that lipid transport proteins (ns-LTPs) participate in modification of the lipidome of cell membranes. Representative of this group, ns-LTP2.8, may be involved in the reaction to abiotic stress of germinating barley plants by mediating the intracellular transport of hydrophobic particles, such as lipids, helping to maintain homeostasis. The ns-LTP2.8 protein was selected for analysis due to its ability to transport not only linear hydrophobic molecules but also compounds with a more complex spatial structure. Moreover, ns-LTP2.8 has been qualified as a member of pathogenesis-related proteins, which makes it particularly important in relation to its high allergenic potential. This paper demonstrates for the first time the influence of various abiotic stresses acting separately as well as in their combinations on the change in the ns-LTP2.8 transcript, ns-LTP2.8 protein and total soluble protein content in the embryonal axes of germinating spring barley genotypes with different ns-LTP2.8 allelic forms and stress tolerance. Tissue localization of ns-LTP2.8 transcript as well as ns-LTP2.8 protein were also examined. Although the impact of abiotic stresses on the regulation of gene transcription and translation processes remains not fully recognized, in this work we managed to demonstrate different impact on applied stresses on the fundamental cellular processes in very little studied tissue of the embryonal axis of barley.

The Musculoskeletal Tumor Society Scoring system is a valid subjective and objective tool to evaluate outcomes of surgical treatment of patients affected by upper and lower extremity tumors.

The main purpose of the present study was to evaluate if there is a difference between objective or subjective administration of the MSTS score in a cohort of patients affected by musculoskeletal oncological diseases. All patients who underwent surgery for bone or soft tissue localization of neoplastic disease in lower or upper limb from June 2015 to June 2020 were considered eligible. In order to administer the score as a PROM, the MSTS was first translated and cross-culturally adapted in Italian. During follow up visits, all patients filled out Italian versions of SF36, TESS and MSTS. Psychometric properties of the Italian version of MSTS were analyzed. Correlation between objective and self-administered MSTS score was assessed through Pearson's coefficient. A finale sample of 110 patients were included: 59 affected by lower extremity involvement and 51 affected by upper extremity involvement. The Italian version of the MSTS score showed good psychometric properties for both lower and upper extremity. The correlation between self-administered and hetero-administered version of the questionnaire was as high as r = 0.97 for lower extremities and r = 0.96 for upper extremities. The Italian version of the MSTS is a valid tool to evaluate outcomes of surgical treatment of patients affected by extremities tumors and it can be used as a subjective tool for both lower and upper extremity.

Wolbachia and Asaia Distribution among Different Mosquito Vectors Is Affected by Tissue Localization and Host Species.

Microbial communities play an important role in the fitness of mosquito hosts. However, the factors shaping microbial communities in wild populations, with regard to interactions among microbial species, are still largely unknown. Previous research has demonstrated that two of the most studied mosquito symbionts, the bacteria Wolbachia and Asaia, seem to compete or not compete, depending on the genetic background of the reference mosquito host. The large diversity of Wolbachia-Asaia strain combinations that infect natural populations of mosquitoes may offer a relevant opportunity to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction. We surveyed Wolbachia and Asaia in 44 mosquito populations belonging to 11 different species of the genera Anopheles, Aedes, and Culex using qualitative PCR. Through quantitative PCR, the amounts of both bacteria were assessed in different mosquito organs, and through metagenomics, we determined the microbiota compositions in some selected mosquito populations. We show that variation in microbial community structure is likely associated with the species/strain of mosquito, its geographical position, and tissue localization. Together, our results shed light on the interactions among different bacterial species in the microbial communities of mosquito vectors, and this can aid the development and/or improvement of methods for symbiotic control of insect vectors.

Rattus norvegicus Glutathione Transferase Omega 1 Localization in Oral Tissues and Interactions with Food Phytochemicals.

Glutathione transferases are xenobiotic-metabolizing enzymes with both glutathione-conjugation and ligandin roles. GSTs are present in chemosensory tissues and fluids of the nasal/oral cavities where they protect tissues from exogenous compounds, including food molecules. In the present study, we explored the presence of the omega-class glutathione transferase (GSTO1) in the rat oral cavity. Using immunohistochemistry, GSTO1 expression was found in taste bud cells of the tongue epithelium and buccal cells of the oral epithelium. Buccal and lingual extracts exhibited thiol-transferase activity (4.9 ± 0.1 and 1.8 ± 0.1 μM/s/mg, respectively). A slight reduction from 4.9 ± 0.1 to 4.2 ± 0.1 μM/s/mg (p < 0.05; Student's t test) was observed in the buccal extract with 100 μM GSTO1-IN-1, a specific inhibitor of GSTO1. RnGSTO1 exhibited the usual activities of omega GSTs, i.e., thiol-transferase (catalytic efficiency of 8.9 × 104 M-1·s-1), and phenacyl-glutathione reductase (catalytic efficiency of 8.9 × 105 M-1·s-1) activities, similar to human GSTO1. RnGSTO1 interacts with food phytochemicals, including bitter compounds such as luteolin (Ki = 3.3 ± 1.9 μM). Crystal structure analysis suggests that luteolin most probably binds to RnGSTO1 ligandin site. Our results suggest that GSTO1 could interact with food phytochemicals in the oral cavity.

Genome-Wide Characterization of Differentially Expressed Scent Genes in the MEP Control Network of the Flower of Lilium 'Sorbonne'.

Fragrance is an important feature of ornamental lilies. Components of volatile substances and important genes for monoterpene synthesis in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway were examined in this study. Twenty volatile compounds (2 in the budding stage, 3 in the initial flowering stage, 7 in the semi-flowering stage, 17 in the full-flowering stage, and 5 in withering stage) were detected in the Oriental lily 'Sorbonne' using gas chromatography-mass spectrometry. The semi- and full-flowering stages were key periods for volatile substance production and enzyme function. Sequence assembly from samples collected during all flowering stages resulted in the detection of 274,849 genes and 129,017 transcripts. RNA sequencing and heatmapping led to the detection of genes in the MEP monoterpene metabolism pathway. Through gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, we extracted key genes (LiDXS2, LiLIS, and LiMYS) and transcription factors (in the bHLH, MYB, HD-ZIP, and NAC families) associated with the MEP pathway. Tissue localization revealed that LiDXS2, LiLIS, and LiMYS were expressed in Lilium 'Sorbonne' petals in the full-flowering stage. Genes regulating the 1-deoxy-D-X-lignone-5-phosphate synthase family of rate-limiting enzymes, involved in the first step of monoterpene synthesis, showed high expression in the semi- and full-flowering stages. LiDXS2 was cloned and localized in chloroplast subcells. The relative expression of terpene-related genes in the MEP and mevalonic acid pathways of wild-type and LiLIS/LiMYS transgenic Arabidopsis thaliana, and changes in chemical composition, confirmed that LiLIS/LiMYS regulates the monoterpene synthesis pathway. The results of this study provide a theoretical basis for the synthesis of lily aromatic substances and the cultivation of new garden flower varieties.

Mucosal host-microbe interactions associate with clinical phenotypes in inflammatory bowel disease

Disrupted host-microbe interactions at the mucosal level are key to the pathophysiology of IBD. This study aimed to comprehensively examine crosstalk between mucosal gene expression and microbiota in patients with IBD. To study tissue-specific interactions, we perform transcriptomic (RNA-seq) and microbial (16S-rRNA-seq) profiling of 697 intestinal biopsies (645 derived from 335 patients with IBD and 52 from 16 non-IBD controls). Mucosal gene expression patterns in IBD are mainly determined by tissue location and inflammation, whereas the mucosal microbiota composition shows a high degree of individual specificity. Analysis of transcript-bacteria interactions identifies six distinct groups of inflammation-related pathways that are associated with intestinal microbiota (adjusted P < 0.05). An increased abundance of Bifidobacterium is associated with higher expression of genes involved in fatty acid metabolism, while Bacteroides correlates with increased metallothionein signaling. In patients with fibrostenosis, a transcriptional network dominated by immunoregulatory genes is associated with Lachnoclostridium bacteria in non-stenotic tissue (adjusted P < 0.05), while being absent in CD without fibrostenosis. In patients using TNF-α-antagonists, a transcriptional network dominated by fatty acid metabolism genes is linked to Ruminococcaceae (adjusted P < 0.05). Mucosal microbiota composition correlates with enrichment of intestinal epithelial cells, macrophages, and NK-cells. Overall, these data demonstrate the presence of context-specific mucosal host-microbe interactions in IBD, revealing significantly altered inflammation-associated gene-taxa modules, particularly in patients with fibrostenotic CD and patients using TNF-α-antagonists. This study provides compelling insights into host–microbe interactions that may guide microbiota-directed precision medicine and fuels the rationale for microbiota-targeted therapeutics as a strategy to alter disease course in IBD.