An enzyme-centric approach for constructing an amperometric l-malate biosensor with a long and programmable linear range.

l-Malate is a key flavor enhancer and acidulant in the food and beverage industry, particularly winemaking. Enzyme-based amperometric biosensors offer convenience for monitoring its concentration. However, only a small number of off-the-shelf malate-oxidizing enzymes have been used in previous devices. These typically have linear ranges poorly suited for the l-malate concentrations found in fruit processing and winemaking, making it necessary to use precisely diluted samples. Here, we describe a pipeline of database-mining, gene synthesis, recombinant expression, and spectrophotometric assays to characterize previously untested enzymes for their suitability in biosensors. The pipeline yielded a bespoke biocatalyst-the Ascaris suum malic enzyme carrying mutation R181Q [AsME(R181Q)]. Our first prototype with AsME(R181Q) had an ultra-wide linear range of 50-200 mM l-malate, corresponding to concentrations found in undiluted fruit juices (including grape). Changing the dication from Mg2+ to Mn2+ increased sensitivity five-fold and adding citrate (100 mM) increased it another six-fold, albeit decreasing the linear range to 1-10 mM. To our knowledge, this is the first time an l-malate biosensor with a tuneable combination of sensitivity and linear range has been described. The sensor response was also tested in the presence of various molecules abundant in juices and wines, with ascorbate shown to be a potent interferent. Interference was mitigated by the addition of ascorbate oxidase, allowing for differential measurements on an undiluted, untreated wine sample that corresponded well with commercial l-malate testing kits. Overall, this work demonstrates the power of an enzyme-centric approach for designing electrochemical biosensors with improved operational parameters and novel functionality.

Enzyme-based amperometric biosensors for malic acid - A review.

Malic acid is a key flavour component of many fruits and vegetables. There is significant interest in technologies for monitoring its concentration, particularly in winemaking. In this review we systematically and comprehensively chart progress in the development of enzyme-based amperometric biosensors for malic acid. We summarise the components and analytical parameters of malic acid sensors that have been reported over the past four decades, discussing their merits and pitfalls in terms of accuracy, sensitivity, linear range, response time and stability. We discuss how advances in electrode materials, electron mediators and the use of coupled enzymes have improved sensitivity and minimised interference, but also uncover a trade-off between sensitivity and linear range. A particular focus of our review is the three types of malate oxidoreductase enzyme that have been used in malic acid biosensors. We describe their different properties and conclude that identifying and/or engineering superior alternatives will be a key future direction for improving the commercial utility of malic acid biosensors.

Outcomes of Primary Reverse Total Shoulder Arthroplasty in Patients Younger Than 65 Years Old.

PURPOSE: We compare clinical outcomes of primary reverse total shoulder arthroplasty (RTSA) in patients 65 years old or younger with a matched control group of patients 70 years old or older. METHODS: Forty-three patients (17 men and 26 women) 65 years old or younger were retrospectively identified. The mean age was 60 years and average follow-up was 4.0 years. The most common surgical indication was rotator cuff arthropathy. Patients were sex- and diagnosis-matched to control patients 70 years old or older with a mean follow-up of 4.1 years. Active range of motion (ROM) and functional outcomes in the 2 groups were evaluated before and after surgery. RESULTS: Patients 65 years old or younger had significantly lower preoperative functional scores; preoperative ROM, however, was similar in the 2 groups. Both groups significantly improved in postoperative ROM and functional scores (with no difference in Shoulder Pain and Disability Index [SPADI]-130, Simple Shoulder Test [SST], University of California-Los Angeles [UCLA], and Constant scores); however, the younger cohort had lower functional scores; American Shoulder and Elbow Surgeons (ASES) and 12-Item Short Form Health Survey (SF-12) were significantly lower after surgery. The change in ROM and outcome measures before to after surgery was similar between groups. Similar complications and notching rates were seen between the groups at final follow-up. CONCLUSIONS: An RTSA in patients 65 years old or younger improves ROM comparably with patients 70 years old or older. Younger patients have lower functional scores before and after surgery. An RTSA in younger patients improves pain and function but is associated with worse perceived outcomes. TYPE OF STUDY AND LEVEL OF EVIDENCE: Therapeutic III.

Diffuse Pigmented Villonodular Synovitis as a Rare Cause of Graft Failure Following Anterior Cruciate Ligament Reconstruction.

This case report describes a 42-year-old woman who was diagnosed with pigmented villonodular synovitis (PVNS) in the knee. The patient had received a bone-patella tendon-bone autograft reconstruction of her anterior cruciate ligament (ACL) 22 years prior to her diagnosis of PVNS. After a traumatic event that tore her ACL graft, she underwent a second surgery to repair the initial reconstruction. However, her pain and joint instability remained unresolved. When radiolucent lesions in her tibia and femur were identified through a radiographic image, the patient was referred to the authors' orthopedic oncology clinic. Additional imaging, including magnetic resonance imaging, revealed PVNS, and she was scheduled for debridement and a complete synovectomy of the knee. After surgery, the patient's pain decreased dramatically. She continues to maintain an active lifestyle despite a relatively minor decrease in range of motion. In this case, PVNS proved to be an unlikely complication after ACL reconstruction. The patient remains at risk for the development of degenerative arthritis. [Orthopedics. 2018; 41(1):e142-e144.].

Potent and Selective Inhibitors of Histone Deacetylase-3 Containing Chiral Oxazoline Capping Groups and a N-(2-Aminophenyl)-benzamide Binding Unit.

A novel series of potent chiral inhibitors of histone deacetylase (HDAC) is described that contains an oxazoline capping group and a N-(2-aminophenyl)-benzamide unit. Among several new inhibitors of this type exhibiting Class I selectivity and potent inhibition of HDAC3-NCoR2, in vitro assays for the inhibition of HDAC1, HDAC2, and HDAC3-NCoR2 by N-(2-aminophenyl)-benzamide 15k gave respective IC50 values of 80, 110, and 6 nM. Weak inhibition of all other HDAC isoforms (HDAC4, 5, 6, 7, and 9: IC50 > 100 000 nM; HDAC8: IC50 = 25 000 nM; HDAC10: IC50 > 4000 nM; HDAC11: IC50 > 2000 nM) confirmed the Class I selectivity of 15k. 2-Aminoimidazolinyl, 2-thioimidazolinyl, and 2-aminooxazolinyl units were shown to be effective replacements for the pyrimidine ring present in many other 2-(aminophenyl)-benzamides previously reported, but the 2-aminooxazolinyl unit was the most potent in inhibiting HDAC3-NCoR2. Many of the new HDAC inhibitors showed higher solubilities and lower binding to human serum albumin than that of Mocetinostat. Increases in histone H3K9 acetylation in the human cell lines U937 and PC-3 was observed for all three oxazolinyl inhibitors evaluated; those HDAC inhibitors also lowered cyclin E expression in U937 cells but not in PC-3 cells, indicating underlying differences in the mechanisms of action of the inhibitors on those two cell lines.

Discovery of potent, isoform-selective inhibitors of histone deacetylase containing chiral heterocyclic capping groups and a N-(2-aminophenyl)benzamide binding unit.

The synthesis of a novel series of potent chiral inhibitors of histone deacetylase (HDAC) is described that contain a heterocyclic capping group and a N-(2-aminophenyl)benzamide unit that binds in the active site. In vitro assays for the inhibition of HDAC1, HDAC2, HDAC3-NCoR1, and HDAC8 by the N-(2-aminophenyl)benzamide 24a gave respective IC50 values of 930, 85, 12, and 4100 nM, exhibiting class I selectivity and potent inhibition of HDAC3-NCoR1. Both imidazolinone and thiazoline rings are shown to be effective replacements for the pyrimidine ring present in many other 2-(aminophenyl)benzamides previously reported, an example of each ring system at 1 µM causing an increase in histone H3K9 acetylation in the human cell lines Jurkat and HeLa and an increase in cell death consistent with induction of apoptosis. Inhibition of the growth of MCF-7, A549, DU145, and HCT116 cell lines by 24a was observed, with respective IC50 values of 5.4, 5.8, 6.4, and 2.2 mM.

Novel chiral skeletons for drug discovery: antibacterial tetramic acids.

Modification of the ring nucleus of tetramic acids derived from serine gives chiral heterocyclic libraries that exhibit antibacterial activity, and correlation with various physicochemical parameters indicates that chiral tetramic acids may provide a potentially valuable non-aromatic skeleton for fragment-based drug discovery.