CO2 Incorporation and 4-Hydroxy-2-Methylbenzoic Acid Formation during Anaerobic Metabolism of m-Cresol by a Methanogenic Consortium

The metabolism of m-cresol by methanogenic cultures enriched from domestic sewage sludge was investigated. In the initial studies, bromoethanesulfonic acid was used to inhibit methane production. This led to the accumulation of 4.0 ± 0.8 mol of acetate per mol of m-cresol metabolized. These results suggested that CO₂ incorporation occurred because each molecule of m-cresol contained seven carbon atoms, whereas four molecules of acetate product contained a total of eight carbon atoms. To verify this, [¹⁴C]bicarbonate was added to bromoethanesulfonic acid-inhibited cultures, and those cultures yielded [¹⁴C]acetate. Of the label recovered as acetate, 89% was found in the carboxyl position. Similar cultures fed [methyl-¹⁴C]m-cresol yielded methyl-labeled acetate. A ¹⁴C-labeled transient intermediate was detected in cultures given either m-cresol and [¹⁴C]bicarbonate or bicarbonate and [methyl-¹⁴C]m-cresol. The intermediate was identified as 4-hydroxy-2-methylbenzoic acid. In addition, another metabolite was detected and identified as 2-methylbenzoic acid. This compound appeared to be produced only sporadically, and it accumulated in the medium, suggesting that the dehydroxylation of 4-hydroxy-2-methylbenzoic acid led to an apparent dead-end product.     

Effect of water potential on seed germination

The response of seed germination to substrate water potential was determined for several plant species of the arctic tundra. Seeds were collected from Cape Thompson and Eagle Summit, Alaska and germinated on dialysis membranes over water solutions of polyethylene glycol with osmotic potentials of 0 to −6 bars. Germination did not occur with potentials below −3 bars, except for three fellfield species. Germination was delayed at lower osmotic potentials. Because the response of most species was similar, substrate water potential is probably not a factor affecting the establishment of most tundra plant species from seeds.     

A structural model for the chromophore-binding domain of ovine rhodopsin

A putative model for the structure of the relatively independent carboxyl-terminal domain of (rhod)opsin has been developed by use of a combination of several secondary structure prediction methods. The validity of this approach was confirmed by comparing the secondary structure for bacteriorhodopsin as predicted by these methods with its known low resolution structure. The resulting predicted structure agreed well with the experimental data. The model obtained for opsin incorporates two transmembrane α-helical rods linked by an intradiscal loop. Each of the helical sections is interrupted by a short irregular region. One of these includes the lysyl residue to which the chromophore 11-cis retinal is attached. The second non-regular segment, almost opposite the first, contains a cysteinyl and a tryptophanyl residue which may be involved in protein—chromophore interaction. The proposed structure of this whole domain could prove instructive in the elucidation of the primary events of visual transduction.     

Serum lipoproteins and albumin in the lecithin: Cholesterol acyltransferase reaction

The unique features of pig ovarian follicular fluids, i.e., presence of high density lipoprotein (HDL) only and lecithin: cholesterol acyltransferase (EC 2.3.1.43; LCAT) activity, provides a good model to study the effect of serum lipoproteins and serum albumin on the LCAT reaction. $\text{In}\text{vitro}$ cholesterol esterification is enhanced when very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions are added, but is inhibited when one or the other of these lipoproteins is absent. High concentrations of HDL₂ result in decreased activation which can be compensated for by the addition of the VLDL-LDL mixture. These findings suggest that the rate of cholesterol esterification in ovarian follicular fluid may be enhanced by providing the exogenous VLDL and LDL as the recipients of HDL-cholesteryl ester. The inhibition of LCAT activity caused by free fatty acid and lysophosphatidylcholine can be partially reversed by the addition of serum albumin, suggesting that serum albumin may regulate the LCAT reaction.     

Intracellular localization of GDP-l-fucose: Glycoprotein and CMP-sialic acid: Apolipoprotein glycosyltransferases in rat and pork livers

A GDP-l-fucose:glycoprotein fucosyltransferase which transfers l-fucose to terminal β-N-acetyl-d-glucosaminyl residues of sialidase-, β-galactosidase-treated α₁-acid glycoprotein and a CMP-sialic acid:glycoprotein sialyltransferase acting on sialidase-treated apolipoprotein-Ala₁ from human very low density lipoprotein have been shown to be concentrated in rat liver Golgi apparatus preparations at enrichments of 40- and 45-fold, respectively, and in pork liver Golgi-rich fractions at enrichments of 35- and 20-fold, respectively. A second fucosyltransferase acting on sialidase-treated α₁-acid glycopretein was absent from rat liver and was enriched only 13-fold in a pork liver Golgi-rich fraction. The smooth-surfaced microsome fraction was the only other rat liver subcellular fraction with appreciable levels of the GDP-l-fucose: β-N-acetyl-d-glucosaminide fucosyltransferase and the lipoprotein sialyltransferase (enrichments of 2.6- and 5.2-fold, respectivley). This enrichment could not be attributed to the plasma membrane content of the smooth microsome fraction since plasma membrane fractions from rat liver were shown to have relatively low concentrations of these two transferases (enrichments of 0.3 or less). Rat liver plasma membrane was also shown to have similarly low relative specific activities for three other glycosyltransferases (sialyl-, galactosyl-, and N-acetylglucosaminyl-). The accurate determination of the glycosyltransferase activities of the plasma membrane fraction required the use of relatively low concentrations of plasma membrane and relatively high concentrations of nucleotide-sugars in order to avoid interference by the high nucleotide-sugar pyrophosphatase and hydrolase activities of this fraction.     

The effect of D-xylose, beta-D-xylosides and beta-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage.

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, beta-D-xylosides and beta-D-galactosides to the incubation medium. Beta-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, beta-D-xylosides and beta-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.     

An allied reprogramming,selection, expansion and differentiation platform for creating hiPSC on microcarriers

ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.Materials and MethodsFive sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.ResultsImprovement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.ConclusionsOur method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

We have developed an allied protocol for reprogramming, selecting, expanding and differentiating human pluripotent stem cells on Microcarriers (designated as RepMC). This method allows faster reprogramming, selecting 30‐50‐fold more candidates for characterization and also allows us to find high quality candidates that differentiate to cardiomyocytes and blood lineages. Mechanistically, this method appears to accelerate the induction, maturation and stabilization phases of reprogramming. Our findings help simplify the process of deriving and expanding iPSCs for therapeutic applications, offering a robust and scalable suspension platform for large‐scale generation of clinical grade iPSCs.     

Focus: Vaccines: Myocarditis Following COVID-19 Vaccination: A Systematic Review of Case Reports

Introduction: COVID-19, the infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), often presents with a spectrum of symptoms at varying levels of severity, ranging from asymptomatic patients to those with fatal complications, such as myocarditis. With increased availability of COVID-19 vaccines, the awareness of possible side effects has expanded as reports surface. This study reviewed cases of myocarditis following COVID-19 vaccination and with existing literature on COVID-19 infection-induced myocarditis to compare clinical courses and analyze possible mechanisms of action. Methods: A systematic review of literature was conducted to identify published case reports (as of February 3, 2022) pertaining to the development of myocarditis following COVID-19 vaccination with either Pfizer or Moderna for an in-depth analysis. Additional subgroup analyses were conducted based on age, past medical history, vaccine manufacturer, and dose number. Results: There were 53 eligible case reports that were included in this study. Patients were mostly male with a median age of 24 years, and the most reported symptom upon presentation was chest pain. Seventy percent of the cases involved the Pfizer vaccine with a majority of myocarditis developing subsequent to second dose. Resolution of symptoms was achieved in all but one patient. Clinical severity, as measured primarily by left ventricular ejection fraction, appeared to be worse among adult patients than pediatric, as well as for patients with comorbidities. Conclusion: This study revealed an observable association between COVID-19 vaccines and myocarditis. However, the clinical course and prognosis seem favorable and less prevalent than those conferred from natural infection.     

Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.