Transposase-mediated transgene integration for rapid generation of high-producing stable cell lines

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Characterization of cellular viability drop during media and platform development

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Dynamic regulation of mitochondrial metabolism as a strategy to maximize mAb production in industrial CHO cell cultures

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Creating a simulation model of INTEGRIS Women's and Children's Services and evaluating needed capacity

The 4th floor of The INTEGRIS Baptist Medical Center in OKC houses Women's and Children's Services, a unit which cares for expecting mothers and women going through labor. This unit operates 24 hours per day and 7 days per week and is operated using three sub-units: Triage, Labor Delivery & Recovery (LDR), and Postpartum. The Senior Design Team (SDT) worked in collaboration with the INTEGRIS Continuous Improvement Team (CIT) to create a simulation model to verify whether the recent recommendations in the capacity allocation of the sub-units, made in response to capacity shortages, adequately service the demand.The SDT began their investigation with a debriefing of previous analysis completed by the CIT. Their study characterized the arrival times, length of stay, and current capacity in each sub-unit. It was found that there was great consistency in admissions, transfers, and discharges both daily and seasonally. The capacity issues experienced by the Women's and Children's Services did not appear to be a result of swings in demand in any one sub-unit. Instead, the sub-units experienced insufficient capacity to meet patient demand. Bottlenecks on the 4th floor caused the typical flow of patients to be altered and compounded capacity issues exhibited in the three sub-units. These observations indicated that there was a need to do a detailed capacity analysis of the 4th floor and to this end we developed a discrete event simulation model.The SDT began the creation of a simulation model by fitting distributions to the data using MATLAB. These distributions were later used to create the simulation model submitted by the SDT. Then, Simio was used to construct a representation of 4th floor operations. The model was verified by peer review and test runs. There were no logical errors in the model and patient flow correctly depicted actual operations. The model was validated by comparing actual demand and length of stay from 2019 data to the results generated by the model. This comparison confirmed that the model accurately represented current operations in Women's and Children's Services.After the simulation model baseline was completed, various alternatives to increase capacity were tested with experiments in Simio. The following alternatives were considered to resolve the capacity issues experienced by the Women's & Children's Services:Triage sub-unito 4-bed option: increased capacity by 1 bedo 5-bed option: increased capacity by 2 bedso 6-bed option: increased capacity by 3 bedsLDR/Postpartum sub-unito Add rooms: added rooms to the LDR/Postpartum sub-unitso LDRP: combined the LDR/Postpartum sub-units by converting all beds in both sub-units to include equipment necessary to care for both LDR and Postpartum patientsAnalysis of the Triage sub-unit showed that the 5-bed option was the most effective method to increase capacity. This alternative decreased wait times by 93%. The 5-bed option incurred a greater cost than the 4-bed option due to physical renovation and equipment acquisition costs. However, unlike the other alternatives, the 5-bed option did not require physical separation of the sub-unit and a subsequent decrease in sub-unit visibility. Reduction in sub-unit visibility had a significantly negative impact on the 4th floor.Analysis of the LDR and Postpartum sub-units showed that a combination of adding rooms and the LDRP alternative was the most effective way to mitigate capacity issues. Significant improvements were realized by implementing this change and adding five additional rooms to the unit. This alternative decreased the number of patients who experience wait times by 92% and decreased patient wait times by 72%. Unfortunately, the Women's & Children's Services did not have the ability to increase capacity in either of these sub-units at all. This restriction made the LDRP alternative the only one available for them to resolve capacity issues. Implementation of this plan will decrease the number of patients who experience wait times by 57% and decrease patient wait times by 49%. Conversion to LDRP rooms was less costly than increasing the overall capacity of the two sub-units separately. Additionally, less time was required to clean and maintain the rooms because patients will not require room transfer during their stay. This additional benefit serves to streamline processes on the 4th floor and will help the unit the meet patient demand

Metabolic engineering of high-productivity CHO host lines for biomanufacturing

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Developing and assessing the feasibility of a home-based preexposure prophylaxis monitoring and support program

We piloted PrEP@Home, a preexposure prophylaxis system of remote laboratory and behavioral monitoring designed to replace routine quarterly follow-up visits with home care to reduce the patient and provider burden. The system was highly acceptable and in-demand for future use, and more than one-third of participants reported greater likelihood of persisting in care if available

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α 2 ββ'ωσ 70 ), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λP R and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of longlived λP R -discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λP R -discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles. RNA polymerase | open complex lifetime | transcription initiation | abortive RNA | hybrid length M any facets of transcription initiation by E. coli RNA polymerase (RNAP; α 2 ββ′ωσ 70 ) have been elucidated, but significant questions remain about the mechanism or mechanisms by which initial transcribing complexes (ITC) with a short RNA-DNA hybrid decide to advance and escape from the promoter to enter elongation mode, or, alternately, to stall, release their short RNA, and reinitiate (abortive cycling). For RNAP to escape, its sequencespecific interactions with promoter DNA in the binary open complex (OC) must be overcome. The open regions of promoter DNA in the binary OC are the −10 region (six residues, with specific interactions between σ 2.2 and the nontemplate strand), the discriminator region (typically six to eight residues with no consensus sequence, the upstream end of which interacts with σ 1.2 ), and the transcription start site (TSS, +1) and adjacent residue (+2), which are in the active site of RNAP What drives promoter escape? Escape involves disrupting all the favorable interactions involved in forming and stabilizing the binary OC as well as σ-core interactions. Escape from these interactions is fundamentally driven by the favorable chemical (free) energy change of RNA synthesis, but this energy must be stored in the ITC in each step before escape. Proposed means of energy storage as the length of the RNA-DNA hybrid increases include the stresses introduced by scrunching distortions of the discriminator regions of the open strands in the cleft (2, 5, 6) and by unfavorable interactions of the RNA-DNA hybrid with the hairpin loop of σ 3.2 (7-10). Scrunching of the discriminator region of the template strand is proposed to be most significant for Significance The enzyme RNA polymerase (RNAP) transcribes DNA genetic information into RNA. Regulation of transcription occurs largely in initiation; these regulatory mechanisms must be understood. Lifetimes of transcription-capable RNAP-promoter open complexes (OCs) vary greatly, dictated largely by the DNA discriminator region, but the significance of OC lifetime for regulation was unknown. We observe that a significantly longer RNA:DNA hybrid is synthesized before RNAP escapes from long-lived λP R -promoter OCs as compared with shorter-lived T7A1 promoter OCs. We quantify the free energy needed to overcome OC stability and allow escape from the promoter and elongation of the nascent RNA, and thereby predict escape points for ribosomal (rrnB P1) and lacUV5 promoters. Longer-lived OCs produce longer abortive RNAs, which likely have specific regulatory roles

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α 2 ββ'ωσ 70 ), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λP R and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of longlived λP R -discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λP R -discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles. RNA polymerase | open complex lifetime | transcription initiation | abortive RNA | hybrid length M any facets of transcription initiation by E. coli RNA polymerase (RNAP; α 2 ββ′ωσ 70 ) have been elucidated, but significant questions remain about the mechanism or mechanisms by which initial transcribing complexes (ITC) with a short RNA-DNA hybrid decide to advance and escape from the promoter to enter elongation mode, or, alternately, to stall, release their short RNA, and reinitiate (abortive cycling). For RNAP to escape, its sequencespecific interactions with promoter DNA in the binary open complex (OC) must be overcome. The open regions of promoter DNA in the binary OC are the −10 region (six residues, with specific interactions between σ 2.2 and the nontemplate strand), the discriminator region (typically six to eight residues with no consensus sequence, the upstream end of which interacts with σ 1.2 ), and the transcription start site (TSS, +1) and adjacent residue (+2), which are in the active site of RNAP What drives promoter escape? Escape involves disrupting all the favorable interactions involved in forming and stabilizing the binary OC as well as σ-core interactions. Escape from these interactions is fundamentally driven by the favorable chemical (free) energy change of RNA synthesis, but this energy must be stored in the ITC in each step before escape. Proposed means of energy storage as the length of the RNA-DNA hybrid increases include the stresses introduced by scrunching distortions of the discriminator regions of the open strands in the cleft (2, 5, 6) and by unfavorable interactions of the RNA-DNA hybrid with the hairpin loop of σ 3.2 (7-10). Scrunching of the discriminator region of the template strand is proposed to be most significant for Significance The enzyme RNA polymerase (RNAP) transcribes DNA genetic information into RNA. Regulation of transcription occurs largely in initiation; these regulatory mechanisms must be understood. Lifetimes of transcription-capable RNAP-promoter open complexes (OCs) vary greatly, dictated largely by the DNA discriminator region, but the significance of OC lifetime for regulation was unknown. We observe that a significantly longer RNA:DNA hybrid is synthesized before RNAP escapes from long-lived λP R -promoter OCs as compared with shorter-lived T7A1 promoter OCs. We quantify the free energy needed to overcome OC stability and allow escape from the promoter and elongation of the nascent RNA, and thereby predict escape points for ribosomal (rrnB P1) and lacUV5 promoters. Longer-lived OCs produce longer abortive RNAs, which likely have specific regulatory roles