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The heart transplantation policy change (PC) has improved outcomes in high-acuity (Old 1A, New 1-3) patients, but the effect on low-priority (Old 1B/2, New 4-6) patients is unknown. We sought to determine if low-priority patient outcomes were compromised by benefits to high-priority patients by evaluating for interaction between PC and priority status (PS). We included adult first-time heart transplant candidates and recipients from the UNOS registry during a 19-month period before and after the PC. We compared clinical characteristics and performed competing risks and survival analyses stratified by PC and PS. There was a dependence of PC and PS on waitlist death/deterioration with an interaction sub-distribution hazard ratio (adjusted sdHR) of 0.59 (0.45-0.78), p-value .001. There was a trend toward a benefit of PC on waitlist death/deterioration (adjusted sdHR: 0.86 [0.73-1.01]; p = .07) and an increase in heart transplantation (adjusted sdHR: 1.08 [1.02-1.14], p = .007) for low-priority patients. There was no difference in 1-year post-transplant survival (log-rank p = .22) when stratifying by PC and PS. PC did not negatively affect waitlisted or transplanted low-priority patients. High-priority, post-PC patients had a targeted reduction in waitlist death/deterioration and did not come at the expense of worse post-transplant survival.

Long-term success of heart transplantation is limited by allograft rejection and cardiac allograft vasculopathy (CAV). Classic management has relied on frequent invasive testing to screen for early features of rejection and CAV to allow for early treatment. In this review, we discuss new developments in the screening and prevention of allograft rejection and CAV.Newer noninvasive screening techniques show excellent sensitivity and specificity for the detection of clinically significant rejection. New biomarkers and treatment targets continue to be identified and await further studies regarding their utility in preventing allograft vasculopathy.Noninvasive imaging and biomarker testing continue to show promise as alternatives to invasive testing for allograft rejection. Continued validation of their effectiveness may lead to new surveillance protocols with reduced frequency of invasive testing. Furthermore, these noninvasive methods will allow for more personalized strategies to reduce the complications of long-term immunosuppression whereas continuing the decline in the overall rate of allograft rejection.

Mortality in patients with STEMI-associated cardiogenic shock (CS) is increasing. Whether a comprehensive ST-elevation myocardial infarction (STEMI) protocol (CSP) can improve their care delivery and mortality is unknown. We evaluated the impact of a CSP on incidence and outcomes in patients with STEMI-associated CS. We implemented a 4-step CSP including: (1) Emergency Department catheterization lab activation; (2) STEMI Safe Handoff Checklist; (3) immediate catheterization lab transfer; (4) and radial-first percutaneous coronary intervention (PCI). We studied 1,272 consecutive STEMI patients who underwent PCI and assessed for CS incidence per National Cardiovascular Data Registry definitions within 24-hours of PCI, care delivery, and mortality before (January 1, 2011, to July 14, 2014; n = 723) and after (July 15, 2014, to December 31, 2016; n = 549) CSP implementation. Following CSP implementation, CS incidence was reduced (13.0% vs 7.8%, p = 0.003). Of 137 CS patients, 43 (31.4%) were in the CSP group. CSP patients had greater IABP-Shock II risk scores (1.9 ± 1.8 vs 2.8 ± 2.2, p = 0.014) with otherwise similar hemodynamic and baseline characteristics, cardiac arrest incidence, and mechanical circulatory support use. Administration of guideline-directed medical therapy was similar (89.4% vs 97.7%, p = 0.172) with significant improvements in trans-radial PCI (9.6% vs 44.2%, p < 0.001) and door-to-balloon time (129.0 [89:160] vs 95.0 [81:116] minutes, p = 0.001) in the CSP group, translating to improvements in infarct size (CK-MB 220.9 ± 156.0 vs 151.5 ± 98.5 ng/ml, p = 0.005), ejection fraction (40.8 ± 14.5% vs 46.7 ± 14.6%, p = 0.037), and in-hospital mortality (30.9% vs 14.0%, p = 0.037). In conclusion, CSP implementation was associated with improvements in CS incidence, infarct size, ejection fraction, and in-hospital mortality in patients with STEMI-associated CS. This strategy offers a potential solution to bridging the historically elusive gap in their care.

Pea protein has attracted the attention of consumers owing to its non-GMO, hypoallergenic, and high nutritional value, however, the beany flavor limits its applicability. Hence, GC-MS and GC-IMS were applied to assess the influence of Saccharomyces cerevisiae and Lactobacillus plantarum on the beany flavor of pea protein. Specifically, 51 and 65 volatiles were detected in pea proteins via GC-MS and GC-IMS, respectively. Odor activity value results showed that the components contributing to the beany flavor mainly included: 2-pentylfuran, (E, E)-2,4-decadienal, hexanal, nonanal, (E, E)-2,4-nonadienal, octanal, (E)-2-nonenal and (E)-2-octenal. S. cerevisiae and L. plantarum fermentation could remove 79.65% and 78.94% of the major beany flavor aldehydes from pea protein, respectively. Principal component analysis and hierarchical cluster analysis indicated that the unfermented and fermented pea protein samples can be distinguished by volatile compounds. These results provide a reference for the removal of beany off-flavors from pea proteins by fermentation.

Background: Chilli-pepper (CP) is well known for its anti-inflammatory, antioxidant, anticancer, and blood glucose regulation effects. However, the impact of CP on all-cause and cardiovascular mortality is not clear. Methods: A comprehensive search was performed using Ovid, Cochrane, Medline, Embase, and Scopus. We screened the studies from inception till January 16, 2020, and reported the outcomes of our interest after consumption of CP (Figure1a). The inclusion criteria were: all observational and randomized controlled trials reporting the outcomes of interest, and pediatric, animal studies, letters/case reports, reviews, abstracts, and book chapters were excluded. All-cause mortality was studied as the primary endpoint. Cardiovascular, cerebrovascular, and cancer-related deaths were studied as secondary outcomes. Results: From 4729 studies screened, four studies including 570,762 subjects met the inclusion criteria. Random-effects pooled analysis showed that there was a 25% reduction in the relative risk of all-cause mortality for regular CP consumers, compared to non-consumers (relative risk (RR): 0.75; 95% confidence intervals (CI): 0.64-0.88, p = 0.0004, Figure1b). Furthermore, there were a 26% and 23% reduction in the RR of death due to cardiovascular causes (RR: 0.74; 95% CI: 0.62-0.88, p=0.0006, Figure1c) and cancer (RR: 0.77; 95% CI: 0.71-0.84, p=0.0001, Figure 1d), respectively. However, there were no statistically significant differences in cerebrovascular accidents (RR: 0.76; 95% CI: 0.36-1.60, p=0.47). Conclusion: In a contemporary meta-analysis of 570,762 subjects, regular CP consumption was associated with a significant reduction in all-cause, cardiovascular, and cancer-related mortalities. Future studies are needed to better understand the potential mechanisms of the mortality benefits of CP consumption.

This paper presents the evolution of completion sand control designs for the Peng Lai field in Bohai Bay, offshore China. A range of sand control designs from open hole gravel packs to open hole standalone screens to cased hole high rate water packs and cased hole frac-packs have been tried over time. Evaluation of these various designs indicates that single-trip multi-zone cased hole frac-pack designs provide the most effective method for developing the thick, low net-to-gross, unconsolidated sandstone reservoirs in this waterflood field. The paper describes the completion design and evaluation process used in the field and shows how field challenges led to innovative frac-pack downhole equipment designs. This paper highlights completion practices and results from a range of well types used to develop the large Peng Lai field, offshore China. Development challenges and productivity and reliability results for alternative completion designs are discussed on the basis of field performance. The resulting preferred design, consisting of cased hole frac-packs using large-bore and small-bore single-trip multi-zone equipment are then discussed in further detail with emphasis on development of an innovative, fit-for-purpose, small-bore design. The installation challenges, solutions and successes experienced during the design and installation process and the actual performance and benefits of the new system are also discussed in this paper. Over the course of Peng Lai field development, a range of alternative completion sand control designs have been tried and evaluated. Cased hole frac-packs provide the most effective completion method for the thick, low net-to-gross, sandstone reservoirs. To enable effective completions in sidetracked wells, a 7" single-trip multi-zone frac pack tool has been developed. This small-bore system has been used to complete 13 wells with 67 zones in the field to date, saving over 43 rig days and $8MM. This paper presents reliability and productivity comparisons for a range of alternative completion sand control designs in a single, large field environment. A new 7" single-trip, fit-for-purpose design has been developed for the field and successfully implemented in multiple well campaigns. Installation time data and cost savings are provided.

Several studies designed to augment high density lipoprotein (HDL) levels have so far been unsuccessful in reducing rates of major adverse cardiovascular and cerebrovascular events (MACCE). In this study, we report the effect of HDL-C levels on overall survival outcomes and rates of MACCE following percutaneous coronary intervention (PCI). We reviewed patients who underwent PCI at the Cleveland Clinic from 2005 to 2017 and followed them through the end of 2018. Restricted cubic splines incorporated into Cox proportional hazard regression models were used to assess the outcomes. The HDL-C level associated with the lowest mortality was used as a reference value.15,633 patients underwent PCI during the study period, of which 70% were male, 81% were white, and 73% were on statins. The mean age at the time of procedure was 65.8 ± 11.8 years. After adjusting for demographics, co-morbidities, lipid profile, statin use, and date of procedure, our model demonstrated a U-shaped association between HDL-C and overall mortality, with HDL-C levels of 30-50 mg/dl associated with the most favorable outcomes, and HDL-C levels30 mg/dl or50 mg/dl associated with worse outcomes. A sensitivity analysis in men yielded a similar U-shaped association. In conclusion, our study shows that both low and high levels of HDL-C are associated with worse overall survival, with no effect on rates of MACCE in PCI patients. Further studies are required to understand the mechanism of this association between elevated HDL-C levels with increased overall mortality in patients with atherosclerotic cardiovascular disease (ASCVD).

Summary A nine–well subsea development project has been completed using casedhole frac packs (CHFPs) for sand control and multizone intelligent–well systems (IWSs) to improve recovery from a series of shallow, low–pressure gas reservoirs. In these wells, CHFPs have been installed to provide reliable sand control over the long, low–net–to–gross–ratio sand/shale target sequence: typically, three to six frac packs per well. This outer CHFP completion is then augmented with a multizone IWS, consisting of isolation seals, surface–controlled zonal–isolation valves, and downhole–pressure/temperature (DHP/T) gauges. The IWS string is run as a separate inner string to provide flow–monitoring capability and allow shutoff of zones producing high water volumes. This critical water–shutoff capability eliminates the risk of one or more high–water–production zones loading up and killing adjacent low–pressure gas zones, with the associated loss of reserves. To date, a total of nine wells have been completed and are being produced from three subsea gas fields. To maximize recovery from the fields’ numerous but relatively thin gas reservoirs, production wells are completed over three to six separate intervals. These frac–packed intervals are then grouped to allow flow control and pressure/temperature monitoring to occur through up to six surface–operated interval control valves (ICVs) and associated downhole gauges. This combination of sand control and intelligent–well control has provided an ability to perform multirate tests (MRTs) and pressure–buildup (PBU) tests on each reservoir interval to detect the start of water production or identify other impending production issues. After approximately 6 years of production service to the October 2018 date of this paper, 16 of the 34 zones completed in the nine–well project have been shut in to eliminate high water production. These water–shutoff actions performed using the surface–controlled ICVs are estimated to have improved gas–recovery factors from 50 to 60% without requiring rig intervention. This paper describes the reservoir challenges addressed and the completion–design and –operating practices used in this successful program.

The journey to Mars will be an ambitious, yet arduous task as it will entail culmination of all the information we have gathered over many decades. While the mission is of utmost importance, preservation of astronaut's well-being is paramount also. To that end, mitigation of radiation risk especially afflicting cardiovascular disease (CVD) is of great interest and challenge. Current data from astronauts on low earth orbit and Apollo missions provides insight on the risk of CVD from radiation exposure. However, data is limited given the small cohort size of astronauts who embarked on just nine prolonged missions. Therefore, a cerebral approach to understanding and mitigating risks are essential. This paper discusses the need for a predictive preclinical model to help understand and mitigate the effects of radiation on astronauts. We will discuss strengths and limitations of preclinical models and the methods of validating and constructing a model to predict human clinical outcomes. Our bedside-bench-bedside approach focuses on adapting the preclinical model through common investigative tools used between humans and animals. The result will be an optimization of preclinical model to a point of being a surrogate clinical model capable of predicting CVD outcomes in astronauts exposed to radiation.

The beany flavor of pea protein limits its application in the food industry. This study aimed at addressing this problem by combining the advantages of solid-based spray drying technique and the ability of cyclodextrins (CD) to entrap volatiles. Pea protein isolates (PPI) was extracted by alkaline extraction-isoelectric precipitation, followed by co-spray drying with CD. The resulted PPI-CD showed no major structure changes. HS-SPME-GC-MS coupled to untargeted metabolomics successfully identified 23 aroma compounds that represent the different odorants among PPI-control, physically mixed PPI-CD, and co-spray dried PPI-CD samples. Heat map analysis also showed a remarkable beany odor mitigation effect upon the addition of CD, which was further proved to be due to CD entrapping aroma compounds during spray drying. In the meantime, the functional attributes of PPI-CD were not adversely impacted by the addition of CD.

The common method used in the food industry to manufacture pea protein isolate (PPI) is extraction by alkaline extraction‒isoelectric precipitation, followed by spray drying. In this study, the effect of adding a co-spraying agent to PPI at a ratio of 10:90, trehalose (Tr), sucrose (Su) or sodium hexametaphosphate (SHMP), on the structure, functionality and aromatic profile of spray-dried PPI was studied. Results showed that the addition of Tr/Su/SHMP induced protein conformational changes in accordance to FTIR spectrum and quantification of free sulfhydryl and disulfide groups. The surface charge (ζ-potential) and surface hydrophobicity of co-spray dried PPI was also modified substantially. Such structural changes exhibited significant impacts on the functionalities of co-spray dried PPI, including solubility, water/oil binding capacity, thermal property, emulsifying capacity and stability, and foaming capacity and stability. The better foaming properties of co-spray dried PPI was also supported by measuring their ability to reduce air‒water surface tension. In addition, the impact of co-spraying agent (Tr/Su/SHMP) on the aromatic profile of PPI was investigated. Twelve compounds were obtained as characteristic compounds that represent the difference among samples. Such difference was visualized by performing principal component analysis and hierarchical cluster analysis. The findings reported in this study provided structural data as the basis for facilitating the development of pea protein products with better functionalities.

d-Psicose is a highly valuable rare sugar because of its excellent physiological properties and commercial potential. d-Psicose 3-epimerase (DPEase) is the key enzyme catalyzing the isomerization of d-fructose to d-psicose. However, the poor thermostability and low catalytic efficiency are serious constraints on industrial application. To address these issues, site-directed mutagenesis of Tyr68 and Gly109 of the Clostridium bolteae DPEase was performed. Compared with the wild-type enzyme, the Y68I variant displayed the highest substrate-binding affinity and catalytic efficiency, and the G109P variant showed the highest thermostability. Furthermore, the double-site Y68I/G109P variant was generated and exhibited excellent enzyme characteristics. The Km value decreased by 17.9%; the kcat/Km increased by 1.2-fold; the t1/2 increased from 156 to 260 min; and the melting temperature (Tm) increased by 2.4 °C. Moreover, Co(2+) enhanced the thermostability significantly, including the t1/2 and Tm values. All of these indicated that the Y68I/G109P variant would be appropriate for the industrial production of d-psicose.

Ketose 3-epimerase displayed an important role in not only the cyclic monosaccharides bioconversion strategy, named Izumoring, but also in the industrial biological production of d -psicose, a novel low-calorie rare sugar widely used in food and medical industries. Since the non-enzymatic side reactions could be reduced in acid conditions, slightly acidic pH optimum is one of the main issues for biological production of d -psicose. In this study, we first characterized an acidic ketose 3-epimerase, the recombinant d -psicose 3-epimerase (DPEase) from Dorea sp. CAG317. The protein exhibited high amino acid sequence identity with other reported DPEases, and was determined as a homotetramer with subunit molecular weight approximately 33 kDa, which was the same as other reported findings. The recombinant DPEase was a metal-dependent enzyme with the optimum metal cofactor as Co2+. In presence of 1 mM of Co2+, the enzyme displayed the maximal activity as 803 U/mg at pH 6.0 and 70 °C. The catalytic efficiency (kcat/Km) was measured to be 412 and 199 mM−1 min−1 toward d -psicose and d -fructose, respectively. The equilibrium ratio between d -fructose and d -psicose was approximately 30:70, and the elevated temperature did not significantly shift the equilibrium toward d -psicose. Compared to other reported DPEases, the recombinant Dorea sp. DPEase displayed significantly higher specific activity at acidic pHs and remarkably higher productivity of d -psicose at pH 6.0, indicating that it was appropriate for use as a new source of d -psicose producing enzyme.

A mutation, D478N, was obtained by an error-prone polymerase chain reaction using the l-arabinose isomerase (l-AI) gene from Alicyclobacillus hesperidum URH17-3-68 as the template. The mutated isomerase showed higher activity for d-galactose isomerization. The mutation site obtained from random mutagenesis was then introduced as a single-site mutation using site-directed mutagenesis. Single-site variants, D478N, D478Q, D478A, D478K, and D478R, were constructed. The optimum temperatures were all higher than 60 °C. D478A, D478N, and D478Q retained more than 80 % of the maximum relative activity of the wild-type l-AI at 75 °C. With the exception of the D478A variant, all variants showed decreased optimum pH values in the acidic range (6.0–6.5). All of the variant l-AIs could be significantly activated by the addition of Co2+ and Mn2+. D478N and D478Q showed higher catalytic efficiencies (k cat/K m) toward d-galactose than that of wild-type l-AI. In addition, the D478N and D478Q variants exhibited a much higher conversion ratio of d-galactose to d-tagatose at 6.0 than the wild-type l-AI. According to the molecular model, residue D478 was located on the surface of the enzyme and distant from the active site. It was supposed that the charged state of residue 478 may influence the optimum pH for substrate binding or isomerization.

Summary This paper covers the development, laboratory testing, and field testing of acid-soluble-plug (ASP) technology as a viable completion alternative to wireline- or tubing-conveyed perforating. The ASPs are installed in a preperforated reservoir liner and dissolve when soaked in acid, allowing access to the reservoir. This allows the technology to be easily applied in reservoirs in which matrix acid jobs or acid-fracturing techniques are used. The ASP technology was developed to reduce risk and cost associated with wireline- and tubing-conveyed perforating. ASPs were designed, manufactured, and field tested in both 5-in. and 7.625-in. reservoir liner sizes for wells in the Ekofisk field. The combination of laboratory testing and large-scale field testing influenced the design of the ASPs as well as the additives used in the acid systems used to dissolve them. From concept to initial field implementation, the process of ASP-engineering development took more than 2 years. The concept was in the beginning field-tested in deviated injectors, with ASPs installed in the deepest section of the reservoir liner. The field tests proved the ASP concept before depending on the technology in a horizontal producer with an uncemented, tubing-in-liner completion solution. The field tests showed that acid soaking dissolved the ASPs in the downhole environment and allowed efficient acid stimulation of the reservoir. It also reduced the number of wireline runs necessary to complete the well. When field tested in a tubing-in-liner completion application, installation of the ASPs in the reservoir liner eliminated tubing-conveyed-perforating runs. The reservoir liner was uncemented with mechanical openhole packers for zonal isolation. The ASPs provided a pressure-tight reservoir liner to set the packers against and eliminated fluid loss during the running of the inner completion string. Optimization of this technology is an ongoing process. The plug design itself continues to evolve as well as the operational steps to minimize the soaking time necessary to dissolve the plugs and to gain access to the reservoir.

A nine well subsea development project has been completed using cased hole frac-packs for sand control and an innovative multi-zone intelligent well system to improve recovery from a series of shallow, low pressure, gas reservoirs. In these wells, cased hole frac-packs have been installed to provide reliable sand control over the long, low net-to-gross, sand/shale target sequence: typically, three to six frac-packs per well. This outer cased hole frac-pack completion is then augmented with a multi-zone intelligent well system, consisting of isolation seals, surface controlled zonal isolation valves and downhole pressure/temperature gauges. The intelligent well system is run as a separate inner string to provide flow monitoring capability and allow shut-off of zones producing high water volumes. This critical water shut-off capability eliminates the risk of one or more high water production zones loading-up and killing adjacent low-pressure gas zones with associated loss of reserves. To date, a total of nine wells have been completed and are being successfully produced from three subsea gas fields. To maximize recovery from the fields' numerous but relatively thin gas reservoirs, production wells are completed over three to six separate intervals. These frac-packed intervals are then grouped to allow flow control and pressure/temperature monitoring to occur through up to six surface operated Interval Control Valves and associated downhole gauges. This combination of sand control and intelligent well control has provided an ability to perform multi-rate flow tests and pressure build-up tests on each reservoir interval to detect the start of water production or identify other impending production issues. After roughly five years of production service to June 2017, four of the 34 zones completed in the nine well project have had to be shut-in to eliminate high water production. These water shut-off actions performed via the surface controlled interval control valves are estimated to have improved gas recovery factors from 50% to 60% without requiring rig intervention. This paper will describe the reservoir challenges, completion design and operating practices employed in this successful program.

Stroke is one of the potential complications of mitral clip (MC) procedure with a reported incidence of 0.2% to 2.8%. We studied the incidence of stroke at 30 days after a mitral clip (MC) by performing meta-analysis from previously published literature. Ovid, Medline, Embase, Scopus and Cochrane

The rare monosaccharide d -tagatose is a low-calorie sugar-substituting sweetener, having 92% of relative sweetness but only 1/3 of energy content of sucrose. Industrial production of d -tagatose is carried out from d -galactose by l -arabinose isomerase ( l -AI). It is generally recognized that commercial l -AI for d -tagatose production requires two important enzymatic properties, thermostability and slightly acidic pH optimum. In this article, a thermostable l -AI was characterized from a novel thermoacidophilic bacterium, Alicyclobacillus hesperidum URH17-3-68, which showed promising thermostability and displayed a relatively wide pH spectrum. The araA gene encoding the Al. hesperidum l -AI was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and ion-exchange chromatography. The enzyme displayed maximal activity at 70 °C and pH 7.0, and showed more than 75% of maximal activity from pH 5.5 to 7.0. Cobalt ion was required as optimum metal cofactor for both activity simulation and thermostability improvement at high temperature. The enzyme retained 93% and 63% of initial activity after 4 and 16 h of incubation, respectively, at 75 °C in the presence of Co2+. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for substrate d -galactose were measured to be 54.7 mM, 68.0 min−1, and 1.2 mM−1 min−1, respectively. The bioconversion yield of d -tagatose by the purified enzyme after 27 h at 70 °C reached 43% and 22%, from 50 and 200 mM of d -galactose, respectively. Due to the promising thermostability and high activity at slight acidic pH, the Al. hesperidum l -AI was appropriate for use as a new source of d -tagatose producing enzyme.

Pharmaceutical excipients contain reactive groups and impurities due to manufacturing processes that can cause decomposition of active drug compounds. The aim of this investigation was to determine if commercially available oral disintegrating tablet (ODT) platforms induce active pharmaceutical ingredient (API) degradation. Benzocaine was selected as the model API due to known degradation through ester and primary amino groups. Benzocaine was either compressed at a constant pressure, 20 kN, or at pressure necessary to produce a set hardness, i.e., where a series of tablets were produced at different compression forces until an average hardness of approximately 100 N was achieved. Tablets were then stored for 6 months under International Conference on Harmonization recommended conditions, 25°C and 60% relative humidity (RH), or under accelerated conditions, 40°C and 75% RH. Benzocaine degradation was monitored by liquid chromatography–mass spectrometry. Regardless of the ODT platform, no degradation of benzocaine was observed in tablets that were kept for 6 months at 25°C and 60% RH. After storage for 30 days under accelerated conditions, benzocaine degradation was observed in a single platform. Qualitative differences in ODT platform behavior were observed in physical appearance of the tablets after storage under different temperature and humidity conditions.

d-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for d-psicose production from d-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a d-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co2+. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using d-psicose as the substrate, the apparent K m, k cat, and catalytic efficiency (k cat/K m) were 27.4 mM, 49 s−1, and 1.78 s−1 mM−1, respectively. Under the optimal conditions, the equilibrium ratio of d-fructose to d-psicose was 69:31. For high production of d-psicose, 216 g/L d-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate d-fructose, indicating that the enzyme was a potential d-psicose producer for industrial production.

The color of active pharmaceutical ingredients (APIs) can be a critical quality attribute for pharmaceutical products. Color variation can be indicative of contaminants, chemical impurities, solid-state form impurities, or degradation products. The color of the pharmaceutical materials is typically specified and determined using visual appearance testing. To avoid the inherent subjectivity of visual appearance testing, to understand the link between the color and the process parameters, and to facilitate quality agreements between customers and vendors of chemical starting materials, quantitative color measurements utilizing visible spectroscopic methods are increasingly needed. In this work, a systematic method development strategy (MDS) is described to assist with the development of robust and rugged quantitative color measurement methods based on a science- and risk-based approach aligned with quality-by-design principles. The MDS is illustrated using typical scenarios encountered in method development through several examples including a method developed for 2,3-dimethyl-2H-indazol-6-amine, a starting material in the commercial synthesis of pazopanib hydrochloride API. Flowcharts and examples are presented to illuminate key decision points in the MDS process including selection of (1) solution-state versus solid-state spectroscopic method, (2) optimal color space for reporting color measurement results, and (3) appropriate method risk assessment and control to ensure successful method implementation. General guidance is also provided to facilitate the discussion for setting quantitative color specifications.

Difructose anhydride III (DFA III), the smallest cyclic disaccharide, consists of two fructose residues. DFA III is a hydrolysate of inulin and is rarely found in nature. Industrial interest in DFA III as a low-calorie sugar substitute is increasing. The present review describes the properties and physiological functions of DFA III as well as its commercial importance. Focus is also given on the biological production of DFA III from inulin, which contains enzyme resources, inulase II properties, and the capacity for mass DFA III production. Inulase II as an industrial enzyme and its molecular evolution are discussed as well. The aim is to better understand commercial-scale DFA III production as a food product.

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.

The soil bacterium Arthrobacter aurescens SK 8.001 produces inulin fructotransferase (IFTase), and liquid chromatography–mass spectrometry (LC–MS) and carbon-13 nuclear magnetic resonance (13C NMR) analysis demonstrated that the main product of the enzyme was difructose anhydride III (DFA III). The IFTase was purified by ethanol precipitation, DEAE Sepharose Fast Flow, and Superdex 200 10/300 GL gel chromatography. Its molecular mass was estimated to be 40 kDa by SDS–PAGE and 35 kDa by gel filtration. The enzyme showed maximum activity at pH 5.5 and 60–70 °C, and retained 86.5% of its initial activity after incubation at 60 °C for 4 h. Chemical modification results suggested that a tryptophan residue is essential to enzyme activity. The N-terminal amino acid sequence was determined as AEGAKASPLNSPNVYDVT. The kinetic values, Km and Vmax, were estimated to be 0.52 mM and 0.3 μmol/ml min. Nystose was observed to be the smallest substrate for the produced IFTase. This IFTase provides a promising way to utilize inulin for the production of DFA III.

Several mass spectrometry (MS) techniques including accurate MS and MS/MS, as well as hydrogen/deuterium (H/D) exchange, were utilized to characterize a pseudo-oxidative reaction by-product (impurity I) in the pharmaceutical synthesis of S-(thiobenzoyl)thioglycolic acid. The negative ion MS/MS data provided complementary structural information to the positive ion MS/MS data. An understanding of the gas-phase Smiles rearrangement upon collision-induced dissociation (CID) in the negative ion MS/MS mode played an important role in structural elucidation of impurity I. The theoretical calculations by density functional theory (DFT) at the B3LYP/6-311G(d,p) level provided insights into the thermochemistry of the Smiles rearrangement reaction. This pseudo-oxidative impurity is proposed to be generated via the base-catalyzed hydrolysis in solution.

A scalable synthesis of a novel pleuromutilin-based antibiotic is reported. The synthesis features the scale-up of an interesting skeletal rearrangement of the pleuromutilin core and isolation of a highly purified product despite starting with relatively impure pleuromutilin. The use of Design of Experiment (DoE) and Principal Component Analysis (PCA) tools to achieve these goals is also discussed. Furthermore, the novel coupling of a carbamate and N-acyl-imidazole to produce the imidodicarbonate portion of the target molecule is described.

The objective of this project was to investigate the potential of Kleptose Linecaps DE17 (KLD) in masking the unpleasant/bitter taste of therapeutic agents by hot melt extrusion (HME). Griseofulvin (GRI) and caffeine anhydrous (CA) were used as a bitter active pharmaceutical ingredient (API) model drugs. Thermogravimetric studies confirmed the stability of GRI, CA, and KLD at the employed extrusion temperatures. The differential scanning calorimetry (DSC) studies revealed a characteristic melting endotherm of GRI at 218–220°C and CA at 230–232°C in the physical mixtures as well as in all extrudates over the period of study, indicating the crystalline nature of drug. HME of KLD was achieved only in the presence of plasticizer. Among the several plasticizers investigated, xylitol showed improved processability of KLD at 15% w/w concentration. Dissolution studies of HME extrudates using simulated salivary medium exhibited ∼threefold less release compared to physical mixture at the end of 5 min (the lesser drug release, better the taste masking efficiency). Furthermore, the results from the sensory evaluation of products in human panel demonstrated strong bitter taste in the case of physical mixture compared to the HME formulation, suggesting the potential of Kleptose Linecaps DE17 as taste masking polymer in melt extruded form.

The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K(d,ThDP) = 4.34 microM and K(m,ThDP) = 11 microM were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1',4'-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.