Temporal Dynamics of Powdery Mildew (Oidium neolycopersici) and its Effects on the Host Growth Dynamics of Tomato

Controlled glasshouse experiments were conducted to investigate the temporal progress of powdery mildew and its effects on host dynamics of tomato, without and with one fungicide application. Healthy tomato transplants (5‐ to 6‐week old) were artificially inoculated with powdery mildew, and disease progress as well as host growth were monitored in both fungicide sprayed and unsprayed treatments and compared with non‐inoculated plants. Actual disease severity on a plant basis increased in unsprayed plants reaching maximum severity in the proportionate range of 0.53–0.83. One fungicide spray significantly reduced the maximum disease severity by two‐ to fourfolds. Despite adjustments for defoliation, declines in the proportion of disease severity between successive assessments were evident. Whereas the estimated growth rates of diseased plants were significantly lower than that of healthy plants, no significant differences were observed in the maximum leaf area formed of inoculated and non‐inoculated plants. A considerable effect of the powdery mildew epidemics was manifested through hastened shrivelling and defoliation of diseased leaves within the tomato canopy. An average of 18–29% and 40–52% of leaves had abscised from the plant canopy at the last date of assessment in sprayed and non‐sprayed plants, respectively. Accordingly, defoliation accounted for 14–33.3% and 58.3–63.1% losses in leaf area of sprayed and non‐sprayed plants, respectively. Duration of healthy leaf area and yield of inoculated plants were also significantly reduced by powdery mildew epidemics.     

An 86Y-labeled mirror-image oligonucleotide: influence of Y-DOTA isomers on the biodistribution in rats

A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.     

A Missing PD-L1/PD-1 Coinhibition Regulates Diabetes Induction by Preproinsulin-Specific CD8 T-Cells in an Epitope-Specific Manner

Coinhibitory PD-1/PD-L1 (B7-H1) interactions provide critical signals for the regulation of autoreactive T-cell responses. We established mouse models, expressing the costimulator molecule B7.1 (CD80) on pancreatic beta cells (RIP-B7.1 tg mice) or are deficient in coinhibitory PD-L1 or PD-1 molecules (PD-L1^−/− and PD-1^−/− mice), to study induction of preproinsulin (ppins)-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD) by DNA-based immunization. RIP-B7.1 tg mice allowed us to identify two CD8 T-cell specificities: pCI/ppins DNA exclusively induced K^b/A_12–21-specific CD8 T-cells and EAD, whereas pCI/ppinsΔA_12–21 DNA (encoding ppins without the COOH-terminal A_12–21 epitope) elicited K^b/B_22–29-specific CD8 T-cells and EAD. Specific expression/processing of mutant ppinsΔA_12–21 (but not ppins) in non-beta cells, targeted by intramuscular DNA-injection, thus facilitated induction of K^b/B_22–29-specific CD8 T-cells. The A_12–21 epitope binds K^b molecules with a very low avidity as compared with B_22–29. Interestingly, immunization of coinhibition-deficient PD-L1^−/− or PD-1^−/− mice with pCI/ppins induced K^b/A_12–21-monospecific CD8 T-cells and EAD but injections with pCI/ppinsΔA_12–21 did neither recruit K^b/B_22–29-specific CD8 T-cells into the pancreatic target tissue nor induce EAD. PpinsΔA_12–21/(K^b/B_22–29)-mediated EAD was efficiently restored in RIP-B7.1⁺/PD-L1^−/− mice, differing from PD-L1^−/− mice only in the tg B7.1 expression in beta cells. Alternatively, an ongoing beta cell destruction and tissue inflammation, initiated by ppins/(K^b/A_12–21)-specific CD8 T-cells in pCI/ppins+pCI/ppinsΔA_12–21 co-immunized PD-L1^−/− mice, facilitated the expansion of ppinsΔA_12–21/(K^b/B_22–29)-specific CD8 T-cells. CD8 T-cells specific for the high-affinity K^b/B_22–29- (but not the low-affinity K^b/A_12–21)-epitope thus require stimulatory ´help from beta cells or inflamed islets to expand in PD-L1-deficient mice. The new PD-1/PD-L1 diabetes models may be valuable tools to study under well controlled experimental conditions distinct hierarchies of autoreactive CD8 T-cell responses, which trigger the initial steps of beta cell destruction or emerge during the pathogenic progression of EAD.     

The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes

Glc7, the yeast protein phosphatase 1, is a component of the cleavage and polyadenylation factor (CPF). Here we show that downregulation of Glc7, or its dissociation from CPF in the absence of CPF subunits Ref2 or Swd2, results in similar snoRNA termination defects. Overexpressing a C-terminal fragment of Sen1, a superfamily I helicase required for snoRNA termination, suppresses the growth and termination defects associated with loss of Swd2 or Ref2, but not Glc7. Suppression by Sen1 requires nuclear localization and direct interaction with Glc7, which can dephosphorylate Sen1 in vitro. The suppressing fragment, and in a similar manner full-length Sen1, copurifies with the snoRNA termination factors Nrd1 and Nab3, suggesting loss of Glc7 from CPF can be compensated by recruiting Glc7 to Nrd1-Nab3 through Sen1. Swd2 is also a subunit of the Set1c histone H3K4 methyltransferase complex and is required for its stability and optimal methyltransferase activity.     

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.     

Treatment-resistant depression: definition,prevalence, detection,management, and investigational interventions

Treatment-resistant depression (TRD) is common and associated with multiple serious public health implications. A consensus definition of TRD with demonstrated predictive utility in terms of clinical decision-making and health outcomes does not currently exist. Instead, a plethora of definitions have been proposed, which vary significantly in their conceptual framework. The absence of a consensus definition hampers precise estimates of the prevalence of TRD, and also belies efforts to identify risk factors, prevention opportunities, and effective interventions. In addition, it results in heterogeneity in clinical practice decision-making, adversely affecting quality of care. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have adopted the most used definition of TRD (i.e., inadequate response to a minimum of two antidepressants despite adequacy of the treatment trial and adherence to treatment). It is currently estimated that at least 30% of persons with depression meet this definition. A significant percentage of persons with TRD are actually pseudo-resistant (e.g., due to inadequacy of treatment trials or non-adherence to treatment). Although multiple sociodemographic, clinical, treatment and contextual factors are known to negatively moderate response in persons with depression, very few factors are regarded as predictive of non-response across multiple modalities of treatment. Intravenous ketamine and intranasal esketamine (co-administered with an antidepressant) are established as efficacious in the management of TRD. Some second-generation antipsychotics (e.g., aripiprazole, brexpiprazole, cariprazine, quetiapine XR) are proven effective as adjunctive treatments to antidepressants in partial responders, but only the olanzapine-fluoxetine combination has been studied in FDA-defined TRD. Repetitive transcranial magnetic stimulation (TMS) is established as effective and FDA-approved for individuals with TRD, with accelerated theta-burst TMS also recently showing efficacy. Electroconvulsive therapy is regarded as an effective acute and maintenance intervention in TRD, with preliminary evidence suggesting non-inferiority to acute intravenous ketamine. Evidence for extending antidepressant trial, medication switching and combining antidepressants is mixed. Manual-based psychotherapies are not established as efficacious on their own in TRD, but offer significant symptomatic relief when added to conventional antidepressants. Digital therapeutics are under study and represent a potential future clinical vista in this population.     

The tryptophan aminotransferase Tam1 catalyses the single biosynthetic step for tryptophan-dependent pigment synthesis in Ustilago maydis

Tryptophan is a precursor for many biologically active secondary metabolites. We have investigated the origin of indole pigments first described in the pityriasis versicolor-associated fungus Malassezia furfur . Some of the identified indole pigments have properties potentially explaining characteristics of the disease. As M. furfur is not amenable to genetic manipulation, we used Ustilago maydis to investigate the pathway leading to pigment production from tryptophan. We show by high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance analysis that the compounds produced by U. maydis include those putatively involved in the etiology of pityriasis versicolor. Using a reverse genetics approach, we demonstrate that the tryptophan aminotransferase Tam1 catalyses pigment biosynthesis by conversion of tryptophan into indolepyruvate. A forward genetics approach led to the identification of mutants incapable of producing the pigments. These mutants were affected in the sir1 gene, presumably encoding a sulphite reductase. In vitro experiments with purified Tam1 showed that 2-oxo 4-methylthio butanoate serves as a substrate linking tryptophan deamination to sulphur metabolism. We provide the first direct evidence that these indole pigments form spontaneously from indolepyruvate and tryptophan without any enzymatic activity. This suggests that compounds with a proposed function in M. furfur -associated disease consist of indolepyruvate-derived spontaneously generated metabolic by-products.     

Aldosterone regulates paracellular pathway resistance in rabbit distal colon

Summary Regulation of the paracellular pathway in rabbit distal colon by the hormone aldosterone was investigated in vitro in Ussing chambers by means of transepithelial and microelectrode techniques. To evaluate the cellular and paracellular resistances an equivalent circuit analysis was used. For the analysis the apical membrane resistance was altered using the antibiotic nystatin. Under control conditions two groups of epithelia were found, each clearly dependent on the light: dark regime. Low-transporting epithelia (LT) were observed in the morning and high-transporting epithelia (HT) in the afternoon. Na⁺ transport was about 3-fold higher in HT than in LT epithelia. Incubating epithelia of both groups with 0.1 mol·1^-1 aldosterone on the serosal side nearly doubled in LT epithelia the short circuit current and transepithelial voltage but the transepithelial resistance was not influenced. Maximal values were reached after 4–5 h of aldosterone treatment. In HT epithelia due to the effect of aldosterone all three transepithelial parameters remained constant over time. Evaluation of the paracellular resistance revealed a significant increase after aldosterone stimulation in both epithelial groups. This increase suggests that tight junctions might have been regulated by aldosterone. The hormonal effect on electrolyte transport was also dependent on the physiological state of the rabbit colon. Since net Na⁺ absorption in distal colon is, in addition to transcellular absorption capacity, also dependent on the permeability of the paracellular pathway, the regulation of tight junctions by aldosterone may be a potent mechanism for improving Na⁺ absorption during hormone-stimulated ion transport.Abbreviations V _t transepithelial potential difference (mV) - R _t transepithelial resistance (·cm²) - G _t transepithelial conductance (mS·cm^-2) - I_sc calculated short circuit current (A·cm^-2) - V _a apical membrane potential difference (mV) - V _bl basolateral membrane potential difference (mV) - voltage divider ratio - R _a apical membrane resistance (·cm²) - R _bl basolateral membrane resistance (·cm²) - R _c cellular resistance ( of apical and basolateral resistance) (·cm²) - R _p resistance of the paracellular pathway (·cm²) - G _a apical membrane conductance (mS·cm^-2) - G _bl basolateral membrane conductance (mS·cm^-2) - G _p paracellular conductance (mS·cm^-2) - G _t transepithelial conductance (mS·cm^-2) - HT _contr high transporting control epithelia - LT _contr low transporting control epithelia - HT _aldo aldosterone incubated high transporting epithelia - LT _aldo aldosterone incubated low transporting epithelia     

A hydroponic culture system for growing<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plantlets under sterile conditions

We developed a hydroponic cultivation system for growingArabidopsis plantlets under sterile, controlled environmental conditions. The system consists of a piece of stainless-steel wire cloth (125 μm mesh size) that is fixed between 2 flat rings and held in place by 3 legs, placed in a commercially-available glass jar, and covered by the original glass lid or a sheet of sterilized cellophane. Sterilized seeds were distributed evenly across the mesh piece, the size of which allowed root growth and kept the seeds in place. After 3 weeks of cultivation, shoot and root tissues were easily harvested without mechanical damage. Proteome and metabolite analyses were performed on root and shoot tissues and demonstrated excellent reproducibility, indicating that the system is advantageous when biological variation is minimized. Induction experiments can be performed by transferring the apparatus (with plants) to a new jar containing a different nutrient solution. The apparatus is reusable and can easily be sterilized by autoclaving or dry heat. The system can be adapted to other small-seed plants by varying the mesh size.     

Fermentative degradation of monohydroxybenzoates by defined syntrophic cocultures

From anaerobic freshwater enrichment cultures with 3-hydroxybenzoate as sole substrate, a slightly curved rod-shaped bacterium was isolated in coculture with Desulfovibrio vulgaris as hydrogen scavenger. The new isolate degraded only 3-hydroxybenzoate or benzoate, and depended on syntrophic cooperation with a hydrogenoxidizing methanogen or sulfate reducer. 3-Hydroxybenzoate was degraded via reductive dehydroxylation to benzoate. With 2-hydroxybenzoate (salicylate), short coccoid rods were enriched from anaerobic freshwater mud samples, and were isolated in defined coculture with D. vulgaris. This isolate also fermented 3-hydroxybenzoate or benzoate in obligate syntrophy with a hydrogen-oxidizing anaerobe. The new isolates were both Gram-negative, non-sporeforming strict anaerobes. They fermented hydroxybenzoate or benzoate to acetate, CO₂, and, presumably, hydrogen which was oxidized by the syntrophic partner organism. With hydroxybenzoates, but not with benzoate, Acetobacterium woodii could also serve as syntrophic partner. Other substrates such as sugars, alcohols, fatty or amino acids were not fermented. External electron acceptors such as sulfate, sulfite, nitrate, or fumarate were not reduced. In enrichment cultures with 4-hydroxybenzoate, decarboxylation to phenol was the initial step in degradation which finally led to acetate, methane and CO₂.