Optimizing and developing a scalable, chemically defined, animal component-free lentiviral vector production process in a fixed-bed bioreactor.

Lentiviral vectors (LVVs) play a critical role in gene delivery for ex vivo gene-modified cell therapies. However, the lack of scalable LVV production methods and the high cost associated with them may limit their use. In this work, we demonstrate the optimization and development of a scalable, chemically defined, animal component-free LVV production process using adherent human embryonic kidney 293T cells in a fixed-bed bioreactor. The initial studies focused on the optimization of the culture process in 2D static cultures. Process changes such as decreasing cell seeding density on day 0 from 2.5 × 104 to 5 × 103 cells/cm2, delaying the transient transfection from 24 to 120 h post-seeding, reducing plasmid DNA to 167 ng/cm2, and adding 5 mM sodium butyrate 6 h post-transfection improved functional LVV titers by 26.9-fold. The optimized animal component-free production process was then transferred to the iCELLis Nano bioreactor, a fixed-bed bioreactor, where titers of 1.2 × 106 TU/cm2 were achieved when it was operated in perfusion. In this work, comparable functional LVV titers were obtained with FreeStyle 293 Expression medium and the conventional Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum both at small and large scale.

Cationic polymer precipitation for enhanced impurity removal in downstream processing.

Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation-depth filtration and the current platform process for the purification of biologics.

Defects in vein valve PROX1/FOXC2 antithrombotic pathway in endothelial cells drive the hypercoagulable state induced by trauma and critical illness.

OBJECTIVES: Deep venous thrombosis (DVT) causes significant morbidity and mortality after trauma. Recently, we have shown that blood flow patterns at vein valves induce oscillatory stress genes, which maintain an anticoagulant endothelial phenotype that inhibits spontaneous clotting at vein valves and sinuses, is lost in the presence of DVT in human pathological samples, and is dependent on expression of the transcription factor FOXC2. We describe an assay, modifying our mouse multiple injury system, which shows evidence of clinically relevant microthrombosis and hypercoagulability applicable to the study of spontaneous DVT in trauma without requiring direct vascular injury or ligation. Finally, we investigated whether these model findings are relevant to a human model of critical illness by examining gene expression changes by quantitative polymerase chain reaction and immunofluorescence in veins collected from critically ill. METHODS: C57/Bl6 mice were subjected to a modified mouse multiple injury model with liver crush injury, crush and pseudofracture of a single lower extremity, and 15% total blood volume hemorrhage. Serum was assayed for d-dimer at 2, 6, 24, and 48 hours after injury by enzyme-linked immunosorbent assay. For the thrombin clotting assay, veins of the leg were exposed, 100 µL of 1 mM rhodamine (6 g) was injected retro-orbitally, and 450 µg/mL thrombin was then applied to the surface of the vein with examination of real-time clot formation via in vivo immunofluorescence microscopy. Images were then examined for percentage area of clot coverage of visible mouse saphenous and common femoral vein. Vein valve specific knockout of FOXC2 was induced with tamoxifen treatment in PROX1 Ert2Cre FOXC2 fl/fl mice as previously described. Animals were then subjected to a modified mouse multiple injury model with liver crush injury, crush and pseudofracture of a single lower extremity, and 15% total blood volume hemorrhage. Twenty-four hours after injury, we examined the valve phenotype in naive versus multiple injury animals, with and without loss of the FOXC2 gene from the vein valve (FOXC2 del ) via the thrombin assay. Images were then examined for proximity of clot formation to the valve present at the junction of the mouse saphenous, tibial, and superficial femoral vein and presence of spontaneous microthrombi present in the veins before exposure to thrombin. Human vein samples were obtained from excess tissue preserved after harvest for elective cardiac surgery and from organ donors after organ procurement. Sections were submitted for paraffin embedding and then assayed by immunofluorescence for PROX1, FOXC2, thrombomodulin, endothelial protein C receptor, and von Willebrand's factor. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee, and all human studies reviewed and approved by the institutional review board. RESULTS: After mouse multiple injuries, enzyme-linked immunosorbent assay for d-dimer showed evidence of products of fibrin breakdown consistent with formation of clot related to injury, fibrinolysis, and/or microthrombosis. The thrombin clotting assay demonstrated higher percentage area of vein covered with clot when exposed to thrombin in the multiple injury animals compared with uninjured (45% vs. 27% p = 0.0002) consistent with a phenotype of hypercoagulable state after trauma in our model system. Unmanipulated FoxC2 knockout mice manifest increased clotting at the vein valve as compared with unmanipulated wild type animals. After multiple injuries, wild type mice manifest increase clotting at the vein after thrombin exposure ( p = 0.0033), and equivalent to that of valvular knockout of FoxC2 (FoxC2del), recapitulating the phenotype seen in FoxC2 knockout animals. The combination of multiple injuries and FoxC2 knockout resulted in spontaneous microthrombi in 50% of the animals, a phenotype not observed with either multiple injuries or FoxC2 deficiency alone (χ 2 , p = 0.017). Finally, human vein samples demonstrated the protective vein valve phenotype of increased FOXC2 and PROX1 and showed decreased expression in the critically ill organ donor population by immunofluorescence imaging in organ donor samples. CONCLUSION: We have established a novel model of posttrauma hypercoagulation that does not require direct restriction of venous flow or direct injury to the vessel endothelium to assay for hypercoagulability and can generate spontaneous microthrombosis when combined with valve-specific FOXC2 knockout. We find that multiple injuries induce a procoagulant phenotype that recapitulates the valvular hypercoagulability seen in FOXC2 knockout and, in critically ill human specimens, find evidence for loss of oscillatory shear stress-induced gene expression of FOXC2 and PROX1 in the valvular endothelium consistent with potential loss of DVT-protective valvular phenotype.

Assessing non-adjunctive CGM safety at home and in new markets (ANSHIN).

INTRODUCTION: Continuous glucose monitoring (CGM) can guide treatment for people with type 1 (T1D) and type 2 diabetes (T2D). The ANSHIN study assessed the impact of non-adjunctive CGM use in adults with diabetes using intensive insulin therapy (IIT). MATERIALS AND METHODS: This single-arm, prospective, interventional study enrolled adults with T1D or T2D who had not used CGM in the prior 6 months. Participants wore blinded CGMs (Dexcom G6) during a 20-day run-in phase, with treatment based on fingerstick glucose values, followed by a 16-week intervention phase and then a randomized 12-week extension phase with treatment based on CGM values. The primary outcome was change in HbA1c. Secondary outcomes were CGM metrics. Safety endpoints were the number of severe hypoglycaemic (SH) and diabetic ketoacidosis (DKA) events. RESULTS: Of the 77 adults enrolled, 63 completed the study. Those enrolled had mean (SD) baseline HbA1c of 9.8% (1.9%), 36% had T1D, and 44% were ≥65 years old. Mean HbA1c decreased by 1.3, 1.0 and 1.0 percentage points for participants with T1D, T2D or age ≥65, respectively (p < .001 for each). CGM-based metrics including time in range also improved significantly. SH events decreased from the run-in period (67.3 per 100 person-years) to the intervention period (17.0 per 100 person-years). Three DKA events unrelated to CGM use occurred during the total intervention period. CONCLUSIONS: Non-adjunctive use of the Dexcom G6 CGM system improved glycaemic control and was safe for adults using IIT.

Medial Patellofemoral Ligament Reconstruction in Obese Patients Results in Low Complication Rates and Improved Subjective Outcomes.

Purpose: To compare outcomes, activity scores, and complication rates of obese and non-obese patients undergoing medial patellofemoral ligament (MPFL) reconstruction. Methods: A retrospective review identified patients undergoing MPFL reconstruction for recurrent patellofemoral instability. Patients were included if they had undergone MPFL reconstruction and had follow-up for a minimum of 6 months. Patients were excluded if they underwent surgery less than 6 months earlier, had no outcome data recorded, or underwent concomitant bony procedures. Patients were divided into 2 groups based on body mass index (BMI): BMI of 30 or greater and BMI less than 30. Presurgical and postsurgical patient-reported outcomes including Knee Injury and Osteoarthritis Outcome Score (KOOS) domains and the Tegner score were collected. Complications requiring reoperation were recorded. P < .05 was defined as a statistically significant difference. Results: A total of 55 patients (57 knees) were included. There were 26 knees with a BMI of 30 or greater and 31 knees with a BMI less than 30. There were no differences in patient demographic characteristics between the 2 groups. Preoperatively, no significant differences were found in KOOS subscores or Tegner scores (P = .21) between groups. At minimum 6-month follow-up (range, 6.1-70.5 months), patients with a BMI of 30 or greater showed statistically significant improvements in the KOOS Pain, Activities of Daily Living, Symptoms, and Sport/Recreation subscores. Patients with a BMI less than 30 showed a statistically significant improvement in the KOOS Quality of Life subscore. The group with a BMI of 30 or greater had significantly lower KOOS Quality of Life (33.34 ± 19.10 vs 54.47 ± 28.00, P = .03) and Tegner (2.56 ± 1.59 vs 4.78 ± 2.68, P = .05) scores. Complication rates were low, with 2 knees (7.69%) requiring reoperation in the cohort with a BMI of 30 or greater and 4 knees (12.90%) requiring reoperation in the cohort with a BMI less than 30, including 1 reoperation for recurrent patellofemoral instability (P = .68). Conclusions: In this study, MPFL reconstruction in obese patients was safe and effective, with low complication rates and improvements in most patient-reported outcomes. Compared with patients with a BMI less than 30, obese patients had lower quality-of-life and activity scores at final follow-up. Level of Evidence: Level III, retrospective cohort study.

Isotherm model discrimination for multimodal chromatography using mechanistic models derived from high-throughput batch isotherm data.

In this work, we have examined an array of isotherm formalisms and characterized them based on their relative complexities and predictive abilities with multimodal chromatography. The set of isotherm models studied were all based on the stoichiometric displacement framework, with considerations for electrostatic interactions, hydrophobic interactions, and thermodynamic activities. Isotherm parameters for each model were first determined through twenty repeated fits to a set of mAb - Capto MMC batch isotherm data spanning a range of loading, ionic strength, and pH as well as a set of mAb - Capto Adhere batch data at constant pH. The batch isotherm data were used in two ways-spanning the full range of loading or consisting of only the high concentration data points. Predictive ability was defined through the model's capacity to capture prominent changes in salt gradient elution behavior with respect to pH for Capto MMC or unique elution patterns and yield losses with respect to gradient slope for Capto Adhere. In both cases, model performance was quantified using a scoring metric based on agreement in peak characteristics for column predictions and accuracy of fit for the batch data. These scores were evaluated for all twenty isotherm fits and their corresponding column predictions, thereby producing a statistical distribution of model performances. Model complexity (number of isotherm parameters) was then considered through use of the Akaike information criterion (AIC) calculated from the score distributions. While model performance for Capto MMC benefitted substantially from removal of low protein concentration data, this was not the case for Capto Adhere; this difference was likely due to the qualitatively different shapes of the isotherms between the two resins. Surprisingly, the top-performing (high accuracy with minimal number of parameters) isotherm model was the same for both resins. The extended steric mass action (SMA) isotherm (containing both protein-salt and protein-protein activity terms) accurately captured both the pH-dependent elution behavior for Capto MMC as well as loss in protein recovery with increasing gradient slope for Capto Adhere. In addition, this isotherm model achieved the highest median score in both resin systems, despite it lacking any explicit hydrophobic stoichiometric terms. The more complex isotherm models, which explicitly accounted for both electrostatic and hydrophobic interaction stoichiometries, were ill-suited for Capto MMC and had lower AIC model likelihoods for Capto Adhere due to their increased complexity. Interestingly, the ability of the extended SMA isotherm to predict the Capto Adhere results was largely due to the protein-salt activity coefficient, as determined via isotherm parameter sensitivity analyses. Further, parametric studies on this parameter demonstrated that it had a major impact on both binding affinity and elution behavior, therein fully capturing the impact of hydrophobic interactions. In summary, we were able to determine the isotherm formalisms most capable of consistently predicting a wide range of column behavior for both a multimodal cation-exchange and multimodal anion-exchange resin with high accuracy, while containing a minimized set of model parameters.

Relationship of Impairments in Associative Learning With Intellectual Disability and Cerebellar Hypoplasia in Autistic Children.

BACKGROUND AND OBJECTIVES: The severity of autism spectrum disorder (ASD) varies widely and is associated with intellectual disability (ID) and brain dysmorphology. We tested the hypothesis that the heterogeneity of ASD can be accounted for, in part, by altered associative learning measured by eye-blink conditioning (EBC) paradigms, used to test for forebrain and cerebellar dysfunction across the full range of ASD severity and intellectual ability. METHODS: Children in this cohort study were diagnosed with ASD or typical development (TD); most children were recruited from a 10-year longitudinal study. Outcome measures were the percentage and timing of conditioned eye-blink responses (CRs) acquired to a tone, recorded photometrically and related to measures of ASD severity, IQ, and age 2 brain morphometry by MRI. A sequence of trace and delay EBC was used. Analysis of variance, t test, and logistic regression (LR) were used. RESULTS: Sixty-two children were studied at school age. Nine children with ASD with ID since age 2 (ASD + ID; IQ = 49 ± 6; 11.9 ± 0.2 years old [±SD]) learned more slowly than 30 children with TD (IQ = 120 ± 16; 10.5 ± 1.5 years old [±SD]) during trace EBC and showed atypically early-onset CRs (1.4 SD pre-TD) related to hypoplasia of the cerebellum at age 2 but not of the amygdala, hippocampus, or cerebral cortex. Conversely, 16 children with ASD with robust intellectual development since age 2 (IQ = 100 ± 3; 12.0 ± 0.4 years old [±SD]) learned typically but showed early-onset CRs only during long-delay EBC (0.8 SD pre-TD) unrelated to hypoplasia of any measured brain area. Using 16 EBC measures, binary LR classified ASD and TD with 80% accuracy (95% CI = 72-88%), 81% sensitivity (95% CI = 69-92%), and 79% specificity (95% CI = 68-91%); multinomial LR more accurately classified children based on ID (94% accuracy, 95% CI = 89-100%) than ASD severity (85% accuracy, 95% CI = 77-93%). Separate analyses of 39 children with MRI (2.1 ± 0.3 years old [±SD]) indicated that cerebellar hypoplasia did not predict ASD + ID over ages 2-4 (Cohen d = 0.3) compared with early-onset CRs during age 11 trace EBC (Cohen d = -1.3). DISCUSSION: Trace EBC reveals the relationship between cerebellar hypoplasia and ASD + ID likely by engaging cerebrocerebellar circuits involved in intellectual ability and implicit timing. Follow-up prospective studies using associative learning can determine whether ID can be predicted in children with early ASD diagnoses.

A high-throughput approach to developing and optimizing mixed-mode membrane chromatography for protein purification.

Membrane chromatography has been established as a viable alternative to packed-bed column chromatography for the purification of therapeutic proteins. Purification via membrane chromatography offers key advantages, including higher productivity and reduced buffer usage. Unlike column chromatography purification, the utilization of high-throughput screening in order to reduce development times and material requirements has been a challenge for membrane chromatography. This research focused on the development of a new, high-throughput screening technique for use in screening membrane chromatography conditions for monoclonal antibody purification. The developed screen utilizes a 96-well plate format, thereby allowing for the screening of multiple different membrane conditions at once. For this study, four mixed-mode cation exchange membranes and one cation exchange membrane were evaluated on the plate. The screen is performed in a similar manner to that of a resin slurry plate screen, however, instead of a single loading step, the antibody feed was loaded in 50 mg/ml increments up to a maximum loading of 450 mg/ml. Performing a similar, incremental loading on a resin plate would be impractical, as mixing times are substantially longer due to pore diffusion limitations. However, due to the significantly faster rate of mass transfer for membranes relative to resin, mixing times could be reduced by up to a factor of sixty on the membrane plate. Additional optimization showed that higher hydrophobicity can potentially lead to slower kinetics and mixing times that may need to be adjusted accordingly. The end result is a screen that has been proven to provide results comparable to those obtained on larger-scale membrane purification runs while also enabling exploration of a much greater operating space and significantly reducing the feed materials required.

Comparisons of Fifth-, Sixth-, and Seventh-Generation Continuous Glucose Monitoring Systems.

BACKGROUND: Between-system differences for continuous glucose monitoring (CGM) devices have important clinical consequences. PURPOSE: Here we review attributes of Dexcom's fifth-, sixth-, and seventh-generation (G5, G6, and G7) CGM systems. METHODS: Accuracy metrics were derived from preapproval trials of the three systems and compared after propensity score adjustments were used to balance baseline demographic characteristics. Metrics included mean absolute relative differences (MARD) between CGM and YSI values and the proportion of CGM values within 20% or 20 mg/dL of the YSI values ("%20/20"). Ease-of-use was evaluated by formal task analysis. CONCLUSIONS: Adjusted MARD and %20/20 agreement rates were 9.0%/93.1% (abdomen-placed G5), 9.9%/92.3% (abdomen-placed G6), 9.1%/93.2% (abdomen-placed G7), and 8.2%/95.3% (arm-placed G7). Task analysis favored G7 over earlier systems. Favorable clinical outcomes such as hemoglobin A1c reduction and hypoglycemia avoidance seen with G5 and G6 are anticipated with G7 use.

Accuracy and Safety of Dexcom G7 Continuous Glucose Monitoring in Adults with Diabetes.

Background: We evaluated the accuracy and safety of a seventh generation (G7) Dexcom continuous glucose monitor (CGM) during 10.5 days of use in adults with diabetes. Methods: Adults with either type 1 or type 2 diabetes (on intensive insulin therapy or not) participated at 12 investigational sites in the United States. In-clinic visits were conducted on days 1 or 2, 4 or 7, and on the second half of day 10 or the first half of day 11 for frequent comparisons with comparator blood glucose measurements obtained with the YSI 2300 Stat Plus glucose analyzer. Participants wore sensors concurrently on the upper arm and abdomen. Accuracy evaluation included the proportion of CGM values within 15% of comparator glucose levels >100 mg/dL or within 15 mg/dL of comparator levels ≤100 mg/dL (%15/15), along with the %20/20 and %30/30 agreement rates. The mean absolute relative difference (MARD) between temporally matched CGM and comparator values was also calculated. Results: Data from 316 participants (619 sensors, 77,774 matched pairs) were analyzed. For arm- and abdomen-placed sensors, overall MARDs were 8.2% and 9.1%, respectively. Overall %15/15, %20/20, and %30/30 agreement rates were 89.6%, 95.3%, and 98.8% for arm-placed sensors and were 85.5%, 93.2%, and 98.1% for abdomen-placed sensors. Across days of wear, glucose concentration ranges, and rates of change, %20/20 agreement rates varied by no more than 9% from the overall %20/20. No serious adverse events were reported. Conclusions: The G7 CGM provides accurate glucose readings with single-digit MARD with arm or abdomen placement in adults with diabetes. Clinicaltrials.gov: NCT04794478.

Propylene Glycol Caprylate-Based Nanoemulsion Formulation of Plumbagin: Development and Characterization of Anticancer Activity.

Plumbagin, a bioactive naphthoquinone, has demonstrated potent antitumor potential. However, plumbagin is a sparingly water-soluble compound; therefore, clinical translation requires and will be facilitated by the development of a new pharmaceutical formulation. We have generated an oil-in-water nanoemulsion formulation of plumbagin using a low-energy spontaneous emulsification process with propylene glycol caprylate (Capryol 90) as an oil phase and Labrasol/Kolliphor RH40 as surfactant and cosurfactant excipients. Formulation studies using Capryol 90/Labrasol/Kolliphor RH40 components, based on pseudoternary diagram and analysis of particle size distribution and polydispersity determined by dynamic light scattering (DLS), identified an optimized composition of excipients for nanoparticle formulation. The nanoemulsion loaded with plumbagin as an active pharmaceutical ingredient had an average hydrodynamic diameter of 30.9 nm with narrow polydispersity. The nanoemulsion exhibited long-term stability, as well as good retention of particle size in simulated physiological environments. Furthermore, plumbagin-loaded nanoemulsion showed an augmented cytotoxicity against prostate cancer cells PTEN-P2 in comparison to free drug. In conclusion, we generated a formulation of plumbagin with high loading drug capacity, robust stability, and scalable production. Novel Capryol 90-based nanoemulsion formulation of plumbagin demonstrated antiproliferative activity against prostate cancer cells, warranting thus further pharmaceutical development.

Mitigation of Rebound Hyperglycemia With Real-Time Continuous Glucose Monitoring Data and Predictive Alerts.

BACKGROUND: Excess carbohydrate intake during hypoglycemia can lead to rebound hyperglycemia (RH). We investigated associations between RH and use of real-time continuous glucose monitoring (rtCGM) and an rtCGM system's predictive alert. METHODS: RH events were series of sensor glucose values (SGVs) >180 mg/dL starting within two hours of an antecedent SGV <70 mg/dL. Events were characterized by their frequency, duration (consecutive SGVs >180 mg/dL × five minutes), and severity (area under the glucose concentration-time curve). To assess the impact of rtCGM, data gathered during the four-week baseline phase (without rtCGM) and four-week follow-up phase (with rtCGM) from 75 participants in the HypoDE clinical trial (NCT02671968) of hypoglycemia-unaware individuals were compared. To assess the impact of predictive alerts, we identified a convenience sample of 24 518 users of an rtCGM system without predictive alerts who transitioned to a system whose predictive alert signals an SGV ≤55 mg/dL within 20 minutes (Dexcom G5 and G6, respectively). RH events from periods of blinded versus unblinded rtCGM wear and from periods of G5 and G6 wear were compared with paired t tests. RESULTS: Compared to RH events in the HypoDE baseline phase, the mean frequency, duration, and severity of events fell by 14%, 12%, and 23%, respectively, in the follow-up phase (all P < .05). Compared to RH events during G5 use, the mean frequency, duration, and severity of events fell by 7%, 8%, and 13%, respectively, during G6 use (all P < .001). CONCLUSIONS: Rebound hypreglycemia can be objectively quantified and mitigated with rtCGM and rtCGM-based predictive alerts.

Synaptic protein interaction networks encode experience by assuming stimulus-specific and brain-region-specific states.

A core network of widely expressed proteins within the glutamatergic post-synapse mediates activity-dependent synaptic plasticity throughout the brain, but the specific proteomic composition of synapses differs between brain regions. Here, we address the question, how does proteomic composition affect activity-dependent protein-protein interaction networks (PINs) downstream of synaptic activity? Using quantitative multiplex co-immunoprecipitation, we compare the PIN response of in vivo or ex vivo neurons derived from different brain regions to activation by different agonists or different forms of eyeblink conditioning. We report that PINs discriminate between incoming stimuli using differential kinetics of overlapping and non-overlapping PIN parameters. Further, these "molecular logic rules" differ by brain region. We conclude that although the PIN of the glutamatergic post-synapse is expressed widely throughout the brain, its activity-dependent dynamics show remarkable stimulus-specific and brain-region-specific diversity. This diversity may help explain the challenges in developing molecule-specific drug therapies for neurological disorders.

Liposome Sterile Filtration Characterization via X-ray Computed Tomography and Confocal Microscopy.

Two high resolution, 3D imaging techniques were applied to visualize and characterize sterilizing grade dual-layer filtration of liposomes, enabling membrane structure to be related with function and performance. Two polyethersulfone membranes with nominal retention ratings of 650 nm and 200 nm were used to filter liposomes of an average diameter of 143 nm and a polydispersity index of 0.1. Operating conditions including differential pressure were evaluated. X-ray computed tomography at a pixel size of 63 nm was capable of resolving the internal geometry of each membrane. The respective asymmetry and symmetry of the upstream and downstream membranes could be measured, with pore network modeling used to identify pore sizes as a function of distance through the imaged volume. Reconstructed 3D digital datasets were the basis of tortuous flow simulation through each porous structure. Confocal microscopy visualized liposome retention within each membrane using fluorescent dyes, with bacterial challenges also performed. It was found that increasing pressure drop from 0.07 MPa to 0.21 MPa resulted in differing fluorescent retention profiles in the upstream membrane. These results highlighted the capability for complementary imaging approaches to deepen understanding of liposome sterilizing grade filtration.

Association between attendance at an American diabetes camp and improvements in glycaemic control and treatment satisfaction.

INTRODUCTION: Few studies have evaluated glycaemic control using continuous glucose monitoring (CGM) in individuals before and after attendance at a diabetes camp or by comparing control groups at home to control groups at camp. METHODS: Youth (6-17 years) with T1D and receiving insulin therapy were enrolled at a week-long diabetes camp. They participated in three clinic visits: at the start of a week at home, by initiating a Dexcom G6 CGM system; at the start of a week at camp, where the home week G6 was removed and a camp week G6 was inserted; and after camp, where the camp week G6 was removed. We administered Problem Areas in Diabetes (PAID) surveys at the second and third visits. Participants with <80% CGM data coverage or who did not complete all PAID surveys were excluded from analysis. We compared glycaemic control and PAID scores between the week at home and week at camp. RESULTS: Of 76 enrolled campers, 69 completed the study and 52 had results that qualified for analysis. The mean participant age was 12.5 ± 2.2 years. Camp was associated with significantly improved treatment satisfaction, time in desired glucose range and insulin sensitivity. Time in hyperglycaemia and basal insulin requirements decreased significantly. CONCLUSIONS: Diabetes camp is associated with significant improvements in diabetes treatment satisfaction and glycaemic control compared to home care.

Remodeling of the Homer-Shank interactome mediates homeostatic plasticity.

Neurons maintain stable levels of excitability using homeostatic synaptic scaling, which adjusts the strength of a neuron's postsynaptic inputs to compensate for extended changes in overall activity. Here, we investigated whether prolonged changes in activity affect network-level protein interactions at the synapse. We assessed a glutamatergic synapse protein interaction network (PIN) composed of 380 binary associations among 21 protein members in mouse neurons. Manipulating the activation of cultured mouse cortical neurons induced widespread bidirectional PIN alterations that reflected rapid rearrangements of glutamate receptor associations involving synaptic scaffold remodeling. Sensory deprivation of the barrel cortex in live mice (by whisker trimming) caused specific PIN rearrangements, including changes in the association between the glutamate receptor mGluR5 and the kinase Fyn. These observations are consistent with emerging models of experience-dependent plasticity involving multiple types of homeostatic responses. However, mice lacking Homer1 or Shank3B did not undergo normal PIN rearrangements, suggesting that the proteins encoded by these autism spectrum disorder-linked genes serve as structural hubs for synaptic homeostasis. Our approach demonstrates how changes in the protein content of synapses during homeostatic plasticity translate into functional PIN alterations that mediate changes in neuron excitability.

Sustainable Use of a Real-Time Continuous Glucose Monitoring System from 2018 to 2020.

We aimed to describe patterns of continuous glucose monitoring (CGM) system use and glycemic outcomes from 2018 to 2020 in a large real-world cohort by analyzing anonymized data from US-based CGM users who transitioned from the G5 to the G6 System (Dexcom) in 2018. The main end points were persistent use, within-day and between-day utilization, hypoglycemia, time in range (TIR, 70-180 mg/dL [3.9-10 mmol/L]), and use of the optional calibration feature in 2019 and 2020. In a cohort of 31,034 individuals, rates of persistent use were high, with 27,932 (90.0%) and 26,861 (86.6%) continuing to upload data in 2019 and 2020, respectively. Compared with G5 use, G6 use was associated with higher device utilization, less hypoglycemia, higher TIR (in 2020), and >80% fewer calibrations in both 2019 and 2020 (P's < 0.001). High persistence and utilization of the G6 system may contribute to sustainable glycemic outcomes and decreased user burden.

Real-Time Continuous Glucose Monitoring During the Coronavirus Disease 2019 Pandemic and Its Impact on Time in Range.

Background: The coronavirus disease 2019 (COVID-19) pandemic disrupted the lives of people with diabetes. Use of real-time continuous glucose monitoring (rtCGM) helped manage diabetes effectively. Some of these disruptions may be reflected in population-scale changes to metrics of glycemic control, such as time-in-range (TIR). Methods: We examined data from 65,067 U.S.-based users of the G6 rtCGM System (Dexcom, Inc., San Diego, CA) who had uploaded data before and during the COVID-19 pandemic. Users associated with three counties that included the cities of Los Angeles, Chicago, and New York or with five regions designated by the Centers for Disease Control and Prevention (CDC) were compared. Public data were used to associate regions with prepandemic and intrapandemic glycemic parameters, COVID-19 mortality, and median household income. Results: Compared with an 8-week prepandemic interval before stay-at-home orders (January 6, 2020, to March 1, 2020), overall mean (standard deviation) TIR improved from 59.0 (20.1)% to 61.0 (20.4)% during the early pandemic period (April 20, 2020 to June 14, 2020, P < 0.001). TIR improvements were noted in all three counties and in all five CDC-designated regions. Higher COVID-19 mortality was associated with higher proportions of individuals experiencing TIR improvements of ≥5 percentage points. Users in economically wealthier zip codes had higher pre- and intrapandemic TIR values and greater relative improvements in TIR. TIR and pandemic-related improvements in TIR varied across CDC-designated regions. Conclusions: Population-level rtCGM data may be used to monitor changes in glycemic control with temporal and geographic specificity. The COVID-19 pandemic is associated with improvements in TIR, which were not evenly distributed across the United States.