Adrenergic agonists with (R)-configuration are active pharmaceutical ingredients, whereas the (S)-enantiomers exhibit reduced activity or even toxicity. However, the asymmetric synthesis of adrenergic agonist precursors is highly challenging due to the instability of the characteristic catechol/phenolic hydroxyl groups and the poor reactivity of their starting materials in adol condensation reactions. In this work, by application of the highly stereoselective L-threonine aldolase (LTA) variant LTA-LN and efficient tyrosine decarboxylase from Enterococcus faecalis (EfaTDC), a bienzymatic cascade system for the asymmetric synthesis of (R)-configured adrenergic agonist precursors was developed. The versatile precursors for most adrenergic agonists were achieved as (R)-enantiomers (88-98% yield; 77-93% enantiomeric excess (ee)), with innocuous CO2 as the sole byproduct. Significantly, the clinical first-line emergency medication (R)-norepinephrine (98% yield, 84% ee) and the precursor for the natural adrenergic agonist (R)-synephrine (95% yield, 93% ee) were synthesized efficiently. Further mechanistic studies revealed that the high stereoselectivity and robust stereochemical control of LTA-LN, coupled with the efficient catalysis by EfaTDC, critically governed reaction outcomes. This bienzymatic system synergistically leverages the strengths of LTA-LN and EfaTDC while compensating for their individual limitations, representing an efficient LTA-mediated catalytic strategy for adrenergic agonist precursors with pharmacologically active (R)-configuration.

In this research, a composite hydrogel microsphere material, SA/P(NVCL-ALS)-N-ZnO/A-Dol-urea (hereafter referred to as SA-PANZ), with dual responsiveness to temperature and pH, was developed by integrating aminated lignosulfonate sodium (ALS), poly(N-vinylcaprolactam) (PNVCL), nano zinc oxide (N-ZnO), and alkali-treated dolomite (A-Dol). The material exhibited excellent controlled-release performance, with a cumulative urea release rate of 67.22% at 40 °C and pH = 4. The Ritger-Peppas model fitted well with the release data, providing strong theoretical support for optimizing the microspheres. Compared to control groups, both the loading efficiency and encapsulation efficiency were significantly improved, achieving 38.50 and 12.83%, respectively. Water retention tests demonstrated that the microspheres maintained 39.92% of their moisture after 5 h, indicating an excellent water-holding capacity. Degradation experiments indicated a degradation rate of 60.14% under acidic conditions, demonstrating excellent biodegradability. Antibacterial assays revealed a clear inhibitory effect against Aspergillus niger, and plant cultivation experiments confirmed that the microspheres promoted pea growth under acidic and high-temperature stress. This research introduced a novel biodegradable microsphere-based soil conditioner with strong potential to improve nutrient use efficiency and soil quality in agricultural environments.

Adol condensation mediated by L-threonine aldolase (LTA) is a promising route to synthesize β-hydroxy-α-amino acids, such as the anti-Parkinson's disease drug L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS; droxidopa). However, the biocatalytic approach generally suffered from poor stereoselectivity at the Cβ chiral center. Herein, several LTAs from diverse microorganisms were examined for the stereoselective synthesis of L-threo-DOPS, and an LTA from Firmicutes bacterium (FbLTA) was identified with relatively high stereoselectivity. A semi-rational design was then employed to further enhance the stereoselectivity, resulting in the identification of beneficial mutations and the generation of beneficial mutants with markedly elevated stereoselectivity (up to 80 % de) over the wild-type (23 % de). Molecular dynamics (MD) simulation revealed that the significant improvement of stereoselectivity could be attributed to the structural reconstitution of substrate tunnels. Finally, the best mutant was applied to a scaled-up reaction, affording 1.3 g/L yield of L-threo-DOPS with 80 % de. This study provides new insights into the stereoselectivity control of LTAs and potential engineered LTAs for the manufacture of L-threo-DOPS.

The Developmental Origins of Health and Disease (DOHaD) concept has been established for three decades. Many studies have shown that, besides pregnancy, other plastic phases (mainly preconception, lactation, and infancy-adolescence) are also sensitive to environmental changes, including nutritional conditions, that can program health or disease later in life. This study compared the susceptibility of the gestation, lactation and adolescence to a high-fat diet (HFD) intervention to program rats into autonomic nervous system imbalance and cardiometabolic dysfunction in adulthood. Four groups of rats were studied: offspring from mothers exposed to a HFD (35% fat) or a standard chow diet (4.5% fat) during gestation (GEST and CONT groups, respectively), offspring from mothers exposed to the HFD during lactation (LAC), and adolescent rats exposed to the HFD (ADOL). Mothers treated during pregnancy exhibited a higher body mass, but nursing mothers presented the highest food energy intake and higher adiposity. Compared to the other groups, the LAC rats showed increased body mass gain, food energy intake, body fat, glucose intolerance and blood pressure. LAC group also showed increased parasympathetic activity. In contrast, LAC sympathetic nerve activity decreased compared with the other groups. The ADOL group exhibited mostly similar responses but of a smaller magnitude. This suggests that the lactation phase is the most sensitive to HFD programming for cardiometabolic dysfunction in adulthood and that early overnutrition may affect neural connections by altering the autonomic nervous system balance.

Fifty-seven Gallid alphaherpesvirus 2 (GaHV-2) isolates, collected during a 30-year period (1990-2019) from commercial poultry flocks affected by Marek's disease (MD), were molecularly characterised. The GaHV-2 meq gene was amplified and sequenced to evaluate the virus virulence, based on the number of PPPPs within the proline-rich repeats (PRRs) of its transactivation domain. The present illustration of virus virulence evaluation on a large scale of field virus isolates by molecular analysis exemplifies the practical benefit and usefulness of the molecular marker in commercial GaVH-2 isolates. The alternative assay of GaVH-2 virulence pathotyping is the classical Gold Standard ADOL method, which is difficult and impossible to employ on a large scale using the Specific Pathogen Free (SPF) chicks of the ADOL strains kept in isolators for two months. The phylogenetic analysis performed in the present study showed that the meq gene amino acid sequences of the 57 Israeli strains divide into 16 phylogenetic branches. The virulence evaluation was performed in comparison with 36 GaHV-2 prototype strains, previously characterised by the in vivo Gold Standard ADOL assay. The results obtained revealed that the GaHV-2 strains circulating in Israel have evolved into a higher virulence potential during the years, as the four-proline stretches number in the meq gene decreased over the investigated period, typically of very virulent virus prototypes. The present study supports the meq gene molecular markers for the assessment of field GaVH-2 strains virulence.

A widely used approach for protein conjugation is through the lysine residues reacting with NHS- or other active esters. However, it is a challenge to precisely control the degree of labeling (DoL) due to the instability of active ester and variability of reaction efficiencies. Here, we provide a protocol for better control of aDoL using existing Copper-free Click Chemistry reagents. It is a two-step reaction with one purification in between. Briefly, proteins of interest were first activated with azide-NHS. After removing unreacted azide-NHS, the protein-N3 is then reacted with a limited amount of complementary click tag. Our studies have shown the click tag will fully react with the protein-N3 after 24 h' incubation, and therefore does not require additional purification steps. As such, the aDoL is equal to the input molar ratio of the click tag and the protein. Furthermore, this approach offers a much simpler and more economical way to perform parallel microscale labeling. Once a protein is pre-activated with N3-NHS, any fluorophore or molecule with the complementary click tag can be attached to the protein by mixing the two ingredients. Quantities of the protein used in the click reaction can be at any desired amount. In one example, we labeled an antibody in parallel with 9 different fluorophores using a total of 0.5 mg of antibody. In another example, we labeled Ab with targeted aDoL value from 2 to 8. In a stability comparison study, we have found the conjugated fluorophore using the suggested click protocol stayed attached to the protein longer than with standard NHS-fluorophore labeling.

Unlike rodent cells, spontaneous immortalization of avian cells and human cells is a very rare event. According to patent publications and current literature, there are no more than 4 spontaneously immortalized chicken embryo fibroblast (CEF) cell lines established up to date. One of those cell lines is ADOL (Avian Disease and Oncology Laboratory) ZS-1 cell line, which was established by continuous passaging of the CEFs derived from the specific pathogen free (SPF) 0.TVB*S1 (commonly known as rapid feathering susceptible or RFS) genetic line of chickens. The RFS genetic line of chickens was developed and has been maintained on the SPF chicken farm of USDA-ARS facility, ADOL, in East Lansing, Michigan, which is known as one of a few lines of chickens that are free of any known avian endogenous virus genes. To explore potential roles that epigenetic factors may play in modulating cellular senescence processes and spontaneous immortalization state, total RNAs extracted from samples of the RFS primary CEFs, RFS CEFs reached the 21st passage, and the ZS-1 cells were subjected to small RNA sequencing. Collectively, a total of 531 miRNAs was identified in the 3 types of samples. In contrast to the primary CEF samples, 50 miRNAs were identified with significantly differential expression only in the 21st passage samples; a different subset of 63 differentially expressed miRNAs was identified only in the ZS-1 samples; the majority of differentially expressed miRNAs identified in both the 21st passage CEF and the ZS-1 samples were more or less directionally consistent. Gene Ontology analysis results suggested that the epigenetic factor, miRNAs, plays a role in modulating the cellular senescence and spontaneous immortalization processes through various bioprocesses and key pathways including ErbB and MAPK signaling pathways. These findings provided the experimental and bioinformatic evidence for a better understanding on the epigenetic factor of miRNAs in association with cellular senescence and spontaneous immortalization process in avian cells.