Exploration of the Performance of a Hybrid Closed Loop Insulin Delivery Algorithm That Includes Insulin Delivery Limits Designed to Protect Against Hypoglycemia.

BACKGROUND: Hypoglycemia remains a risk for closed loop insulin delivery particularly following exercise or if the glucose sensor is inaccurate. The aim of this study was to test whether an algorithm that includes a limit to insulin delivery is effective at protecting against hypoglycemia under those circumstances. METHODS: An observational study on 8 participants with type 1 diabetes was conducted, where a hybrid closed loop system (HCL) (Medtronic™ 670G) was challenged with hypoglycemic stimuli: exercise and an overreading glucose sensor. RESULTS: There was no overnight or exercise-induced hypoglycemia during HCL insulin delivery. All daytime hypoglycemia was attributable to postmeal bolused insulin in those participants with a more aggressive carbohydrate factor. CONCLUSION: HCL systems rely on accurate carbohydrate ratios and carbohydrate counting to avoid hypoglycemia. The algorithm that was tested against moderate exercise and an overreading glucose sensor performed well in terms of hypoglycemia avoidance. Algorithm refinement continues in preparation for long-term outpatient trials.

Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil.

BACKGROUND: The ability to modulate levels of individual fatty acids within soybean oil has potential to increase shelf-life and frying stability and to improve nutritional characteristics. Commodity soybean oil contains high levels of polyunsaturated linoleic and linolenic acid, which contribute to oxidative instability - a problem that has been addressed through partial hydrogenation. However, partial hydrogenation increases levels of trans-fatty acids, which have been associated with cardiovascular disease. Previously, we generated soybean lines with knockout mutations within fatty acid desaturase 2-1A (FAD2-1A) and FAD2-1B genes, resulting in oil with increased levels of monounsaturated oleic acid (18:1) and decreased levels of linoleic (18:2) and linolenic acid (18:3). Here, we stack mutations within FAD2-1A and FAD2-1B with mutations in fatty acid desaturase 3A (FAD3A) to further decrease levels of linolenic acid. Mutations were introduced into FAD3A by directly delivering TALENs into fad2-1a fad2-1b soybean plants. RESULTS: Oil from fad2-1a fad2-1b fad3a plants had significantly lower levels of linolenic acid (2.5 %), as compared to fad2-1a fad2-1b plants (4.7 %). Furthermore, oil had significantly lower levels of linoleic acid (2.7 % compared to 5.1 %) and significantly higher levels of oleic acid (82.2 % compared to 77.5 %). Transgene-free fad2-1a fad2-1b fad3a soybean lines were identified. CONCLUSIONS: The methods presented here provide an efficient means for using sequence-specific nucleases to stack quality traits in soybean. The resulting product comprised oleic acid levels above 80 % and linoleic and linolenic acid levels below 3 %.

Continuous Glucose Monitoring Adherence: Lessons From a Clinical Trial to Predict Outpatient Behavior.

AIMS: This study reports continuous glucose monitoring (CGM) adherence patterns and contributing factors in patients who were part of a 6-month clinical trial using sensor augmented pump therapy with low glucose insulin suspension. METHODS: CGM data from 38 patients using sensor augmented pump therapy for 6 months were analyzed. CGM adherence was defined by having a working sensor available and determined by the time it was switched on as a proportion of available time for the 6 month study period with allowance for practical CGM use. Age, gender, HbA1c, duration of diagnosis, capillary blood glucose testing frequency, sensor accuracy, and insulin pump alarm frequency were characterized and examined for an association with CGM adherence. RESULTS: Overall CGM adherence was 75% (range: 35% to 96%), CGM adherence was demonstrated to fall after 9 to 11 weeks before reaching a steady rate. CGM adherence patterns showed substantial variation. Mean adherence differed (P < .01) between age groups 72% (<12 years), 69% (12-18 years), and 88% (≥18 years). Sensor accuracy predicted adherence, where every 1% decline in mean absolute difference in a given week was associated with a 0.5% decline in sensor adherence (P < .01). Gender, HbA1c, duration of diagnosis, capillary blood glucose testing frequency, and insulin pump alarm frequency were not associated with CGM adherence. CONCLUSIONS: CGM adherence and patterns of use are individualized. However, a predictable fall in adherence at 9 to 11 weeks may present an opportunity for timed interventions to increase CGM use. Adolescent age and sensor accuracy predict CGM adherence.

Effect of Exercise Intensity on Glucose Requirements to Maintain Euglycemia During Exercise in Type 1 Diabetes.

CONTEXT: No recommendations exist to inform the carbohydrate amount required to prevent hypoglycemia associated with exercise of different intensities in individuals with type 1 diabetes (T1D). OBJECTIVE: The relationship between exercise intensity and carbohydrate requirements to maintain stable euglycemia in individuals with T1D remains to be determined. It was predicted that an "inverted-U" relationship exists between exercise intensity and the amount of glucose required to prevent hypoglycemia during exercise at basal insulinemia. Our objective was to investigate this relationship and elucidate the underlying glucoregulatory mechanisms. DESIGN, PARTICIPANTS, AND INTERVENTION: We subjected nine individuals (mean ± SD age, 21.5 ± 4.0 years; duration of disease, 11.4 ± 6.4 years; glycated hemoglobin, 7.9 ± 0.8% [60 mmol/mol]; body mass index, 25.4 ± 5.5 kg/m(2); VO2peak, 34.8 ± 5.1 mL·kg(-1)·min(-1); and lactate threshold, 59.9 ± 5.9% VO2peak) with T1D to a euglycemic clamp, whereby euglycemia was maintained by infusing basal insulin rates with concomitant infusion of [6,6-(2)H2]glucose for determining glucose kinetics. Glucose was infused to maintain euglycemia during and for 2 hours after exercise of different intensities (35, 50, 65, and 80% VO2peak). MAIN OUTCOME MEASURES: The glucose infusion rate (GIR), levels of glucoregulatory hormones, and rates of endogenous glucose appearance and disappearance were compared between conditions. RESULTS: The mean GIR to maintain euglycemia during exercise increased with intensity up to 50% (4.0 ± 1.6 g/h; P < .05) and 65% (4.1 ± 1.7 g/h), but no glucose was required at 80% VO2peak. Glucose rate of appearance and disappearance increased with intensity and, together with plasma catecholamines, reached higher levels at 80% VO2peak. CONCLUSION: Our findings support the predicted inverted-U relationship between exercise intensity and glucose requirement. However, the relationship between iv and oral glucose requirements needs to be investigated to translate these GIR data to clinical practice.

Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production.

Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and ß(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two ß(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked ß(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and ß(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

Improving cold storage and processing traits in potato through targeted gene knockout.

Cold storage of potato tubers is commonly used to reduce sprouting and extend postharvest shelf life. However, cold temperature stimulates the accumulation of reducing sugars in potato tubers. Upon high-temperature processing, these reducing sugars react with free amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide--a potential carcinogen. To minimize the accumulation of reducing sugars, RNA interference (RNAi) technology was used to silence the vacuolar invertase gene (VInv), which encodes a protein that breaks down sucrose to glucose and fructose. Because RNAi often results in incomplete gene silencing and requires the plant to be transgenic, here we used transcription activator-like effector nucleases (TALENs) to knockout VInv within the commercial potato variety, Ranger Russet. We isolated 18 plants containing mutations in at least one VInv allele, and five of these plants had mutations in all VInv alleles. Tubers from full VInv-knockout plants had undetectable levels of reducing sugars, and processed chips contained reduced levels of acrylamide and were lightly coloured. Furthermore, seven of the 18 modified plant lines appeared to contain no TALEN DNA insertions in the potato genome. These results provide a framework for using TALENs to quickly improve traits in commercially relevant autotetraploid potato lines.

Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family.

Soybean oil is high in polyunsaturated fats and is often partially hydrogenated to increase its shelf life and improve oxidative stability. The trans-fatty acids produced through hydrogenation pose a health threat. Soybean lines that are low in polyunsaturated fats were generated by introducing mutations in two fatty acid desaturase 2 genes (FAD2-1A and FAD2-1B), which in the seed convert the monounsaturated fat, oleic acid, to the polyunsaturated fat, linoleic acid. Transcription activator-like effector nucleases (TALENs) were engineered to recognize and cleave conserved DNA sequences in both genes. In four of 19 transgenic soybean lines expressing the TALENs, mutations in FAD2-1A and FAD2-1B were observed in DNA extracted from leaf tissue; three of the four lines transmitted heritable FAD2-1 mutations to the next generation. The fatty acid profile of the seed was dramatically changed in plants homozygous for mutations in both FAD2-1A and FAD2-1B: oleic acid increased from 20% to 80% and linoleic acid decreased from 50% to under 4%. Further, mutant plants were identified that lacked the TALEN transgene and only carried the targeted mutations. The ability to create a valuable trait in a single generation through targeted modification of a gene family demonstrates the power of TALENs for genome engineering and crop improvement.

Antecedent hypoglycaemia does not diminish the glycaemia-increasing effect and glucoregulatory responses of a 10 s sprint in people with type 1 diabetes.

AIMS/HYPOTHESIS: A 10 s sprint has been reported to provide a means to prevent acute post-exercise hypoglycaemia in young adults with type 1 diabetes because of its glycaemia-raising effect, but it is unclear whether this effect is impaired by antecedent hypoglycaemia. The purpose of this study was to investigate whether antecedent hypoglycaemia impairs the glycaemia-raising effect of a 10 s sprint in individuals with type 1 diabetes. METHODS: Eight individuals underwent a hyperinsulinaemic-hypoglycaemic or hyperinsulinaemic-euglycaemic clamp on two separate mornings. Thereafter, the participants underwent a basal insulin-euglycaemic clamp before performing a 10 s sprint on a cycle ergometer. The levels of blood glucose and glucoregulatory hormones and rates of glucose appearance (Ra) and disappearance (Rd) were compared between conditions. RESULTS: During the morning clamps, blood glucose levels were significantly different between conditions of hypoglycaemia (2.8 ± 0.1 mmol/l) and euglycaemia (5.4 ± 0.2 mmol/l; p < 0.001). Mean glycaemia prior to sprinting was similar (5.6 ± 0.4 and 5.5 ± 0.3 mmol/l for hypoglycaemic and euglycaemic conditions, respectively; p = 0.83). In response to the afternoon sprint, the pattern of increase in blood glucose levels did not differ between conditions, reaching similar maximal levels 45 min after exercise (6.5 ± 0.4 and 6.6 ± 0.3 mmol/l, respectively; p = 0.43). The early post-exercise patterns in glucose Ra and Rd and increases in plasma adrenaline (epinephrine), growth hormone and cortisol levels did not differ between conditions. CONCLUSIONS/INTERPRETATION: Hypoglycaemia in the morning does not diminish the glycaemia-raising effect of an afternoon 10 s sprint in young adults with type 1 diabetes, suggesting that sprinting is a useful strategy for opposing hypoglycaemia, regardless of prior hypoglycaemia.

Effect of sensor-augmented insulin pump therapy and automated insulin suspension vs standard insulin pump therapy on hypoglycemia in patients with type 1 diabetes: a randomized clinical trial.

IMPORTANCE: Hypoglycemia is a critical obstacle to the care of patients with type 1 diabetes. Sensor-augmented insulin pump with automated low-glucose insulin suspension has the potential to reduce the incidence of major hypoglycemic events. OBJECTIVE: To determine the incidence of severe and moderate hypoglycemia with sensor-augmented pump with low-glucose suspension compared with standard insulin pump therapy. DESIGN, SETTING, AND PARTICIPANTS: A randomized clinical trial involving 95 patients with type 1 diabetes, recruited from December 2009 to January 2012 in Australia. INTERVENTIONS: Patients were randomized to insulin pump only or automated insulin suspension for 6 months. MAIN OUTCOMES AND MEASURES: The primary outcome was the combined incidence of severe (hypoglycemic seizure or coma) and moderate hypoglycemia (an event requiring assistance for treatment). In a subgroup, counterregulatory hormone responses to hypoglycemia were assessed using the hypoglycemic clamp technique. RESULTS: Of the 95 patients randomized, 49 were assigned to the standard-pump (pump-only) therapy and 46 to the low-glucose suspension group. The mean (SD) age was 18.6 (11.8) years; duration of diabetes, 11.0 (8.9) years; and duration of pump therapy, 4.1 (3.4) years. The baseline rate of severe and moderate hypoglycemic events in the pump-only group was 20.7 vs 129.6 events per 100 patient months in the low-glucose suspension group. After 6 months of treatment, the event rates decreased from 28 to 16 in the pump-only group vs 175 to 35 in the low-glucose suspension group. The adjusted incidence rate per 100 patient-months was 34.2 (95% CI, 22.0-53.3) for the pump-only group vs 9.5 (95% CI, 5.2-17.4) for the low-glucose suspension group. The incidence rate ratio was 3.6 (95% CI, 1.7-7.5; P <.001). There was no change in glycated hemoglobin in either group: mean, 7.4 (95% CI, 7.2-7.6) to 7.4 (95% CI, 7.2-7.7) in the pump-only group vs mean, 7.6 (95%, CI, 7.4-7.9) to 7.5 (95% CI, 7.3-7.7) in the low-glucose suspension group. Counterregulatory hormone responses to hypoglycemia were not changed. There were no episodes of diabetic ketoacidosis or hyperglycemia with ketosis. CONCLUSIONS AND RELEVANCE: Sensor-augmented pump therapy with automated insulin suspension reduced the combined rate of severe and moderate hypoglycemia in patients with type 1 diabetes. TRIAL REGISTRATION: anzctr.org.au Identifier: ACTRN12610000024044.

The use of an automated, portable glucose control system for overnight glucose control in adolescents and young adults with type 1 diabetes.

OBJECTIVE: A key milestone in progress towards providing an efficacious and safe closed-loop artificial pancreas system for outpatient use is the development of fully automated, portable devices with fault detection capabilities to ensure patient safety. The ability to remotely monitor the operation of the closed-loop system would facilitate future physician-supervised home studies. RESEARCH DESIGN AND METHODS: This study was designed to investigate the efficacy and safety of a fully automated, portable, closed-loop system. The Medtronic Portable Glucose Control System (PGCS) consists of two subcutaneous glucose sensors, a control algorithm based on proportional-integral-derivative with insulin feedback operating from a BlackBerry Storm smartphone platform, Bluetooth radiofrequency translator, and an off-the-shelf Medtronic Paradigm Veo insulin pump. Participants with type 1 diabetes using insulin pump therapy underwent two consecutive nights of in-clinic, overnight, closed-loop control after a baseline open-loop assessment. RESULTS: Eight participants attended for 16 overnight studies. The PGCS maintained mean overnight plasma glucose levels of 6.4 ± 1.7 mmol/L (115 ± 31 mg/dL). The proportion of time with venous plasma glucose <3.9, between 3.9 and 8 (70 and 144 mg/dL), and >8 mmol/L was 7, 78, and 15%, respectively. The proportion of time the sensor glucose values were maintained between 3.9 and 8 mmol/L was greater for closed-loop than open-loop (84.5 vs. 46.7%; P < 0.0001), and time spent <3.3 mmol/L was also reduced (0.9 vs. 3%; P < 0.0001). CONCLUSIONS: These results suggest that the PGCS, an automated closed-loop device, is safe and effective in achieving overnight glucose control in patients with type 1 diabetes.

Analysis of glucose responses to automated insulin suspension with sensor-augmented pump therapy.

OBJECTIVE: The advent of sensor-augmented pump therapy with a low-glucose suspend (LGS) function (Medtronic Paradigm Veo System), allowing insulin to be automatically suspended for up to 2 h when sensor glucose falls below a preset threshold, has the potential to reduce the duration of hypoglycemia. In this article, we analyzed blood glucose profiles following a full 2-h insulin suspension activated by the LGS function, as well as examined different patterns of use among patients. RESEARCH DESIGN AND METHODS: Data from a cohort of participants using the Veo System for up to 6 months were analyzed to determine the time and duration of insulin suspension activated by the LGS function. We further evaluated overnight suspend events with no patient response occurring prior to 3:00 a.m., which allowed us to determine the pattern of sensor glucose values with no patient intervention during and after the period of insulin suspension. RESULTS: There were 3,128 LGS events during the 2,493 days evaluated. The median duration was 11.2 min, and 36% of events occurred overnight. There were 126 full 2-h suspend events that occurred overnight with no patient response, occurring before 3:00 a.m. For these events, the mean sensor glucose at the end of the 2-h suspend period was 99 ± 6 mg/dL ([means ± SE] 5.5 ± 0.3 mmol/L). The mean sensor glucose 2 h after insulin delivery resumed was 155 ± 10 mg/dL (8.6 ± 0.6 mmol/L). There were no episodes of severe hypoglycemia or diabetic ketoacidosis. CONCLUSIONS: Analyses of sensor glucose patterns following insulin suspension activated by LGS suggest that this technology is safe and unlikely to be associated with adverse outcomes.