bluuebunny/crossref_metadata_2025 huggingface dataset:
Disrupted BKCa Channel β1 Subunit Gene Contributes to Vascular Dysfunction in Pulmonary Hypertension
Authors: Barnes Elizabeth A , Chen Chih‐hsin , Lee Lori , Barnes Shayna L , Cornfield David N
Published: November 2023

BACKGROUND

The BKCa channel is a calcium‐sensitive potassium channel fundamental to the control of smooth muscle cell (SMC) tone. Loss of BKCa channel function leads to membrane depolarization and vasoconstriction. Previous reports have shown that mice with a disrupted BKCa channel β1 subunit have an increase in blood pressure and arterial tone in cerebral arteries. In addition, a gain‐of‐function β1 mutation, E65K, has been associated with low prevalence of diastolic hypertension, suggesting that the β1 subunit of the BKCa channel is a key molecular component involved in vasoregulation.

HYPOTHESIS

It is unknown as to the role of the β1 subunit in modulating pulmonary vascular tone. To assess the contribution of the BKCa channel β1 subunit in pulmonary vasoregulation, mice with a disrupted β1 gene (KCNMB1−/−) were examined.

RESULTS

KCNMB1−/− mice exhibited significantly higher right ventricular systolic pressure (RVSP) compared to wild‐type (WT) littermates with exposure to acute or chronic hypoxia in the absence of histologic evidence of accentuated vascular remodeling. The β1 subunit was predominantly expressed in pulmonary arteriole SMC. Isolated peripheral PASMC promoted collagen gel contraction irrespective of β1 expression. Isolated KCNMB1−/− PASMC exhibited significant increases in focal adhesions and Rho kinase expression compared to WT cells.

CONCLUSIONS

Compromised PASMC β1 function may contribute to the heightened vasoconstriction in the microvasculature that characterizes pulmonary hypertension.
5‐Flurouracil disrupts nuclear transport during apoptosis in a calcium dependent manner
Authors: Koper Jarrett Abram , Higby Kelly , Foltz Lauren , Resendes Karen
Published: November 2023

Regulation of nuclear transport is an essential component of apoptosis. As chemotherapy induced cell death progresses, nuclear transport and the nuclear pore complex (NPC) are slowly disrupted and dismantled. 5‐fluorouracil (5‐FU) and the camptothecin derivative topotecan, are linked to altered nuclear transport of specific proteins; however, their general effects on the NPC and transport during apoptosis have not been characterized. The effects of these chemotherapies on the NPC and nuclear transport was analyzed using immunofluorescence of Ran, a key component of nuclear transport events and FG nucleoporins at the NPC. We demonstrate that 5‐FU, but not topotecan, disrupts Ran‐mediated nuclear transport before the disruption of the NPC. This Ran disruption correlates with increased NPC permeability, determined using the RGG2.2 cell line, which expresses a cytoplasmically restricted 70kD GFP tagged protein that only diffuses into the nucleus with an increase in NPC permeability limit above its 40kD baseline. Immunoblotting indicates that this change in the NPC occurs before caspase activation. Furthermore, we show that the increased NPC permeability is dependent on increased cellular calcium, as the Ca2+ chelator BAPTA‐AM, abolished the effects of 5‐FU on the NPC. Increased calcium alone induced by thapasigargin, which inhibits the uptake of calcium in the cell, was sufficient to disrupt the Ran gradient, and addition of BAPTA‐AM helped to reset the Ran gradient. In sum, our results indicate that 5‐FU disrupts nuclear transport early in apoptosis in a calcium dependent manner, a new mechanism of action for this therapeutic during apoptosis. This result opens the door to other potential combination chemotherapies that employ 5‐FU as a calcium mediated inhibitor of nuclear export, particularly in Crm1 overexpressing cells, where the excess nuclear export of several pro‐apoptotic factors has been identified as a potential trigger for cancerous transformation, as well as a mechanism for chemotherapy resistance.
Fluorescence Investigations of the Rate‐Limiting Step in the HCN Ion Channel Deactivation Pathway
Authors: Magee Kaylee EA , Claydon Thomas W , Young Edgar C
Published: November 2023

Hyperpolarization‐ and cyclic nucleotide‐activated (HCN) ion channels support electrical oscillations of neuronal networks and help prevent absence seizures. The S4 helix of the voltage‐sensor (VS) region in HCN channels moves inwards upon hyperpolarization and this movement is coupled to pore opening. An open HCN channel can further be stabilized by cAMP binding to its C‐terminal region. Modifying the speed of the rate‐limiting step for HCN channel gating might help treat neuropathies, but the steps in the gating pathway are unclear. Specifically, it has been proposed that the depolarization‐dependent deactivation pathway of HCN channels includes a voltage‐independent pore closure step that becomes rate‐limiting at strong depolarizations. To clarify which step is rate‐limiting at different depolarizations, we performed voltage clamp fluorometry on mouse HCN2 channel derivatives expressed in Xenopus oocytes using a fluorophore attached to the N‐terminal region of the S4 helix. These results represent the first direct measurement of VS movements of an HCN2 channel. The fluorophore tracked depolarization‐dependent VS movements during deactivation that preceded pore closure significantly at strong depolarizations; the current decay rate was approximately 4‐fold slower than the fluorescence decay rate at +20 mV. We call this ratio of decay rates the “transmembrane coupling quotient”. At weaker depolarizations, VS movements still preceded pore closure, but less dramatically (transmembrane coupling quotient approximately 1.5‐fold at ‐60 mV). This voltage dependence of the transmembrane coupling quotient was cAMP‐independent, i.e., it remained similar even when cAMP binding was prevented by a mutation in the C‐terminal region. The voltage dependence of the transmembrane coupling quotient is consistent with a model of mammalian HCN channel deactivation where the VS undergoes movements that have a greater voltage dependence compared to pore closure movements. VS movements may serve as the rate‐limiting step for deactivation at weak depolarizations, but another step – perhaps pore closure – is the rate‐limiting step at strong depolarizations. The temporal association between VS movements and pore movements would thus be loose at strong depolarizations, and tight at weak depolarizations.

Support or Funding Information

Natural Sciences and Engineering Research Council (NSERC) Postgraduate Scholarship to K.E.A.M; Natural Sciences and Engineering Research Council (NSERC) Discovery Grant to E.C.Y
Identification of Accessory Proteins Impacting the Function of the Epithelial Sodium Channel (ENaC)
Authors: Adewunmi Toyosi , Booth Rachell
Published: November 2023

The epithelial sodium channel (ENaC) plays a crucial role in ensuring proper Na+ balance in the body. ENaC is a heterotrimeric channel located mainly in tight or high resistance epithelia and allows for the passive transport of Na+ into the epithelial cell from the lumen. In the nephron, ENaC regulates the final 3–5% reabsorption of Na+ and is a limiting factor for Na+ reabsorption in the kidneys. Because of its role in the kidneys, ENaC is thought to be a critical component in the control of blood pressure. Though there have been several accessory proteins known to regulate ENaC function, there are still questions to be answered about the role other proteins play in ENaC trafficking and assembly. Understanding the mechanisms behind ENaC regulation will potentially provide insight into certain defects in the trafficking or assembly of the channel, and identify possible therapeutic targets. Through survival dilution growth assays in yeast deletion strains, several genes were shown to effect ENaC function in a high salt growth media. Yeast deletion strains expressing αENaC but lacking ER degradation enhancing alpha‐mannosidase like protein 3 (EDEM3) or nuclear protein localization 4 (NPL4) showed an increase in the growth of yeast cells compared to wildtype indicating a loss in ENaC function. A syntaxin 3 yeast deletion strain expressing αENaC resulted in decreased cell growth indicating a gain in ENaC function. Preliminary studies on the knockdown of EDEM3 using a CRISPR/Cas9 knockout plasmid in a M‐1 mammalian cell line endogenously expressing αβγENaC, demonstrated a decrease of ENaC protein expression levels.

Support or Funding Information

This research was supported by National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number R15GM086798 (R. Booth and W. David) and R25GM10783 (K. Oyajobi, N. Blake, R. Booth, and R. Walter).
Enhanced Aggressiveness of Bystander Cells in an Anti‐tumor Photodynamic Therapy Model: Role of Nitric Oxide Produced by Targeted Cells
Authors: Girotti Albert , Bazak Jerzy , Fahey Jonathan , Korytowski Witold
Published: November 2023

The bystander effects of anti‐cancer ionizing radiation have been widely studied, but far less is known about such effects in the case of non‐ionizing photodynamic therapy (PDT). In the present study, we tested the hypothesis that photodynamically‐stressed prostate cancer PC3 cells can elicit nitric oxide (NO)‐mediated pro‐growth/migration responses in non‐stressed bystander cells. A novel approach was used whereby both cell populations existed on a culture dish, but made no physical contact with one other. Visible light irradiation of target cells sensitized with 5‐aminolevulinic acid‐induced protoporphyrin IX resulted in a striking upregulation of inducible nitric oxide synthase (iNOS) along with NO, the level of which increased after irradiation. Slower and less pronounced iNOS/NO upregulation was also observed in bystander cells. Activation of transcription factor NF‐κB was implicated in iNOS induction in both targeted and bystander cells. Like surviving targeted cells, bystanders exhibited a significant increase in growth and migration rate, both responses being strongly attenuated by an iNOS inhibitor (1400W), a NO scavenger (cPTIO), or iNOS knockdown. Incubating bystander cells with conditioned medium from targeted cells failed to stimulate growth/migration, ruling out involvement of relatively long‐lived stimulants. The following post‐irradiation changes in pro‐survival/pro‐growth proteins were observed in bystander cells: upregulation of COX‐2 and activation of protein kinases Akt and ERK1/2, NO again playing a key role. This is the first reported evidence for NO‐enhanced bystander aggressiveness in the context of PDT. In the clinical setting, such effects could be averted through pharmacologic use of iNOS inhibitors as PDT adjuvants.

Support or Funding Information

NIH/NCI Grant CA70823
H/D exchange mass spectrometry reveals calmodulin‐controlled regulatory interactions in neuronal nitric oxide synthase
Authors: Carley Jeffrey R , Barrett Morgan F , Gilbreath Tyler J , Underbakke Eric S
Published: November 2023

Neuronal nitric oxide synthase (nNOS) produces nitric oxide (NO), a unique signaling gas molecule involved in retrograde neuronal signaling. Homodimeric nNOS is composed of a reductase and oxidase subdomain, connected by a mobile FMN subdomain. The reductase domain delivers reducing equivalents from NADPH through FAD and FMN to the heme of the oxidase domain, where NO is generated from arginine. Delivery of reducing equivalents is tightly regulated by Ca2+ via the intermediary Ca2+‐sensor calmodulin (CaM). We examined conformational changes induced by CaM‐binding that encourage the inter‐domain delivery using hydrogen‐deuterium exchange mass spectrometry (HDX‐MS).

HDX‐MS revealed critical regulatory surfaces on the reductase domain and FMN subdomain. In particular, two highly localized regions exhibited CaM‐induced increase in exchange rates. Changes in the reductase domain were most pronounced in a regulatory beta‐finger, a structural element unique to the CaM‐regulated isoforms of NOS. Interestingly, the CaM‐perturbed regions encompass two serine residues that are subject to phosphorylation, a modification reported to tune nNOS activity. Changes in the mobile FMN subdomain were more distributed largely localized to the distal surface opposite the FMN cofactor. This surface includes an auto‐inhibitory insert involved in docking the FMN subdomain to the reductase domain. Taken together, the HDX‐MS results define several inter‐domain interaction surfaces that are released upon Ca2+/CaM‐induced activation.
Effect of Methyglyoxal and Curcumin on PC12 and RINm5f Cells
Authors: de Castro Victoria Puccini , Keys Steven , Su Jing , Mungre Sue
Published: November 2023

Uncontrolled diabetes can lead to several serious complications such as kidney failure, atherosclerosis and neuropathy. Methylglyoxal (MG), a highly reactive compound derived from glucose and fructose metabolism has been implicated as the causative agent in these complications. Our work with neuronal PC12 cells has shown that MG treatment caused death of 60% of PC12 cells within 24 hours. It also increased the activity of inducible nitric oxide synthase (iNOS) and caspase‐3, suggesting that MG causes apoptotic cell death via oxidative stress. Curcumin, an antioxidant from the spice Curcuma longa, prevented cell death and the inhibited the activity of iNOS and caspase‐3. Oxidative stress is also reported to cause ER stress resulting in unfolded protein response (UPR). To determine whether it occurs in PC12 cells, we studied the effect of MG on the expression of CHOP and Bip, genes involved in UPR using qPCR. MG treatment increased the expression of CHOP by 20 fold and that of BiP by 10 fold. Treatment with curcumin partially reversed the effect, indicating that MG caused ER stress in PC12 cells. We extended these studies to RINm5f, a pancreatic beta cell line. Diabetic conditions have been reported to cause dysfunction of beta cells. Our preliminary studies show that there was 60–70% death in MG treated RINm5f cells, suggesting that similar oxidative stress/ER stress mechanism may be involved.

Support or Funding Information

COR grant from Northeastern Illinois University
Analysis of the functional relationship between the Kaposi Sarcoma‐associated Herpesvirus (KSHV) vBcl‐2 protein and the pro‐apoptotic host protein, BIK
Authors: Hixon Kimberlee , Roecklein‐Canfield Jennifer
Published: November 2023

Kaposi's Sarcoma‐Associated Herpesvirus (KSHV) is a harmful oncogenic virus that leads to skin cancer commonly found in immunocompromised individuals. KSHV evades host defense systems by targeting signals transduction pathways to suppress autophagy and apoptosis. The Bcl‐2 family of proteins controls commitment to apoptosis by regulating mitochondrial outer membrane permeabilization (MOMP). Research has been conducted examining the specific mechanisms and localization of different proteins in the Bcl‐2 family such as Bak and Bax, but other proteins like Bik (Bcl‐2 interacting killer) have been far less studied. Bik is a pro‐apoptotic tumor suppressor that is critical in apoptosis induced by cytokines, toxins and viral invasion. Anti‐apoptotic Bcl‐2 family members competitively bind pro‐apoptotic proteins, like BIK, and suppress apoptosis. Although BIK is a known therapeutic target, the localization and non‐apoptotic function, along with the mechanism of action and localization in the presence of the KSHV protein, vBcl‐2, is not well understood. Wildtype plasmids, BIK, human Bcl‐2 and vBcl‐2 proteins were cloned, amplified, and transfected into HEK239‐T cells. Fluorescence Complementation was used to detect the cellular localization of BIK in the presence and absence of the vBcl‐2 protein. An apoptosis assay was used to determine if MOMP is triggered in the presence and absence of vBcl‐2. Definitively elucidating the mechanism, localization, and protein interactions in apoptotic and non‐apoptotic cell death in the presence and absence of vBcl‐2 will guide future therapeutic approaches against KSHV and possibly elucidate other non‐resistant mechanisms to kill cancer cells.
Pro‐death Bax Has an Intrinsic Capability to Induce Aggregate‐dependent Caspase 8‐mediated Cell Death
Authors: Manas Adriana , Wang Sheng , Li Jiajun , Nelson Adam , Davis Aislinn , Lamerand Sydney , Zhang Huaiyuan , Xiang Jialing
Published: November 2023

BaxΔ2 is a unique functional Bax isoform resulted from the combination of a mononucleotide deletion and an alternative splicing in the N‐terminus. The rest of the sequence is the same between Baxα and BaxΔ2. However, BaxΔ2 is unable to target mitochondria due to the loss of helix α1, encoded by exon 2. Instead, it forms cytosolic aggregates to activate caspase 8‐dependent death pathway. The functional domains involved in BaxΔ2 aggregation and cytotoxicity remained elusive. Using computational modeling in combination with biochemical and cellular analysis, we show here that the drastic changes in the BaxΔ2 N‐terminus have no significant impact on either aggregation or caspase 8 activation. In addition, the hallmark BH3‐killing domain is necessary but not sufficient for either aggregation or cell death. The intact core region of BaxΔ2 encoded by exons 3, 4, and 5 is required for the formation of large aggregates, which are essential for BaxΔ2 cytotoxicity. Surprisingly, aggregation by itself is still not sufficient to trigger cell death. The C‐terminal end encoded by exon 6 is essential for activation of caspase 8. Finally, the C‐terminal conformation, but not the primary sequence, appears to be responsible for the aggregate‐mediated caspase 8 activation and cell death. As Baxα with deletion of exon 2 behaves the same as BaxΔ2, and also shares the same C‐terminus sequence or conformational motifs with most Bax‐isoform family members, these results imply that Bax isoforms may have an intrinsic ability to activate caspase 8 if they fail to target mitochondria.

Support or Funding Information

This work is supported by NIH R15CA195526 grant
Leveraging Synthetic Lethality to Target Convergent Therapeutic Resistance
Authors: Wood Kris C
Published: November 2023

Our laboratory uses genomic and pharmacological methods to define the pathways of resistance to targeted anticancer therapies. Toward this end, we recently developed a technique in which engineered lentiviral cDNAs encoding activators of major oncogenic signaling pathways are introduced into cells and then profiled to identify those capable of conferring resistance to drugs (Science Signaling 2014, 7, ra121). In parallel, we have also developed analogous loss‐of‐function screening approaches based on CRISPR/Cas9 gene editing, and have used these approaches to map the pathways supporting intrinsic resistance to targeted therapies. Using these tools in conjunction with data and mouse models derived from human tumors, we recently discovered that major pathways of resistance frequently converge onto common downstream effectors, the targeting of which can forestall resistance. Here, I will describe the discovery of common resistance effectors in JAK2‐driven myeloproliferative neoplasms, EGFR‐driven colorectal cancers, and BRAF‐driven melanomas (Science Signaling 2014, 7, ra122; Nature Communications 2015, 6, 8305). Focusing particularly on melanoma, I will describe the discovery that major pathways of resistance converge to activate the transcription factor c‐MYC (MYC). MYC expression and gene signatures were suppressed following initial drug treatment, then rebounded during tumor progression in each member of a large panel of human patients, independent of the upstream pathway driving resistance. MYC suppression using either genetic approaches or BET bromodomain inhibition was sufficient to both resensitize diverse BRAFi/MEKi‐resistant models and delay resistance in treatment naïve models. Although direct pharmacological inhibition of MYC remains difficult, the BRAFi/MEKi‐resistant, MYC‐activated state harbors synthetic lethal signaling and metabolic dependencies, including those involving SRC family and c‐KIT tyrosine kinases and glucose, glutamine, and serine pathways, that can be targeted to create combination therapies that uniquely select against resistance evolution.

Support or Funding Information

NIH Building Interdisciplinary Research Careers in Women's Health Program (K12HD043446)Stewart Trust Fellowship (K.C.W.)V Scholar Award from the V Foundation for Cancer Research (K.C.W.)DoD Breast Cancer Research Program Breakthrough Award (BC151664) (K.C.W) NIH/NCI award R01CA207083 (K.C.W.)
Differential Inhibitory effects of Nocodazole on Human Hematopoietic and Hepatocytic cells and Their Stem/Progenitors in Culture
Authors: Baquier Jocelyn , Darrell Henry , Brumaire Sebastien , Schoonover Laura , Hu Tang
Published: November 2023

Nocodazole is known as a cell cycle inhibitor that inhibits polymerization of microtubules. However, its effect on apoptosis and differentiation of human myeloid leukemia cells and cancerous hepatocytes, especially on their stem/progenitors is largely unknown. In this study, we investigated the effect of Nocodazole on the growth of two human myeloid leukemia cell lines, TF‐1a and MV4‐11 and one liver cancer cell line, HEP‐G2. Addition of Nocodazole at concentrations of 5μM significantly inhibited the growth of MV4‐11, TF‐1a, and HEP‐G2 cells in culture, with 50% (MV4‐11), 45% (HEP‐G2), and 40% (TF‐1a) inhibition being observed, respectively, on day 3 after treatment with Nocodazole. The inhibitions were dose‐dependent with maximal inhibition being observed at a concentration of 10uM of Nocodazole. Less than 1 μM had no significant inhibitory effect on the growth of the cells, measured by XTT proliferation assay after the cells were treated with Nocodazole. Paralleled to the changes in the cell proliferation, expression of cdc2 and cdk2 were also inhibited, indicating that the inhibition was linked to the cell cycle arrest. However, Trypan blue stain and cell morphology analysis suggested that some cells displayed apoptotic‐like cell death in the presence of Nocodazole, which was confirmed by activation of caspase proteins. We were surprised to find that the stem/progenitors of MV4‐11, HEP‐G2 and TF‐1a cells showed a higher sensitivity to Nocodazole treatment measured by counting colony forming cells in semi‐solid (soft agar or soft agar/agarose) culture. At a concentration of 0.01uM, Nocodazole inhibited 80–90% stem/progenitors derived from MV4‐11, HEP and TF‐1a. The Giemsa and methylene blue/eosin stain showed that Nocodazole also induced differentiation in about 30% TF‐1a cells, evidenced by reduced nucleus/cytoplasm ratio. Our data demonstrated that Nocodazole induced‐inhibition of the cells tested is the result of its multiple functions including the cell cycle arrest, apoptosis and differentiation, which are cell type dependent. The high sensitivity of hematopoietic stem/progenitor cells to Nocodazole makes this agent a potential drug for disrupting damaged bone marrow stem cells before receiving healthy bone marrow transplantation.

Support or Funding Information

Supported by NIH‐NIGMS RISE Grant, R25 GM059244‐16 Barry University.
Genetic mapping of strain specific differences in autophagy effector recruitment to the Toxoplasma gondii parasitophorous vacuole
Authors: Radke Joshua B , Sibley L. David
Published: November 2023

Autophagy is a highly conserved cellular process by which cytoplasmic constituents such as damaged organelles and mis‐folded proteins are recycled. Additionally, autophagy or autophagy related genes play a critical role in the innate immune response by associating with intracellular pathogens and targeting them for elimination. Recently, our laboratory identified an innate, noncanonical autophagy (ATG) pathway that regulates T. gondii infection in a strain‐dependent manner in IFN‐γ activated human cells. The core set of ATG16L1, ATG12 and ATG5 proteins are involved in recruitment of LC3 to the PV, and yet upstream activators (i.e. Beclin and Atg14) and downstream degradation function (i.e. lysosomal fusion) are not required for this pathway. This core set of ATG protein is also necessary for the IFN‐γ‐dependent control of T. gondii strains in mouse and human cells. In humans, parasitophorous vacuoles (PV) containing susceptible strains (type II and III) of T. gondii are ubiquitinated, followed by ligation of the ATG adapters p62 and NDP52, and finally engulfed in multiple layers of host membrane resulting in the stunting of parasite growth. However, type I strains of T. gondii (type I) are largely resistant to ATG mediated growth restriction. Here we interrogated the genetic differences in autophagy effector recruitment between the resistant type I (GT1‐SNF) strain and the susceptible type III (CTG‐ARA) strain of T. gondii using 34 unique progeny isolated from a genetic cross (IxIII). We evaluated all progeny for the hIFN‐γ‐dependent recruitment of GFP‐LC3 to the PV. Genome wide analysis identified two quantitative trait loci (QTL) associated with the autophagy effector recruitment phenotype, one on Chr. II (LOD 3.35) and a second on Chr. VIII (LOD 4.05). In total, 373 genes fall within these loci (95% CI) and the two QTL account for 77% of the phenotypic variance identified in the progeny. We evaluated polymorphism, the presence of signal peptides and/or transmembrane domains and gene annotation to prioritize 10 candidate genes for further evaluation. We have evaluated subcellular localization for all priority genes and found 2 genes that localize to the PV. Current genetic studies are focused on inducible gene knockdown strategies (i.e the plant auxin inducible degron system) and genetic ablation using CRISPR/Cas9 to evaluate the role for both genes in the autophagy recruitment phenotype. Identifying the cellular basis for avoidance of ATG recruitment may reveal mechanisms of enhanced pathogenicity of T. gondii strains, and allow for selective enhancement of this pathway to affect pathogen clearance.
Molecular Characterization and Pathogenicity of Meloidogyne incognita on Tomato Cultivars (Solanum lycopersicon L.)
Authors: ABDULAI MUNTALA , Cole Biodun Claudius , Fawole Bamidele
Published: November 2023

The study was conducted to determine the distribution of the common Meloidogyne species in and around the Ibadan area in Oyo State, south‐west Nigeria, West Africa using Polymerase Chain Reaction (PCR), as well as determine the effect of Meloidogyne incognita on five tomato cultivars. The locations included Akobo 7°25′49.13″N; 3°56′ 55.44″E (a peri‐urban vegetable farm), Ijaye 7°50′48.60″N; 3°34′11.23″E, National Institute for Horticultural Research (NIHORT) 7°24′19.13″N; 3°50′57.01″E, International Institute of Tropical Agriculture(IITA), Ibadan 7°29′52.05″N; 3°54′ 19.16″E and inoculum plots of the Department of Crop Protection and Environmental Biology, University of Ibadan 7°27′4.14″N; 3°53′ 49.19″E. DNA was extracted from the bulk juveniles, according to modified (Dellaporta et al., 1983). Identification of species was carried out using Meloidogyne species universal primer 194/195 and specific SCAR primers MIF/MIR. The nucleic acid was size‐fractioned in 1% agarose gel, stained with 4 μl ethidium bromide and captured by the ultraviolet light. Meloidogyne incognita produced amplification fragments of 720‐bp and 999‐bp for 194/195 and MIF/MIR primers, respectively. Meloidogyne incognita and javanica were the predominant species of root‐knot nematodes identified from the collected samples. The pathogenicity of Meloidogyne incognita was evaluated at different egg mass inoculum levels on five tomato cultivars in the screenhouse in (IITA). All growth parameters such as plant height, leaf number, and yield responded negatively with increasing inoculum levels for all the cultivars except Small Fry VFN and Celebrity cultivars which were found to be resistant based on (Soriano et al., 2000) resistance rating. The results also showed that the Ibadan Local cultivar was the most susceptible to M. incognita. The reproductive factor of the nematode (RF) was determined according to (Oostenbrink, 1966). The root galling index was rated on a 0–5 scale, where 0=0% galled roots; 1= < 10%, 2= 10–25%, 3= 25–50%,4=50–75% and 5= > 75% galled roots (Soriano et al., 2000). All data were analyzed by analysis of variance using SAS 9.2 version and means were separated using Least Significant Difference (LSD). In conclusion, the common meloidogyne species distribution in south‐west Nigeria was determined and this is very important in the formulation of management tactics against this pathogen.

Support or Funding Information

The research was supported by Alliance For A Green Revolution in Africa (AGRA)
Effects of Over‐Expression of RecA on Transformation in Bacillus subtilis
Authors: Penumutchu Swathi , Korry Benjamin , Belenky Peter
Published: November 2023

Antibiotics are used heavily in modern medical practices and they often affect microorganisms in unanticipated ways. One such effect of antibiotics on bacteria is the transfer of antibiotic resistance. These resistant strains of bacteria pose a threat to current medical practices because their growth cannot be controlled. In the gram‐positive bacteria, Bacillus subtilis, the development of antibiotic resistance occurs through several methods, one being horizontal gene transfer (HGT). Exogenous DNA is taken up by cells, resulting in transformed cells with integrated DNA that confers antibiotic resistance. Transformation in bacteria relies on competence and generates genetic diversity through integrating DNA present in the environment. The SOS response in some bacterial cells is responsible for correcting DNA damage. Genes in the SOS box such as RecA are involved in the processes of homologous recombination and nucleotide excision repair. We hypothesize that the RecA levels increase in response to DNA damage, aiding homologous recombination and transformation in cells. According to preliminary experiments conducted, the addition of antibiotic stress causes an increase in the number of transformed cells and an increase in RecA expression. It is proposed that the over‐expression of RecA will allow cells to better integrate exogenous DNA and increase transformation efficiency levels. This study has significant implications into the understanding of how antibiotics effect various populations of bacteria and the development of pools of genetically diverse bacteria.

Support or Funding Information

Research sponsored by National Science Foundation, Division of Integrative Organismal Systems, IOS‐1249925
Genesis of Antibiotic Resistance XXI Non‐Existent F‐Tag‐428 implementation in developing countries, exacerbate genesis of antibiotic resistance (AR)
Authors: Carbajal Carlos , Fuentes Esai , Wickham Cheyenne , Lopez Christian , Johnson Dennie , Kannan Subburaj
Published: November 2023

As per the American Society of Consultant Pharmacists (ASCP) mandated guidelines, the F428: Category = Quality of Care, drug regimen review is a prime requirement to avoid prescription drug induced and unforeseen clinical outcomes in patient care. The clinical directive by the ASCP is that the drug regimen of each resident must be reviewed at least once a month by a licensed pharmacist. Surveys and data analysis conducted in developing countries in regard to rural patient care, rehabilitation centers, hospice care, and physicians in private practice concerning the relevance of tag F‐428 and the antibiogram of each patient show that F‐428 is an almost fictitious concept. Prominent pharmacies and pharmacists along the border regions of the United States and Mexico and in developing countries such as India and Nigeria that are strongly influenced by United Kingdom, acknowledge a genuine lack awareness of F‐tag‐428 principles and the necessity for its implementation. The survey data indicates that pharmacists in these developing regions are not part of the clinical decision making process for antibiotic prescription and physicians who are in private practice are not mandated to keep record of antibiotic prescriptions. Information concerning individual culture sensitivity assay results are on file. There do exist prevailing and synergistically compounding factors that make implementation of F‐428 a futile exercise in developing countries. The following factors are considered derogatory to F‐428 implementation; the inappropriate prescription of antibiotics by physicians, poor compliance by patients, the over‐the‐counter availability of antimicrobials as a nonprescription commodity, patient ‐ time pressures, purulent discharge, diagnostic and treatment uncertainties, no availability of a rapid and low cost antibiotics, culture and sensitivity, lack of facilities for conducting antibiogram in rural settings, lack of antibiotic guidelines, inadequacy or absence of regional laboratories to provide periodic and continuing information concerning antibiotic resistance, patient requests and expectations, patient literacy regarding antibiotics (principally women), patient pressure for antibiotics prescriptions for the abatement and expeditious relief from infectious disease, and tenuous pathways for maintaining the continuing medical education requirement for physicians (Medicos with MBBS and M.D.) F428 implementation goals contrasted with the above program frustration, and in consideration of present data analysis irrefutably indicate that antibiotic usage in developing countries poses imminent risk factor for Antibiotic Resistance Pandemic (ARP). Data analysis and adequate literature sources will be presented at EB 2017.

Support or Funding Information

Professional development funds to Subburaj Kannan
Determining the Rhodoquinone Biosynthetic Pathway in Rhodospirillum rubrum Using Gene Knock‐outs
Authors: Martin Amanda , Shepherd Jennifer
Published: November 2023

Many parasitic helminths require the metabolic cofactor rhodoquinone (RQ) for anaerobic respiration. Inhibition of RQ biosynthesis may shut down the anaerobic respiration pathway and provide a new target for anti‐helminthic drug design. The bacteria Rhodospirillum rubrum (R. rubrum) is a good model organism for RQ biosynthesis because it utilizes the same respiration pathway as the helminths when grown under anaerobic conditions. Previous studies have shown that ubiquinone (Q) is a required precursor to RQ in R. rubrum, and that the gene, rquA, is required for this conversion. Since this process likely requires additional enzymes, further investigation has identified potential gene targets within the R. rubrum genome that may be involved in the pathway. This project involved completing a collection of knockout mutant strains, with the deletion of ten genes from the R. rubrum genome that are hypothesized to be involved in the RQ biosynthetic pathway. RQ production was analyzed using liquid chromatography mass spectroscopy in each of the mutant strains, alongside wild type. The gene expression of rquA was also monitored using real‐time quantitative PCR.

Support or Funding Information

National Institutes of Health (1R15GM096398‐01) and Howard Hughes Medical Institute (award to Gonzaga University).
Increased N‐acetyltaurine in the skeletal muscle after endurance exercise
Authors: Miyazaki Teruo , Nakamura Yuho , Ebina Kei , Ra Song‐Gyu , Ishikura Keisuke , Ohmori Hajime , Ikegami Tadashi , Matsuzaki Yasushi , Honda Akira
Published: November 2023

Aim

Acetate produced from acetyl‐CoA in the liver is utilized for energy production in the skeletal muscle after reconversion to acetyl‐CoA during endurance exercise. However, excess acetate in the muscle might become a nuisance that disturbs normalizing of energy metabolism after the exercise. Therefore, it is suggested that acetate may be metabolized to a novel metabolite N‐acetyltaurine (NAT) by reacting with taurine abundantly contained in the muscle for facilitation of urinary acetate excretion. This study aimed to evaluate NAT in serum, urine, and muscle in human and rats after endurance exercise, and in the cultured muscle cell.

Method

NAT and taurine levels in serum and urine from healthy volunteers after a transient endurance running and in the soleus muscle from rats after a treadmill running to exhaustion were measured by HPLC‐MS/MS system. The muscular ability of NAT synthesis was also evaluated in C2C12 myotube treated with acetate and taurine.

Result

Serum taurine and NAT levels were significantly increased immediately after the exercise, and recovered to the basic levels after one day. Urinary excretions of taurine and NAT were markedly higher in the post 24‐hour period. In the rat muscle, NAT concentration was significantly increased correlated with its level in serum after the exercise. When taurine was co‐treated with acetate to the myotube, the significant inhibition of intracellular acetyl‐CoA accumulation and the significant facilitation of extracellular NAT excretion were observed.

Conclusion

These findings suggest that NAT might be synthesized to excrete the excess acetate from the skeletal muscle into the urine after endurance exercise in order to prevent intramuscular excess accumulation of acetyl‐CoA that might induce a metabolic imbalance after the exercise.

Support or Funding Information

This study was supported in part by Kakenhi grants (15K09026, 15K08319, 16K01734) from the Japan Society for the Promotion of Science.
Elucidating the Biosynthetic Pathway of Rhodoquinone in Caenorhabditis elegans
Authors: Babcock Shannon , Shepherd Jennifer
Published: November 2023

Parasitic helminthes are worm‐like, obligate endoparasites that can use humans and livestock as their definitive host. The WHO reports about 24% of the world population is infected with a soil‐transmitted parasitic helminth, especially in tropical, third‐world countries where sanitation and hygiene are poor. These infections can cause economical and sociological effects along with negative or even detrimental health effects on the host. There are currently anti‐helminthic drugs available; however, the increased resistance to those drugs and lack of specificity has called for a new drug target. Parasitic helminthes utilize the fumarate reductase pathway for anaerobic respiration while inside of the host. Rhodoquinone (RQ), the final electron transporter in this pathway, is a potential new target for anti‐helminthic drugs because it is required for metabolism while in the host, and humans don't utilize or make RQ. However, the biosynthetic pathway of RQ is not currently known. This study aimed to elucidate the biosynthetic pathway of RQ in the model organism, Caenorhabditis elegans, using RNA interference (RNAi) to knockdown four potential genes involved in the pathway. C. elegans was used as the model organism because of the similar physiology and close phylogeny to parasitic helminthes. Liquid chromatography‐mass spectrometry (LC‐MS) and real time quantitative polymerase chain reaction (RT‐qPCR) were used to quantify RQ levels in C. elegans after RNAi feeding experiments.

Support or Funding Information

National Institutes of Health (1R15GM096398‐01) and Howard Hughes Medical Institute (award to Gonzaga University)
Fruit Extract of Thaumatococcus daniellii Reduces Oxidative Stress in Rats
Authors: Iheagwam Franklyn N. , Chinedu Shalom N. , Emiloju Opeyemi C. , Okenmuo Augustine C.
Published: November 2023

Thaumatococcus daniellii (Benn.) Benth is a tropical rhizomatous herb recognized as the natural source of the protein sweetner, thaumatin. Thaumatin is extracted from the fruit arils, which constitutes about 4% of the fruit leaving a large chunk as waste in the extraction process. In this study, we examined the effect of ethanolic extract of T. daniellii fruit pulp on some oxidative stress parameters in male Wistar rats. The fruit pulp was extracted with 80% ethanol and a graded concentration was administered orally to the rats for 14 days. The liver and kidney of the rats were harvested; in‐vivo antioxidant activity and liver histopathology were determined. Result were statistically analysed using one way analysis of variance (ANOVA) supplemented with Duncan multiple range test (DMRT). Results showed a significant (p<0.05) decrease in MDA levels and a significant increase (p<0.05) in SOD activity and GSH concentration compared to the normal and positive controls. Histopathological examinations indicated no pathologic abnormality. The antioxidant boosting activities of the T. daniellii fruit extract may be due to the presence of phytochemicals such as flavonoids and tannins. The study suggests the medicinal potential of the fruit in reducing oxidative stress. The fruit could be incorporated into animal feed for its nutritive and phytochemical constituents.
Lactotransferrin Prevents Hepatic Steatosis through Inhibition of Hepatic Dietary Lipid Uptake in Hormone Disturbance Non‐alcoholic Fatty Liver Disease Model
Authors: Lee Sungmin , Son Beomseok , Park Gaeul , Kim Wanyeon , Youn HyeSook , Youn BuHyun
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is a globally emerging metabolic disease which even could develop to hepatic cirrhosis and hepatoma. Although many studies suggested the mechanism and therapeutic targets, development of NAFLD is not completely understood. In this study, we established IR (Ionizing radiation)‐induced fatty liver mouse model which is observed to develop hepatic lipid accumulation, an initial step of the NAFLD without alteration of caloric intake. Through transcriptome analysis, we found that hepatic secretory protein lactotransferrin (Ltf) was down‐regulated in the model and expression of Ltf was modulated by growth hormone (GH). IR‐induced fatty liver mouse showed lower serum GH levels. Moreover, we investigated the role of Ltf in prevention of chylomicron uptake and lipid accumulation in the hepatocytes. Notably, we uncovered that coumestrol as a stimulant for expression of hepatic Ltf and therapeutic compound of fatty liver. Taken together, we suggest the novel fatty liver mouse model, the role of Ltf in inhibiting hepatic lipid accumulation and novel natural compound for improvement of NAFLD.

Support or Funding Information

The Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (20131610101840)
Niclosamide blocks glucagon phosphorylation of serine 552 on β‐catenin leading to decrease target genes expression and improve glucose metabolism via PKA signalling pathway
Authors: Chowdhury Md. Kamrul Hasan , Morris Margaret , Turner Nigel , Wu Lindsay , Shepherd Peter , Smith Greg
Published: November 2023

It has been found that glucagon is able to activate the β‐catenin signalling pathway leading to increased cyclin D1 and c‐Myc expression in liver. Therefore, the main aim of this study is to determine if the effect of glucagon activating β‐catenin signalling leading to increased target gene expression is mediated through cAMP activation of protein kinase A. Primary rat hepatocytes were incubated with insulin, glucagon or epinephrine and a range of inhibitors of PI 3‐kinase, Wnt, mitochondrial uncoupler (niclosamide) or PKA inhibitors to dissection out the pathway leading to increased serine 552 phosphorylation of β‐catenin following glucagon exposure. In primary rat liver cells, we found that short exposure of glucagon or epinephrine caused a rapid increase in serine 552 phosphorylation on β‐catenin that leads to increased cyclin D1 and c‐Myc expression. Both glucose and insulin had no effect on this pathway. A range of PI 3‐kinase and Wnt inhibitors were unable to block the effect of glucagon phosphorylating β‐catenin. Interestingly, both niclosamide and the PKA inhibitor H89 blocked the glucagon effect on β‐catenin signalling leading to a reduction in the target genes expression. Our study also provide evidence that inhibition of the glucagon signalling pathway contributes to the beneficial effects of niclosamide (both forms of niclosamide) on whole body glucose metabolism. We have identified a new pathway via glucagon signalling that leads to increased β‐catenin activity that can be reversed with the antihelminthic drug niclosamide, and decrease blood glucose levels, which has recently shown promise as a potential treatment of type 2 diabetes (T2D). This novel finding could be a useful adjunctive therapeutic strategy to treat T2D, as hepatic glucose output is elevated in people with T2D and in liver cancer treatment, particularly in the context of T2D with increased β‐catenin activity.

Support or Funding Information

‐ UNSW Australia, PhD scholarship

‐ Diabetes Australian Research Grant
Potential role of dietary fat‐ and obesity‐sensitive adipose PKCbeta signaling in pathophysiology of adipose dysfunction
Authors: Mehta Neil K , Huang Wei
Published: November 2023

Excess delivery of nutrients to adipose tissue in obesity results in an increase in adipose tissue mass, followed by an increase in stress signals, and thereby an altered production of proinflammatory adipokines, ultimately leading to the progression of chronic inflammation and development of insulin‐resistance. Although the features of chronic inflammation in obese adipose tissue are clearly defined, the signals and mechanisms that trigger chronic inflammation (origin or mechanism of initiation of inflammatory signaling) and maintenance are not well understood and remains a critical area of research. There is clear evidence that the inflammatory trigger is metabolic and a strong correlation between adipocyte size and macrophage infiltration has also been reported. Importantly, specialized metabolic cells (such as adipocytes) are the cells that sustain the insult and whose expansion begins the inflammatory program. Accordingly, adipocyte hypertrophy and local hypoxia are implicated in immune cell infiltration, cytokine secretion and disruption of different signaling cascades. Our recent study suggested that protein kinase C beta (PKC beta) might be a pivotal protein regulating triglyceride storage and could be a target for the pathogenesis of adipose dysfunction in response to nutritional changes. Accordingly, a systemic PKC beta deficiency protects mice from diet‐induced adiposity and development of insulin‐resistance. To determine how PKC beta deficiency exerts its effect, we determined high‐fat diet‐PKC beta interaction and the impact of PKC beta deficiency on relevant adipose signaling pathways and metabolism. Here, we report that dietary fat strongly increased adipose PKC beta expression in a time‐dependent manner. The induction was observed at 2–6 weeks, peaked at 10 weeks, and remained elevated for at least 16 weeks (the last time point measured). We and others have reported involvement of PKC beta in regulating phosphorylation of retinoblastoma, I kappa b kinase, insulin‐receptor substrate, and p66shc, as well as dictates autophagy levels, we therefore compared changes in phosphorylation of these proteins and autophagy levels in different adipose tissues of above mice. The time‐dependent changes were also compared using adipose tissues from PKC beta‐deficient mice fed same high‐fat diet for identical periods. The available results support the possibility that adipocyte PKC beta is a key sensor to detect dangerous dietary signal, whose activation initiates adipose inflammation by sequential events consisting of PKC beta‐induced changes, thus mediating the interface between metabolic input and inflammatory output in the expanding adipose tissue. This study opens the possibility that modulation of PKC beta activity in the adipose tissue may be an effective approach to attenuate adipose dysfunction.

Support or Funding Information

HL079091