SUMMARY Exercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor β-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of a bone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS). L-BAIBA was as or more protective than estrogen or N-acetyl cysteine, signaling through the Mas-Related G Protein-Coupled Receptor Type D (MRGPRD) to prevent the breakdown of mitochondria due to ROS. BAIBA supplied in drinking water prevented bone loss and loss of muscle function in the murine hindlimb unloading model, a model of osteocyte apoptosis. The protective effect of BAIBA was lost with age, not due to loss of the muscle capacity to produce BAIBA but likely to reduced Mrgprd expression with aging. This has implications for understanding the attenuated effect of exercise on bone with aging.
3D printing has the potential to deliver personalized implants and devices for obstetric and gynecologic applications. The aim of this study is to engineer customizable and biodegradable 3D printed implant materials that can elute estrogen and/or progesterone. All 3D constructs were printed using polycaprolactone (PCL) biodegradable polymer laden with estrogen or progesterone and were subjected to hormone-release profile studies using ELISA kits. Material thermal properties were tested using thermogravimetric analysis and differential scanning calorimetry. The 3D printed constructs showed extended hormonal release over a one week period. Cytocompatibility and bioactivity were assessed using a luciferase assay. The hormone-laden 3D printed constructs demonstrated an increase in luciferase activity and without any deleterious effects. Thermal properties of the PCL and hormones showed degradation temperatures above that of the temperature used in the additive manufacturing process–suggesting that 3D printing can be achieved below the degradation temperatures of the hormones. Sample constructs in the shape of surgical meshes, subdermal rods, intrauterine devices and pessaries were designed and printed. 3D printing of estrogen and progesterone-eluting constructs was feasible in this proof of concept study. These custom designs have the potential to act as a form of personalized medicine for drug delivery and optimized fit based on patient-specific anatomy.
Isomeric molecules present a challenge for analytical resolution and quantification, even with MS-based detection. The eight-aminobutyric acid (ABA) isomers are of interest for their various biological activities, particularly γ-aminobutyric acid (GABA) and the d- and l-isomers of β-aminoisobutyric acid (β-AIBA; BAIBA). This study aimed to investigate LC-MS/MS-based resolution of these ABA isomers as their Marfey's (Mar) reagent derivatives. HPLC was able to separate three Mar-ABA isomers l-β-ABA (l-BABA), and l- and d-α-ABA (AABA) completely, with three isomers (GABA, and d/l-BAIBA) in one chromatographic cluster, and two isomers (α-AIBA (AAIBA) and d-BABA) in a second cluster. Partially separated cluster components were deconvoluted using Gaussian peak fitting except for GABA and d-BAIBA. MS/MS detection of Marfey's derivatized ABA isomers provided six MS/MS fragments, with substantially different intensity profiles between structural isomers. This allowed linear deconvolution of ABA isomer peaks. Combining HPLC separation with linear and Gaussian deconvolution allowed resolution of all eight ABA isomers. Application to human serum found a substantial level of l-AABA (13 μM), an intermediate level of l-BAIBA (0.8 μM), and low but detectable levels (<0.2 μM) of GABA, l-BABA, AAIBA, d-BAIBA, and d-AABA. This approach should be useful for LC-MS/MS deconvolution of other challenging groups of isomeric molecules.
Cytoplasmic peptidoglycan (PG) precursor levels were determined in methicillin-resistant (MRSA) after exposure to several cell wall-targeting antibiotics. Three experiments were performed: (i) exposure to 4× MIC levels (acute); (ii) exposure to sub-MIC levels (subacute); (iii) a time course experiment of the effect of vancomycin. In acute exposure experiments, fosfomycin increased UDP-GlcNAc, as expected, and resulted in substantially lower levels of total UDP-linked metabolite accumulation relative to other pathway inhibitors, indicating reduced entry into this pathway. Upstream inhibitors (fosfomycin, d-cycloserine, or d-boroalanine) reduced UDP-MurNAc-pentapeptide levels by more than fourfold. Alanine branch inhibitors (d-cycloserine and d-boroalanine) reduced d-Ala-d-Ala levels only modestly (up to 4-fold) but increased UDP-MurNAc-tripeptide levels up to 3,000-fold. Downstream pathway inhibitors (vancomycin, bacitracin, moenomycin, and oxacillin) increased UDP-MurNAc-pentapeptide levels up to 350-fold and UDP-MurNAc-l-Ala levels up to 80-fold, suggesting reduced MurD activity by downstream inhibitor action. Sub-MIC exposures demonstrated effects even at 1/8× MIC which strongly paralleled acute exposure changes. Time course data demonstrated that UDP-linked intermediate levels respond rapidly to vancomycin exposure, with several intermediates increasing three- to sixfold within minutes. UDP-linked intermediate level changes were also multiphasic, with some increasing, some decreasing, and some increasing and then decreasing. The total (summed) UDP-linked intermediate pool increased by 1,475 μM/min during the first 10 min after vancomycin exposure, providing a revised estimate of flux in this pathway during logarithmic growth. These observations outline the complexity of PG precursor response to antibiotic exposure in MRSA and indicate likely sites of regulation (entry and MurD).
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