Observed Changes In Phenotype Research Articles

Evaluating Physiological and Organizational Properties of the Tight Junction ZO-1 ZU-5 Domain in MDCK Cells

Zonula occludens-1 (ZO-1) is an essential TJ component contributing to junctional formation by mediating interactions between junctional proteins and the actomyosin cytoskeleton. MDCK II cells, a model epithelial cell line, with ZO-1 knocked-out (ZO-1 KO) have observable phenotypic changes such as alterations in the actin cytoskeletal organization and physiological barrier properties. This study focuses on the ZO-1-ZU5 domain by rescuing ZO-1 with full-length and a panel of ZO-1-ZU5 mutants. ZO-1-ZU-5 is known to bind cingulin and through this interaction ZO-1 adopts an extended conformation. We examined actomyosin cytoskeleton organization, ZO-1 mobility, gene expression and barrier function to determine whether the ZO-1-ZU5 mutants rescue full-length activity. We found that the ZO-1- ZU5 truncation mutant cell line rescued transepithelial electrical resistance (TER) but did not rescue tight junction distribution or the amount of total gamma-actin. We will present evaluation of additional ZU5 mutants. This study expands our understanding of how extended ZO-1 regulates the mechanical, genetic, and phenotypic properties of epithelial cells. Trinity University Biology Summer Undergraduate Research Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

RNAi-Mediated Knockdown of Acidic Ribosomal Stalk Protein P1 Arrests Egg Development in Adult Female Yellow Fever Mosquitoes, Aedes aegypti.

After taking a blood meal, the fat body of the adult female yellow fever mosquito, Aedes aegypti, switches from a previtellogenic state of arrest to an active state of synthesizing large quantities of yolk protein precursors (YPPs) that are crucial for egg development. The synthesis of YPPs is regulated at both the transcriptional and translational levels. Previously, we identified the cytoplasmic protein general control nonderepressible 1 (GCN1) as a part of the translational regulatory pathway for YPP synthesis. In the current study, we used the C-terminal end of GCN1 to screen for protein-protein interactions and identified 60S acidic ribosomal protein P1 (P1). An expression analysis and RNAi-mediated knockdown of P1 was performed to further investigate the role of P1 in mosquito reproduction. We showed that in unfed (absence of a blood meal) adult A. aegypti mosquitoes, P1 was expressed ubiquitously in the mosquito organs and tissues tested. We also showed that the RNAi-mediated knockdown of P1 in unfed adult female mosquitoes resulted in a strong, transient knockdown with observable phenotypic changes in ovary length and egg deposition. Our results suggest that 60S acidic ribosomal protein P1 is necessary for mosquito reproduction and is a promising target for mosquito population control.

Evaluating Phenotypic and Transcriptomic Responses Induced by Low-Level VOCs in Zebrafish: Benzene as an Example.

Urban environments are plagued by complex mixtures of anthropogenic volatile organic compounds (VOCs), such as mixtures of benzene, toluene, ethylene, and xylene (BTEX). Sources of BTEX that drive human exposure include vehicle exhaust, industrial emissions, off-gassing of building material, as well as oil spillage and leakage. Among the BTEX mixture, benzene is the most volatile compound and has been linked to numerous adverse health outcomes. However, few studies have focused on the effects of low-level benzene on exposure during early development, which is a susceptible window when hematological, immune, metabolic, and detoxification systems are immature. In this study, we used zebrafish to conduct a VOC exposure model and evaluated phenotypic and transcriptomic responses following 0.1 and 1 ppm benzene exposure during the first five days of embryogenesis (n = 740 per treatment). The benzene body burden was 2 mg/kg in 1 ppm-exposed larval zebrafish pools and under the detection limit in 0.1 ppm-exposed fish. No observable phenotypic changes were found in both larvae except for significant skeletal deformities in 0.1 ppm-exposed fish (p = 0.01) compared with unexposed fish. Based on transcriptomic responses, 1 ppm benzene dysregulated genes that were implicated with the development of hematological system, and the regulation of oxidative stress response, fatty acid metabolism, immune system, and inflammatory response, including apob, nfkbiaa, serpinf1, foxa1, cyp2k6, and cyp2n13 from the cytochrome P450 gene family. Key genes including pik3c2b, pltp, and chia.2 were differentially expressed in both 1 and 0.1 ppm exposures. However, fewer transcriptomic changes were induced by 0.1 ppm compared with 1 ppm. Future studies are needed to determine if these transcriptomic responses during embryogenesis have long-term consequences at levels equal to or lower than 1 ppm.

Examination of the Interplay between Membrane Tension and the Tight Junction Protein, ZO‐1

Zonula occluden‐1 (ZO‐1) has been implicated as a major regulator of tight junction formation, functioning as a scaffolding protein and mediator between the junction and the actomyosin cytoskeleton. MDCK II cells that have ZO‐1 knocked‐down (ZO‐1 KD) or knocked‐out (ZO‐1 KO) have observable phenotypic changes, including increased apical actin accumulation, increased cell height, and linearized junctions. The apical actin accumulation, resulting from the dysregulation of myosin II, is suspected to cause an increase in membrane tension seen in the ZO‐1 KO, which may explain the phenotypic changes. The function of ZO‐1 as a regulator of myosin II was confirmed by exposing the ZO‐1 KO to blebbistatin, a myosin II inhibitor, which restored the cell height to that of wild‐type MDCK II cells (WT) and the curvature of the junctions. Flipper‐TR, which is a live cell fluorescent membrane tension probe, along with fluorescence‐lifetime imaging microscopy (FLIM) analysis, are being used to test the membrane tension of the ZO‐1 KO cells compared to WT cells. The Flipper‐TR probe works by inserting itself into a lipid membrane where it undergoes conformational changes induced by membrane tension. The relationship between membrane tension and actin was examined using latrunculin A (LatA), which causes the depolymerization of actin filaments. When both the ZO‐1 KO and WT cells were incubated with LatA, the membrane tension of both decreased to a similar extent. The preliminary results of this testing have shown an increased membrane tension in the ZO‐1 KO cells. To measure the response of ZO‐1 KO cells to increased tension, the expression of tension‐related genes is being measured using quantitative real‐time polymerase chain reaction (qRT‐PCR). These tension‐related genes include the ERM (ezrin, radixin, and moesin) proteins, with a primary interest in ezrin given its known activity as a major regulator of membrane tension in epithelial cells. This study aims to understand the mechanism between ZO‐1 loss and regulation of membrane tension.

BuqiTongluo Granule for Ischemic Stroke, Stable Angina Pectoris, Diabetic Peripheral Neuropathy with Qi Deficiency and Blood Stasis Syndrome: Rationale and Novel Basket Design.

Background: BuqiTongluo (BQTL) granules are herbal phenotypic drugs for Qi deficiency and blood stasis (QDBS) syndrome. Its discovery relied primarily on knowledge of observable phenotypic changes associated with diseases. Although BQTL granules have been widely advocated by Chinese Medicine (CM) practitioners, its use lacks empirical support. Aim of the study: In this basket trial, the efficacy of BQTL granules in multiple diseases that have the QDBS syndrome in common will be compared with placebo. Materials and Methods: The BuqiTongluo granule for Qi deficiency and blood stasis syndrome (BOSS) study is a basket herbal trial (ClinicalTrials.gov, NCT04408261). It will be a double-blinded, randomized, placebo-controlled, parallel, multicenter, clinical trial. In total, 432 patients (1:1:1 ischemic stroke, stable angina pectoris, and diabetic peripheral neuropathy), who meet the operationalized diagnostic criteria for QDBS syndrome, have been recruited and randomized in a ratio of 1:1 to receive 6 weeks’ treatment with BQTL granules or placebo. The primary outcome is the change in the QDBS syndrome score at week 6 from baseline. Secondary outcomes include objective outcome measures for the three diseases and adverse events. Omics will help to understand these responses by molecular events. Conclusion: QDBS syndrome is a common phenotypic marker that was hypothesized to predict whether patients with multiple diseases would respond to this targeted therapy. No previous basket trial has assessed the potential efficacy of an herbal intervention for multiple diseases. The unique promise of the trial is its ability to exploit a disease phenotype to discover novel treatments for three diseases for which the root cause is unknown, complex, or multifactorial, and for which scientific understanding is insufficient to provide valid molecular targets.

Effects of corexit 9500A and Corexit-crude oil mixtures on transcriptomic pathways and developmental toxicity in early life stage mahi-mahi (Coryphaena hippurus)

Crude oil and polycyclic aromatic hydrocarbon (PAH) exposure in early life stage fish has been well-characterized to induce phenotypic malformations such as altered heart development and other morphological impacts. The effects of chemical oil dispersants on toxicity are more controversial. To better understand how chemical dispersion of oil can impact toxicity in pelagic fish, embryos of mahi-mahi (Coryphaena hippurus) were exposed to three concentrations of the chemical dispersant Corexit 9500A, or Corexit 9500A-oil mixtures (chemically enhanced water accommodated fractions: CEWAF) of Deepwater Horizon crude oil for 48 h. RNA sequencing, gene ontology enrichment, and phenotypic measurements were conducted to assess toxicity. Exposure to Corexit 9500A altered expression of less than 50 genes at all concentrations (2.5, 5, and 10 mg/L nominal concentration) and did not induce acute mortality or phenotypic malformations, corroborating other studies showing minimal effects of Corexit 9500A on developing mahi-mahi embryos. CEWAF preparations contained environmentally relevant ∑PAH concentrations ranging from 1.4 to 3.1 μg/L and similarly did not alter larval morphology. Differentially expressed genes and significantly altered pathways related to cardiotoxicity, visual impairments, and Ca2+ homeostasis reinforced previous work that expression of genes associated with the heart and eye are highly sensitive molecular endpoints in oil-exposed early life stage fish. Differential expression and gene ontology pathways were similar across the three CEWAF treatments, indicating that increased chemical dispersion did not alter molecular outcomes within the range tested here. In addition, significant sublethal molecular responses occurred in the absence of observable phenotypic changes to the heart, indicating that effects of oil on early life stage fish may not be completely dependent on cardiac function.

Gain-of-function mutation of AtDICE1, encoding a putative endoplasmic reticulum-localized membrane protein, causes defects in anisotropic cell elongation by disturbing cell wall integrity in Arabidopsis.

Anisotropic cell elongation depends on cell wall relaxation and cellulose microfibril arrangement. The aim of this study was to characterize the molecular function of AtDICE1 encoding a novel transmembrane protein involved in anisotropic cell elongation in Arabidopsis. Phenotypic characterizations of transgenic Arabidopsis plants mis-regulating AtDICE1 expression with different pharmacological treatments were made, and biochemical, cell biological and transcriptome analyses were performed. Upregulation of AtDICE1 in Arabidopsis (35S::AtDICE1) resulted in severe dwarfism, probably caused by defects in anisotropic cell elongation. Epidermal cell swelling was evident in all tissues, and abnormal secondary wall thickenings were observed in pith cells of stems. These phenotypes were reproduced not only by inducible expression of AtDICE1 but also by overexpression of its poplar homologue in Arabidopsis. RNA interference suppression lines of AtDICE1 resulted in no observable phenotypic changes. Interestingly, wild-type plants treated with isoxaben, a cellulose biosynthesis inhibitor, phenocopied the 35S::AtDICE1 plants, suggesting that cellulose biosynthesis was compromised in the 35S::AtDICE1 plants. Indeed, disturbed cortical microtubule arrangements in 35S::AtDICE1/GFP-TuA6 plants were observed, and the cellulose content was significantly reduced in 35S::AtDICE1 plants. A promoter::GUS analysis showed that AtDICE1 is mainly expressed in vascular tissue, and transient expression of GFP:AtDICE1 in tobacco suggests that AtDICE1 is probably localized in the endoplasmic reticulum (ER). In addition, the external N-terminal conserved domain of AtDICE1 was found to be necessary for AtDICE1 function. Whole transcriptome analyses of 35S::AtDICE1 revealed that many genes involved in cell wall modification and stress/defence responses were mis-regulated. AtDICE1, a novel ER-localized transmembrane protein, may contribute to anisotropic cell elongation in the formation of vascular tissue by affecting cellulose biosynthesis.

Developmental and Reproductive Effects of Iron Oxide Nanoparticles in Arabidopsis thaliana.

Increasing use of iron oxide nanoparticles in medicine and environmental remediation has led to concerns regarding exposure of these nanoparticles to the public. However, limited studies are available to evaluate their effects on the environment, in particular on plants and food crops. Here, we investigated the effects of positive (PC) and negative (NC) charged iron oxide (Fe2O3) nanoparticles (IONPs) on the physiology and reproductive capacity of Arabidopsis thaliana at concentrations of 3 and 25 mg/L. The 3 mg/L treated plants did not show evident effects on seeding and root length. However, the 25 mg/L treatment resulted in reduced seedling (positive-20% and negative-3.6%) and root (positive-48% and negative-negligible) length. Interestingly, treatment with polyethylenimine (PEI; IONP-PC coating) also resulted in reduced root length (39%) but no change was observed with polyacrylic acid (PAA; IONP-NC coating) treatment alone. However, treatment with IONPs at 3 mg/L did lead to an almost 5% increase in aborted pollen, a 2%–6% reduction in pollen viability and up to an 11% reduction in seed yield depending on the number of treatments. Interestingly, the treated plants did not show any observable phenotypic changes in overall size or general plant structure, indicating that environmental nanoparticle contamination could go dangerously unnoticed.

Loss of Hif-2α Rescues the Hif-1α Deletion Phenotype of Neonatal Respiratory Distress In Mice.

Hypoxia is a state of decreased oxygen reaching the tissues of the body. During prenatal development, the fetus experiences localized occurrences of hypoxia that are essential for proper organogenesis and survival. The response to decreased oxygen availability is primarily regulated by hypoxia-inducible factors (HIFs), a family of transcription factors that modulate the expression of key genes involved in glycolysis, angiogenesis, and erythropoiesis. HIF-1α and HIF-2α, two key isoforms, are important in embryonic development, and likely are involved in lung morphogenesis. We have recently shown that the inducible loss of Hif-1α in lung epithelium starting at E4.5 leads to death within an hour of parturition, with symptoms similar to neonatal respiratory distress syndrome (RDS). In addition to Hif-1α, Hif-2α is also expressed in the developing lung, although the overlapping roles of Hif-1α and Hif-2α in this context are not fully understood. To further investigate the independent role of Hif-2α in lung epithelium and its ability to alter Hif-1α-mediated lung maturation, we generated two additional lung-specific inducible Hif-α knockout models (Hif-2α and Hif-1α+Hif-2α). The intrauterine loss of Hif-2α in the lungs does not lead to decreased viability or observable phenotypic changes in the lung. More interestingly, survivability observed after the loss of both Hif-1α and Hif-2α suggests that the loss of Hif-2α is capable of rescuing the neonatal RDS phenotype seen in Hif-1α-deficient pups. Microarray analyses of lung tissue from these three genotypes identified several factors, such as Scd1, Retlnγ, and Il-1r2, which are differentially regulated by the two HIF-α isoforms. Moreover, network analysis suggests that modulation of hormone-mediated, NF-κB, C/EBPα, and c-MYC signaling are central to HIF-mediated changes in lung development.

Epilepsy spectrum disorders: A concept in need of validation or refutation

Episodic psychiatric symptoms are not uncommon and range from panic attacks to repeated violent acts. Some evidence has accumulated over the years that at least in a subset of patients exhibiting these symptoms there may be evidence for the presence of focal cortical/subcortical hyperexcitability. In these cases the condition could be conceptualized as an epilepsy spectrum disorder (ESD) with significant treatment implications. There is currently no clear demarcation of this category of symptoms, their prevalence, an understanding of how these symptoms occur, what is appropriate work up and possible treatments. In this article, we propose that milder degrees of increased neural excitability (i.e., a subthreshold excitation insufficient to cause seizures) may nonetheless be capable of causing observable phenotypic changes. The observable phenotypic changes depend on the degree of hyperexcitability and the location of the hyperexcitable neural tissue. The location of the abnormal neural tissue may dictate the initial manifestation of an attack resulting from activation of the hyperexcitable tissue, but the anatomical connectivity of the abnormal region will dictate the breadth of manifestations. We provide some evidence, derived mainly from either electroencephalography studies of these populations or clinical reports of response to anti-epilepsy treatment, for the assumption and propose methods to test the advanced hypothesis.

An Influence of Pretreatment Conditions on Mutagen Binding of Lactobacillus paracasei subsp. tolerans JG22 against MNNG and 2-NF

Abstract The objectives of this study were to investigate theeffect of Lactobacillus paracasei subsp. tolerans JG22 isolatedfrom pepper leaf jangajji on the mutagenic activity of N-methyl,N'-nitro, N-nitrosoguanidine (MNNG) and 2-nitrofluene (2-NF)and to evaluate the effect of physico-chemical pretreatment on theantimutagenic activity of the strain. The viable cells of JG22 straindisplayed a significantly high (p <0.05) antimutagenic activityagainst both mutagens tested. The antimutagenic effect of JG22strain seems to be positively correlated with the amounts of thecells in the incubation time. This strain produced the antimutagenicactivity of the maximum levels after preincubation for 30 min.The binding of this strain against the mutagenic compounds mightbe mainly present in the cell wall fraction rather than the cytosolfraction. Pretreatment with proteolytic enzymes and simulatedgastric and intestinal juices and at different pH values had nosignificant effect on two mutagens removal by the viable cells.However, the binding activity of the mutagen by the strain seemsto be affected by heating, enzymes including α-amylase andlysozyme, divalent ions, and sodium metaperiodate. Thus,carbohydrates consisting of the cell walls may be importantelements responsible for the binding of MNNG and 2-NF by thisstrain. In conclusion, the binding of the mutagens to cells of JG 22strain may play a vital role in suppressing the process ofmutagenesis induced by mutagens.Keywords ames test · antimutagenic activity · Lactobacillus paracasei subspecies tolerans · mutagen bindingIntroductionA mutagen is an agent of substance that causes observablephenotypic changes of DNA such as nucleotide alteration andchromosomal aberration. There are three main types of mutagensclassifying by their sources: chemical (5-bromouracil, 2-aminopurine,etc.), biological (rubella and hepatitis B virus), and physical (highenergy radiation and UV light) mutagens (Kodym and Afza,2003). Among chemical mutagens, alkylating agent leads to cross-link DNA strands and chromosome breaks by two reactive alkylgroups. Deaminating agent converts amino groups to keto groupsand changes hydrogen-bonding potential through oxidativedeamination of amino groups in adenine, guanine, and cytosine.Intercalating agent such as acridine dyes increases rigidity andalters conformation of double helix and base analogs derivativesof the normal bases incorporated in DNA, altering base pairingproperties (Singer and Kusmierek, 1982). Many mutagens arisefrom cigarette smoke, automotive exhaust gas, X-ray irradiation,hazardous chemicals, cooking or food processing, (un)intentionaladditives, and environmental contaminants (Ferguson, 2002).Presence of mutagens is not only involved in the probability ofincidence for colorectal cancer but do also play a role in thepathogenesis of other chronic degenerative diseases, includingatherosclerosis and heart diseases (Gaubatz, 1997).According to previous report, the mutagenicity of direct andindirect mutagens was significantly decreased by probiotic strainsincluding Bifidibacterium ssp., Lactobacillus bulgaricus,Enterococcus faecium, and Streptococcus thermophilus thatshowed the greatest protective effect against DNA damage (Burnsand Rowland, 2004). Probiotics that provide numerous health-promoting effects mainly through improving intestinal microbialbalance of human and animal have been widely used intherapeutic applications (Gomes and Malcata, 1999). Severalreports clearly show that lactic acid bacteria (LAB) andbifidobacteria strains isolated from dairy products are able toreduce enzymatic activities relates to mutagen formation andinhibit the activity of mutagenic chemical compounds in in vivo as

Evidence that RNA silencing functions as an antiviral defense mechanism in fungi

The role of RNA silencing as an antiviral defense mechanism in fungi was examined by testing the effect of dicer gene disruptions on mycovirus infection of the chestnut blight fungus Cryphonectria parasitica. C. parasitica dicer-like genes dcl-1 and dcl-2 were cloned and shown to share a high level of predicted amino acid sequence identity with the corresponding dicer-like genes from Neurospora crassa [Ncdcl-1 (50.5%); Ncdcl-2 (38.0%)] and Magnaporthe oryzae [MDL-1 (45.6%); MDL-2 (38.0%)], respectively. Disruption of dcl-1 and dcl-2 resulted in no observable phenotypic changes relative to wild-type C. parasitica. Infection of Deltadcl-1 strains with hypovirus CHV1-EP713 or reovirus MyRV1-Cp9B21 resulted in phenotypic changes that were indistinguishable from that exhibited by wild-type strain C. parasitica EP155 infected with these same viruses. In stark contrast, the Deltadcl-2 and Deltadcl-1/Deltadcl-2 mutant strains were highly susceptible to mycovirus infection, with CHV1-EP713-infected mutant strains becoming severely debilitated. Increased viral RNA levels were observed in the Deltadcl-2 mutant strains for a hypovirus CHV1-EP713 mutant lacking the suppressor of RNA silencing p29 and for wild-type reovirus MyRV1-Cp9B21. Complementation of the Deltadcl-2 strain with the wild-type dcl-2 gene resulted in reversion to the wild-type response to virus infection. These results provide direct evidence that a fungal dicer-like gene functions to regulate virus infection.

Origin of Mutations Under Selection: The Adaptive Mutation Controversy

Growth under selection causes new genotypes to predominate in a population. It is difficult to determine whether selection stimulates formation of new mutations or merely allows faster growth of mutants that arise independent of selection. In the practice of microbial genetics, selection is used to detect and enumerate pre-existing mutants; stringent conditions prevent growth of the parent and allow only the pre-existing mutants to grow. Used in this way, selection detects rare mutations that cause large, easily observable phenotypic changes. In natural populations, selection is imposed on growing cells and can detect the more common mutations that cause small growth improvements. As slightly improved clones expand, they can acquire additional mutational improvements. Selected sequential clonal expansions have huge power to produce new genotypes and have been suggested to underlie tumor progression. We suggest that the adaptive mutation controversy has persisted because the distinction between these two uses of selection has not been appreciated.