Assessing Genotoxicity of Ten Different Engineered Nanomaterials by the Novel Semi-Automated FADU Assay and the Alkaline Comet Assay

Increased engineered nanomaterial (ENM) production and incorporation in consumer and biomedical products has raised concerns about the potential adverse effects. The DNA damaging capacity is of particular importance since damaged genetic material can lead to carcinogenesis. Consequently, reliable and robust in vitro studies assessing ENM genotoxicity are of great value. We utilized two complementary assays based on different measurement principles: (1) comet assay and (2) FADU (fluorimetric detection of alkaline DNA unwinding) assay. Assessing cell viability ruled out false-positive results due to DNA fragmentation during cell death. Potential structure–activity relationships of 10 ENMs were investigated: three silica nanoparticles (SiO2-NP) with varying degrees of porosity, titanium dioxide (TiO2-NP), polystyrene (PS-NP), zinc oxide (ZnO-NP), gold (Au-NP), graphene oxide (GO) and two multi-walled carbon nanotubes (MWNT). SiO2-NPs, TiO2-NP and GO were neither cytotoxic nor genotoxic to Jurkat E6-I cells. Quantitative interference corrections derived from GO results can make the FADU assay a promising screening tool for a variety of ENMs. MWNT merely induced cytotoxicity, while dose- and time-dependent cytotoxicity of PS-NP was accompanied by DNA fragmentation. Hence, PS-NP served to benchmark threshold levels of cytotoxicity at which DNA fragmentation was expected. Considering all controls revealed the true genotoxicity for Au-NP and ZnO-NP at early time points.

The 14-3-3 Proteins Bmh1 and Bmh2 Are Key Regulators of Meiotic Commitment

Induction of meiosis requires exogenous signals that activate internal gene regulatory networks. Meiotic commitment ensures the irreversible continuation of meiosis, even upon withdrawal of the meiosis inducing signals. Budding yeast cells have a unique property in that cells enter meiosis when starved, but if given nutrient-rich medium prior to the commitment point, they exit meiosis and enter mitosis. After the meiotic commitment point in prometaphase I, cells remain in meiosis even with addition of nutrients. Despite the importance of meiotic commitment in ensuring the production of gametes, only a few genes involved in the commitment process are known. We performed a genome-scale screen in budding yeast and discovered new regulators of meiotic commitment including Bcy1, which is involved in nutrient sensing, the meiosis-specific kinase Ime2, Polo kinase Cdc5, and the 14-3-3 proteins Bmh1 and Bmh2. Importantly, we found that Bmh1 and Bmh2 are involved in multiple processes throughout meiosis including the maintenance of the middle meiosis transcription factor Ndt80, activation of Cdc5, and interaction with an RNA-binding protein Pes4, which is important for regulating the timing of translation of several mRNAs in meiosis II. This study identifies a meiotic commitment regulatory network with the 14-3-3 proteins functioning as central regulators.

Immunohistochemical Characteristics of the Human Carotid Body in the Antenatal and Postnatal Periods of Development

The evolutionary and ontogenetic development of the carotid body is still understudied. Research aimed at studying the comparative morphology of the organ at different periods in the individual development of various animal species should play a crucial role in understanding the physiology of the carotid body. However, despite more than two centuries of study, the human carotid body remains poorly understood. There are many knowledge gaps in particular related to the antenatal development of this structure. The aim of our work is to study the morphological and immunohistochemical characteristics of the human carotid body in the antenatal and postnatal periods of development. We investigated the human carotid bodies from 1 embryo, 20 fetuses and 13 adults of different ages using samples obtained at autopsy. Immunohistochemistry revealed expression of βIII-tubulin and tyrosine hydroxylase in the type I cells and nerve fibers at all periods of ontogenesis; synaptophysin and PGP9.5 in the type I cells in some of the antenatal cases and all of the postnatal cases; 200 kDa neurofilaments in nerve fibers in some of the antenatal cases and all of the postnatal cases; and GFAP and S100 in the type II cells and Schwann cells in some of the antenatal cases and all of the postnatal cases. A high level of tyrosine hydroxylase in the type I cells was a distinctive feature of the antenatal carotid bodies. On the contrary, in the type I cells of adults, the expression of tyrosine hydroxylase was significantly lower. Our data suggest that the human carotid body may perform an endocrine function in the antenatal period, while in the postnatal period of development, it loses this function and becomes a chemosensory organ.

Histo-Morphology Study of the Respiratory Portion of Goat Lung (Capra Hircus) in Baghdad Province

The Histo-mrphology were directed on the pneumonic alveoli of 6 male goats. The respiratory portion is composed of typical cuboidal epithelial cells with Clara cell, however, alveolar ducts are lined by simple squamous epithelium and alveoli were generally circular structures that opened into the alveolar conduits and alveolar sacs or respiratory bronchioles. Alveoli were made out of two kinds of cells for example Type-I pneumocytes and Type-II pneumocytes. Previous framed the mainlining epithelial cells of alveoli which were squamous in sort having noticeable perinuclear territory and central found the core, while the last were cuboidal fit as a fiddle with the midway found core and periodically found among the Sort I cells in the alveolar epithelium. The lung pulmonary parenchyma was enveloped by the mesothelium (squamous epithelium) layer of visceral pleura.

Expression of Cholecystokinin and its Receptors in the Intestinal Tract of Type 2 Diabetes Patients and Healthy Controls

Background Cholecystokinin (CCK) is a gut hormone originally known for its effects on gallbladder contraction and release of digestive enzymes. CCK, however, also mediates satiety and stimulate insulin secretion. Knowledge of the distribution of CCK-producing enteroendocrine cells (I cells) in humans is sparse. The general notion, based on animal data, is that I cells are present mainly in the proximal small intestine. We examined the occurrence of I cells (immunohistochemically) and the expression of CCK mRNA as well as CCK1 and CCK2 receptor mRNA along the intestines in healthy individuals and patients with type 2 diabetes. Methods Mucosal biopsies were collected with 30 cm intervals in the small intestine and from seven anatomical locations in the large intestine (using double-balloon enteroscopy) from 12 patients with type 2 diabetes and 12 gender, age and body mass index-matched healthy individuals. The biopsies were analyzed using mRNA sequencing and immunohistochemical staining. Results We observed a gradual decrease in CCK mRNA expression and density of CCK-immunoreactive cells from duodenum to ileum. Very few CCK-immunoreactive cells and nearly undetectable CCK mRNA expression were found in the large intestine. No significant differences were seen between the groups. Expression of CCK receptors was observed in the duodenum of both groups Conclusions Both density of CCK cells and expression of CCK mRNA decreased through the small intestine in both groups with low levels in the large intestine. Patients with type 2 diabetes did not have altered density of CCK cells or expression of CCK mRNA in intestinal mucosa.

Unraveling the mechanisms of surround suppression in early visual processing

This paper uses mathematical modeling to study the mechanisms of surround suppression in the primate visual cortex. We present a large-scale neural circuit alistic modeling work are used. The remaining parameters are chosen to produce model outputs that emulate experimentally observed size-tuning curves. Our two main results are: (i) we discovered the character of the long-range connections in Layer 6 responsible for surround effects in the input layers; and (ii) we showed that a net-inhibitory feedback, i.e., feedback that excites I-cells more than E-cells, from Layer 6 to Layer 4 is conducive to producing surround properties consistent with experimental data. These results are obtained through parameter selection and model analysis. The effects of nonlinear recurrent excitation and inhibition are also discussed. A feature that distinguishes our model from previous modeling work on surround suppression is that we have tried to reproduce realistic lengthscales that are crucial for quantitative comparison with data. Due to its size and the large number of unknown parameters, the model is computationally challenging. We demonstrate a strategy that involves first locating baseline values for relevant parameters using a linear model, followed by the introduction of nonlinearities where needed. We find such a methodology effective, and propose it as a possibility in the modeling of complex biological systems.

Soluble N-Acetylgalactosamine-Modified Antigens Enhance Hepatocyte-Dependent Antigen Cross-Presentation and Result in Antigen-Specific CD8+ T Cell Tolerance Development

Hepatocytes compose up to 80% of the total liver and have been indicated as important players in the induction of immunologic tolerance in this organ. We show that hepatocytes possess the molecular machinery required for the cross-presentation of extracellular antigens. Using a derivative of the model antigen ovalbumin (OVA) covalently modified with a polymer containing multiple N-acetylgalactosamine residues (pGal-OVA) that enhance extracellular antigen uptake by mimicking the glycome of apoptotic debris, we show efficient hepatocyte-dependent induction of cross-tolerance of both adoptively transferred OT-I cells and endogenous OVA-specific CD8+ T lymphocytes, for example inducing tolerance to OVA-expressing skin transplants. Our study confirms that hepatocytes are capable of inducing peripheral tolerogenesis and provides proof of concept that they may be a valuable candidate for in vivo targeted tolerogenic treatments.

Carotid Body Chemoreceptors: Physiology, Pathology, and Implications for Health and Disease

The carotid body (CB) is the main peripheral chemoreceptor for arterial respiratory gases O2 and CO2, and pH, eliciting reflex ventilatory, cardiovascular and humoral responses to maintain homeostasis. This review examines the fundamental biology underlying CB chemoreceptor function, its contribution to integrated physiologic responses, and its role in maintaining health and potentiating disease. Emphasis will be placed on: i) Transduction mechanisms in chemoreceptor (type I) cells, highlighting the role played by the hypoxic inhibition of O2-dependent K+ channels and mitochondrial oxidative metabolism, and their modification by intracellular molecules and other ionic channels; ii) Synaptic mechanisms linking type I cells and petrosal nerve terminals, focusing on the role played by the main proposed transmitters and modulatory gases, and the participation of glial cells in regulation of the chemosensory process; iii) Integrated reflex responses to CB activation, emphasizing that the responses differ dramatically depending on the nature of the physiological, pathological or environmental challenges, and the interactions of the chemoreceptor reflex with other reflexes in optimizing oxygen delivery to the tissues; and iv) The contribution of enhanced CB chemosensory discharge to autonomic and cardiorespiratory pathophysiology in obstructive sleep apnea, congestive heart failure, resistant hypertension and metabolic diseases, and how modulation of enhanced CB reactivity in disease conditions may attenuate pathophysiology.

High expression of CD38 and MHC class II on CD8+ T cells during severe influenza disease reflects bystander activation and trogocytosis

Although co-expression of CD38 and HLA-DR on CD8+ T cells reflects activation during influenza, SARS-CoV-2, Dengue, Ebola and HIV-1 viral infections, high and prolonged CD38+HLA-DR+ expression can be associated with severe and fatal disease outcomes. As the expression of CD38+HLA-DR+ is poorly understood, we used mouse models of influenza A/H7N9, A/H3N2 and A/H1N1 infection to investigate the mechanisms underpinning CD38+MHC-II+ phenotype on CD8+ T-cells. Our analysis of influenza-specific immunodominant DbNP366+CD8+ T-cell responses showed that CD38+MHC-II+ co-expression was detected on both virus-specific and bystander CD8+ T-cells, with increased numbers of both CD38+MHC-II+CD8+ T-cell populations observed in the respiratory tract during severe infection. To understand the mechanisms underlying CD38 and MHC-II expression, we also used adoptively-transferred transgenic OT-I CD8+ T-cells recognising the ovalbumin-derived KbSIINFEKL epitope and A/H1N1-SIINKEKL infection. Strikingly, we found that OT-I cells adoptively-transferred into MHC-II−/− mice did not display MHC-II after influenza virus infection, suggesting that MHC-II was acquired via trogocytosis in wild-type mice. Additionally, detection of CD19 on CD38+MHC II+ OT-I cells further supports that MHC-II was acquired by trogocytosis, at least partially, sourced from B-cells. Our results also revealed that co-expression of CD38+MHC II+ on CD8+ T-cells was needed for the optimal recall ability following secondary viral challenge. Overall, our study provides evidence that both virus-specific and bystander CD38+MHC-II+ CD8+ T-cells are recruited to the site of infection during severe disease, and that MHC-II expression occurs via trogocytosis from antigen-presenting cells. Our findings also highlight the importance of the CD38+MHC II+ phenotype for CD8+ T-cell memory establishment and recall.SummaryCo-expression of CD38 and MHC-II on CD8+ T cells is recognized as a classical hallmark of activation during viral infections. High and prolonged CD38+HLA-DR+ expression, however, can be associated with severe disease outcomes and the mechanisms are unclear. Using our established influenza wild-type and transgenic mouse models, we determined how disease severity affected the activation of influenza-specific CD38+MHC-II+CD8+ T cell responses in vivo and the antigenic determinants that drive their activation and expansion. Overall, our study provides evidence that both virus-specific and bystander CD38+MHC-II+ CD8+ T-cells are recruited to the site of infection during severe disease, and that MHC-II expression occurs, at least in part, via trogocytosis from antigen-presenting cells. Our findings also highlight the importance of the CD38+MHC II+ phenotype for CD8+ T-cell memory establishment and recall.