Plant carboxylesterases involved in pesticide hydrolysis

Many herbicides are applied in the form of carboxylic esters to increase their hydrophobicity and hence aid their passage through the waxy cuticle. Hydrolysis in planta of these pro-herbicide esters releases the active acid or alcohol and the rate of this cleavage can be a factor in determining herbicide selectivity. Protein extracts from 13 crop and weed species were assayed for carboxylesterase activity toward multiple xenobiotic and pesticide ester substrates, including 2,4-D-methyl, aryloxyphenoxypropionate esters and p-nitrophenyl esters. A diversity of activities was exhibited by the different species, with Arabidopsis thaliana extracts showing high hydrolytic activity toward several xenobiotic esters, particularly 2,4-D- methyl.The major 2,4-D-methyl hydrolysing enzyme in arabidopsis cell cultures was purified through three rounds of chromatography, then selectively labelled with a biotinylated fluorophosphonate probe (FP-biotin). Following streptavidin affinity purification, the labelled protein was identified by proteomics as the previously uncharacterised serine hydrolase AtCXE12. Recombinant AtCXE12 was subsequently confirmed to effectively hydrolyse 2,4-D-methyl.A T-DNA insertion knockout line that did not express AtCXE12 was identified and characterised. Protein from the knockout plants did not contain AtCXE12 and was found to have a reduced rate of 2,4-D-methyl hydrolysis compared to wild-type plant extracts. This translated into a higher tolerance of 2,4-D-methyl in young atcxel2 plants, due to a lower rate of bioactivation of the pro-herbicide.The fluorophosphonate-based chemical probe was subsequently used to identify other major serine hydrolases in arabidopsis. AtCXE12 and three previously uncharacterised hydrolases were identified, each belonging to a distinct enzyme family

One-Pot Phosphate-Mediated Synthesis of Novel 1,3,5-Trisubstituted Pyridinium Salts: A New Family of S. aureus Inhibitors

Polysubstituted pyridinium salts are valuable pharmacophores found in many biologically active molecules. Their synthesis typically involves the use of multistep procedures or harsh reaction conditions. Here, we report water-based phosphate mediated reaction conditions that promote the condensation of arylacetaldehydes with amines to give 1,3,5-pyridinium salts. The reaction, carried out at pH 6, provides conditions suitable for the use of less stable aldehydes and amines in this Chichibabin pyridine condensation. The evaluation of selected 1,3,5-trisubstituted pyridinium salts highlighted that they can inhibit the growth of S. aureus in the low μg/mL range. The synthetic accessibility of these compounds and preliminary growth inhibition data may pave the way towards the discovery of new anti-bacterials based on the 1,3,5-trisubstituted pyridinium scaffold

Genes and Pathways Associated with Skeletal Sagittal Malocclusions: A Systematic Review

Skeletal class II and III malocclusions are craniofacial disorders that negatively impact people’s quality of life worldwide. Unfortunately, the growth patterns of skeletal malocclusions and their clinical correction prognoses are difficult to predict largely due to lack of knowledge of their precise etiology. Inspired by the strong inheritance pattern of a specific type of skeletal malocclusion, previous genome-wide association studies (GWAS) were reanalyzed, resulting in the identification of 19 skeletal class II malocclusion-associated and 53 skeletal class III malocclusion-associated genes. Functional enrichment of these genes created a signal pathway atlas in which most of the genes were associated with bone and cartilage growth and development, as expected, while some were characterized by functions related to skeletal muscle maturation and construction. Interestingly, several genes and enriched pathways are involved in both skeletal class II and III malocclusions, indicating the key regulatory effects of these genes and pathways in craniofacial development. There is no doubt that further investigation is necessary to validate these recognized genes’ and pathways’ specific function(s) related to maxillary and mandibular development. In summary, this systematic review provides initial insight on developing novel gene-based treatment strategies for skeletal malocclusions and paves the path for precision medicine where dental care providers can make an accurate prediction of the craniofacial growth of an individual patient based on his/her genetic profile. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

'Dopamine-first' mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile

Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the Pictet–Spengler condensation of dopamine and an aldehyde, forming a substituted (S)-tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both in vitro biocatalysis and in vivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the ‘dopamine-first’ mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity

Mass Cytometry profiling of the peripheral blood immunome in patients with psoriasis and psoriatic arthritis uncovers potential biomarkers related to disease progression

Cutaneous psoriasis (PsC) is an auto-immune disorder affecting 60 million people globally, among 30% of whom progress to psoriatic arthritis (PsA), a disease with poorly understood etiology, making diagnosis and treatment difficult. Indeed, a complete systemic immune profile of PsA has yet to be performed. In the study herein, we collected peripheral blood samples from patients with PsC, PsA with or without systemic therapy, and healthy controls (HC), and utilized mass cytometry by time of flight (CyTOF) to acquire immune cell profiles of major leukocyte subsets. We found that patients with PsC and/or PsA exhibited increased frequencies of intermediate (CD14+CD16+) and nonclassical (CD14-CD16+) monocytes as well as regulatory T cells. Separation of our heterogenous patient population revealed distinct immune profiles according to ethnicity and sex in patient groups. Analysis of homing markers revealed upregulation of CCR4, CCR7, and CXCR3 on Classical Monocytes and/or Naïve CD8+ T cells in PsC and/or PsA patients. Moreover, analysis of functional markers revealed upregulation of CD38, CD28, and CD25 on Tregs and EM CD4+ T cells in PsC and/or PsA patients. Unbiased machine learning algorithms (CITRUS) revealed upregulation of Classical Monocytes in PsC and PsA compared to HC patients. Lastly, CITRUS revealed upregulated Intermediate Monocytes in PsA compared to PsC patients, and upregulated Classical Monocytes in treated PsA compared to untreated PsA patients. Therefore, we provide a comprehensive profile of immune cell population frequencies and phenotypes in patients with PsC and PsA, highlighting Monocytes and Tregs as potential biomarkers for early diagnosis of PsA

Hyperbaric Oxygen Therapy Facilitates Healing of Chronic Foot Ulcers in Patients With Diabetes

OBJECTIVE: Chronic diabetic foot ulcers are a source of major concern for both patients and health care systems. The aim of this study was to evaluate the effect of hyperbaric oxygen therapy (HBOT) in the management of chronic diabetic foot ulcers. RESEARCH DESIGN AND METHODS: The Hyperbaric Oxygen Therapy in Diabetics with Chronic Foot Ulcers (HODFU) study was a randomized, single-center, double-blinded, placebo-controlled clinical trial. The outcomes for the group receiving HBOT were compared with those of the group receiving treatment with hyperbaric air. Treatments were given in a multi-place hyperbaric chamber for 85-min daily (session duration 95 min), five days a week for eight weeks (40 treatment sessions). The study was performed in an ambulatory setting. RESULTS: Ninety-four patients with Wagner grade 2, 3, or 4 ulcers, which had been present for >3 months, were studied. In the intention-to-treat analysis, complete healing of the index ulcer was achieved in 37 patients at 1-year of follow-up: 25/48 (52%) in the HBOT group and 12/42 (29%) in the placebo group (P = 0.03). In a sub-analysis of those patients completing >35 HBOT sessions, healing of the index ulcer occurred in 23/38 (61%) in the HBOT group and 10/37 (27%) in the placebo group (P = 0.009). The frequency of adverse events was low. CONCLUSIONS: The HODFU study showed that adjunctive treatment with HBOT facilitates healing of chronic foot ulcers in selected patients with diabetes

Secretion by Overexpression and Purification of the Water-Soluble Streptomyces K15 Dd-Transpeptidase/Penicillin-Binding Protein

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity

RNA Sequencing Reveals Diverse Functions of Amniotic Fluid Neutrophils and Monocytes/Macrophages in Intra-Amniotic Infection

Intra-amniotic infection, the invasion of microbes into the amniotic cavity resulting in inflammation, is a clinical condition that can lead to adverse pregnancy outcomes for the mother and fetus as well as severe long-term neonatal morbidities. Despite much research focused on the consequences of intra-amniotic infection, there remains little knowledge about the innate immune cells that respond to invading microbes. We performed RNA-seq of sorted amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection to determine the transcriptomic differences between these innate immune cells. Further, we sought to identify specific transcriptomic pathways that were significantly altered by the maternal or fetal origin of amniotic fluid neutrophils and monocytes/macrophages, the presence of a severe fetal inflammatory response, and pregnancy outcome (i.e., preterm or term delivery). We show that significant transcriptomic differences exist between amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection, indicating the distinct roles these cells play. The transcriptome of amniotic fluid immune cells varies based on their maternal or fetal origin, and the significant transcriptomic differences between fetal and maternal monocytes/macrophages imply that those of fetal origin exhibit impaired functions. Notably, transcriptomic changes in amniotic fluid monocytes/macrophages suggest that these immune cells collaborate with neutrophils in the trafficking of fetal leukocytes throughout the umbilical cord (i.e., funisitis). Finally, amniotic fluid neutrophils and monocytes/macrophages from preterm deliveries display enhanced transcriptional activity compared to those from term deliveries, highlighting the protective role of these cells during this vulnerable period. Collectively, these findings demonstrate the underlying complexity of local innate immune responses in women with intra-amniotic infection and provide new insights into the functions of neutrophils and monocytes/macrophages in the amniotic cavity. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements