Neuronal innervation regulates the secretion of neurotrophic myokines and exosomes from skeletal muscle.

Myokines and exosomes, originating from skeletal muscle, are shown to play a significant role in maintaining brain homeostasis. While exercise has been reported to promote muscle secretion, little is known about the effects of neuronal innervation and activity on the yield and molecular composition of biologically active molecules from muscle. As neuromuscular diseases and disabilities associated with denervation impact muscle metabolism, we hypothesize that neuronal innervation and firing may play a pivotal role in regulating secretion activities of skeletal muscles. We examined this hypothesis using an engineered neuromuscular tissue model consisting of skeletal muscles innervated by motor neurons. The innervated muscles displayed elevated expression of mRNAs encoding neurotrophic myokines, such as interleukin-6, brain-derived neurotrophic factor, and FDNC5, as well as the mRNA of peroxisome-proliferator-activated receptor γ coactivator 1α, a key regulator of muscle metabolism. Upon glutamate stimulation, the innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs than neuron-free muscles. Consequently, biological factors secreted by innervated muscles enhanced branching, axonal transport, and, ultimately, spontaneous network activities of primary hippocampal neurons in vitro. Overall, these results reveal the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggest that the engineered neuromuscular tissue model holds significant promise as a platform for producing neurotrophic molecules.

Patient and ward related risk factors in a multi-ward nosocomial outbreak of COVID-19: Outbreak investigation and matched case-control study.

BACKGROUND: Risk factors for nosocomial COVID-19 outbreaks continue to evolve. The aim of this study was to investigate a multi-ward nosocomial outbreak of COVID-19 between 1st September and 15th November 2020, occurring in a setting without vaccination for any healthcare workers or patients. METHODS: Outbreak report and retrospective, matched case-control study using incidence density sampling in three cardiac wards in an 1100-bed tertiary teaching hospital in Calgary, Alberta, Canada. Patients were confirmed/probable COVID-19 cases and contemporaneous control patients without COVID-19. COVID-19 outbreak definitions were based on Public Health guidelines. Clinical and environmental specimens were tested by RT-PCR and as applicable quantitative viral cultures and whole genome sequencing were conducted. Controls were inpatients on the cardiac wards during the study period confirmed to be without COVID-19, matched to outbreak cases by time of symptom onset dates, age within ± 15 years and were admitted in hospital for at least 2 days. Demographics, Braden Score, baseline medications, laboratory measures, co-morbidities, and hospitalization characteristics were collected on cases and controls. Univariate and multivariate conditional logistical regression was used to identify independent risk factors for nosocomial COVID-19. RESULTS: The outbreak involved 42 healthcare workers and 39 patients. The strongest independent risk factor for nosocomial COVID-19 (IRR 3.21, 95% CI 1.47-7.02) was exposure in a multi-bedded room. Of 45 strains successfully sequenced, 44 (97.8%) were B.1.128 and differed from the most common circulating community lineages. SARS-CoV-2 positive cultures were detected in 56.7% (34/60) of clinical and environmental specimens. The multidisciplinary outbreak team observed eleven contributing events to transmission during the outbreak. CONCLUSIONS: Transmission routes of SARS-CoV-2 in hospital outbreaks are complex; however multi-bedded rooms play a significant role in the transmission of SARS-CoV-2.

Asymptomatic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in adults is uncommon using rigorous symptom characterization and follow-up in an acute-care adult hospital outbreak.

Asymptomatic coronavirus disease 2019 (COVID-19) has been reported as a significant driver of COVID-19 outbreaks. Our hospital ward outbreak analysis suggests that comprehensive symptoms and signs assessment, in combination with adequate follow-up, allows a more precise determination of COVID-19 symptoms. Asymptomatic infection was quite uncommon among adults in this setting.

Homeostatic regulation of extracellular signal-regulated kinase 1/2 activity and axonal Kv7.3 expression by prolonged blockade of hippocampal neuronal activity.

Homeostatic plasticity encompasses the mechanisms by which neurons stabilize their synaptic strength and excitability in response to prolonged and destabilizing changes in their network activity. Prolonged activity blockade leads to homeostatic scaling of action potential (AP) firing rate in hippocampal neurons in part by decreased activity of N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes including KCNQ3 which encodes Kv7.3. Neuronal Kv7 channels are mostly heterotetramers of Kv7.2 and Kv7.3 subunits and are highly enriched at the axon initial segment (AIS) where their current potently inhibits repetitive and burst firing of APs. However, whether a decrease in Kv7.3 expression occurs at the AIS during homeostatic scaling of intrinsic excitability and what signaling pathway reduces KCNQ3 transcript upon prolonged activity blockade remain unknown. Here, we report that prolonged activity blockade in cultured hippocampal neurons reduces the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) followed by a decrease in the activation of brain-derived neurotrophic factor (BDNF) receptor, Tropomyosin receptor kinase B (TrkB). Furthermore, both prolonged activity blockade and prolonged pharmacological inhibition of ERK1/2 decrease KCNQ3 and BDNF transcripts as well as the density of Kv7.3 and ankyrin-G at the AIS. Collectively, our findings suggest that a reduction in the ERK1/2 activity and subsequent transcriptional down-regulation may serve as a potential signaling pathway that links prolonged activity blockade to homeostatic control of BDNF-TrkB signaling and Kv7.3 density at the AIS during homeostatic scaling of AP firing rate.

Digital White Racial Socialization: Social Media and the Case of Whiteness.

The Internet has become a ubiquitous central element in the lives of adolescents. In this conceptual paper, we focus on digital white racial socialization (D-WRS), arguing: (1) for an expanded conceptualization of WRS as doings, and (2) that social media may be changing processes of WRS through an extension of traditional settings and through the creation of unique social contexts. We highlight the uniqueness of social media contexts due to the designed normalization of whiteness, weak-tie racism, social media affordances, and racialized pedagogical zones allowing adolescents to practice doing race. We introduce a conceptual framework for D-WRS and end with an expressed need for conceptually guided research on the multidimensional relationship between social media and WRS processes.

4D flow MRI for the assessment of renal transplant dysfunction: initial results.

OBJECTIVES: (1) Determine inter-observer reproducibility and test-retest repeatability of 4D flow parameters in renal allograft vessels; (2) determine if 4D flow measurements in the renal artery (RA) and renal vein (RV) can distinguish between functional and dysfunctional allografts; (3) correlate haemodynamic parameters with estimated glomerular filtration rate (eGFR), perfusion measured with dynamic contrast-enhanced MRI (DCE-MRI) and histopathology. METHODS: Twenty-five prospectively recruited renal transplant patients (stable function/chronic renal allograft dysfunction, 12/13) underwent 4D flow MRI at 1.5 T. 4D flow coronal oblique acquisitions were performed in the transplant renal artery (RA) (velocity encoding parameter, VENC = 120 cm/s) and renal vein (RV) (VENC = 45 cm/s). Test-retest repeatability (n = 3) and inter-observer reproducibility (n = 10) were assessed by Cohen's kappa, coefficient of variation (CoV) and Bland-Altman statistics. Haemodynamic parameters were compared between patients and correlated to the estimated glomerular filtration rate, DCE-MRI parameters (n = 10) and histopathology from allograft biopsies (n = 15). RESULTS: For inter-observer reproducibility, kappa was > 0.99 and 0.62 and CoV of flow was 12.6% and 7.8% for RA and RV, respectively. For test-retest repeatability, kappa was > 0.99 and 0.5 and CoV of flow was 27.3% and 59.4%, for RA and RV, respectively. RA (p = 0.039) and RV (p = 0.019) flow were both significantly reduced in dysfunctional allografts. Both identified chronic allograft dysfunction with good diagnostic performance (RA: AUC = 0.76, p = 0.036; RV: AUC = 0.8, p = 0.018). RA flow correlated negatively with histopathologic interstitial fibrosis score ci (ρ = - 0.6, p = 0.03). CONCLUSIONS: 4D flow parameters had better repeatability in the RA than in the RV. RA and RV flow can identify chronic renal allograft dysfunction, with RA flow correlating with histopathologic interstitial fibrosis score. KEY POINTS: • Inter-observer reproducibility of 4D flow measurements was acceptable in both the transplant renal artery and vein, but test-retest repeatability was better in the renal artery than in the renal vein. • Blood flow measurements obtained with 4D flow MRI in the renal artery and renal vein are significantly reduced in dysfunctional renal transplants. • Renal transplant artery flow correlated negatively with histopathologic interstitial fibrosis score.

Gadoxetate disodium-enhanced MRI: Assessment of arterial phase artifacts and hepatobiliary uptake in a large series.

PURPOSE: To report the quality of gadoxetate disodium MRI in a large series by assessing the prevalence of: 1) arterial phase (AP) artifacts and its predictive factors, 2) decreased hepatic contrast uptake during the hepatobiliary phase (HBP). METHODS: This retrospective single center study included 851 patients (M/F:537/314, mean age: 63y) with gadoxetate disodium MRI. The MRI protocol included unenhanced, dual arterial [early and late arterial phases (AP)], portal venous, transitional and hepatobiliary phases. Three radiologists graded dynamic images using a 5-scale score (1: no motion, 5: severe, nondiagnostic) for assessment of transient severe motion (TSM, defined as a score ≥4 during at least one AP with a score ≤3 during other phases). HBP uptake was assessed using a 3-scale score (based on portal vein/hepatic signal). The association between demographic, clinical and acquisition parameters with TSM was tested in uni- and multivariate logistic regression. RESULTS: TSM was observed in 103/851 patients (12.1 %): 83 (9.8 %) in one AP and 20 (2.3 %) in both APs. A score of 5 (nondiagnostic) was assigned in 7 patients in one AP (0.8 %) and none in both. Presence of TSM was significantly associated with age (p = 0.002) and liver disease (p = 0.033) in univariate but not in multivariate analysis (p > 0.05). No association was found between acquisition parameters and TSM occurrence. Limited or severely limited HBP contrast uptake was observed in 87 patients (10.2 %), and TSM was never associated with severely limited HBP contrast uptake. CONCLUSION: TSM was present in approximately 12 % of gadoxetate disodium MRIs, rarely on both APs (2.3 %), and was poorly predicted. TSM was never associated with severely limited HBP contrast uptake.

The VSV matrix protein inhibits NF-κB and the interferon response independently in mouse L929 cells.

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in immune evasion. While VSV has been thought to suppress the interferon (IFN) response primarily by inhibiting host cell transcription and translation, our recent findings indicate that the M protein also targets NF-κB activation. Therefore, the M protein may utilize two distinct mechanisms to limit expression of antiviral genes, inhibiting both host gene expression and NF-κB activation. Here we characterize a recently reported mutation in the M protein [M(D52G)] of VSV isolate 22-20, which suppressed IFN mRNA and protein production despite activating NF-κB. 22-20 inhibited reporter gene expression from multiple promoters, suggesting that 22-20 suppressed the IFN response via M-mediated inhibition of host cell transcription. We propose that suppression of the IFN response and regulation of NF-κB are independent, genetically separable functions of the VSV M protein.

Transcription Dynamics Regulate Poly(A) Tails and Expression of the RNA Degradation Machinery to Balance mRNA Levels.

Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.

Contrast enhanced ultrasound: A review of radiology applications.

Ultrasound contrast agents have been used for decades in Europe and Asia for cardiac and abdominal imaging and are now being more commonly utilized in the United States for radiology applications. Our article reviews the basics of contrast-enhanced ultrasound including how the contrast agent works, advantages and disadvantages, as well as pearls and pitfalls to help the radiologist efficiently integrate this technology into day-to-day clinical practice. We also discuss the diagnosis of focal hepatic lesions as well as off-label applications such as evaluation of renal masses.

Multiparametric magnetic resonance imaging shows promising results to assess renal transplant dysfunction with fibrosis.

Here we assessed the diagnostic value of a quantitative multiparametric magnetic resonance imaging (mpMRI) protocol for evaluation of renal allograft dysfunction with fibrosis. Twenty-seven renal transplant patients, including 15 with stable functional allografts (eGFR mean 71.5 ml/min/1.73m2), and 12 with chronic dysfunction/established fibrosis (eGFR mean 30.1 ml/min/1.73m2), were enrolled in this prospective single-center study. Sixteen of the patients had renal biopsy (mean 150 days) before the MRI. All patients underwent mpMRI at 1.5T including intravoxel-incoherent motion diffusion-weighted imaging, diffusion tensor imaging, blood oxygen level dependent (BOLD R2*) and T1 quantification. True diffusion D, pseudodiffusion D*, perfusion fraction PF, apparent diffusion coefficient (ADC), fractional anisotropy (FA), R2* and T1 were calculated for cortex and medulla. ΔT1 was calculated as (100x(T1 Cortex-T1 Medulla)/T1 Cortex). Test-retest repeatability and inter-observer reproducibility were assessed in four and ten patients, respectively. mpMRI parameters had substantial test-retest and interobserver repeatability (coefficient of variation under 15%), except for medullary PF and D* (coefficient of variation over 25%). Cortical ADC, D, medullary ADC and ΔT1 were all significantly decreased, while cortical T1 was significantly elevated in fibrotic allografts. Cortical T1 showed positive correlation to the Banff fibrosis and tubular atrophy scores. The combination of ΔT1 and cortical ADC had excellent cross-validated diagnostic performance for detection of chronic dysfunction with fibrosis. Cortical ADC and T1 had good performance for predicting eGFR decline at 18 months (4 or more ml/min/1.73m2/year). Thus, the combination of cortical ADC and T1 measurements shows promising results for the non-invasive assessment of renal allograft histology and outcomes.

SELfies and CELLfies: Whole Genome Sequencing and Annotation of Five Antibiotic Resistant Bacteria Isolated from the Surfaces of Smartphones, An Inquiry Based Laboratory Exercise in a Genomics Undergraduate Course at the Rochester Institute of Technology.

Are touchscreen devices a public health risk for the transmission of pathogenic bacteria, especially those that are resistant to antibiotics? To investigate this, we embarked on a project aimed at isolating and identifying bacteria that are resistant to antibiotics from the screens of smartphones. Touchscreen devices have become ubiquitous in society, and it is important to evaluate the potential risks they pose towards public health, especially as it pertains to the harboring and transmission of pathogenic bacteria that are resistant to antibiotics. Sixteen bacteria were initially isolated of which five were unique (four Staphylococcus species and one Micrococcus species). The genomes of the five unique isolates were subsequently sequenced and annotated. The genomes were analyzed using in silico tools to predict the synthesis of antibiotics and secondary metabolites using the antibiotics and Secondary Metabolite Analysis SHell (antiSMASH) tool in addition to the presence of gene clusters that denote resistance to antibiotics using the Resistance Gene Identifier (RGI) tool. In vivo analysis was also done to assess resistance/susceptibility to four antibiotics that are commonly used in a research laboratory setting. The data presented in this manuscript is the result of a semester-long inquiry based laboratory exercise in the genomics course (BIOL340) in the Thomas H. Gosnell School of Life Sciences/College of Science at the Rochester Institute of Technology.

An Interesting Observation Regarding Retrograde Vertebral Artery Flow in Patients With Dialysis Access Fistulas.

OBJECTIVES: Retrograde vertebral artery flow, the steal phenomenon, is most frequently caused by a flow-limiting stenosis of the proximal subclavian artery. The reversal of flow can be incomplete, resulting in bidirectional flow: retrograde in systole and antegrade in diastole. Less often, retrograde vertebral artery flow is the consequence of increased subclavian flow, as might occur with a well-functioning dialysis access fistula. Our objective was to evaluate bidirectional vertebral artery flow associated with dialysis access fistulas. METHODS: We retrospectively reviewed the direction of flow through the vertebral artery in systole and diastole of 335 patients with dialysis fistulas who had undergone extracranial cerebral vascular Doppler examinations. RESULTS: Fifteen patients had retrograde flow in their vertebral artery ipsilateral with the side of their fistula. There was completely reversed flow in 1 patient and bidirectional flow in the other 14. For each of these 14, the flow was antegrade in early systole and retrograde in diastole. Compression of the fistula restored the antegrade flow. CONCLUSIONS: Under conditions of reduced subclavian artery flow, bidirectional vertebral artery flow will be retrograde in early systole and antegrade in diastole. Under conditions of increased subclavian artery flow, bidirectional flow through the vertebral artery will be antegrade in early systole and retrograde in diastole.

Carotid Ultrasound Examinations: Indications Correlated With Abnormal Findings.

Our purpose was to evaluate the clinical indications for carotid duplex ultrasonography and determine for each indication how often carotid artery disease was identified.We retrospectively reviewed the consecutive reports for 3191 carotid ultrasound examinations. We tracked 14 indications to determine how often examinations were requested for each indication and correlated each indication with the finding of carotid artery disease.We found 26.5% of all examinations were abnormal; 17.5% of patients showed internal carotid artery disease; 10.9%, a moderate stenosis; 5.2%, a severe stenosis; and 1.4%, a total occlusion. For each of the 14 individual indications, with 1 exception, carotid ultrasound examination showed a 19.7% or greater chance of an abnormal result and an 11.6% or greater chance of finding a flow-limiting stenosis of an internal carotid artery.Our results validate the collaborative position of the American College of Radiology, the American Institute of Ultrasound in Medicine, the Society for Pediatric Radiology, and the Society of Radiologists in Ultrasound that, overall and for almost all indications they enumerate, carotid ultrasound examinations are a proven and useful procedure for evaluating extracranial carotid artery disease.

Prolonged seizure activity causes caspase dependent cleavage and dysfunction of G-protein activated inwardly rectifying potassium channels.

Recurrent high-frequency epileptic seizures cause progressive hippocampal sclerosis, which is associated with caspase-3 activation and NMDA receptor-dependent excitotoxicity. However, the identity of caspase-3 substrates that contribute to seizure-induced hippocampal atrophy remains largely unknown. Here, we show that prolonged high-frequency epileptiform discharges in cultured hippocampal neurons leads to caspase-dependent cleavage of GIRK1 and GIRK2, the major subunits of neuronal G protein-activated inwardly rectifying potassium (GIRK) channels that mediate membrane hyperpolarization and synaptic inhibition in the brain. We have identified caspase-3 cleavage sites in GIRK1 (387ECLD390) and GIRK2 (349YEVD352). The YEVD motif is highly conserved in GIRK2-4, and located within their C-terminal binding sites for Gßγ proteins that mediate membrane-delimited GIRK activation. Indeed, the cleaved GIRK2 displays reduced binding to Gßγ and cannot coassemble with GIRK1. Loss of an ER export motif upon cleavage of GIRK2 abolishes surface and current expression of GIRK2 homotetramic channels. Lastly, kainate-induced status epilepticus causes GIRK1 and GIRK2 cleavage in the hippocampus in vivo. Our findings are the first to show direct cleavage of GIRK1 and GIRK2 subunits by caspase-3, and suggest the possible role of caspase-3 mediated down-regulation of GIRK channel function and expression in hippocampal neuronal injury during prolonged epileptic seizures.

cAMP-dependent recruitment of acidic organelles for Ca2+ signaling in the salivary gland.

Autonomic neural activation of intracellular Ca2+ release in parotid acinar cells induces the secretion of the fluid and protein components of primary saliva critical for maintaining overall oral homeostasis. In the current study, we profiled the role of acidic organelles in shaping the Ca2+ signals of parotid acini using a variety of imaging and pharmacological approaches. Results demonstrate that zymogen granules predominate as an apically polarized population of acidic organelles that contributes to the initial Ca2+ release. Moreover, we provide evidence that indicates a role for the intracellular messenger NAADP in the release of Ca2+ from acidic organelles following elevation of cAMP. Our data are consistent with the "trigger" hypothesis where localized release of Ca2+ sensitizes canonical intracellular Ca2+ channels to enhance signals from the endoplasmic reticulum. Release from acidic stores may be important for initiating saliva secretion at low levels of stimulation and a potential therapeutic target to augment secretory activity in hypofunctioning salivary glands.

Radiologist, obstetric patient, and emergency department provider survey: radiologist-patient interaction in the emergency department setting.

The aim of this study was to evaluate the feasibility and acceptance of a model of direct interaction between radiologist and patients in the emergency department (ED) setting. The study population was comprised of pregnant patients accrued in a non-consecutive prospective manner from June 2014 to September 2015, who had an obstetrical ultrasound performed in the radiology department of an inner-city tertiary care hospital at the request of the ED. The feasibility and approval of direct communication between radiologist and patient were evaluated by means of a questionnaire presented by an independent observer to the ED provider, patient, and radiologist. The exam enrolled 54 patients. Ultrasound (US) exam results were divided into (31) normal live intrauterine gestation (group 1), (7) abnormal failed intrauterine gestation or ectopic pregnancy (group 2), and (16) indeterminate pregnancies that could not be placed in the former categories and may require a follow-up exam (group 3). Forty-five (83 %) ED providers approved of the radiologist's direct communication with patients. Fifty (93 %) patients stated a better understanding of the radiologist's role in their care after than before the interaction. The radiologists found the interaction with patients to be positive in 52 (96 %) cases. Direct communication between radiologist and patient yielded a good acceptance by the radiologist, ED provider, and patient. More importantly, after the encounter, the vast majority of patients reported a better understanding of the radiologist's role in their care.

Integration of host strain bioengineering and bioprocess development using ultra-scale down studies to select the optimum combination: an antibody fragment primary recovery case study.

An ultra scale-down primary recovery sequence was established for a platform E. coli Fab production process. It was used to evaluate the process robustness of various bioengineered strains. Centrifugal discharge in the initial dewatering stage was determined to be the major cause of cell breakage. The ability of cells to resist breakage was dependant on a combination of factors including host strain, vector, and fermentation strategy. Periplasmic extraction studies were conducted in shake flasks and it was demonstrated that key performance parameters such as Fab titre and nucleic acid concentrations were mimicked. The shake flask system also captured particle aggregation effects seen in a large scale stirred vessel, reproducing the fine particle size distribution that impacts the final centrifugal clarification stage. The use of scale-down primary recovery process sequences can be used to screen a larger number of engineered strains. This can lead to closer integration with and better feedback between strain development, fermentation development, and primary recovery studies.

Association between linear skull fractures and intracranial hemorrhage in children with minor head trauma.

BACKGROUND: To determine whether skull fractures can be used to associate intracranial hemorrhage with minor head trauma (MHT). OBJECTIVE: We conducted a retrospective study evaluating the association between linear skull fractures and intracranial hemorrhage among children with MHT. Furthermore, we evaluated the significance of small intracranial hemorrhages by assessing the need for neurosurgical interventions. MATERIALS AND METHODS: The case group included 114 children with a diagnosis of a linear skull fracture and the control group included 125 children without the diagnosis. We conducted multivariable logistic regression analyses to estimate the odds ratio (OR) between linear skull fractures and intracranial bleeding. RESULTS: Among the cases, 29 of 114 (25%) children were diagnosed with an intracranial hemorrhage on CT, compared to only 14 of 125 (11%) among the controls. The multivariable OR for intracranial hemorrhages comparing cases and controls adjusted for age and gender was 2.17 (95% confidence interval [CI]: 1.01, 4.68). All the intracranial hemorrhages were small (3.8 +/- 2.3 mm) and none of them required any neurosurgical intervention. CONCLUSION: The presence of a linear skull fracture is an independent risk factor for intracranial hemorrhage. However, all the intracranial hemorrhages associated with the skull fractures were small and did not require any neurosurgical interventions.

Production of fuculose-1-phosphate aldolase using operator-repressor titration for plasmid maintenance in high cell density Escherichia coli fermentations.

We report a novel application for the operator-repressor titration (ORT) plasmid maintenance system. The ability of ORT to maintain a plasmid during production of DNA has been demonstrated previously. In this study, we have used the ORT system to maintain a plasmid during high cell density cultivation and expression of a recombinant protein. No evidence of plasmid loss was seen during protein expression at high cell densities. In addition, the quantity of protein produced using this system was similar to traditional plasmid maintenance systems.