Biocatalytic Cascades toward Iminosugar Scaffolds Reveal Promiscuous Activity of Shikimate Dehydrogenases.

Iminosugar scaffolds are highly sought-after pharmaceutical targets, but their chemical synthesis is lengthy and can suffer from poor scalability and purification. Here we report protecting-group-free chemoenzymatic and biocatalytic cascades to synthesize iminosugars from sugar-derived aminopolyols in two steps. Using galactose oxidase variant F2 followed by a chemical or enzymatic reduction provided an efficient one-pot route to these targets, with product formation >70%. Key to success of this strategy was the application of genome mining, which identified bacterial shikimate dehydrogenases as promiscuous iminosugar reductases. The cell-free protocols allowed for isolation of highly polar iminosugar products from biotransformations in a single step through development of a gradient-elution cation exchange purification. The two-step pathway provides a short synthetic route that can be used as a cell-free platform for broader iminosugar synthesis.

Stable binding to phosphatidylserine-containing membranes requires conserved arginine residues in tandem C domains of blood coagulation factor VIII.

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic "tenase" complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through ß-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

Three-Component Stereoselective Enzymatic Synthesis of Amino-Diols and Amino-Polyols.

Amino-polyols represent attractive chemical building blocks but can be challenging to synthesize because of the high density of asymmetric functionalities and the need for extensive protecting-group strategies. Here we present a three-component strategy for the stereoselective enzymatic synthesis of amino-diols and amino-polyols using a diverse set of prochiral aldehydes, hydroxy ketones, and amines as starting materials. We were able to combine biocatalytic aldol reactions, using variants of d-fructose-6-phosphate aldolase (FSA), with reductive aminations catalyzed by IRED-259, identified from a metagenomic library. A two-step process, without the need for intermediate isolation, was developed to avoid cross-reactivity of the carbonyl components. Stereoselective formation of the 2R,3R,4R enantiomers of amino-polyols was observed and confirmed by X-ray crystallography.

Synthesis and Characterization of Tunable, pH-Responsive Nanoparticle-Microgel Composites for Surface-Enhanced Raman Scattering Detection.

The synthesis of microgels with pH-tunable swelling leads to adjustable and pH-responsive substrates for surface-enhanced Raman scattering (SERS)-active nanoparticles (NPs). Sterically stabilized and cross-linked latexes were synthesized from random copolymers of styrene (S) and 2-vinylpyridine (2VP). The pH-dependent latex-to-microgel transition and swellability were tuned based on their hydrophobic-to-hydrophilic content established by the S/2VP ratio. The electrostatic loading of polystyrene/poly(2-vinylpyridine) microgels [PS x P2VP y (M)] with anions such as tetrachloroaurate (AuCl4 -) and borate-capped Ag NPs was quantified. The PS x P2VP y (M) can load ∼0.3 equiv of AuCl4 - and the subsequent photoreduction results in Au NP-loaded PS x P2VP y (M) with NPs located throughout the structure. Loading PS x P2VP y (M) with borate-capped Ag NPs produces PS x P2VP y (M) with NPs located on the surface of the microgels, where the Ag content is set by S/2VP. The pH-responsive SERS activity is also reported for these Ag NP-loaded microgels. Analytical enhancement factors for dissolved crystal violet are high (i.e., 109 to 1010) and are set by S/2VP. The Ag NP-loaded microgels with ∼80 wt % 2VP exhibited the most stable pH dependent response.

Re-evaluating neonatal-age models for ungulates: does model choice affect survival estimates?

New-hoof growth is regarded as the most reliable metric for predicting age of newborn ungulates, but variation in estimated age among hoof-growth equations that have been developed may affect estimates of survival in staggered-entry models. We used known-age newborns to evaluate variation in age estimates among existing hoof-growth equations and to determine the consequences of that variation on survival estimates. During 2001-2009, we captured and radiocollared 174 newborn (≤24-hrs old) ungulates: 76 white-tailed deer (Odocoileus virginianus) in Minnesota and South Dakota, 61 mule deer (O. hemionus) in California, and 37 pronghorn (Antilocapra americana) in South Dakota. Estimated age of known-age newborns differed among hoof-growth models and varied by >15 days for white-tailed deer, >20 days for mule deer, and >10 days for pronghorn. Accuracy (i.e., the proportion of neonates assigned to the correct age) in aging newborns using published equations ranged from 0.0% to 39.4% in white-tailed deer, 0.0% to 3.3% in mule deer, and was 0.0% for pronghorns. Results of survival modeling indicated that variability in estimates of age-at-capture affected short-term estimates of survival (i.e., 30 days) for white-tailed deer and mule deer, and survival estimates over a longer time frame (i.e., 120 days) for mule deer. Conversely, survival estimates for pronghorn were not affected by estimates of age. Our analyses indicate that modeling survival in daily intervals is too fine a temporal scale when age-at-capture is unknown given the potential inaccuracies among equations used to estimate age of neonates. Instead, weekly survival intervals are more appropriate because most models accurately predicted ages within 1 week of the known age. Variation among results of neonatal-age models on short- and long-term estimates of survival for known-age young emphasizes the importance of selecting an appropriate hoof-growth equation and appropriately defining intervals (i.e., weekly versus daily) for estimating survival.

Is formal decompression necessary for parameniscal cysts associated with meniscal tears?

BACKGROUND: Meniscal tears and associated parameniscal cysts have good outcomes after partial meniscectomy and cyst evacuation. Good outcomes have been noted after partial meniscectomy with recurrent cysts. This investigation asks if partial meniscectomy without meniscal cyst decompression is sufficient for positive patient outcome. METHODS: Sixteen patients treated between 2005 and 2010 for a meniscal cyst and concomitant meniscal tear. Inclusion criteria were meniscal tear and parameniscal cyst per MRI, no prior surgery on the affected knee, and Outerbridge classification of I/II. Two patients were excluded. Fourteen patients completed the study until final follow-up. Lysholm knee scores were documented at 6 months post-operatively and at final follow-up. Re-evaluation or second surgery of the treated knee was documented. RESULTS: Eight lateral cysts and six medial cysts were diagnosed. Eight cysts were associated with a horizontal cleavage tear, while six menisci had a complex tear with a horizontal cleavage component. The average cyst size was 1.3 cm (0.5 to 3.5) at the largest diameter. At 6 months, the average Lysholm knee score was 94.1. At mean 5 years, the average score was 89.1. Patients with medial cysts, cysts greater than 1.0 cm, horizontal cleavage tears, or without simultaneous chondroplasty scored higher in the short and medium-term than their respective counterparts. CONCLUSIONS: Excellent short and medium-term outcomes can be achieved following partial meniscectomy without cyst decompression for patients with meniscal cysts and associated meniscal tears. LEVEL OF EVIDENCE: Level IV.

Improving recording accuracy, transparency, and performance for obstetric quality measures in a community hospital-based obstetrics department.

BACKGROUND: The obstetric arena has been typically ignored in the race to determine hospital quality measures due primarily to the fact that a large majority of patients do not have Medicare federal insurance, which has been the focus of hospital measures of quality. With "normal vaginal delivery" being the number one hospital discharge diagnosis and cesarean sections rates varying greatly between hospitals, national organizations are taking greater interest in determining differences in quality. METHODS: Sutter Medical Center, Sacramento (California) chartered a multidisciplinary Perinatal Data Committee to improve and simplify data capture for six obstetric quality measures. RESULTS: All six quality measures showed significantly improved trends from 2010 through 2012, with elective delivery < 39 weeks decreasing (15.3% to 2.3%, p < .001), nulliparous term singleton vertex cesarean (NTSV) delivery rate decreasing (31.3% to 24.7%, p < .001), episiotomy rates decreasing (4.7% to 2.3%, p < .001), antenatal steroid documentation increasing (80.0% to 100%, p <.01), exclusive breastfeeding at hospital discharge increasing (57.9% to 69.9%, p <.001), and deep vein prophylaxis at cesarean increasing (95.4% to 98.2%,p < .001). CONCLUSION: That performance on all six quality measures improved suggests that the improvement approach was effective and perhaps reproducible in other clinical situations to improve hospital quality outcomes. A key contributor to success was that the dashboard of results was shared with the department's physicians and the hospital administration on a monthly basis. Reinforcement of good results helped keep the project front and center with the hospital, particularly more recently, given that data reporting for four of the six measures is soon to be required.

Postoperative pain control after supracondylar humerus fracture fixation.

BACKGROUND: Postoperative pain control in pediatric patients has become a priority for all institutions. There is a paucity of literature on pain control after orthopedic procedures in the pediatric population. The purpose of this study is to compare the efficacy of acetaminophen with narcotic analgesics, specifically, acetaminophen/codeine and morphine, for pain management after closed reduction and percutaneous pinning of displaced supracondylar humerus fractures in children. METHODS: We retrospectively evaluated 217 patients who received closed reduction and percutaneous pinning of type II or III supracondylar humerus fractures at our institution from 2003 to 2009. Hospital charts were reviewed to obtain demographic data. Patients were divided into narcotic and non-narcotic groups. The Oucher and FLACC scales were used to quantify the effectiveness of the pain control that was delivered. RESULTS: A total of 174 patients were treated with non-narcotic pain medications and 43 patients received narcotics. The average age of these patients was 5.45 years. The mean postoperative pain score for the non-narcotic group was 1.9, whereas the mean postoperative pain score for the narcotic group was 2.2. This difference was not statistically significant. To account for the difference of age in patients and severity of fracture type, we created an age-matched cohort of patients with only type III supracondylar fractures. The average age of this group was 6.22 years. The mean pain score for the acetaminophen subgroup was 2.1 compared with a mean pain score of 2.4 for the narcotic subgroup. This difference was not statistically significant. Severe nausea or vomiting attributed to either class of medication was not observed. In addition, no patients developed a compartment syndrome. CONCLUSIONS: Acetaminophen is as effective as narcotic analgesics for providing pain control after supracondylar fracture surgery in children and is historically associated with fewer side effects. It is our recommendation to use acetaminophen alone for postoperative pain control in these patients. LEVEL OF EVIDENCE: III.

Selenium status and antibodies to selected pathogens in white-tailed deer (Odocoileus virginianus) in Southern Minnesota.

To determine exposure to a variety of infectious diseases potentially important for native ungulates, livestock, and humans, serum samples from 114 (94 adults, 20 fawns) female white-tailed deer (Odocoileus virginianus) were collected during January 2000-03 from multiple locations in southeast (SE) and southwest (SW) Minnesota. Antibody prevalence was determined for the following pathogens: Mycobacterium avium subsp. paratuberculosis, Leptospira interrogans (six serovars), Anaplasma marginale, Borrelia burgdorferi, Brucella abortus, epizootic hemorrhagic disease virus, and bovine viral diarrhea virus (BVDV) types 1 and 2. Samples collected in 2001 were screened for antibodies against Anaplasma phagocytophilum, and whole blood was submitted for polymerase chain reaction (PCR) testing for A. phagocytophilum and B. burgdorferi. In addition, serum selenium concentrations were evaluated for samples collected during 2001-03. Antibody prevalence and selenium concentration were compared by age-class and geographic region. Antibodies to all of the infectious agents except A. marginale and B. abortus were detected; when detected, antibody prevalence was highest in adults. Deer collected from SE Minnesota had a higher antibody prevalence to B. burgdorferi than SW deer. Blood culture and PCR results for A. phagocytophilum and B. burgdorferi were negative. Antibodies against BVDV (combined types 1 and 2) were more prevalent (chi(2) = 3.617, P< or = 0.029) in deer collected in SW (41%) than in SE (25%) Minnesota. No statistically significant differences in serum selenium concentrations were detected when data were analyzed by age-class or by geographic location.

Tonsillar biopsy test for chronic wasting disease: Two sampling approaches in mule deer and white-tailed deer.

Preclinical antemortem testing of deer (Odocoileus spp.) for chronic wasting disease (CWD) can be important for determining prevalence rates and removing infected individuals from wild populations. Because samples with high numbers of tonsillar follicles are likely to provide earlier detection of CWD than samples with fewer follicles, the method of obtaining follicular samples may be critical when investigating disease prevalence. Between January 2003 and January 2005, white-tailed deer (O. virginianus) in southeast and southwest Minnesota and white-tailed and mule deer (O. hemionus) in Wind Cave National Park, South Dakota, were sampled using dorso-lateral and ventral-medial approaches for collecting tonsillar follicles. We obtained significantly more follicles using a dorso-lateral (median number of follicles = 19) rather than a ventral-medial (median number of follicles = 5.5) approach. No differences were observed in collection of tonsillar follicles that were related to sex, age class, or species of deer. We recommend the dorso-lateral approach for assessing CWD prevalence in deer populations.