Prescreening of biological and biorational fungicides against common hemp pathogens using in vitro analyses

Upon reintroduction of hemp in 2014 and legalization in 2018, labeled pesticides have remained limited. Further, consumer demand has aimed the market toward organic or chemical-free production systems. In efforts to manage diseases and pests in fields and greenhouses, producers turn toward biological and biorational formulations. Efficacy of these fungicides against common aerial diseases of hemp is largely unpublished. Challenges of efficacy testing, however, further delay or discourage research. In this study, we evaluated screening methods against some common biological products. The aim was to test a screening model in order to examine products against fungal pathogens and to identify demonstrable differences under controlled conditions. Thus, in this study, we prescreen 11 biological and biorational fungicides against four common fungal leaf and flower pathogens using three bioassays. Confirmation that the major modes of action for these products have measurable activity against major pathogens of hemp serves as a first step toward more complex field studies.

Fusion of Chitin-Binding Domain From Chitinolyticbacter meiyuanensis SYBC-H1 to the Leaf-Branch Compost Cutinase for Enhanced PET Hydrolysis

Polyethylene terephthalate (PET) is a mass-produced petroleum-based non-biodegradable plastic that contributes to the global plastic pollution. Recently, biocatalytic degradation has emerged as a viable recycling approach for PET waste, especially with thermophilic polyester hydrolases such as a cutinase (LCC) isolated from a leaf-branch compost metagenome and its variants. To improve the enzymatic PET hydrolysis performance, we fused a chitin-binding domain (ChBD) from Chitinolyticbacter meiyuanensis SYBC-H1 to the C-terminus of the previously reported LCCICCG variant, demonstrating higher adsorption to PET substrates and, as a result, improved degradation performance by up to 19.6% compared to with its precursor enzyme without the binding module. For compare hydrolysis with different binding module, the catalytic activity of LCCICCG-ChBD, LCCICCG-CBM, LCCICCG-PBM and LCCICCG-HFB4 were further investigated with PET substrates of various crystallinity and it showed measurable activity on high crystalline PET with 40% crystallinity. These results indicated that fusing a polymer-binding module to LCCICCG is a promising method stimulating the enzymatic hydrolysis of PET.

APPLICATION OF A SCINTILLATION GAMMA-SPECTROMETER FOR DETERMINATION OF RADON CONTENT IN WATER

Experimental studies have been carried out to determine the sensitivity of a mobile scintillation gamma-spectrometer to radon-222 in mineral water samples for the selected measurement geometry and the minimum measurable activity of radon-222 in such samples. The measurement results of radon content in mineral water samples obtained using such gamma-spectrometer are presented too.

Tamoxifen Derivatives Alter Retromer-Dependent Endosomal Tubulation and Sorting to Block Retrograde Trafficking of Shiga Toxins

Shiga toxin 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol of cells where they target ribosomes. As retrograde trafficking is essential for disease, inhibiting STx1/STx2 trafficking is therapeutically promising. Recently, we discovered that the chemotherapeutic drug tamoxifen potently inhibits the trafficking of STx1/STx2 at the critical early endosome-to-Golgi step. We further reported that the activity of tamoxifen against STx1/STx2 is independent of its selective estrogen receptor modulator (SERM) property and instead depends on its weakly basic chemical nature, which allows tamoxifen to increase endolysosomal pH and alter the recruitment of retromer to endosomes. The goal of the current work was to obtain a better understanding of the mechanism of action of tamoxifen against the more disease-relevant toxin STx2, and to differentiate between the roles of changes in endolysosomal pH and retromer function. Structure activity relationship (SAR) analyses revealed that a weakly basic amine group was essential for anti-STx2 activity. However, ability to deacidify endolysosomes was not obligatorily necessary because a tamoxifen derivative that did not increase endolysosomal pH exerted reduced, but measurable, activity. Additional assays demonstrated that protective derivatives inhibited the formation of retromer-dependent, Golgi-directed, endosomal tubules, which mediate endosome-to-Golgi transport, and the sorting of STx2 into these tubules. These results identify retromer-mediated endosomal tubulation and sorting to be fundamental processes impacted by tamoxifen; provide an explanation for the inhibitory effect of tamoxifen on STx2; and have important implications for the therapeutic use of tamoxifen, including its development for treating Shiga toxicosis.

Benefits and Drawbacks of Harboring Plasmid pP32BP2, Identified in Arctic Psychrophilic Bacterium Psychrobacter sp. DAB_AL32B

Psychrobacter sp. DAB_AL32B, originating from Spitsbergen island (Arctic), carries the large plasmid pP32BP2 (54,438 bp). Analysis of the pP32BP2 nucleotide sequence revealed the presence of three predicted phenotypic modules that comprise nearly 30% of the plasmid genome. These modules appear to be involved in fimbriae synthesis via the chaperone-usher pathway (FIM module) and the aerobic and anaerobic metabolism of carnitine (CAR and CAI modules, respectively). The FIM module was found to be functional in diverse hosts since it facilitated the attachment of bacterial cells to abiotic surfaces, enhancing biofilm formation. The CAI module did not show measurable activity in any of the tested strains. Interestingly, the CAR module enabled the enzymatic breakdown of carnitine, but this led to the formation of the toxic by-product trimethylamine, which inhibited bacterial growth. Thus, on the one hand, pP32BP2 can enhance biofilm formation, a highly advantageous feature in cold environments, while on the other, it may prevent bacterial growth under certain environmental conditions. The detrimental effect of harboring pP32BP2 (and its CAR module) seems to be conditional, since this replicon may also confer the ability to use carnitine as an alternative carbon source, although a pathway to utilize trimethylamine is most probably necessary to make this beneficial. Therefore, the phenotype determined by this CAR-containing plasmid depends on the metabolic background of the host strain.

Engineering glycoside hydrolase stability by the introduction of zinc binding

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.

Active and non-active agents: residents’ agency in assisted living

ABSTRACTCulturally, institutional care has been seen to strip older people of their status as full adult members of society and turn them into ‘have-nots’ in terms of agency. The substantial emphasis in gerontology of measuring the activity and functional ability of the elderly has unintentionally fostered these stereotypes, as have traditional definitions of agency that emphasise individuals’ choices and capacities. The aim of this paper is to discover what kind of opportunities to feel agentic exist for people who have reduced functional abilities and therefore reside in assisted living. In this paper, agency is approached empirically from the viewpoint of Finnish sheltered housing residents. The data were gathered using participant observation and thematic interviews. This study suggests that even people with substantial declines in their functional abilities may feel more or less agentic depending on their functional and material surroundings and the support they receive from the staff, relatives and other residents. The perception that residents’ agency in assisted living cannot be reduced to measurable activity has methodological implications for gerontological research on agency. Care providers can utilise our findings in reasserting their residents’ quality of life.

Ecology- and Bioassay-Guided Drug Discovery for Treatments of Tropical Parasitic Disease: 5α,8α-Epidioxycholest-6-en-3β-ol Isolated from the Mollusk Dolabrifera dolabrifera Shows Significant Activity against Leishmania donovani

An ecology- and bioassay-guided search employed to discover compounds with activity against tropical parasitic diseases and cancer from the opisthobranch mollusk, Dolabrifera dolabrifera, led to the discovery of antileishmanial properties in the known compound, 5α,8α-epidioxycholest-6-en-3β-ol (1). Compound 1 was identified through nuclear magnetic resonance spectroscopy (1H, 13C) and mass spectrometry. The compound was concentrated in the digestive gland of D. dolabrifera, but was not detected in other body parts, fecal matter or mucus. Compound 1 showed an IC50 of 4.9 μM towards the amastigote form of Leishmania donovani compared with an IC50 of 281 μM towards the control Vero cell line, a 57.3-fold difference, and demonstrated no measurable activity against Plasmodium falciparum, Trypanosoma cruzi, and the breast cancer cell line, MCF-7.

Identification and Characterization of a Novel HIV-1 Nucleotide-Competing Reverse Transcriptase Inhibitor Series

ABSTRACTSeveral groups have recently reported on the identification of nucleotide-competing reverse transcriptase inhibitors (NcRTIs), a new class of RT inhibitors. NcRTIs reversibly inhibit binding of the incoming nucleotide to the RT active site but do not act as chain terminators, unlike the nucleos(t)ide reverse transcriptase inhibitor (NRTI) class. We identified a novel benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI chemical series. Structure-activity relationship evaluation of this series with both RT and viral replication assays led to the identification of compound A, a new NcRTI. Compound A inhibited HIV-1 RT in a primer extension assay (50% inhibitory concentration, 2.6 nM) but had no measurable activity against human DNA polymerase γ at 10 μM. It potently inhibited HIV-1 replicationin vitro(50% effective concentration, 1.5 nM). The antiviral potency of compound A was unaffected by the presence of nonnucleotide RT inhibitor (NNRTI) mutations tested (L100I, K103N/Y181C, V106A, or Y188L). Notably, viruses encoding K65R were hypersusceptible to inhibition by compound A. Compound A also retained full activity against viruses encoding M184V.In vitroselection for resistant virus to compound A led to the selection of a single substitution within RT: W153L. A recombinant virus encoding the RT W153L was highly resistant to compound A (fold change, 160). W153 is a highly conserved residue in HIV RT and has not been previously associated with drug resistance. In summary, a novel NcRTI series with optimized antiviral activity, minimal cross-resistance to existing RT inhibitor classes, and a distinct resistance profile has been discovered. These results further establish NcRTIs as an emerging class of antiretroviral agents.

Purification of a Single Peptide with Vitamin K Epoxide to Vitamin K and Vitamin K to Vitamin KH2 Activity.

More than 21 million prescriptions for warfarin are written yearly in the US. Yet, in spite of its importance, vitamin K epoxide reductase (VKOR), the target of warfarin, has resisted purification since its identification in 1972. We report the first successful purification and reconstitution of activity of a recombinant human vitamin K epoxide reductase. A series of detergents were screened to determine that best for solubilization of VKOR from microsomes. Detergents tested that were effective in solubilization of VKOR also led to loss of measurable activity. This loss of activity supports our previous prediction that VKOR is embedded in and requires a membrane environment for enzymatic activity. The short-chain phospholipid, DHPC (1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine) was the detergent of choice to efficiently extract VKOR from the microsomes, even though this reagent completely inhibited enzyme activity. Partial reconstitution was achieved on-column by washing with 0.4 % dioleoylphosphatidylcholine/0.4% deoxycholate. Complete recovery of activity was achieved by removing the deoxycholate through dialysis in the presence of the reducing reagent, THP (Tris(hydroxypropyl)phosphine). During dialysis, the solution became cloudy indicating the formation of membrane-like structure. Purified recombinant VKOR is ~21 kDa (~18.5 kDa + tag); fully active; and over 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported and assumed that VKOR is a multi-subunit enzyme. Our results, however, suggest that a single peptide can accomplish the reaction. The trace amounts of contaminating proteins were identified by mass spectrometry; however, none are apparently relevant to the VKOR reaction. Moreover, the turn-over number of purified VKOR (0.25 sec-1 is approximately two-fold higher than microsomes and about 10 fold higher than the turnover number of gamma-glutamyl carboxylase for CO2 addition. In addition to the vitamin K epoxide to vitamin K reaction, our results also indicate that VKOR can efficiently convert vitamin K to vitamin K epoxide. Our results suggest that ancillary proteins (other than a thioredoxin-like enzyme) are not necessary for full VKOR activity. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.