De novo design of diverse small molecule binders and sensors using Shape Complementary Pseudocycles.

A general method for designing proteins to bind and sense any small molecule of interest would be widely useful. Due to the small number of atoms to interact with, binding to small molecules with high affinity requires highly shape complementary pockets, and transducing binding events into signals is challenging. Here we describe an integrated deep learning and energy based approach for designing high shape complementarity binders to small molecules that are poised for downstream sensing applications. We employ deep learning generated psuedocycles with repeating structural units surrounding central pockets; depending on the geometry of the structural unit and repeat number, these pockets span wide ranges of sizes and shapes. For a small molecule target of interest, we extensively sample high shape complementarity pseudocycles to generate large numbers of customized potential binding pockets; the ligand binding poses and the interacting interfaces are then optimized for high affinity binding. We computationally design binders to four diverse molecules, including for the first time polar flexible molecules such as methotrexate and thyroxine, which are expressed at high levels and have nanomolar affinities straight out of the computer. Co-crystal structures are nearly identical to the design models. Taking advantage of the modular repeating structure of pseudocycles and central location of the binding pockets, we constructed low noise nanopore sensors and chemically induced dimerization systems by splitting the binders into domains which assemble into the original pseudocycle pocket upon target molecule addition.

Molecular Characterization and Antimicrobial Resistance in Neisseria gonorrhoeae, Nunavut Region of Inuit Nunangat, Canada, 2018-2019.

We assessed antimicrobial resistance (AMR) in Neisseria gonorrhoeae in Nunavut, Canada, using remnant gonorrhea nucleic acid amplification test-positive urine specimens. This study confirms the feasibility of conducting N. gonorrhoeae AMR surveillance and highlights the diversity of gonococcal sequence types and geographic variation of AMR patterns in the territory.

Mycobacterium tuberculosis resistance in pulmonary TB patients in Cameroon: a phenotypic susceptibility assay.

OBJECTIVE: To determine the resistance of Mycobacterium tuberculosis to first- and second-line agents in adult pulmonary tuberculosis (TB) patients in Cameroon using a novel phenotypic assay. SETTING: Samples were collected from TB patients at Bamenda Hospital in Bamenda, Cameroon. DESIGN: Samples were collected consecutively from adult pulmonary TB patients over a 2-month period. TREK Sensititre(TM) MYCOTB panels were used to perform phenotypic drug susceptibility testing (DST). Susceptibility/resistance was determined by comparing minimum inhibitory concentrations to standard critical concentrations established for first- and second-line anti-tuberculosis drugs. RESULTS: Of 103 sputum samples processed, growth on Löwenstein-Jensen media was confirmed in 78 samples, 65 of which were suitable for DST. Thirty-nine strains (60%) were susceptible to all first- and second-line drugs. Five strains (8%) were categorized as multidrug-resistant TB. Two strains (3%) were classified as pre-extensively drug-resistant TB. Of those isolates susceptible to first-line drugs, 20% were resistant to at least one second-line drug. CONCLUSION: Antimicrobial resistance may be higher than assumed in TB strains in Cameroon, especially with regard to second-line drugs. There remains a need for rapid, comprehensive DST.

Postoperative pain assessment in the neonatal intensive care unit.

OBJECTIVES: To compare the convergent validity of two measures of pain (premature infant pain profile (PIPP) and crying, requires oxygen, increased vital signs, expression, and sleepless (CRIES)) in real life postoperative pain assessment in infants. METHODS: This study was a prospective, repeated measures, correlational design. Two staff nurses were randomly assigned either the PIPP or CRIES measure. An expert rater assessed each infant after surgery, and once a day using the visual analogue scale (VAS). SETTING: A level III neonatal intensive care unit in a metropolitan university affiliated paediatric hospital. RESULTS: Pain was assessed in 51 neonates (28-42 weeks of gestational age) after surgery. There was no significant difference in the rates of change between the pain assessment measures across time using repeated measures analysis of variance (F(50,2) = 0.62, p = 0.540), indicating correlation between the measures. Convergent validity analysis using intraclass correlation showed correlation, most evident in the first 24 hours (immediately, 4, 8, 20, and 24 hours after the operation). Correlations were more divergent at 40 and 72 hours after surgery. No significant interactions were found between gestational age and measure (F(304,4) = 0.75, p = 0.563) and surgical group and measure (F(304,2) = 0.39, p = 0.680). CONCLUSIONS: PIPP and CRIES are valid measures that correlate with pain for the first 72 hours after surgery in term and preterm infants. Both measures would provide healthcare professionals with an objective measure of a neonatal patient's pain.

Validation of the premature infant pain profile in the clinical setting.

OBJECTIVE: The Premature Infant Pain Profile (PIPP) is a 7-indicator composite measure developed to assess acute pain in preterm and term neonates. It has been validated in studies using synchronized videotaping of infants undergoing procedures. The purpose of this study was to establish (a) construct validity of the PIPP and (b) inter- and intrarater reliability of the PIPP prospectively in the clinical environment. DESIGN: A randomized, crossover design was used. SETTING: The study was conducted in a Level III outborn neonatal intensive care unit. PARTICIPANTS: A convenience sample of 43 neonates, stratified by gestational age, was studied. INTERVENTIONS: Each infant experienced three separate, randomly ordered events: baseline, a painful event, and a nonpain event. Infants were videotaped and scored at the bedside using the PIPP by the nurse caring for the infant and the clinical nurse specialist who bad expertise in infant pain. The videotapes were later reviewed by two additional experts; one in real time and one using a second-to-second stop frame technique. RESULTS: Repeated-measures analysis of the main effects and interactions yielded a statistically significant main effect for event (pain, nonpain, baseline), thus differentiating pain from nonpain and baseline events (F = 48, p = 0.0001) and establishing construct validity. Interrater reliability analysis of individual event scores of the PIPP yielded reliability coefficients of 0.93-0.96. Intrarater reliability coefficients analysis for individual events were equally high at 0.94-0.98. CONCLUSIONS: This study demonstrates that the PIPP is a pain measure with good construct validity and excellent inter- and intrarater reliability for the assessment of procedural pain of preterm and term infants in clinical settings.

Seasonal changes in energetics and torpor patterns in the subtropical blossom-bat Syconycteris australis (Megachiroptera).

Little is known about how animals from tropical and subtropical climates adjust their energy expenditure to cope with seasonal changes of climate and food availability. To provide such information, we studied the thermal physiology, torpor patterns and energetics of the nocturnal blossom-bat (Syconycteris australis 18 g) from a subtropical habitat in both summer and winter. In both seasons, S. australis frequently entered daily torpor at ambient temperatures between 12 and 25°C when food and water were withheld. Unlike patterns observed in temperate animals, mean minimum metabolic rates during torpor were lower in summer (0.47 ± 0.07 ml O2 g-1 h-1) than in winter (0.75 ± 0.11 ml O2 g-1 h-1). Body temperatures during torpor were regulated at 19.3 ± 1.0°C in summer and at 23.4 ± 2.0°C in winter. Torpor bout duration was significantly longer in summer (7.3 ± 0.6 h) than in winter (5.5 ± 0.3 h), but in both seasons, bout duration was not affected by ambient temperature. Consequently, average daily metabolic rates were also significantly lower in summer than in winter. Body temperatures and metabolic rates in normothermic bats did not change with season. Our findings on seasonal changes of torpor in this bat from the subtropics are opposite to those made for many species from cold climates which generally show deeper and longer torpor in winter and are often entirely homeothermic in summer. More pronounced torpor in subtropical S. australis in summer may be due to low or unpredictable nectar availability, short nights which limit the time available for foraging, and long days without access to food. Thus, the reversed seasonal response of this subtropical bat in comparison to temperate species may be an appropriate response to ecological constraints.

Transport characterization of membranes for immunoisolation.

This study relates to the diffusive transport characterization of hollow fibre membranes used in implantable bio-hybrid organs and other immunoisolatory devices. Techniques were developed to accurately determine the mass transfer coefficients for diffusing species in the 10(2)-10(5) MW range, validated and then used to study one membrane type known to effectively immunoisolate both allografts and xenografts in vivo. Low-molecular-weight diffusing markers included glucose, vitamin B12 and cytochrome C; higher-molecular-weight molecules were bovine serum albumin, immunoglobulin G, apoferritin and a range of fluorescein-tagged dextrans. Overall and fractional mass transfer coefficients through the hollow fibres were determined using a resistance-in-series model for transport. A flowing dialysis-type apparatus was used for the small-molecular-weight diffusants, whereas a static diffusion chamber was used for large-molecular-weight markers. For diffusion measurements of small-molecular-weight solutes, convective artefacts were minimized and the effect of boundary layers on both sides of the membrane were accounted for in the model. In measuring diffusion coefficients of large-molecular-weight species, boundary layer effects were shown to be negligible. Results showed that for small-molecular-weight species (< 13,000 MW) the diffusion coefficient in the membrane was reduced relative to diffusion in water by two to four times. The diffusion rate of large-molecular-weight species was hindered by several thousand-fold over their rate of diffusion in water.

Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans.

The effect of pO2s reduced below physiological levels on GSIR by isolated islets of Langerhans was investigated with a microperifusion apparatus that provided control of pO2 and rapid dynamic response. Second-phase insulin secretion was reduced substantially by hypoxia. The response to lower pO2 was rapid and reversible. Although the steady, normoxic (pO2 = 142 mmHg) second-phase secretion rate varied widely from one islet preparation to another, the ratio of Sx to S142 for each preparation could be represented by a single curve that exhibited a continuous reduction with decreasing pO2. For rat islets perifused 1 day after isolation, the secretion rate was nearly 100% of the normoxic value at a pO2 of 60 mmHg, 50% at 27 mmHg (P50, the pO2 at which the S142 is reduced by 50%), and approximately 2% at 5 mmHg. Oxygen sensitivity of second-phase secretion rate declined after 1 wk of in vitro culture: P50 was 13 mmHg after 1 wk and remained at 10 mmHg after 2-5 wk of culture. Canine islets exhibited a P50 of 16 mmHg after 1 wk of culture. The reduction in insulin secretion is thought to be associated with the existence of pO2 gradients outside and inside the isolated islets, resulting in exposure of islet cells to low pO2 levels that decrease radially from the periphery to the core. We hypothesize that the effect of low pO2 on S is manifested through depletion of the energy stores of the beta-cells. The effect of hypoxia on S may be an important factor in some in vitro secretion studies and may play a critical role in the effectiveness of transplanted islets before their revascularization and of immunoisolated islet implantation devices.

Maintenance of normoglycemia in diabetic mice by subcutaneous xenografts of encapsulated islets.

The goal of islet transplantation in human diabetes is to maintain the islet grafts in the recipients without the use of immunosuppression. One approach is to encapsulate the donor islets in permselective membranes. Hollow fibers fabricated from an acrylic copolymer were used to encapsulate small numbers of rat islets that were immobilized in an alginate hydrogel for transplantation in diabetic mice. The fibers were biocompatible, prevented rejection, and maintained normoglycemia when transplanted intraperitoneally; hyperglycemia returned when the fibers were removed at 60 days. Normoglycemia was also maintained by subcutaneous implants that had an appropriately constructed outer surface on the fibers.

A microperifusion system with environmental control for studying insulin secretion by pancreatic tissue.

A continuous flow reactor (perifusion system) was fabricated and tested for measuring the kinetics of insulin secretion from isolated pancreatic islets of Langerhans in response to step changes in the glucose concentration and oxygen partial pressure in the perfusate flowing around the islets. The system was capable of making rapid changes in perfusate glucose concentration and pO2, had rapid dynamic response for measuring the change in insulin secretion rate as a result of these changes in perfusate, and was suitable for studying very small volumes of tissue. Initial experiments with this system demonstrated that (1) the response of isolated rat islets to glucose stimulation was very fast, with the first phase peak occurring in as little as about 10 s, (2) bulk perfusate oxygen partial pressure levels of 30 mmHg or less reduced the second-phase insulin secretion rate in graded fashion, (3) the reduction in secretion rate began within 1 min following an oxygen partial pressure decrease, and (4) the reduction in secretion rate was reversible, with a burst of insulin secretion occurring during the first minute after partial pressure restoration.

Effect of oxygen on isolated pancreatic tissue.

One approach to insulin replacement therapy is transplantation of islets of Langerhans immunoisolated from host tissue by a semipermeable membrane. In this state, islets depend on diffusion of nutrients and wastes to and from the beta-cell to provide a suitable environment for survival and secretion. A perifusion system was constructed to test glucose-stimulated (100-300 mg/100 ml) insulin secretion from whole islets, or small (5-10 cell) aggregates, under controlled pO2. First phase insulin secretion from adult rat islets was unaffected by hypoxic levels of pO2, but second phase secretion was rapidly reduced at pO2 levels below 60 mmHg in the bulk media. Secretion from single-cell aggregates was unaffected until pO2 levels dropped to 12 mmHg, at which point secretion progressively decreased with falling pO2. A theoretical reaction/diffusion model was developed to correlate intraislet pO2 with reduced insulin secretion. Oxygen limited secretion was reversible, and not a result of decreased cell viability, as ascertained by both long-term static culture and trypan blue staining. Insulin secretion is more sensitive to hypoxia than is cell viability, in part because O2 uptake increases with glucose stimulation. These results indicate that O2 may be the limiting factor in the ability of immunoisolated islets to respond to blood glucose changes. We conclude that maintenance of a sufficiently high islet pO2 for maximal insulin secretion may be an important issue for graft design and implant site selection.