Individual Tissues Research Articles

The biological process of lysine-tRNA charging is therapeutically targetable in liver cancer.

Background & AimsMature transfer RNAs (tRNA) charged with amino acids decode mRNA to synthesize proteins. Dysregulation of translational machineries has a fundamental impact on cancer biology. This study aims to map the tRNAome landscape in liver cancer patients and to explore potential therapeutic targets at the interface of charging amino acid with tRNA.MethodsResected tumour and paired tumour‐free (TFL) tissues from hepatocellular carcinoma (HCC) patients (n = 69), and healthy liver tissues from organ transplant donors (n = 21), HCC cell lines, and cholangiocarcinoma (CC) patient‐derived tumour organoids were used.ResultsThe expression levels of different mature tRNAs were highly correlated and closely clustered within individual tissues, suggesting that different members of the tRNAome function cooperatively in protein translation. Interestingly, high expression of tRNA‐Lys‐CUU in HCC tumours was associated with more tumour recurrence (HR 1.1; P = .022) and worse patient survival (HR 1.1; P = .0037). The expression of Lysyl‐tRNA Synthetase (KARS), the enzyme catalysing the charge of lysine to tRNA‐Lys‐CUU, was significantly upregulated in HCC tumour tissues compared to tumour‐free liver tissues. In HCC cell lines, lysine deprivation, KARS knockdown or treatment with the KARS inhibitor cladosporin effectively inhibited overall cell growth, single cell‐based colony formation and cell migration. This was mechanistically mediated by cell cycling arrest and induction of apoptosis. Finally, these inhibitory effects were confirmed in 3D cultured patient‐derived CC organoids.ConclusionsThe biological process of charging tRNA‐Lys‐CUU with lysine sustains liver cancer cell growth and migration, and is clinically relevant in HCC patients. This process can be therapeutically targeted and represents an unexplored territory for developing novel treatment strategies against liver cancer.

Tooth-Supporting Hard Tissue Regeneration Using Biopolymeric Material Fabrication Strategies.

The mineralized tissues (alveolar bone and cementum) are the major components of periodontal tissues and play a critical role to anchor periodontal ligament (PDL) to tooth-root surfaces. The integrated multiple tissues could generate biological or physiological responses to transmitted biomechanical forces by mastication or occlusion. However, due to periodontitis or traumatic injuries, affect destruction or progressive damage of periodontal hard tissues including PDL could be affected and consequently lead to tooth loss. Conventional tissue engineering approaches have been developed to regenerate or repair periodontium but, engineered periodontal tissue formation is still challenging because there are still limitations to control spatial compartmentalization for individual tissues and provide optimal 3D constructs for tooth-supporting tissue regeneration and maturation. Here, we present the recently developed strategies to induce osteogenesis and cementogenesis by the fabrication of 3D architectures or the chemical modifications of biopolymeric materials. These techniques in tooth-supporting hard tissue engineering are highly promising to promote the periodontal regeneration and advance the interfacial tissue formation for tissue integrations of PDL fibrous connective tissue bundles (alveolar bone-to-PDL or PDL-to-cementum) for functioning restorations of the periodontal complex.

Identifying Pathways and Networks Associated With the SARS-CoV-2 Cell Receptor ACE2 Based on Gene Expression Profiles in Normal and SARS-CoV-2-Infected Human Tissues.

Because ACE2 is a host cell receptor of the SARS-CoV-2, an investigation of ACE2 expression in normal and virus-infected human tissues is crucial for understanding the mechanism of SARS-CoV-2 infection. We identified pathways associated with ACE2 expression and gene co-expression networks of ACE2 in pan-tissue based on the gene expression profiles in normal human tissues. We found that the pathways significantly associated with ACE2 upregulation were mainly involved in immune, stromal signature, metabolism, cell growth and proliferation, and cancer and other diseases. The number of genes having a significant positive expression correlation with ACE2 in females far exceeded that in males. The estrogen receptors (ESR1 and ESR2) and androgen receptor (AR) genes had a significant positive expression correlation with ACE2. Meanwhile, the enrichment levels of immune cells were positively associated with the expression levels of ESR1 and ESR2, while they were inversely associated with the expression levels of AR in pan-tissue and multiple individual tissues. It suggests that females are likely to have a more robust immune defense system against SARS-CoV-2 than males. ACE2 was upregulated in SARS-CoV-2-infected tissues relative to normal tissues and in SARS-CoV-2-infected males relative to females, while its expression levels had no significant difference between healthy females and males. Numerous immune-related pathways were highly enriched in SARS-CoV-2-infected males relative to females. These data indicate that males are more susceptible and more likely to have an excessive immune response to SARS-CoV-2 infection than females. This study furnishes potentially cues explaining why females have better clinical outcomes of SARS-CoV-2 infections than males and warrant further investigation for understanding the mechanism of SARS-CoV-2 infection.

Tissue Engineering – The Current Scenario & Innovations

Tissue engineering is an emerging field in medical arena which combines the knowledge of science and engineering to accomplish the increasing demands to aid the damaged tissues or even a whole organ. With time various methods of tissue engineering such as traditional scaffold method ,advanced 3D bioprinting technology and the use of bio ink (the extracellular matrix materials) have become popular at various medical levels. Scaffold is a 3D structure which results in tissue formation by providing space for cells to attach, to proliferate in various directions & by secreting extracellular matrix. Also, the recent development is the use of decellularised extracellular material i.e. dECM as bio-ink to generate vascular organs like Kidney & Heart. Textiles have been playing an indispensable role in tissue engineering as it provide superior methods over other ways to fabricate scaffold. The use of smart biomaterial based scaffolds costs less and is more effective which gives advantage to tailor the tissues according to individual's tissue structure . This paper reviews the application of textiles technology in tissue engineering, various approaches of tissue engineering from traditional to the currently used approach , recent advances and its indications.

Alterations in allocation and composition of lipid classes in Euonymus fruits and seeds.

The spindle tree (Euonymus europaeus L.) is a much-branched deciduous shrub or small tree. Its fruit capsules contain seeds with remarkably high content of oil interesting for industry, especially the 3-acetyl-1,2-diacyl-sn-glycerols (AcDAG) synthesized by a specific acetyl-CoA diacylglycerol acetyltransferase. The distribution and amount of individual triacylglycerols (TAG) and especially acetyl-triacylglycerols (AcDAG) in Euonymus fruit have previously been assigned to specific tissues. Using anatomical and microscopical observations, we studied the fruit morphology, and for the first time, we identified a more detailed allocation of oil bodies in individual tissue structures. Thin layer chromatography separation of extracts from immature and mature fruits confirmed TAG and AcDAG as the most abundant lipid classes in both endosperm and embryo, followed by fatty acids and polar lipids. The abundance of fatty acids was further studied in the TAG and AcDAG fractions using gas chromatography. Data revealed particular FAs in both fractions allocated in tissue-specific manner and/or as indicators of maturation of E. europaeus seeds. While the abundance of cis-vaccenic-, linoleic as well as α-linolenic acids in the AcDAG structures generally drop with maturation in both embryo and endosperm, content of oleic acid increases. Abundance of cis-vaccenic acid in TAG was recorded in immature endosperm. For embryo, the abundance of stearic acid in AcDAG and oleic acid in TAG fraction was distinctive. Deeper understanding of underlying metabolic processes will be essential for targeted metabolic engineering and/or application for oilseed crops.

Preliminary development of guided template of middle and upper thoracic percutaneous vertebroplasty in thoracic pedicle approach due to three dimentional soft tissue print technique

To investigate the feasibility of a drill template for the placement of guided template of middle and upper thoracic percutaneous vertebroplasty in thoracic pedicle approach on digital design and 3D printing technology. The preoperative CT images of 20 patients with thoracic fracture were collected retrospectively. With the 3D soft tissue printing technology, the data was reconstructed by 3D imaging reconstruction software to produce 1∶1 three dimensional soft tissue model. The pedicle screw channel and the digital template were designed by the 3-matic module of Mimics15.0 software. After guide template was printed by 3D printer and three dimensional template was fixed on the model, 2.0 mm Kirschner was placed and the accuracy of a drill template was observed by CT scans, bone cement was injected through the puncture tube and verified with images. The time of nail guide design, guide template production and cost were recorded. The effectiveness of three dimensional thoracic model and digital guided template of middle and upper thoracic percutaneous vertebroplasty of thoracic fractures in thoracic pedicle approach was confirmed. Kirschner was placed and the accuracy of screw placement was confirmed with CT scanning. Template and the corresponding anatomical landmark fitted well, bone cement had showed good filling. The average printing time of upper thoracic spine model with soft tissue, the mean time of nail guide design, guide template production and cost were (719.00±3.03) min, (12.30±1.01) min, (55.50±10.30) min and RMB 3 150 yuan on average respectively. By means of individual design and 3D soft tissue printingtechnology, accurate placement of guided template of middle and upper thoracic percutaneous vertebroplasty could be realized.

A mouse tissue atlas of small noncoding RNA

Small noncoding RNAs (ncRNAs) play a vital role in a broad range of biological processes both in health and disease. A comprehensive quantitative reference of small ncRNA expression would significantly advance our understanding of ncRNA roles in shaping tissue functions. Here, we systematically profiled the levels of five ncRNA classes (microRNA [miRNA], small nucleolar RNA [snoRNA], small nuclear RNA [snRNA], small Cajal body-specific RNA [scaRNA], and transfer RNA [tRNA] fragments) across 11 mouse tissues by deep sequencing. Using 14 biological replicates spanning both sexes, we identified that ∼30% of small ncRNAs are distributed across the body in a tissue-specific manner with some also being sexually dimorphic. We found that some miRNAs are subject to "arm switching" between healthy tissues and that tRNA fragments are retained within tissues in both a gene- and a tissue-specific manner. Out of 11 profiled tissues, we confirmed that brain contains the largest number of unique small ncRNA transcripts, some of which were previously annotated while others are identified in this study. Furthermore, by combining these findings with single-cell chromatin accessibility (scATAC-seq) data, we were able to connect identified brain-specific ncRNAs with their cell types of origin. These results yield the most comprehensive characterization of specific and ubiquitous small RNAs in individual murine tissues to date, and we expect that these data will be a resource for the further identification of ncRNAs involved in tissue function in health and dysfunction in disease.

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach.

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

The effect of simulation strategies on prediction of power deposition in the tissue around electronic implants during magnetic resonance imaging

Numerical simulations are increasingly employed in safety assessment of high-field magnetic resonance imaging (MRI) in patients with conductive medical implants such as those with deep brain stimulation (DBS) devices. Performing numerical simulations with realistic patient models and implant geometry is the preferred method as it provides the most accurate results; however, in many cases such an approach is infeasible due to limitation of computational resources. The difficulties in reconstructing realistic patient and device models and obtaining accurate electrical properties of tissue have compelled researchers to adopt compromises, either to exceedingly simplify implant structure and geometry, or the complexity of the body model. This study examines the effect of variations in anatomical details of the human body model and implant geometry on predicted values of specific absorption rate (SAR) values during MRI in a patient with a DBS implant. We used a patient-derived model of a fully implanted DBS implant and performed numerical simulations to calculate the maximum SAR during MRI at 1.5T (64 MHz) and 3T (127 MHz). We then assessed the effect of uncertainties in dielectric properties of tissue, complexity of body model, truncation of body/DBS model, and DBS lead geometry on SAR. Our results showed that 40% variation in the conductivity of individual tissues in a heterogeneous body model caused a peak of 7% variation in maximum SAR value at 64 MHz, and 10.6% variation in SAR at 127 MHz. SAR predictions from a homogeneous body model with a conductivity range of could cover the full range of SAR variations predicted by the heterogeneous body model. Truncation of body model below the implanted pulse generator changed the predicted SAR by 16% at 1.5T and 32% at 3T while saving 250% and 148% in computational time and memory allocation, respectively. In contrast, variation in DBS lead geometry significantly changed the SAR by up to 51% at 64 MHz and 67% at 127 MHz. These results suggest that the error introduced by simplifying the implant’s geometry could negate the benefit of using a realistic body model, should such model be used at the expense of oversimplifying implant geometry.

Stable isotope ecology in insects: a review

1. The use of stable isotope analysis (SIA) in ecological research has dramatically increased in recent years largely because it allows researchers to investigate ecological questions that have been previously difficult to address.2. Ecological applications of SIA include estimating fundamental niche space and overlap, evaluating trophic or species level interactions, and investigating food web structure. Increasingly, researchers have been incorporating SIA in studies of animal migration, disease transmission, diet composition, nutrient assimilation, and body condition among others.3. Studies using SIA to evaluate the ecology of terrestrial insects have lagged behind other taxonomic groups. This poor representation of stable isotope studies in publications likely stems from a lack of familiarity of entomologists with this technique.4. An improved understanding of SIA, as well as the advantages and disadvantages specifically related to insect research, will benefit the field of entomology. In addition, insect‐model systems provide unique opportunities for entomologists to incorporate SIA in their research to advance our knowledge of insect biology and the stable isotope ecology of insects.5. We provide background information on stable isotopes, explain sources of isotopic variation, describe the processes of how isotopes are differentially routed and incorporated into an individual's tissues, explain the principles that influence isotopic fractionation and discrimination, highlight different methods and advancements in SIA, review innovative stable isotope studies, and provide an overview of common mistakes, considerations, and future directions entomologists can explore.

Правове регулювання відносин у біомедичній сфері в контексті євроінтеграції

Стаття присвячена аналізу проблемних питань приведення у відповідність законодавства України до європейських стандартів у контексті діяльності зі створення та використання біобанків в Україні. На підставі аналізу положень відповідних Директив ЄС і їх зіставлення із чинним законодавством України визначено ступінь імплементації норм права ЄС у національне законодавство України та виокремлено питання, які ще потребують уніфікації.
 Автор наголошує на важливості прийняття нової редакції Закону про трансплантацію, як нормативно-правового кроку для імплементації Директив в українське законодавство. Виключення ст. 3 Закону про трансплантацію зі сфери його регулювання питання трансплантації репродуктивних клітин, аутотран-сплантації та діяльності банків пуповинної крові, що входить в обсяг регулювання Директиви, не сприяє процесу адаптації українського законодавства до європейських стандартів.
 Аналізуючи правове регулювання репродуктивної медицини одного із видів діяльності управителів біо-банків в Україні, зроблено наголос на тому, що сьогодні ані в чинному законодавстві, ані в законопроєктах не сформовано дієвих правових механізмів для вирішення питання щодо забезпечення захисту ембріонів, зокрема від їхнього створення із дослідницькою чи комерційною метою, не встановлено чітких умов і порядку подальшого використання / застосування невикористаних із репродуктивною метою ембріонів і їхньої утилізації.
 Зроблено висновок про те, що українське законодавство необхідно привести у відповідність до Директив в тому, що стосується запровадження системи якості в установах, які працюють із людськими тканинами та клітинами, включаючи навчання персоналу, процедури повідомлення про серйозні негативні випадки та реакції, інформування донорів і їх обстеження перед забором тканин або клітин. Також необхідно доповнити критерії відбору донорів, доопрацювати стандарти підготовки тканин і клітин, їхньої обробки та розподілу прямої поставки окремих тканин і клітин реципієнту.

Universal Nature of Drug Treatment Responses in Drug-Tissue-Wide Model-Animal Experiments Using Tensor Decomposition-Based Unsupervised Feature Extraction.

Gene expression profiles of tissues treated with drugs have recently been used to infer clinical outcomes. Although this method is often successful from the application point of view, gene expression altered by drugs is rarely analyzed in detail, because of the extremely large number of genes involved. Here, we applied tensor decomposition (TD)-based unsupervised feature extraction (FE) to the gene expression profiles of 24 mouse tissues treated with 15 drugs. TD-based unsupervised FE enabled identification of the common effects of 15 drugs including an interesting universal feature: these drugs affect genes in a gene-group-wide manner and were dependent on three tissue types (neuronal, muscular, and gastroenterological). For each tissue group, TD-based unsupervised FE enabled identification of a few tens to a few hundreds of genes affected by the drug treatment. These genes are distinctly expressed between drug treatments and controls as well as between tissues in individual tissue groups and other tissues. We also validated the assignment of genes to individual tissue groups using multiple enrichment analyses. We conclude that TD-based unsupervised FE is a promising method for integrated analysis of gene expression profiles from multiple tissues treated with multiple drugs in a completely unsupervised manner.

Abstract 6712: Analysis of VEGF as a potential complimentary target with immunotherapy

Abstract Background: Angiogenesis is a hallmark of malignancy and greatly contributes to tumor growth and dissemination to metastatic sites. Vascular endothelial growth factors (VEGFs) are major members of angiogenesis signaling and have been shown to have immunosuppressive function. Bevacizumab (Bev), a VEGF inhibitor, in combination with immune checkpoint inhibitor (ICI) therapy and chemotherapy, has been shown in a previous clinical trial to improve outcomes in lung cancer versus chemotherapy plus Bev, even in PDL1-low and negative tumors. (IMPOWER150). Tumor mutation burden (TMB) has been established as a biomarker of ICI response, independent of PDL1. We set out to explore the relationship between VEGF signaling and TMB. Methods: We performed a retrospective analysis of data from the NantHealth database of 2,737 commercial cases where matched WGS or WES and RNA-Seq were performed. Primary and metastatic breast tumors were included in the analysis. TMB was computed as the count of SNVs per tumor sample. Bowtie2, RSEM, and custom software are used for alignment, transcript quantification and variant analysis. Per-tumor type analysis was performed to find VEGF signaling correlates with TMB and a panel of immune markers (n = 9). Results: Overall TMB does not correlate with PDL1, PDL2, CTLA4 and other immune markers' expression (spearman range [-0.01 - 0.15]). We also observed that overall TMB does not exhibit strong correlations with VEGF signaling markers (spearman range [-.17 - .17]), however when limited to TMB-low tumors VEGFA and VEGFD positively correlate with TMB (p = 1e-5 and 4.1e-2) and VEGFE negatively correlates with TMB (p = 1.6e-4). We then limited our analysis to PDL1-low tumors, of which the top three categories by sample counts are breast (n = 103), colon (n = 85), and sarcoma (n = 78). In whole-cohort analysis, we observed significantly higher expression of VEGFE and VEGFR2 (p = 1.4e-2 and 2.8e-2) in TMB-low tumors. When performing the same analysis in individual tissue types we observed significantly differentially higher expression of VEGFA and PGF (p = 2.1e-2 and 3.3e-2) in TMB-high colon samples, of VEGFR2 (p = 1.5e-2) in TMB-low NSCLC tumors, and of VEGFR2, FOS, and FOXO1(p = 1.9e-2, 3.4e-2, 6.2e-3) in TMB-low uterine endometrial tumors. Conclusions: We provide evidence that targeting VEGF signaling in some tumor types, especially in tumors with low TMB, might provide a boost to therapeutic effects of checkpoint inhibitors. Our findings warrant further translational interrogation of the potential efficacy with a combination of VEGF inhibitors and ICI in various cancer types. For example, NCT03971474, a trial of ramucirumab (anti-VEGFR2 MAb) plus pembrolizumab in NSCLC, may produce differential activity in TMB-High v TMB-Low cohorts. Citation Format: Yulia Newton, Christopher W. Szeto, Stephen C. Benz, Sandeep K. Reddy. Analysis of VEGF as a potential complimentary target with immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6712.

Exploiting the circuit breaker cancer evolution model in human clear cell renal cell carcinoma.

The incessant interactions between susceptible humans and their respective macro/microenvironments registered throughout their lifetime result in the ultimate manifestation of individual cancers. With the average lifespan exceeding 50 years of age in humans since the beginning of 20th century, aging – the “time” factor – has played an ever-increasing role alongside host and environmental factors in cancer incidences. Cancer is a genetic/epigenetic disease due to gain-of-function mutations in cancer-causing genes (oncogene; OG) and/or loss-of-function mutations in tumor-suppressing genes (tumor suppressor genes; TSG). In addition to their integral relationship with cancer, a timely deployment of specific OG and/or TSG is in fact needed for higher organisms like human to cope with respective physiological and pathological conditions. Over the past decade, extensive human kidney cancer genomics have been performed and novel mouse models recapitulating human kidney cancer pathobiology have been generated. With new genomic, genetic, mechanistic, clinical and therapeutic insights accumulated from studying clear cell renal cell carcinoma (ccRCC)–the most common type of kidney cancer, we conceived a cancer evolution model built upon the OG-TSG signaling pair analogous to the electrical circuit breaker (CB) that permits necessary signaling output and at the same time prevent detrimental signaling overdrive. Hence, this viewpoint aims at providing a step-by-step mechanistic explanation/illustration concerning how inherent OG-TSG CBs intricately operate in concert for the organism's wellbeing; and how somatic mutations, the essential component for genetic adaptability, inadvertently triggers a sequential outage of specific sets of CBs that normally function to maintain and protect and individual tissue homeostasis.

New developments and possibilities in the field of post-mortem medicine mortui vivos docent

The body as a whole, along with individual organs and tissues, is the source for many examinations and measures relating to both teaching and research in medicine. There is no possibility of having an alternative model for the body in the near future. When it comes to organ and tissue donation activities, the human body gives other people another chance in life and indicates a better quality of life for many. Therefore, interventions on the deceased are carried out at the Institute of Legal Medicine in Hamburg (with the declaration of consent being taken from the person during lifetime or the relatives) for teaching, research, and clinical practices other than judicial autopsies. This includes scientific investigations in clinical research projects and surgical interventions within the framework of the Post-mortem Learning Centre and tissue donation, especially cornea transplantation in Germany and abroad. For clinical research projects, especially regarding quality assurance and innovation, post-mortem procedures on corpses are increasingly becoming the focus of attention.

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants.

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Disease Progression in Lake Trout (Salvelinus namaycush) Experimentally Infected With Epizootic Epitheliotropic Disease Virus (Salmonid Herpesvirus-3).

Epizootic epitheliotropic disease virus (salmonid herpesvirus-3; EEDV) is responsible for the death of millions of hatchery-raised lake trout (Salvelinus namaycush) in the Laurentian Great Lakes Basin. However, little is known about its biology, pathology, tropism, and host interactions. In this study, the presence and disease progression of EEDV were evaluated following exposure of naïve juvenile lake trout to EEDV via bath immersion under controlled laboratory conditions (n = 84 infected; n = 44 control). Individual tissues (n = 10 per fish), collected over 6 weeks, were analyzed for viral load by quantitative polymerase chain reaction, gross and histopathologic changes, and virus cellular targets using in situ hybridization. Skin, fin, and ocular tissues were the earliest viral targets and yielded the highest viral loads throughout the course of infection. Early gross lesions included exophthalmia, ocular hemorrhage, fin congestion, and hyperemia of visceral blood vessels. Advanced disease was characterized by multifocal to coalescing erosions and ulcerations of the skin, and congestion of visceral organs. Microscopically, there was cellular degeneration and necrosis in the epidermis and spleen, and lymphohistiocytic perivasculitis of the dermis, omentum, and the epicardium. EEDV DNA was first detected by in situ hybridization in epithelial cells of the epidermis, with subsequent labeling in the epithelial lining of primary and secondary gill lamellae. During advanced disease, EEDV was detected in endothelial and dendritic cells as well as blood monocytes. This study characterized EEDV tissue tropism and associated pathologic features, to guide research aimed at understanding EEDV disease ecology and improving strategies for disease control.

Metabolic Systemic Effects Triiodothyronine

Triiodothyronine (T3, 3,5,3’-L-triiodothyronine) is a thyroid hormone (thyroid), the secretion of which is carried out directly both by the gland (to a lesser extent) and outside it (the main amount; as a result of peripheral deiodination of thyroxine (T4)). Getting into the nuclei of cells, T3 interacts with specific nuclear receptors of target tissues, which determines its biological activity. This interaction leads to the activation of transcription of a number of genes.In the pituitary gland and peripheral tissues, the action of thyroid hormones is modulated by local deiodinases, which convert T4 to more active T3, the molecular effects of which in individual tissues depend on subtypes of T3 receptors and their interaction with other ligands, coactivators and corepressors, as well as on the activation or repression of specific genes.The reason for the lack of T3 production is primarily a deficiency of iodine in the diet, less often, a defect in the genes encoding the proteins that are involved in T3 biosynthesis. As a result of the low intake of iodide in the body, the so-called adaptive mechanism is activated, which consists in increasing the proportion of synthesized T3, which increases the metabolic efficiency of thyroid homones. With a deficiency in the diet of such a trace element as selenium, the conversion of T4 to T3 is reduced.Thyroid hormones play a vital role in the regulation of homeostasis and the metabolic rate of cells and tissues of humans and mammals. They are necessary for physical and mental development. Their insufficient production at the stage of formation of the internal organs of the fetus and in childhood can lead to various pathologies, primarily to pathology of the central nervous system, and as a result, growth retardation and mental retardation. In adulthood, hypothyroidism leads to a decrease in metabolism, memory impairment, depressive disorders, impaired fertility. Many discussions and ambiguous conclusions have been obtained regarding combination drugs (sodium levothyroxine + lyothironon) for the treatment of hypothyroidism. This article will examine the metabolic effects of T3, the thyroid hormone with the highest activity.

Transcriptomic profiling of the medicinal plant Clitoria ternatea: identification of potential genes in cyclotide biosynthesis

Clitoria ternatea a perennial climber of the Fabaceae family, is well known for its agricultural and medical applications. It is also currently the only known member of the Fabaceae family that produces abundant amounts of the ultra-stable macrocyclic peptides, cyclotides, across all tissues. Cyclotides are a class of gene-encoded, disulphide-rich, macrocyclic peptides (26–37 residues) acting as defensive metabolites in several plant species. Previous transcriptomic studies have demonstrated the genetic origin of cyclotides from the Fabaceae plant family to be embedded in the albumin-1 genes, unlike its counterparts in other plant families. However, the complete mechanism of its biosynthesis and the repertoire of enzymes involved in cyclotide folding and processing remains to be understood. In this study, using RNA-Seq data and de novo transcriptome assembly of Clitoria ternatea, we have identified 71 precursor genes of cyclotides. Out of 71 unique cyclotide precursor genes obtained, 51 sequences display unique cyclotide domains, of which 26 are novel cyclotide sequences, arising from four individual tissues. MALDI-TOF mass spectrometry analysis of fractions from different tissue extracts, coupled with precursor protein sequences obtained from transcriptomic data, established the cyclotide diversity in this plant species. Special focus in this study has also been on identifying possible enzymes responsible for proper folding and processing of cyclotides in the cell. Transcriptomic mining for oxidative folding enzymes such as protein-disulphide isomerases (PDI), ER oxidoreductin-1 (ERO1) and peptidylprolyl cis-trans isomerases (PPIases)/cyclophilins, and their levels of expression are also reported. In particular, it was observed that the CtPDI genes formed plant-specific clusters among PDI genes as compared to those from other plant species. Collectively, this work provides insights into the biogenesis of the medicinally important cyclotides and establishes the expression of certain key enzymes participating in peptide biosynthesis. Also, several novel cyclotide sequences are reported and precursor sequences are analysed in detail. In the absence of a published reference genome, a comprehensive transcriptomics approach was adopted to provide an overview of diverse properties and constituents of C. ternatea.