Downloaded via UNIV OF SHARJAH on February 25, 2024 at 21:05:23 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ACS! Biomaterials SCIENCE & ENGINEERING pubs.acs.org/journal/abseba Engineering a Cortisol Sensing Enteric Probiotic Vaughn Litteral,* Rebecca Migliozzi, David Metzger, Craig McPherson, and Roland Saldanha Cite This: ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 Metrics & More ACCESSI ABSTRACT: Chronic stress can lead to prolonged adrenal gland secretion of cortisol, resulting in human ailments such as anxiety, post-traumatic stress disorder, metabolic syndrome, diabetes, immunosuppression, and cardiomyopathy. Real time monitoring of chronic increases in cortisol and intervening therapies to minimize the physiological effects of stress would be beneficial to prevent these endocrine related illnesses. Gut microbiota have shown the ability to secrete, respond, and even regulate endocrine hormones. One such microbe, Clostridium scindens, responds transcriptionally to cortisol. We engineered these cortisol responsive genetic elements from C. scindens into an enteric probiotic, E. coli Nissle 1917, to drive the expression of a fluorescent reporter allowing for the designing, testing, and building of a robust and physiologically relevant novel cortisol probiotic sensor. This smart probiotic was further engineered to be more sensitive and to respond to elevated cortisol by expressing tryptophan decarboxylase, thereby bestowing the ability to generate tryptamine and serotonin. Here we show that upon cortisol treatment the smart probiotic produces measurable amounts of tryptamine. Accumulated levels of these neuromodulators should improve mood, anxiety, and depression and drive down cortisol levels. Importantly, this work can serve as a model for the engineering of a sense-and-respond probiotic to modulate the gut-brain axis. KEYWORDS: engineered probiotic, probiotics, smart probiotic, gut brain axis, Escherichia coli Nissle 1917, EcN, cortisol, glucocorticoid, 5-alpha-tetrahydrocortisol, adrenal, chronic stress, tryptophan, tryptophan decarboxylase, 5-HT, serotonin, trypta lysR transcription factor, bacterial sensor, Clostridium scindens, bacterial targeted gene integration, biosensor, sense and respond circuit INTRODUCTION The prevalence of anxiety, stress, and depression is ever increasing in modern societies. Mitigating the endocrine response in times of chronic anxiety, stress, and depression has historically been a challenge due to the endocrine system's intimate role in physiological homeostasis. Neuman et al.² demonstrated that endogenous microbial sentinels have been shown to both monitor and influence the endocrine system and ultimately human physiology. Probiotics show promise in restoring homeostasis in endocrine and immunological dysregulation. More recently, engineering probiotics as living diagnostics and therapeutics has allowed for a whole cell therapeutic approach with a beneficial metabolic potential that seems unlimited. Decades of research into the interplay between the gastrointestinal tract and the nervous system referred to as the gut brain axis (GBA) has revealed an intricate communication network shown to have a great impact in human health, development, and physiology. The GBA is a bidirectional communication system between the nervous system (central nervous system, autonomic nervous system, and enteric nervous system), and hypothalamus pituitary adrenal (HPA) axis with gut microbiota symbionts. Sudo et al. demonstrated that microbiota were essential for HPA develop- ACS Publications © 2023 The Authors. Published by American Chemical Society Read Online Article Recommendations 5163 T SI Supporting Information E. coli Nissle 1917 Ato/C:lysRtdc Chromosomal DNA lysR tryptophan tdc Received: November 1, 2022 Accepted: April 17, 2023 Published: August 30, 2023 LysR tryptophan decarboxylase (TDC) Article an tryptamine. Special Issue: Design and Evaluation of Engineered Probiotics Cortisol ment in germ free mice. Stressors, such as from the host inflammatory response and environmental factors, activate the HPA through the hippocampus release of CRF (corticotropin releasing factor), which in turn stimulates the pituitary to release ACTH (adrenal corticotropic hormone). ACTH stimulates specific cellular receptors, resulting in adrenal secretion of the stress hormone cortisol. While acute stress can be beneficial, chronic stress stimuli can lead to chronic cortisol secretion with detriments to human health and performance (i.e., anxiety, stress, post-traumatic stress disorder, diabetes, immunosuppression, cardiomyopathy, weight gain, and alcoholism). About 10% of cortisol is in active form and present in the blood circulation, whereas the remainder is bound to albumin or cortisol binding globulin protein. Cortisol and associated glucocorticoid metabolites regulate nearly every cell type in the body through the glucocorticoid 15 TOIC SACS materials https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering 16 and mineralocorticoid receptors acting as seemingly global transcription factors in gene expression. Traditional ther- apeutic interventions into ameliorating the endocrine response is met with many adverse events in off target tissues as cortisol exerts this global but tissue specific gene expression response. ¹7 18-20 Noninvasive monitoring of free cortisol in sweat and saliva has evolved in recent years as an interest in the human performance and health monitoring industry as well as the United States Department of Defense. Cortisol in saliva, sweat and tears is indicative of blood levels and is a predictive biomarker in determining real time psychological stress.ª Gastro-intestinal glucocorticoid levels have been difficult to ascertain, wherein bacterial secondary metabolism of cortisol and resultant desmolysis occurs beginning in the large intestine. Ridlon et al. employed RNA-seq to investigate cortisol metabolism and its regulated gene expression in one such gut bacterium, Clostridium scindens. C. scindens demon- strated robust response in gene expression to the stress hormone, cortisol, and observed side chain cleavage of cortisol resulting in androgen gut metabolites. These studies showed further evidence that the gut microbiota have the innate ability to monitor the host endocrine stress molecule, cortisol, and influence the metabolic fate of these endocrine hormones. 21 23-25 26,27 In addition, it has been increasingly demonstrated that gut microbiota have additional and significant neuroendocrine roles in the GBA through secondary metabolism.²2 For example, the metabolic fate in the gut of ingested tryptophan, Trp, an essential amino acid, results in indoles, serotonin (5- hydroxytryptamine, 5-HT), tryptamines, melatonin, and kynurenine. All of these metabolites have a profound effect on host mood, anxiety, sleep, and/or stress. Trp metabolism is accomplished primarily through the kynurenine pathway (95%) resulting in nicotinamide dinucleotide (NAD +) production, while only minor amounts (1%) are converted to 5-HT. Dysregulation of the kynurenine pathway can lead to aging, various mental and neurodegenerative disorders, and chronic fatigue syndrome." Whereas 5-HT has been researched for more the 70 years, its intimate role in gut physiology is just recently being understood. 28 Cortisol stimulates 5-HT reuptake in peripheral circulation through Serotonin-selective Re-uptake Transporters (SERT) further affecting 5-HT levels.30 Ninety percent of 5-HT production occurs in intestinal enterochromaffin cells (EC), and 5-HT synthesis can be stimulated through 5-HTR4 activation by the trace amine tryptamine, wherein the gut-produced 5-HT is absorbed, stored, and distributed by platelets.³ Five of the seven known classes of serotonergic receptors (5-HT1 thru 5- HT7) are expressed throughout the gut. Activation of 5-HT2a receptor by intestinal 5-HT results in a block of TNFa and subsequent increase in SERT.³ The 5-HT is distributed by platelets throughout the circulation and plays an important role in hemostasis, peristaltic reflex, and gut physiology.³5 While 5- HT does not have the ability to cross the blood brain barrier, Trp does have the ability to cross and therefore directly contributes to central and peripheral 5-HT and melatonin levels. 35,36 Sequestering of Trp through microbial secondary metabolism in the gut leads to decreased Trp in the brain and can greatly affect its bioavailability for neuroendocrine roles. Corynebacterium spp., Streptococcus spp., and Escherichia coli have been shown to synthesize 5-HT in culture. Williams et al. evaluated bacterial tryptophan decarboxylase activity (a rare functionality for gut bacteria). In fact, there is an approximately 10% prevalence in the population as discovered by Fishbach in 31,32 33,34 35 37 29 5164 Article pubs.acs.org/journal/abseba 38 39 analyzing Human Microbiome Project data. Allowing for the tryptophan decarboxylase activity in the gut would result in an increase in tryptamine and subsequent stimulation of 5-HT production from EC cells, as well as biotransformations of 5- hydroxytryptophan directly to 5-HT. In the case of chronic stress, both in animal models and human studies, subjects greatly benefited through decreasing circulating cortisol levels by increasing 5-HT levels with SRIs (Serotonin Reuptake Inhibitors).36,40 41 42 In order to mitigate the debilitating effects of chronic stress, a promising transient strategy herein was conceived to restore normal physiology and function in the gut with a whole cell engineering approach. This study utilizes a well-characterized probiotic chassis that is able to withstand and colonize the harsh gastric intestinal environment. The only commercially available Gram-negative probiotic to date, E. coli Nissle 1917, EcN, was originally isolated from a World War I soldier that was able to surprisingly survive with dysentery.4¹ The EcN strain was also recently shown to increase 5-HT extracellular concentrations in an in vitro gut tissue model." An EcN smart probiotic with elements of Clostridium scindens to sense active glucocorticoids and respond by contributing the rare Trp decarboxylase activity (similar to Clostridium sporogenes) resulted in decarboxylase activity with some substrate promiscuity toward 5-hydroxytryptophan that would result in 5-HT metabolites as well as dietary tryptophan biotransforma- tion to tryptamine. In periods of chronic stress and elevated cortisol production, we predict the engineered probiotic in the gut would increase tryptamine and ultimately intestinal 5-HT production and distribution via induced promiscuous tryptophan decarboxylase activity. The intestinal and periph- erally distributed 5-HT would drive down circulating cortisol levels and likely would improve mood, anxiety, and depression. 43 Importantly, this effort models a stress-sensing and potentially neuro-modulating probiotic in in vitro systems that prove to mimic human physiology. 44 MATERIALS AND METHODS Materials. Restriction, DNA modifying enzymes, and polymerases were from New England Biolabs (Ipswich, MA, United States). Plasmid miniprep and PCR purification kits were from QIAGEN (Germantown, MD, United States). Synthetic DNA and oligonucleo- tides were obtained from Integrated DNA Technologies (IDT; Coralville, IA, United States). Unless otherwise indicated, all other chemicals and reagents were from Sigma (St. Louis, MO, United States) or Thermo Fisher Scientific (Waltham, MA, United States). 52 Bacterial Strains and Culture Conditions. Table S1 lists the E. coli strains used in this study. Figures S9-S11 display the workflow utilized for recombinant strain construction as further detailed by Yang et al. The wild type E. coli strain Nissle 1917, referenced herein as EcN (Mutaflor, DSM 6601, and serotype 06:K5:H1), was kindly supplied by Dr. A. Breedon. BW2511, JW0451-2, and JW5503-1 were obtained from the CGSC (Yale University, Dept. of MCB), E. coli NEBSalpha (cat# C2987H) strains were obtained from NEB. The E. coli strains were routinely maintained in Luria-Bertani (LB) or M9 minimal salts medium with or without 1.5% Bacto Agar (Difco Laboratories, Detroit, Mich., United States). Electrocompetent cells were prepared using Biorad's MicroPulser (BIORAD Hercules, CA) protocol for the preparation of E. coli electrocompetent cells; see Aususbel et al. and Miller and Nickoloff. 56,57 Ampicillin (50 ug/mL), apramycin (50-100 ug/uL), chloramphenicol (30 ug/mL), and kanamycin 25-50 ug/mL) were used for antibiotic selection in this study. Biotek Plate Assay. Mid log E. coli strains were used to inoculate a fresh culture in early log (OD 600 = 0.02). Glucocorticoids https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering dissolved in culture media in 14 mm Falcon tubes (cat# 14-959-11B) with normalized DMSO 0.02-0.2% as a cosolvent and samples exposed to serially diluted glucocorticoid doses or DMSO and respective antibiotic selection. A kinetic plate assay was carried out with black/clear bottom Costar plates (cat# 3601) on a Biotek Neo2 multimodal plate reader. Both the OD 600 and the fluorescent wavelength(s) were monitored. The glucocorticoid dose response was normalized by OD600 and the EC50 was calculated with a nonlinear regression model. Data analysis was performed in Graphpad Prism 9.0. Tryptophan and Tryptamine Extraction. Following induction of tryptamine production, the culture samples were centrifuged at 16 000g for 1 min and then filtered through a 0.2 um filter, then the filtrate was treated with a 2x volume of 4N sodium hydroxide at pH 11. Next, phase extraction was performed with 1:1 with ethyl acetate, followed by mixing and centrifugation at 16 000g for 1 min to allow for phase separation. The resulting aqueous phase contained tryptophan and the organic phase captured tryptamine. Samples were subsequently prepared for HPLC analysis. Cloning and Construct Assembly. The overall cloning strategy used 2-5 DNA fragments produced from either DNA synthesis (gblocks), oligos or high-fidelity polymerase generated amplicons purified though Qiagen PCR or Qiaex gel extraction protocols. Following purification, fragments were assembled with NEB Builder HiFi Gibson Assembly using a total reaction volume of 5-10 µL. 10% of the HiFi reaction was transformed into NEB5a chemical competent cells and plated on selective media. After the plates were incubated for 18-24 h at 37 °C, individual colonies were selected for evaluation Bacterial Plasmid Construction. pLysE2Cr. The E2 crimson fluorescent reporter was designed to be under the control of LysR using the synthesized lysR with its bidirectional promoter (Genbank WP_004606447.1) included in the 250 bp upstream sequence (Figure S1) all cloned into the supplied pIDT bacterial expression vector, pIDT (kan¹), resulting in plasmid pLysRE2Cr. The lysR open reading frame (ORF) was codon optimized for E. coli expression using online in silico analysis tools provided by Integrated DNA Technologies (IDT, Coralville, IA). pLysRsfGFP. This plasmid was a gift from the U.S. Army Lab (Dr. Steve Blum) and was created by swapping the ORF of the sfGFP reporter for that of the E2 crimson reporter in plasmid pLysRE2Cr. pLysRTDC. The pLysRE2Cr plasmid was used as a PCR template for a 900bp fragment of the lysR gene (C. scindens), primed with lysR- F and lysR-R and amplified with Q5 high fidelity polymerase (NEB #M0491S). The tdc-1 (Tryptophan decarboxylase-1 from Oryza sativa) gene was synthesized by IDT as a 1.5kb fragment (see the supplemental sequence list for details) and was likewise used as a PCR template and amplified with Q5 high fidelity polymerase. The lysR and tdc-1 containing fragments were assembled via an NEB Builder HiFi Assembly kit into the XhoI/NotI sites of pAME200 (this plasmid was a gift from Dr. A. Breedon, USAF RHB) resulting in plasmid pLysTDC. PUC19lysRsfGFP-Plasmid. pLysRsfGFP was used as template with oligos to generate a linear fragment both assembled with either 200 bp or 1000bp of flanking tolC homology gblocks (See the supplemental sequence information for gblocks from IDT) assembled into the pUC19 BamH1/EcoR1 vector. PUC19lysRE2Crimson Plasmid. pLysRE2Crimson was used as templates with oligos to generate a linear fragment both assembled with either 200bp or 1000bp of flanking tolC homology gblocks (see supplemental sequence information for gblocks from IDT) assembled into the pUC19 BamH1/EcoR1 vector. PUC19lysRTDC Plasmids. pLysRE2Crimson was used as templates with oligos to generate a linear fragment both assembled with either 200 bp or 1000bp of flanking tolC homology gblocks (See Supplemental Sequence information for gblocks from IDT) assembled into the pUC19 BamH1/EcoR1 vector. Colony PCR. Colony PCR was performed by touching a standard 10 ul pipet tip into the center of the bacteria colony and transferred to the bottom of a 96-well PCR plate well. NEB Quick load 2X Taq PCR 5165 Article pubs.acs.org/journal/abseba master mix was added to each well on a cooling block and once the denaturation temperature of 95 °C was reached the samples were added to the thermocycler. The initial denaturation of the PCR was conducted for 2 min. NEB Quick load 2X Taq polymerase manufacture recommendations were followed. Standard PCR. Standard PCR employed either the NEB Quick load 2X Taq polymerase protocol for routine usage or the NEB HiFi Q5 polymerase protocol for cloning and assembly efforts in this study. Flow Cytometry Analysis. Following growth and glucocorticoid treatment, E. coli strains were diluted 1:40 in phosphate buffered saline (pH 7.4) with 20mM Hoechst 33342 DNA stain (Fisher Cat. No. H21492) and incubated at 37 °C for 10 min. Following Hoechst DNA staining, all cells were stored on ice until flow cytometry data acquisition. Samples were analyzed on a 5-laser BD FACSAria II cell sorter (BD Biosciences, San Jose, CA) with instrument setup performed using Cytometer Setup and Tracking Software (BD Biosciences). Hoechst was excited with a 355 nm laser and detected with a 450/50 filter. GFP was excited with a 488 nm laser and detected with a 525/50 nm filter. E2 crimson was excited with 561 nM laser and detected with a 670/30 nm filter. During acquisition, all parameters were collected in log mode. Data were collected and analyzed using FACSDiva 8.0 software (BD Biosciences). To reduce noise, the bacteria were cultured in sterile-filtered media and diluted in sterile-filtered PBS. Prior to acquisition, the background noise of the instrument was evaluated by analyzing water-only, sheath fluid-only, and PBS-only blank samples. Forward and side scatter gates were set to exclude noise and debris; the bacterial population, identified as Hoechst- positive cells, was further analyzed to identify single cells (FSC-height vs FSC-width, followed by SSC-height vs SSC-width). Positivity gates for the different fluorescent parameters were set after analyzing multiple controls (unstained/untreated culture, unstained/treated culture, stained/ untreated culture). Flow cytometry analyses are in the supplementary. 53 Gene Knockout Generation. Ato/C:CAT Knockout in E. coli Nissle 1917. A knockout strain was constructed using a traditional Red Recombination protocol by Datsenko and Wanner. The chloramphenicol acetyltransferase, CAT, gene in pKD3 was utilized as template to generate a CAT flanking 50 bp tolC homology PCR cassette (See Figure 4) using oligos C1 and C2. Electrocompetent EN1917 cells expressing the red recombinase helper plasmid, pKD46, were transformed with the resultant PCR product targeting the tolC gene. Cells recovered for 1 h and selection was performed on LB chloramphenicol plates (15 ug/mL). AtolC:Ap' Knockout in E. coli Nissle 1917. The apramycin resistance gene, Ap', in pMDIAI was utilized as a PCR template primed with oligos A1 and A2 to generate an Ap¹ PCR fragment with flanking 50 bp homology to tolC. According to a published protocol by Yang et al.,52 electrocompetent EcN cells expressing the pREDTKI plasmid were transformed with the PCR product targeting the tolC gene. Cells were recovered in 1 h and plated on LB apramycin selection plates (50 ug/mL) and individual colonies were selected for further analysis. 52 Targeted Gene Integration. Integration of donor cassettes from lysRsf GFP, lysRE2Crimson, and lysRtdc-1 into the tolC locus was accomplished according to published protocols by Yang et al.5² The intermediate strain AtolC:Ap¹ EcN was used as a recipient strain for targeted integration into the tolC locus. See Figures S11, S12, and S13 for workflow and an example of the screening and validation. Tryptamine Production. Early log (OD600 = 0.02) strains were grown in M9 media with 1% LB media and 2% glucose with 6 mM L- tryptophan and were maintained under selection of chloramphenicol 30ug/mL. Following an OD600 = 0.2, cultures were induced with the stated quantities of cortisol. Cultured samples were collected at the stated times and centrifuged at 16 0000g for 1 min to remove biomass and were subsequently prepared for chemical analysis. 1. RESULTS AND DISCUSSION 2.1. Designing a Novel Cortisol Biosensor. To design a real-time probiotic stress sensor, the bacterial chassis, E. coli https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering 45-47 Nissle 1917 was employed, due to the probiotic's ability to survive the challenging gastrointestinal environment and its reported use as a framework for synthetic biology. The use of a whole cell approach affords the ability to utilize a transcription factor responsive to cortisol that can activate a downstream transcriptional response (i.e., reporter gene or stress-reducing factor). Published RNA-seq data for C. scindens ATCC 35704 demonstrated an effect for its neighboring regulon upon exposure to cortisol (Figure S1).³ LysR-type transcriptional regulators (LTTRs) are the largest prokaryotic class of transcription regulators. In C. scindens, LysR regulates its own transcription as well as its native divergently transcribed gene product. When LysR disassociates from its own promoter, lysR transcription is active, whereas when bound to the coinducer (i.e., cortisol), promoter activation results in transcription of the divergently located gene (Figure 1). Based on this design, the lysR nucleotide sequence was LysR lysR LysR LysR I Co LysR Co lysR Figure 1. Schematic of the LysR regulation of transcription in Clostridium sp. placed under native control elements for expression in E. coli K-12 and EcN strains and evaluated activation of various LysR responsive reporter genes upon cortisol induction. 2.2. Employing a Novel Cortisol Biological Recog- nition Element, BRE. In Clostridium sp. Ridlon et al. demonstrated that the lysR and its divergently transcribed gene product is transcriptionally active in the presence of cortisol, based on RNA-seq data analysis.³ Preliminary analysis was performed in order to determine if the lysR operon had the ability to function as a glucocorticoid transcriptional driven sensor, by employing the Clostridium scindens lysR native control elements driving the bidirectional expression of the E2 Crimson fluorescent reporter protein. The E2 Crimson fluorescent protein has an excitation maximum at 611 nm and emission maximum at 646 nm, is nontoxic to bacteria and performs well with live animal imaging.* The pLysE2Cr plasmid bears the kanamycin resistance gene and the pMB1 origin of replication. Chemical transformed E. coli K-12 and EcN bearing the plasmid pLysE2Cr following mid log growth were evaluated for their dose response to cortisol by measuring fluorescence with a plate reader and flow cytometry. In Figure 2, the schematic demonstrates that the LysR negatively regulates its own transcription (gold) and positively regulates the divergent reporter gene (green) with putative LysR responsive promoters (blue). Exposing the EcN probiotic bearing the pLysE2Cr to varying doses of cortisol (coinducer in Figure 2) at mid log demonstrated a definitive dose response 48 5166 pubs.acs.org/journal/abseba curve with an EC50 of 158 µM and is supra-physiological, whereas the normal human physiological cortisol concen- trations in plasma observe a diurnal variation and range between 80 and 700 nM, and the water solubility of cortisol is 772 μM. Multiparametric flow cytometry analysis was used in this study, since it affords reproducible and accurate functioning at the single cell level and fluorescent reporter proteins afford accurate representations of isogenic cultures. In the EcN wild type strain bearing the pLysE2Cr plasmid, E2 Crimson fluorescent protein expression was evaluated via flow cytometry following incubation with varying concentrations of cortisol. In Figure 2, the top panel/density plots, show a dose- dependent shift of the cell population into the E2 Crimson positive gate, indicating an increased percentage of cells expressing E2 Crimson in response to cortisol. In Figure 2, the bottom panel histograms show a dose-dependent increase in the population's median fluorescence intensity with increasing cortisol concentration. Evaluation of additional reporters such as sfGFP and iLuX (Figure S3) gave similar results. 2.3. Cortisol BRE Selectivity for a-THF, an Active Cortisol Metabolite. In an effort to determine the glucocorticoid specificity of the sensor, varying concentrations of cortisol and its associated metabolites were evaluated on early growth phase EcN bearing the engineered pLysE2Cr plasmid. Both cortisol and its active metabolite 5-alpha- tetrahydrocortisol (THF) were able to transactivate LysR and drive E2Cr reporter expression in similar percentage of the cell population (73% and 59%, respectively) as demonstrated in the density and histogram plot in Figure 3B. Furthermore, cortisol and THF demonstrated robust dose responses with similar EC50 values (213 µM and 150 μM respectively, Figure 3C). The inactive cortisol metabolite of cortisone, 5-beta- tetrahydrocortisone (THE), was unable to initiate the E2Cr reporter in a dose dependent fashion (Figure S4), further indicating the selectivity of the pLysE2Cr sensor for active glucocorticoids. Article 49 The lumen of the gut is a harsh environment with additional cortisol analogs present, such as bile salts. Bile salts are amphipathic steroid molecules found exclusively in the G.I. tract and bile salts are critical for the absorption of dietary lipophilic foodstuffs in the lumen of the small intestine because of their capacity to form micelles spontaneously in a concentration dependent manner. Deoxycholate (DOC), a secondary bile salt (typically present at 20 µM to 100 µM in the small intestines) was investigated to see if DOC would elicit a response with the LysR sensor. DOC afforded a dose response (Figure S5) with an EC50 of 1.5 mM, although these supra-physiological (>1 mM) quantities of DOC proved deleterious to ECN growth (Figure S5).5⁰ 2.4. Targeted Ablation of Glucocorticoid Efflux in E. coli Nissle 1917 to Improve Glucocorticoid Sensor Sensitivity. Although the LysR sensor is able to selectively recognize cortisol (Figure 3), the limit of cortisol detection by the LysR sensor in the wild type EcN host is not physiological relevant (EC50 = 158 µM) and requires further improvements. Available acrAB and tolC mutants (Keio collection mutants JW0451 and JW5503) generated in the wild type parental strain E. coli BW25113 were tested for cortisol sensitivity. Both acrAB and tolC mutants demonstrated a 2-log improvement in cortisol sensitivity (Figures S6 and S7). Following this, the tolC locus was ablated in EcN by directing the chloramphenicol acetyltransferase gene (CAT) gene to the tolC locus using Red homologous recombination (data not shown). PCR- https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering A. B. C. 530/30 Blue-A 10² 10³ 104 D. LysR family TF Regulator -57 plysE2 Crimson lysR ΟμΜ Singlets 0 10 -81 %E2Cr+, Normalized Response 10 0 10 10 105 E2Crimson 670/30 YG-A Singlets 0% E2Crimson+ E2Crimson MdFI 10⁹ 10 10° E2Crimson 670/30 YG-A 100 50 10⁰ 530/30 Blue-A -65 -65 10² 62.5 μΜ Singlets 0 10² 10 Singlets pubs.acs.org/journal/abseba 10 10 E2Crimson 670/30 YG-A verified AtolC:CAT ECN mutants (data not shown) were transformed with the pLysE2Cr plasmid (Figure 4A) and 10 0.6% E2Crimson MdFI 0 10² 10ª 105 E2Crimson 670/30 YG-A E2Crimson+ 5167 reporter/response E2 Crimson 530/30 Blue-A 10² 10³ 104 105 106 Count 500 1.000 1.500 2,000 46 pMB1 ori 0 125 μM Singlets TIMME TIITING. ITIINI 10 10ª 10$ E2Crimson 670/30 YG-A 28% E2Crimson+ Singlets E2Crimson MdFI 10⁰ E2Crimson 670/30 YG-A 10² 10ª 10⁰ 158μM = EC50 104 [Cortisol nM] Figure 2. A novel cortisol responsive transcription factor drives reporter expression. (A) Schematic of the LysR operational unit, where LysR negatively regulates its own promoter and lysR (gold) and drives the expression of the reporter or response gene (green). (B) Plasmid map of pLysE2 Crimson. (C) Flow cytometric analysis of pLysE2 Crimson in EcN with 0, 62.5, and 125 uM of cortisol. Top row, density plots: the gates are indicative of positive E2 Crimson expression. Bottom row, histograms: the interval gates measure the MdFI, median fluorescence intensity. (D) Cortisol dose response curve (EC50 = 158 uM) of pLys E2Crimson in wild type EcN. Article evaluated in mid log cultures for a cortisol dose response (Figure 4C). The AtolC:CAT mutation in EcN enhanced https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175/n

Solved: Downloaded via UNIV OF SHARJAH on February 25, 2024 at 21:05:23 (UTC)

Home
questions and answers
downloaded via univ of sharjah on february 25 2024 at 21 05 23 utc see

Question

Downloaded via UNIV OF SHARJAH on February 25, 2024 at 21:05:23 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ACS! Biomaterials SCIENCE & ENGINEERING pubs.acs.org/journal/abseba Engineering a Cortisol Sensing Enteric Probiotic Vaughn Litteral,* Rebecca M

This question hasn’t solved by tutor

Solution unavailable? No problem! Generate answers instantly with our AI tool, or receive a tailored solution from our expert tutors.

Found 10 similar results for your question:

Make a graph of column F in the isometric weights spreadsheet (50 lb column). What part of the typical processing of EMG does the signal in the F column look like?

In an experiment the transport of the neurotransmitter serotonin into cultured mammalian cells was measured. Batches of cells were incubated with solutions of different concentrations of radio labelled [3H]-serotonin for a defined time, cells were then separated from the incubation buffer, and serotonin accumulated into the cells was determined by scintillation counting. The rate of transport is given as dpm/min* (see table below). In order to elucidate the transport mechanism, the experiment was conducted under two different conditions, i.e., in condition A normal transport buffer was used, and in condition B the transport buffer did not contain any Na* ions.[Notes: * dpm/min means disintegrations per minute and is a measure of the intensity of-radioactivity of a radio-labelled compound; it is directly related to the rate of transport for this compound] 11. Draw a graph (by using Excel or similar), plotting the serotonin concentration against the-transport rate under both conditions. (5 Marks). 12. What transport rates for each condition would you expect if the experiment was repeated-using 12μM [³H] serotonin? Add your estimate into the table above. (5 Marks). 13. Examine the plot obtained for condition A; what can you conclude with regards to-whether or not the transport of serotonin into these cells requires a protein? Explain your conclusion. (10 Marks). 14. Compare the plots of the transport rates for both conditions. What is most likely mechanism of transport for serotonin into these cells? Explain your interpretation. (10Marks).

1- Write an essay on the importance of anthropometry and how anthropometric data can be used in the design of workplaces to protect the workers from work-related musculoskeletal disorders.

1. Why is it necessary to maintain aseptic conditions in microbiology? 2. Explain why the following steps are necessary during sub culturing: dFlaming the inoculating loop before and after each inoculation (= b. Cooling the inoculating loop before touching the inoculum/culture ( Flaming the neck of tubes/bottles after capping and before re-capping ( Can pure culture be prepared directly from a mixed culture? Explain your answer. 4. You have been asked to determine the bacterial count in a sample of water. The-number of CFU from 1 ml of a 106 dilution was 43 and 49 on duplicate agar plates.per ml of the original sample of water? (5What is the total number of microorganisms-marks)

5. [Total: 8 pts] Muscle stretch reflex The figure below shows a simplified model of muscle stretch reflex in an experimental anesthetized animal. The model describes how the muscle stretch reflex regulates the muscle length. All variables shown have been linearized and represent fluctuations around its mean value under normal resting condition. Z represents external neural frequency stimulating the spinal cord. fa represents the total change in afferent neural frequency. The spinal cord, represented by the gain Gc, converts the incoming neural input to the neural output fe- The changes in the efferent neural frequency, fe, are relayed through motor neurons to the muscle. The muscle converts the efferent neural frequency to changes in muscle length L. The gain of this conversion is-G. Note that the gain is negative because increases in efferent neural frequency lead to decreases in muscle length. The change in the muscle length is sensed by the muscle spindle. The muscle spindle relays the neural output, fs, to the spinal cord through sensory neurons. This process is represented by the gain Gg. fa fe Gc Z -GM Gs fs L a) [4 pts] Suppose that animal A is injected with a drug that disrupts neurotransmission, blocking the sensory neurons. Under such condition, is the system operating as a negative feedback system? Explain your answer. Please also state which gain(s) is affected by the sensory nerve blockade. b) [4 pts] Under which condition between normal and sensory nerve blockade would result in stronger muscle contraction for the same level of the external stimulation Z? Explain your answer.

2. [15 pts] Assume the metabolic hyperbola for CO₂ given by the following equation: 863 - Vcoz V₁ PACO2 Pico2+ where the normal steady-state CO₂ production is 235 mL/min and the inspired CO₂ concentration (or volumetric fraction) is 0. Also, assume a dead-space ventilation rate of 1 L/min. Also, suppose the steady-state ventilatory response to CO₂ is given by the following equation: 32 Vc = Drive external +(1.46 + (Paco2-37) and Vc 20 Pa02-38.6) Note that the above equation is slightly modified from what we derived in class in that a constant term, Drive external is added to the equation. This drive represents an additional source of control that affects ventilatory response e.g., sleep-wake state, alertness, etc. At normal state (resting and awake), let Drive external = 0. a) [5 pts] Use MATLAB to plot the metabolic hyperbola for CO₂ and the ventilatory response to CO₂ under normal condition. Show both graphs on the same figure (see hold function). Label both axes with appropriate units. Also indicate which graph represents the me the metabolic hyperbola for CO₂ and which graph represents the ventilatory response to CO₂ (see legend function). The plot should show the range of ventilation from 0-20 L/min and the range of PACO₂ from 30-60 mmHg (see xlim and ylim functions). Determine the normal steady-state values of ventilation and Pacoz from the plot (e.g., click on the plot where you want to obtain the x-y coordinates). Note: For the metabolic hyperbola for CO₂, please plot V against PACO₂ i.e., you would take the dead-space ventilation into consideration. b) [4 pts] The onset of sleep causes the external drive to breathe to drop by -5 L/min. State which parameter changes and to what value. Show the graphs representing this situation and report the steady-state values of ventilation and Pacoz. c) [4 pts] Follows from b), how would inhalation of a gas mixture containing 3% CO₂ in air (i.e., volumetric fraction of 0.03) during sleep affect the steady-state values of ventilation and Pacoz? State which parameter changes and to what value. Show the graphs representing this situation and report the steady-state values of ventilation and Pacoz. You may assume that the subject is at sea level. d) [2pt] Copy and paste the MATLAB code you used in this question (all parts). You may take a picture of your MATLAB script, but please make sure that the code is clearly readable from the image.

- *S5: Discuss appropriate and informed career management choices and entrepreneurial issues* - Consider including a section on the practical applications of studying flaviviruses and pseudotyped viruses in terms of career opportunities and entrepreneurship.

1. [5 pts] Construct the glucose-insulin regulation model in Simulink as outlined in Fig. 3. The parameter values shown in Fig. 3 are the values of a normal adult. Set the Simulink model to run simulations for a total duration of 24 hours. Although Simulink generally assumes time to be measured in seconds, the units of the model parameters give time in hours-so, you can treat the durations you enter into Simulink as hours (instead of seconds). The initial conditions of normal blood glucose and insulin concentrations are 0.81 mg/mL and 0.055 mU/mL, respectively. These initial values of blood glucose and insulin concentrations should be set in the respective Integrator blocks (shown as in Simulink). To set the initial values in the integrator block, double click the block to open the Block Parameters window. Enter the initial value (e.g., 0.81 for glucose) in the Initial Condition field, and click OK. Run a simulation for a normal adult (Subject N) over 24 hours without any external glucose input (i.e., U(t) = 0). Show the graph of glucose and insulin concentrations as a function of time as 2-by-1 subplots i.e., the glucose concentration is the top subplot and the insulin concentration is the bottom subplot. Label all axes. You may plot using the Scope in Simulink or plot in MATLAB using the simulation results that are sent to MATLAB Workspace. Save this Simulink model as "glucose_N.slx".

Create a 15-slide PowerPoint presentation to include: Visuals and graphs of hospital data and coding data - as your dashboard. For each graph, provide a summary (at least 2 paragraphs) to explain what the information reflects, and why it is a valuable tool for HIM services and managers. Evaluate the use of benchmarking and comparative data. Based on your interpretation of the hospital and coding data results, what are your predictions or assumptions about the data. Evaluate the report using various statistical methods. Justify your interpretation based on the summary of data and references. Recommend how you intend to use the data for making decisions for HIM managers and coding supervisors across the hospital system. Provide a summary of your references. Grading Criteria

(A) Reverse engineer a piece of medical technology. This must include: 1. A brief history of the science leading to its development, and the development of the technology. 2. A careful, physical analysis of how the device works. This must include a schematic or diagram and the math describing the physics. 3. Evaluation of how the device is applied, the impact it has on humanity, and how it could be improved in the future. Citations of strong sources are required DEVICE: Defibrillator for each question