Higher Dose Noninvasive Transcutaneous Auricular Vagus Nerve Stimulation Increases Feeding Volumes and White Matter Microstructural Complexity in Open-Label Study of Infants Slated for Gastrostomy Tube.

OBJECTIVE: To determine whether transcutaneous auricular vagus nerve stimulation (taVNS) paired with twice daily bottle feeding increases the volume of oral feeds and white matter neuroplasticity in term-age-equivalent infants failing oral feeds and determined to need a gastrostomy tube. STUDY DESIGN: In this prospective, open-label study, 21 infants received taVNS paired with 2 bottle feeds for 2 - 3 weeks (2x). We compared 1) increase oral feeding volumes with 2x taVNS and previously reported once daily taVNS (1x) to determine a dose response, 2) number of infants who attained full oral feeding volumes, and 3) diffusional kurtosis imaging and magnetic resonance spectroscopy before and after treatment by paired t tests. RESULTS: All 2x taVNS treated infants significantly increased their feeding volumes compared with 10 days before treatment. Over 50% of 2x taVNS infants achieved full oral feeds but in a shorter time than 1x cohort (median 7 days [2x], 12.5 days [1x], P < .05). Infants attaining full oral feeds showed greater increase in radial kurtosis in the right corticospinal tract at the cerebellar peduncle and external capsule. Notably, 75% of infants of diabetic mothers failed full oral feeds, and their glutathione concentrations in the basal ganglia, a measure of central nervous system oxidative stress, were significantly associated with feeding outcome. CONCLUSIONS: In infants with feeding difficulty, increasing the number of daily taVNS-paired feeding sessions to twice-daily significantly accelerates response time but not the overall response rate of treatment. taVNS was associated with white matter motor tract plasticity in infants able to attain full oral feeds. TRIAL REGISTRATION: Clinicaltrials.gov (NCT04643808).

Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme.

The gene encoding tyrosine aminotransferase (TAT, EC 2.6.1.5) from the parasitic protozoan Trypanosoma cruzi was amplified from genomic DNA, cloned into the pET24a expression vector and functionally expressed as a C-terminally His-tagged protein in Escherichia coli BL21(DE3)pLysS. Purified recombinant TAT exhibited identical electrophoretic and enzymatic properties as the authentic enzyme from T. cruzi. Both recombinant and authentic T. cruzi TATs were highly resistant to limited tryptic cleavage and contained no disulfide bonds. Comprehensive analysis of its substrate specificity demonstrated TAT to be a broad substrate aminotransferase, with leucine, methionine as well as tyrosine, phenylalanine, tryptophan and alanine being utilized efficiently as amino donors. Valine, isoleucine and dicarboxylic amino acids served as poor substrates while polar aliphatic amino acids could not be transaminated. TAT also accepted several 2-oxoacids, including 2-oxoisocaproate and 2-oxomethiobutyrate, in addition to pyruvate, oxaloacetate and 2-oxoglutarate. The functionality of the expression system was confirmed by constructing two variants; one (Arg389) being a completely inactive enzyme; the other (Arg283) retaining its full activity, as predicted from the recently solved three-dimensional structure of T. cruzi TAT. Thus, only one of the two strictly conserved arginines which are essential for the enzymatic activity of subfamily Ialpha aspartate and aromatic aminotransferases is critical for T. cruzi's TAT activity.

Tetrameric and dimeric malate dehydrogenase isoenzymes in Trypanosoma cruzi epimastigotes.

Two malate dehydrogenase isoforms, named MDH1 and MDH2, have been purified to homogeneity from Trypanosoma cruzi epimastigotes. Both enzymes consist of subunits with a molecular mass close to 33 kDa; native molecular mass determination by gel filtration, however, indicated that MDH1 is a dimer, whereas MDH2 is a tetramer. Both isoforms did not cross-react immunologically. The N-termini of both MDH isoforms and several tryptic peptides of MDH1 (amounting to about one third of the complete molecule) have been sequenced by automated Edman degradation. The tryptic digests of both enzymes have also been analysed by mass spectrometry (MALDI-TOF MS). The apparent Km values in both directions of the reaction have been determined, as well as the possible inhibition by excess of the substrate oxaloacetate. The sequence data, together with the pI values and the presence or absence of oxaloacetate inhibition indicate that the dimeric MDH1 is the mitochondrial isoenzyme, whereas the tetrameric MDH2 is the glycosomal isoenzyme. No evidence was found for the presence of a cytosolic isoform.

Crystallization and preliminary X-ray analysis of tyrosine aminotransferase from Trypanosoma cruzi epimastigotes.

Tyrosine aminotransferase from Trypanosoma cruzi has been crystallized from PEG 4000 at pH 6.8. The crystals belong to the monoclinic space group P21 and have lattice constants of a = 59.1, b = 103.0, c = 77.8 A, beta = 113.1 degrees for a data set measured at 138 K. The presence of a non-crystallographic twofold axis together with a Matthews parameter Vm of 2.5 A3 Da-1 indicates that the asymmetric unit contains one dimeric molecule. The crystals diffract to at least 2.7 A and are stable in the X-ray beam in a shock-frozen state. Native data sets have been collected at temperatures of 285 and 138 K using a Siemens X1000 detector on a rotating-anode generator.