bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating the Regulation of Guanylate‐Binding Protein1 Expression during Adipose Tissue Inflammation
Authors: Qiu Xiaoxue , Chen Xiaoli
Published: November 2023

Guanylate‐binding proteins (Gbps) are large interferon (IFN) γ‐inducible GTPase, mainly functioning in innate immunity to protect against pathogen infections by boosting autophagic machinery and phagocyte oxidase. Attentions have been mostly given to understanding roles of Gbps in host defense against intracellular bacteria in macrophages. Little is known about the involvement of Gbps in adipose tissue inflammation in obesity. Infiltration of IFNγ‐secreting T helper type 1 cells into adipose tissue during obesity has been known to promote macrophage recruitment and polarization, leading to a shift of macrophages from a non‐inflammatory M2 toward a pro‐inflammatory M1 phenotype. We found that Gbp1 gene expression was significantly decreased in inguinal white adipose tissue of high fat diet‐induced obese mice. Therefore, our first aim was to identify the Gbp1‐expressing and IFNγ responsive cell types in adipose tissue. To achieve this aim, we determined the regulation of Gbp1 expression in both adipocytes and macrophages by different cytokines and fatty acids. Fully differentiated 3T3‐L1 adipocytes as well as Raw264 macrophages were treated with various above‐mentioned factors for 24h. Our results showed that Gbp1 was not highly expressed in 3T3‐L1 adipocytes, while Gbp1 protein levels were markedly increased in macrophages when treated with IFNγ. However, tumor necrosis factor (TNF) α and interleukin (IL)‐1β failed to induce Gbp1 expression in macrophages; lipopolysaccharide (LPS) was able to induce the expression of Gbp1 at a much lower level compared to IFNγ treatment. Additionally, Gbp1 protein expression levels were not altered in macrophages in response to the treatment with various fatty acids, including palmitic, oleic, linoleic, eicosapentaenoic and docosahexaenoic acids. Furthermore, we found that Gbp1 proteins were detected in both cytosol and mitochondrial fractions. Our data suggest that IFNγ is a specific and potent inducer of Gbp1 expression; macrophages but not adipocytes are the primary Gbp1‐expressing and IFNγ responsive cells in adipose tissue. Due to the mitochondrial localization of Gbp1 protein, further studies will focus on the role of Gbp1 in regulating mitochondrial oxidative metabolism and M2‐M1 polarization in macrophages.

Support or Funding Information

NIDDK‐funded Minnesota Obesity Center (P30DK050456)
Effects of thymol on 3T3‐L1 differentiation and lipase activity
Authors: Dinsmore Olivia , Wells Emily , Aulthouse Amy , Stockert Amy
Published: November 2023

The antibacterial properties of thyme extracts have been well documented, however the effects of thyme on mammalian cells are less well studied and of great importance. Evidence suggests that the antibacterial activities are in part due to the effects of thymol on the membrane structure, effectively altering permeability. As varied permeability and fluidity in the membrane are commonly linked to insulin sensitivity, the exploration of its effects on cell morphology and differentiation in adipose tissue is essential. As a cell model system to explore obesity and diabetes, 3T3‐L1 pre‐adipocytes are well suited due to the ability to examine both adipogenesis and differentiation. Pre‐adipocytes were grown in monolayer in expansion media containing DMEM supplemented with 10% bovine calf serum, as suggested by the supplier. Differentiation was chemically induced following trypsinization of 70% confluent cells with DMEM, 10% fetal bovine serum (FBS), 1 μM Dexamethasone, 0.5 mM methylisobutylxanthine, and 1 μg/ml insulin. Pre‐adipocytes were also treated with thymol in DMEM with 10% FBS minus the chemical induction components dexamethasone, methylisobutylxanthine, and insulin for comparison. Differentiation was monitored at 12, 18, 24, 36, and 48 hours by fixing and staining with oil red O. Some cells were allowed to grow to maturity at 2 weeks and stained for comparison. Cultures were replicated multiple times, allowing testing for cell death using the trypan blue exclusion assay as well as an assay of lipase activity in live unfixed cultures. Lipid droplet volume was estimated based on droplet size and count relative to the cell size. Lipid volume was also assayed using dye extraction and quantitation. Results suggest that, like other phenolic compounds, thymol enhances differentiation from pre‐adipocyte to adipocyte at certain concentrations. However, like other phenolic compounds, this effect is highly sensitive to concentration. The lipase activity was expected to increase where differentiation had occurred, but more data is needed to determine significant changes in lipase activity. A moderate change in cell size was also observed, which can more appropriately monitored further in three‐dimensional culture.
Invivo efficacy and durability of anti‐infective foley catheters functionalized with silver nano particle and antibiotics
Authors: Srinivasakumar KP , Mala R , Annie Aglin A , Kiruthika S , VazagaPriya C
Published: November 2023

Urinary catheters are an inevitable medical device in health care unit. Complications of urinary catheterization are dreadful. To circumvent the drawbacks associated with existing catheters, the present investigation is aimed to impregnate urinary catheter with a synergistic combination of silver nanoparticle and antibiotics. The functionalized catheter was evaluated by invitro and invivo methods for its anti‐infectious property. Silver nanoparticle was synthesized using Spirulina platensis and it was characterized by SEM and FTIR. Susceptibility of uropathogenic E.coli to antibiotics and silver nano particle was evaluated by agar well diffusion method. MIC and MBC of silver nano particles and antibiotics against the uropathogen was determined by serial twofold dilution method. Invitro antimicrobial property of functionalized catheter was assessed by serial plate transfer. Invitro antiadhesive property of the functionalized catheter was evaluated by challenging catheter with multidrug resistant uropathogenic E.coli (105cfu/ml). Invivio efficacy of the functionalized catheter was assessed in mice according to the clearance of Institutional Animal Ethical Committee. After catheterization in mice, urine sample was collected regularly for a period of 15 days and it was assessed for the presence of pathogens. Liver function test and kidney function tests of mice were analyzed to understand the host toxicity. The stability of the catheter was evaluated for a period of 18 months post primary studies to validate the stability. The uropathogenic E.coli was identified to be multidrug resistant.It was susceptible to amikacin and nitrofurantoin. Size of the nanoparticle was 42–75nm. UV‐Vis spectrum exhibited an absorbance maximum at 408nm. The MIC value of nitrofurantoin and amikacin were 2 mg/ml and 150×10−3 mg/ml respectively. Amikacin (40μg/ml) and nitrofurantoin (10μg/ml) showed synergistic effect with 1.176μM of SNP. Invitro anti‐infectious property was evaluated in unfunctionalized catheter, catheter impregnated with amikacin, nitrofurantoin, silver nanoparticles and synergistic combination of antibiotics and silver nanoparticle. All catheters were colonized by the uropathogen except in the synergistic combination. It retained its property till 14 days. In invivo study, the catheter colonization was nil in mice catheterized with the synergistic combination till 15 days. Liver function test and kidney function tests in mice were normal. After 18 months of aseptic preservation, the efficacy was evaluated by invitro methods. It showed an effective zero colonization. Hence, the present study has revealed that impregnation of urinary catheter with a synergistic combination of silver nano particle and antibiotics is an efficient and promising method for preventing Catheter Associated Urinary Tract infection.
Patterns of 16S rRNA Microbial Regions in Aggressor‐Exposed Mice with Features of Post‐Traumatic Stress Disorder
Authors: Gautam Aarti , Hoke Allison , Kumar Raina , Chakraborty Nabarun , Muhie Seid , Hammamieh Rasha , Jett Marti
Published: November 2023

We evaluated repeated exposures of mice to a trained aggressor mouse as a model for aspects of post‐traumatic stress disorder (PTSD), which was adapted from “social stress” models of traumatic stress, and used a “cage‐within‐cage resident‐intruder” protocol where subject C57BL/6J mice were exposed to aggressor mice for 10 days. With the increased awareness of the significance of the symbiotic influence of microbiota on the nervous system and behavior, this longitudinal study focused on the effect of social stress on the microbiome. Fecal samples were collected daily during the 10 day exposure time‐course from control and aggressor‐exposed (Agg‐E) mice. DNA was isolated; followed by amplification of variable V3 and V4 region of the 16S ribosomal RNA (rRNA) amplicon using the Illumina 16S Metagenomics Library Preparation Protocol. The samples were barcoded with Nextera index primers and pooled prior to sequencing on the Illumina MiSeq platform. Our results indicate on day 4 in Agg‐E mice, the phylum Bacteriodetes shows lower relative abundance, whereas phyla Firmicutes and Verrucomicrobia exhibit higher abundance. The predicted functional enrichment of abundant bacterial composition corresponds with the PPAR and Adipocytokine signaling pathways being highly activated in Agg‐E mice in comparison to control mice.

DISCLAIMERS: Research was conducted in compliance with the Animal Welfare Act and all other Federal Requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.

Support or Funding Information

Research supported by CRADA # W81XWH‐11‐0076.
Genesis of Antibiotic Resistance XIX: An Alternative to incineration of Prescription Drug collection &[uml]en masse”
Authors: Kannan Subburaj , Echeverria Leticia , Munoz Janet , Luna Jennifer , Rosales Ramiro , Rodriguez Jennifer , Ruiz Vanessa , Velez Micaela , Rodriguez Lizzeth , Chavez Dariela , Subburaj Prabavathy , Villarreal‐Moreno Raquel
Published: November 2023

The grandeur of “Prescription Drug Take Back Program” (including Pharmaceutical, Personal Care Products (PPCP)) administered by the Drug Enforcement Administration, division of United States Department of Justice (DEA‐US‐DOJ), though it is a stellar success, frill will be flounced by adopting Bioremediation for its disposal. It is essential to point out that the medications of PPCP should be categorized according to their chemical potency and possible contaminants prior to the incineration process. At this time, it is not clear whether DEA‐US‐DOJ or commercial vendors are performing this task prior to the incineration. The DEA‐US‐DOJ's strategy of mass incinerating drugs taken by the aforesaid program, carry a heavy burden of acting as a source of known and unknown pollutants damaging the public health by contaminating ground water table, water reservoirs, land and air. By adopting an appropriate bioremediation method (aerobic or anaerobic) encompassing several specific modus operandi (Cometabolism, biostimulation biosorption, bioleaching, bioaccumulation, bioaugumentation) will definitely alleviate the concerns over contaminating the sand, water and air by the pollutants generated by the current disposal methods, however a multitude of factors such as: competition with/among microorganisms, insufficient supply with essential substrates, unfavorable external conditions (aeration, moisture, pH, temperature), and low bioavailability of the pollutant, biodegradation would potentially act as a limiting factor for choosing an appropriate method for the disposal process. Systematic analysis of merits and demerits of all available methods for disposing the PPCP shall be presented in Experimental Biology 2016.

Support or Funding Information

Supported by professional development funds by SWTJC to S.Kannan for 2014–2015
Genesis of Antibiotic Resistance XI: Critical Appraisal of Disposal Method of National Prescription Drug Take‐Back Day
Authors: KANNAN SUBBURAJ , ROBLES LIZET , MOODY NADINE , Delgado Mayra , Garcia Valerie , Moore Kelly , Echeverria Leticia , Ruiz Pauline , Martinez Consuelo
Published: November 2023

A systematic analysis of the data pertaining to the disposal method of the drugs collected by National Prescription Drug Take‐Back Day has been performed in association with United States Drug Enforcement Administration's (US‐DEA's) – Department of Justice (DOJ), DEA Public Affairs, Washington D.C. Based on the data collected from the National Prescription Drug Take‐Back Day all across United States by US DEA–DOJ, as of September 5, 2014, an estimated 4.1 million pounds (2,123 tons) of prescription medications have been removed from the circulation. As per the US DEA b– DOJ public affairs, the drugs that are collected by this program were disposed by incineration facility monitored and regulated by the United States – Environmental Protection Agency (US‐EPA). The entire disposal of the drugs that are collected by National Prescription Drug Take ‐ Back day is completed by a vendor destined by US DEA ‐DOJ. Further analysis of the disposal process reveal following pitfalls of this process: i. There are no data on file regarding pollutants generated from the incineration of Drugs from the take back programs by US DEA ‐ DOJ, 2. The practice of incineration at the rate of en mass is a potential resource for reaction intermediates, such as dioxins, fly ash, and other unknown pollutants, 3. The practice of not sorting the drugs according to the chemical toxicity prior to incineration potential harm to the environment and biota. Specific information on the class of the medication as per the chemical potencies for generating combustion based pollutants are investigated. Data analysis on the Impact of the disposal practices of drugs from take back day will be presented in Experimental Biology 2016.

Support or Funding Information

Professional Development Funds to S.Kannan at SWTJC for 2014–2015
Genesis of Antibiotic Resistance (AR) XIV: Inter agency anomaly in data sharing debilitate the mitigation of AR Pandemic
Authors: Kannan Subburaj , Villarreal Ana , Valdez Edith , Villarreal‐Moreno Raquel , Sanchez Jema , Serna Gysel , Salazar Ketsia , Rodriguez Victor , Padilla Maria , Navejas Crystal
Published: November 2023

Multitude of agencies within the United States – Department of Health and Human Services, Center for Disease Control and Prevention (US‐DHH‐CDC&P), as identified in the Frieden's annual report on AR, shoulder the responsibility of data collection, analysis, and implementing new strategies for prevention of AR pandemic. We have identified that Frieden's report falls short to incorporate the specific details on the categories of drugs including the antibiotics collected through the National drug Take back day organized by the Drug Enforcement Administration sections of the US Department of Justice (DEA‐US‐DOJ). Regarding the antibiotic prescribing/stewardship, the role of prescription drug monitoring program (PDMP) which is an integral component of National Alliance for Model State Drug Laws (NAMSDL), falls short of the annual progress report. PDMP is a statewide electronic database which collects designated data on substances dispensed including antibiotics / antimicrobials in each of 50 states and also United States Territories. Whereas Prescription Monitoring Information Exchange (PMIX), on the other hand allows the stakeholders to freely exchange the information pertaining to the prescription data at a patent level, including antibiotics and anti‐microbials. Neither the physicians in private practice, local pharmacies, nor the Maverick County Health District officials are aware of PDMP, and PMIX, by CDC&P. Further inquiry confirms that there is a communication gap between CDC & P with District 8 of Texas DHH on PDMP‐PMIX. Frieden's report on AR falls short of details on agencies and current status of data (Quantitative details/refills/duration of being on the specific antibiotics/ antibiotic‐ Drug interaction [blood pressure / diuretics / weight loss medication / anti‐psychotic / illicit drugs ] subsequent emergency room visits / Mortality ratio) sharing on annual rate of prescription of antibiotics in each state and also in the United States Territories. It is of pivotal concern that the integration of data on multinational effort in mitigating the AR along side of CDC&P's annual report on AR is of global significance. Prospects of an enhanced prediction and analysis of morbidity and mortality rate due to the misuse or unaccounted use of antibiotics, in AR pandemic shall be presented in EB2016. (http://www.cdc.gov/drugresistance/threat‐report‐2013/pdf/ar‐threats‐2013‐508.pdf)

Support or Funding Information

Professional development funds provided by the SWTJC to S.Kannan for 2014–2015
Genesis of Antibiotic Resistance (AR) XV: Genetic (Modification) Roulette impairs response to antibiotics – An Insidious AR Pandemic Phenomenon.
Authors: Kannan Subburaj , Alvarez Angelica , Hernandez Guadalupe , Hernandez Martin , Garza Jasmin , Carrizales Tania , Carrizales Diana , Crabtree Cynthia , Subburaj Prabavathy
Published: November 2023

Molecular plant breeding techniques utilize antibiotic resistance genes (ARG) are used as a selection marker in the gene‐transfer construct for genetic modification (GM). It is known that the naked viral DNA promoter that regulate ARG, are hotspots for recombination with host DNA inducing AR via horizontal gene transfer (HGT). For example the npt II gene confers resistance to Neomycin, Kanamycin and other antibiotics, while aad‐3 gene confers resistance to antibiotics, Streptomycin and Spectinomycin (Goldstein et al., 2005). Clinical response to antibiotic therapy performed by Indiana State University and University of California San Diego found that hospitals observed decreased effectiveness of antibiotic drugs. Accordingly, the National Summary Data presented in Antibiotic Threats in U.S., by Friedan of CDC&P predicts that at least 2,049,442 illness and 23, 000 deaths due to AR. Clostridium difficile, Carbapenum resistant enterobacteriaceae, drug resistant Neisseria gonorrhoeae, Salmonella serotype typhi, multi‐drug resistant M. tuberculosis pose imminent threat to the public health. Genetic Roulette implies that increase in an untreatable infectious diseases is a multi factorial event inclusive of persistence of antibiotic r(resistance) genes, and resistome, (Davies and Davies 2010). Neither the Frieden's report on AR threat level (http://www.cdc.gov/drugresistance/pdf/ar‐threats‐2013‐508.pdf) from CDC & P, Atlanta, GA, United States) nor TATFAR report ( http://www.cdc.gov/drugresistance/pdf/tatfar‐progress_report_2014.pdf) nor National Action Plan For Combating Antibiotic‐Resistant Bacteria, (https://www.whitehouse.gov/sites/default/files/docs/national_action_plan_for_combating_antibotic‐resistant_bacteria.pdf) addresses this concern in light looming AR pandemic. Morbidity and mortality rate due to AR induced by nptII, aad‐3 and other antibiotics will be presented in EB2016.

Support or Funding Information

Professional development funds provided by SWTJC to S. Kannan for 2014–2015
Genesis Of Antibiotic Resistance VIII: “Auto Medication”, an universal practice That Contravenes Hardy‐ Weinberg ‐ Castle Principle (HWCP), Escalates as a Serious Risk Factor In Mitigating AR Pandemic.§
Authors: Kannan Subburaj , Carrizales Diana , Carrizales Tania , Alvarez Angelica , Jouregui Suzzette
Published: November 2023

“Auto ‐ Medication” is a universal practice especially in low‐income, un insured, lack adequate health care(rural setting) facility, lack literacy on antibiotic therapy and antibiotic resistance. Based on the population dynamics, poverty level, health care provider per 1000 people, India has been chosen for this case study. A retrospective systematic review for auto‐medication of antibiotics and morbidity vs. mortality data on antibiotic resistance cases during 2013, and 2014 has been completed using the annual report of public health foundation of India. In essence, the public health report is void of the surveillance data on i. reported cases of antibiotic resistance among the emergency department visit, ii. Morbidity vs. mortality rate due to antibiotic resistance among the hospital acquired infections, and iii. Morbidity vs. mortality rate due to Clostridium diffcile infections. An effort to perform the country wide (India) data base analysis on the total number of cases in terms of morbidity vs. mortality rate due to antibiotic resistance is inconclusive. The reason for this inconsistency is due to the fact that there is no reporting mechanism on total number of cases due to antibiotic resistance to local authorities, state and national registry. Statistical analysis on incidence of antibiotic resistance among population of 24 states is incomplete. CDC (Frieden's) report on AR and United States presidential executive strategy to mitigate AR pandemic fail to account the role of immigrants from regions where the prevalence of unregulated antibiotic use with potential role in AR pandemic. Though “Auto Medication”, is a universal practice, global migratory pattern of the population from regions like India, and Mexico contravenes Hardy‐ Weinberg ‐ Castle Principle (HWCP), could pose as a serious risk factor for AR Pandemic.

Support or Funding Information

Supported by Professional Development Funds.
Lactobacillus plantarum Improves Metabolic Outcomes and Alters the Colonic Immune State in Mice Fed a High Fat Diet
Authors: Heeney Dustin , Barouei Javad , Hsieh Yu‐hsin , Martinic Alice , Mishchuk Darya , Kieffer Dorothy , Slupsky Carolyn , Martin Roy , Marco Maria
Published: November 2023

Obesity, type 2 diabetes mellitus, and metabolic syndrome are associated with a dysbiotic microbiome and unregulated immune activation. Thus, identifying dietary strategies enriching for members of the microbiota that correct for these deficiencies in the indigenous microbiota is useful for managing and preventing those chronic conditions. This approach holds particular promise for consumption of strains of Lactobacillus species commonly consumed in fermented foods. Herein, we investigated the effects of regular consumption of Lactobacillus plantarum (LP) on both treatment, and prevention of diet‐induced obesity. Study 1 Treatment: Male C57BL/6J mice received a high‐fat (HF) diet (45.9% energy from fat) for 9 weeks and then were maintained on either the HF diet or HF diet supplemented with 109 cells LP, feeding every other day (n=10/grp), for another 6 weeks. Study 2 Prevention: Male C57BL/6J mice were fed a HF diet with or without feeding 109 cells LP every other day, for nine weeks (n=10/grp). For both studies, ingestion of LP resulted in higher levels of Lactobacillus in the digestive tract but did not result in global alterations to the intestinal microbiome. In study 1, supplementation with LP significantly increased colonic production of the T‐cell proliferative factor IL‐2 (p = 0.010) and the lymphoid organ maintenance signal TNF‐beta (p = 0.0298). In study 2, supplementation with LP significantly increased colonic production of the T‐reg promoting cytokine IL‐27 (p = 0.0325). In both studies IL‐23, a driver of antibacterial and mucosal barrier maintenance, was significantly upregulated in LP supplemented mice compared to HF diet controls (p = 0.0056, 0.0457, for study 1 and 2 respectively). In Study 2, LP‐fed mice were partially protected from weight gain compared with the HF diet controls (p = 0.0310) and showed improved oral glucose tolerance. The reduction in weight gain was not associated with significant changes in food intake. Results from our study provide novel evidence of the capabilities of LP to inform the maintenance of the colonic immune system and that dietary supplementation with this strain can be beneficial even while maintaining a HF diet.

Support or Funding Information

American Diabetes Association
Mechanism and Interactions of Human Pyruvate Dehydrogenase Complex with its Kinase1
Authors: Guevara Elena L , Nemeria Natalia S , Yang Luying , Jordan Frank
Published: November 2023

The human pyruvate dehydrogenase complex (PDC) consist of three principal catalytic components for its mission: E1, E2, and E3. The core of the complex is a strong subcomplex between E2 and an E3‐binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1–4), and inactivation reversed by the action of two phosphatases (PDP1 and 2). Specifically, the regulation of PDC via phosphorylation of E1 by PDK1 is the focus of these studies, as PDK1 has been strongly implicated as an overexpressed culprit in multiple cancer cells; hence inhibition of the interaction of PDK1 with the E1 or E2•E3BP, could provide novel targets for the design of anticancer agents. Since the PDKs reside on (have a stronger binding to) the E2•E3BP core of the entire complex, specific inhibition of the PDK1‐E2•E3BP interaction provides a possible new approach to treatment of cancer. Inactivation of E1 via phosphorylation by PDK1‐4 was initially studied to determine PDK interaction and therefore activation by E2•E3BP or its C‐terminally truncated proteins. The studies suggest that the individual PDK isoforms require different degrees of activation: PDK2 appears to be the most active and not highly selective whereas PDK4 is the least active never fully inactivating E1. Recently, the interaction loci between PDK1 (PDK2) and the E2•E3BP core was defined by the Jordan group by two complementary methods, HD‐exchange MS (HDX‐MS) and NMR1. Interrogation of the sites on E2•E3BP identified to interact with PDK1 (PDK2) was accomplished by site‐directed mutagenesis studies in order to validate the identified ‘hot spots’. To date, there is evidence of the variants on the L1L2S region of E2 effecting inactivation of E1 via phosphorylation by PDK1. Further studies will be performed to identify the protein‐protein interaction of these L1L2S variants with PDK1 and PDK2 using HDX‐MS, isothermal titration calorimetry (ITC) and fluorescence titrations. These measurements will confirm whether the ‘hot spots’ interrogated indeed are important for the interaction of E2•E3BP with the PDK1, and whether it is specific to PDK1 by comparing such measurements with PDK2. These experiments will identify ‘real’ hot spots, against which rational drug design could be undertaken.

Support or Funding Information

This research was supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award GM113601‐01A1 from the National Institute of General Medical Sciences.
Covalent inhibition of ERK docking interactions
Authors: Kaoud Tamer Saad , Johnson William H. , Piserchio Andrea , Ebelt Nancy D , Warthaka Mangalika , Cano Micael , Sammons Rachel , Wang Qiantao , Ren Pengyu , Ghose Ranajeet , Dalby Kevin N.
Published: November 2023

The extracellular‐regulated kinase (ERK) signaling pathway has been implicated in the regulation of several cellular functions, including cell differentiation, proliferation, and inflammatory responses. Upon stimulation, ERKs have been reported to phosphorylate a large number of substrates localized either in the cytoplasm or the nucleus. Taking advantage of our understanding of ERK docking interactions, we tried to design/discover substrate‐selective inhibitors that target the protein‐binding site of ERK. Here we report the discovery of the first irreversible ERK inhibitor that targets its protein‐docking site. Protein NMR, mass spectrometry, mutagenesis and molecular docking studies showed that the compound forms a covalent bond with a conserved cysteine residue C‐159. Extensive biochemical studies have been employed to estimate its kinetic parameters and its kinase‐selectivity profile. Finally, the new ERK inhibitor was shown to inhibit both ERK phosphorylation and its ability to phosphorylate downstream substrates (e.g. p90RSK and Elk‐1) in HEK293 cells. The inhibitor showed dose‐dependent inhibitory effects on HEK293 and melanoma A375 cell proliferation and viability. Targeting ERK in cells was confirmed using a chemical‐genetic approach where the C159A mutant was used to rescue the effects of this compound on ERK signaling. The discovered compound represents a new class of ERK inhibitor that uniqely labels its protein binding site. Further development may result in a pharmacologically useful inhibitor.

Support or Funding Information

This research has been supported by the following grants from the National Institutes of Health: GM059802 (to K.N.D.),GM084278 (to R.G.), and G12 MD007603 (towards partial support of the NMR facilities at The City College of New York). R.G. and K.D. are members of the New York Structural Biology Center, NYSTAR facility. T.S.K. acknowledges Cancer Prevention and Research Institute of Texas Postdoctoral Training Award (RP140108).
Single Molecule Analysis of Allosteric Interactions in Protein Kinase A
Authors: Hao Yuxin , England Jeneffer , Taylor Susan S. , Maillard Rodrigo A
Published: November 2023

Protein Kinase A (PKA) is composed of Catalytic (C) and Regulatory (R) subunits that assemble as an inactive holoenzyme. The allosteric activation of PKA is triggered by the cooperative binding of cAMP to two cyclic‐nucleotide binding domains (CNB‐A and CNB‐B) located in the R subunit. The goal of this study is to establish a quantitative relationship between protein stability, conformation and the observed cooperativity between CNB domains. We used optical tweezers force spectroscopy to probe at the single molecule level the stability and conformation of the CNB domains, either as isolated structures or together as found in the R subunit. Moreover, we investigated the effect of cAMP binding on the stability of each CNB domain and on the interaction energy between them. We found that in the absence of cAMP, the CNB‐A unfolds at forces higher than those observed for the CNB‐B (~10pN and ~6pN, respectively). Interestingly, however, the CNB domains as part of the R subunit unfold at similar forces (~10pN), indicating that the CNB‐B is stabilized by the presence of CNB‐A due to inter‐domain interactions. Binding of cAMP further stabilizes the interaction between the CNB domains, as evidenced by higher unfolding forces near ~16pN. The mutant R241A, a hotspot residue position for allosteric interactions, reduces the folding probability and the mean unfolding force of the CNB‐B, and severely disrupts the interaction between the CNB domains in the R subunit. Altogether, our experimental strategy allowed us to trace and dissect the energetic bases associated with the allosteric interactions triggered by cAMP binding and by inter‐domain interactions between CNB domains. Moreover, this study enabled the identification and quantitative characterization of aberrant allosteric events associated with mutations observed in disease states.
Substrate selective mechanisms in protein kinases: Allostery between nucleotide and substrate in PKCα
Authors: Sommese Ruth , Swanson Carter , Sivaramakrishnan Sivaraj
Published: November 2023

The human kinome comprises over 300 distinct kinases, each of which has multiple substrates. The specificity of kinase‐substrate pairings is mainly attributed to conserved sequence motifs flanking the substrate phosphorylation site. These recognition motifs, though, cannot be generalized across all kinase‐substrate pairings, and in general kinases display greater promiscuity towards substrates in vitro. In the cell, co‐localization with substrates is another mechanism for specificity, but multiple kinase‐substrate pairings are possible in the same subcellular microenvironment. Hence, additional mechanisms likely contribute to the substrate selectivity observed in phosphorylation networks. Here, we present a novel class of FRET‐based sensors that probe the allostery between nucleotide and substrate interactions in the PKCα catalytic cycle. We provide evidence for coordination between nucleotide and substrate states that lead to enhanced catalytic efficiency. This coordination differentially influences substrate interactions to dictate substrate selectivity. In line with our findings, potent inhibitors of PKCα not only displace nucleotide but also disrupt substrate binding. Overall, this work presents a novel assay platform for identifying substrate‐selective kinase inhibitors.
Phosphorylation of PKA Regulatory Subunit RIα by PKG: a Mechanism for “Desensitizing” the Type I PKA Holoenzyme
Authors: Haushalter Kristofer J. , Casteel Darren E. , Stefan Eduard , Taylor Susan S.
Published: November 2023

The catalytic subunit of protein kinase A (PKA‐C) is a critical signaling protein in eukaryotes, requiring a high degree of regulation to control its phosphorylation activity under both homeostatic and stress conditions in the cell. The inhibitory mechanism for the four functionally non‐redundant regulatory subunits of PKA (RIα, RIβ, RIIα and RIIβ) has been well characterized with regard to their ability to keep PKA‐C in an inactive “holoenzyme” complex until activation with cAMP. However, recent evidence suggests that unique features within Type I PKA holoenzymes may be alternatively regulated via non‐canonical signaling mechanisms, such as regulatory subunit phosphorylation and oxidation. Previous work demonstrated that serine 101 of the RIα regulatory subunit can be phosphorylated by cGMP‐dependent protein kinase (PKG) in vitro, whereupon phospho‐RIα no longer inhibited PKA‐C at normal (1:1) stoichiometric ratios. In this study, we validated this phosphorylation mechanism under in vivo conditions to determine whether this phosphorylation event could lead to a physiologically relevant “desensitization” of PKA‐C in the context of cellular stress. Here, we show that PKG is indeed able to incorporate phosphate onto S101 of RIα under both in vitro and in vivo conditions, and moreover that phosphorylation does not occur upon RIα where Ser101 is substituted with Ala (i.e. S101A). The PKG site is not accessible in the holoenzyme prior to activation by cAMP which thus creates an ordered pathway for crosstalk between PKA and PKG. Using a luciferase protein complementation assay, we show that the phospho‐mimetic mutation (i.e. S101E) decreases holoenzyme formation in cells, and this is accompanied by increased downstream activation of PKA‐C. Because PKG signaling can be activated by ROS (i.e. nitric oxide) in cells, ongoing experiments are aimed toward determining if this phosphorylation mechanism may serve as an alternate pathway for “priming” the PKA holoenzyme for activation within the context of oxidative stress. Taken together, these findings support the proposed mechanism for Type I holoenzyme “desensitization” as a potential in vivo pathway for establishing signaling crosstalk between PKA and PKG signaling.

Support or Funding Information

Support for this work was funded by NIH GM34921 to sst.
Three‐dimensional quantification of the spatiotemporal co‐evolution of vascular and neuronal networks within intact eyes
Authors: Singh Jasmine , Seedorf Gregory J. , Brzezinski Joseph A. , Shepherd Douglas P.
Published: November 2023

Retinopathy of prematurity (ROP) is a disease that affects blood vessel development and distribution in the eyes of infants born prematurely. A common method used to investigate structure‐function relationships in ROP is serial sectioning followed by two‐dimensional image analysis. Often, serial sectioning is fraught with inconsistencies due to tissue tearing and folding which may introduce optical artifacts. Of note are the errors that occur when multiple physical sections are computationally reconstructed to quantify the spatial location of fluorescent labels within the original three‐dimensional tissue. In this work we utilize a technique, CLARITY, which renders tissue optically transparent through the establishment of a monomer hydrogel matrix and removal of light scattering lipids to generate optically transparent eyes. To measure fluorescent labels within these intact eyes we have developed a unique digital scanned light sheet microscope (DSLM) specifically designed to quantify fluorescently labeled signaling molecules and structures within CLARITY treated samples. These two techniques combined provide a methodology to quantify the three‐dimensional distribution of key signaling molecules and structures during development of the eye, bypassing issues inherent in serial sectioning, two dimensional imaging and computational reconstruction. Here, we report quantification of the vascular and neuronal network structures in intact control and ROP model rat eyes. Using eyes harvested at multiple time points in development, we provide a comparative analysis evaluating both two dimensional and three dimensional imaging techniques and find that network features in developing eyes are more accurately quantified using our three‐dimensional imaging approach. This paves the way for future studies of the exact timing of changes of signaling and structural development, in three‐dimensions, during the pathogenesis of ROP.
Tracking Dynamics of Vascular Development in Time and Space using Sox18 Dimer split‐Fluorescent Protein Biosensors
Authors: Giles Nichole , McCann Alex , Moustaqil Mehdi , Overman Jeroen , Fontaine Franck , Francois Mathias , Sierecki Emma , Gambin Yann
Published: November 2023

The temporal and milieu‐specific control of cell fate is dependent on the fine‐tuning of the activity of transcription factors. One way this is achieved is by varying the composition of specific protein complexes that binds cooperatively at DNA enhancer regions. SOX18 is a key transcription factor that is transiently expressed in nascent endothelial cells during embryogenesis and adult neovascularization. Mutations of human SOX18 cause Hypotrichosis‐Lymphedema‐Telangietasia and the protein is overexpressed in gastric and pancreatic cancer cell lines. Recently, we showed that SOX18 could act as a heterodimer and more importantly demonstrated that SOX18 can homodimerize in vitro. This is further supported by the identification of DNA binding sites specific to the homodimer and subsets of gene under the control of the homodimer only. These observations suggest that SOX18 heterodimer and homodimer may play different roles during vascular development.

Here, we developed a SOX18 dimer split‐fluorescent protein (FP) biosensor to track the dynamics of SOX18 dimerization and its influence on different gene expression during vascular development in zebrafish embryo.

However, the major obstacle to the general use of these biosensors, especially in living organisms, is the amount of optimisation required, as the complementation depends on multiple parameters: the nature of the fluorescent protein, the relative position of the two parts, the length and flexibility of the linker regions… To shorten the process, the initial screening and optimization of split‐FP biosensors with emission ranging from blue (BFP) to red (mCherry) was realized using a cell‐free expression platform.

Once optimised, the SOX18 dimer biosensor was expressed in an endothelial cell line (HUVEC) for validation and imaging optimization. The sensor was then adapted to a zebrafish embryogenesis model where expression is under the control of endothelial specific fli1 promoter.

The study will be extended to the creation of reporters for different heterodimers of SOX18 to identify the partners driving the different steps of vasculature development.

Support or Funding Information

EMBL Australia Single Molecule Centre Sydney Node
Super‐resolution Imaging of the HER Family of Receptor Tyrosine Kinases
Authors: van Lengerich Bettina , Puchner Elias , Huang Bo , Jura Natalia
Published: November 2023

The human epidermal growth factor receptor (HER/ErbB) family of receptor tyrosine kinases encompasses transmembrane signaling proteins important for cellular growth, differentiation, and survival. The four members of this family (EGFR, HER2, HER4 and the HER3 pseudokinase receptor) are able to bind to numerous different growth factor ligands, leading to receptor homo and hetero‐oligomerization and subsequent activation. This combinatorial potential gives rise to diverse signaling outputs, the extent of which is still not fully understood due to the lack of precise knowledge of how HER receptors interact with each other upon ligand binding. We seek to understand HER receptor interactions at the membrane by directly imaging the receptor molecules at a single molecule level. We use a technique that can accurately count absolute numbers of molecules at the membrane by employing a correction for fluorophore blinking in conjunction with stochastic optical reconstruction microscopy (STORM). This reveals the spatial organization and extent of oligomerization of receptors with sub‐diffraction limit resolution. We then probe how this organization and oligomerization evolve as a function of growth factor ligand stimulation. We also examine the effects of mutations that disrupt key interfaces in active HER receptor dimers, as well as of activating HER receptor mutations that are prevalent in cancer. Finally, we determine the role that over‐expression of a partner co‐receptor has on the extent of receptor oligomerization, and how ligand stimulation influences these interactions. This work aims to contribute to the fundamental understanding of the activation mechanism of HER receptors and to help advance development of new therapeutics targeting aberrant cross‐talk among HER receptors in human disease.

Support or Funding Information

This work is funded by an American Cancer Society post‐doctoral fellowship to BvL.
Phenotype analysis and gene expression profiling in a yeast model of Cystinosis
Authors: Walnofer April E. , Vitiello Seasson
Published: November 2023

CTNS is a gene that encodes the protein Cystinosin, a lysosomal cystine transporter. When CTNS is nonfunctional, it results in the disease cystinosis, which has a hallmark sign of cystine accumulation in the lysosome and cell death. CTNS has an orthologous gene in Saccharomyces cerevisiae, ERS1. When ERS1 is deleted, ers1‐Δ cells do not accumulate cystine. It has been shown that sixteen modifier genes in S. cerevisiae are differentially expressed between parental ERS1+ and ers1‐Δ, which may contribute to compensation of the lack of cystine accumulation. Gene expression profiling and phenotype analysis of ERS1 has been done based on characterization of the sixteen modifier genes. This includes looking at respiration, redox regulation, genetic interactions of modifier genes with ERS1, and sulfur homeostasis.

Support or Funding Information

Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health [P20GM103443] and the Cystinosis Research Foundation.
Functional morphology and evolution of the human trunk musculature ‐ Old and new features
Authors: Schilling Nadja
Published: November 2023

The axial musculoskeletal system is important for the static and dynamic control of the body during both locomotor and non‐locomotor behaviors. It contributes to the production of work, provides the foundation for the production of mechanical work by the limbs, is central to the control of body posture and integrates limb and trunk actions. As a consequence, major evolutionary changes in locomotor and positional habits of a species must be reflected by morpho‐functional adaptations of its axial system. Among mammals, humans are unique in having a striding bipedal gait and a permanent orthograde trunk posture. During locomotion, rotations about the long‐axis of the body dominate, in contrast to the distinct sagittal bending of other mammals. Due to the upright trunk posture and the small contribution of trunk motions to body propulsion, the functional demands on the human trunk musculature appear to be different from other mammals. To better understand the evolution of the human axial muscles and discern possible morpho‐functional changes that were associated with the evolution of orthogrady and a bipedal gait, we 1) compared the activation patterns of the two major epaxial muscles in dogs and humans as we manipulated the moments acting on the trunk and pelvis and 2) analyzed the 3D distribution of slow and fast muscle fibers in the lumbar perivertebral muscles of various non‐primate, lemuriform and hominoid primate species. Our results indicate that the epaxial muscles primarily stabilize the pelvis against a) the vertical components of hindlimb retractor muscles and b) the horizontal components of the hindlimb protractor and retractor muscles in both dogs and humans. Similar to previously studied quadrupedal mammals, the lemuriform primates exhibited a morpho‐functional dichotomy between deep slow contracting local stabilizer muscles and superficial fast contracting global mobilizers and stabilizers and thus retained the fibre distribution pattern typical for quadrupedal non‐primates. In contrast, the hominoid primates showed no regionalization of the fibre types, similar to previous observations in humans. We suggest that this homogeneous fibre composition is associated with the high functional versatility of the axial musculature that was brought about by the evolution of orthograde behaviors and reflects the broad range of mechanical demands acting on the trunk in orthograde hominoids. Because orthogrady is a derived character of euhominoids, the uniform fibre type distribution is hypothesized to coincide with the evolution of orthograde behaviors.