The UBA2 domain functions as an intrinsic stabilization signal that protects Rad23 from proteasomal degradation

The proteasome-interacting protein Rad23 is a long-lived protein. Interaction between Rad23 and the proteasome is required for Rad23's functions in nucleotide excision repair and ubiquitin-dependent degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting, transferable stabilization signal that protects Rad23 from proteasomal degradation. Disruption of the UBA2 domain converts Rad23 into a short-lived protein that is targeted for degradation through its N-terminal ubiquitin-like domain. UBA2-dependent stabilization is required for Rad23 function because a yeast strain expressing a mutant Rad23 that lacks a functional UBA2 domain shows increased sensitivity to UV light and, in the absence of Rpn10, severe growth defects. The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is an evolutionarily conserved stabilization signal that allows Rad23 to interact with the proteasome without facing destruction.     

A deer (subfamily Cervinae) genetic linkage map and the evolution of ruminant genomes

Comparative maps between ruminant species and humans are increasingly important tools for the discovery of genes underlying economically important traits. In this article we present a primary linkage map of the deer genome derived from an interspecies hybrid between red deer (Cervus elaphus) and Père David's deer (Elaphurus davidianus). The map is approximately 2500 cM long and contains >600 markers including both evolutionary conserved type I markers and highly polymorphic type II markers (microsatellites). Comparative mapping by annotation and sequence similarity (COMPASS) was demonstrated to be a useful tool for mapping bovine and ovine ESTs in deer. Using marker order as a phylogenetic character and comparative map information from human, mouse, deer, cattle, and sheep, we reconstructed the karyotype of the ancestral Pecoran mammal and identified the chromosome rearrangements that have occurred in the sheep, cattle, and deer lineages. The deer map and interspecies hybrid pedigrees described here are a valuable resource for (1) predicting the location of orthologs to human genes in ruminants, (2) mapping QTL in farmed and wild deer populations, and (3) ruminant phylogenetic studies.     

Response of Black-Capped Chickadees to House Finch Mycoplasma gallisepticum

Tests for the presence of pathogen DNA or antibodies are routinely used to survey for current or past infections. In diseases that emerge following a host jump estimates of infection rate might be under- or overestimated. We here examine whether observed rates of infection are biased for a non-focal host species in a model system. The bacterium Mycoplasma gallisepticum is a widespread pathogen in house finches (Haemorhous mexicanus), a fringillid finch, but an unknown proportion of individuals of other songbird species are also infected. Our goal is to determine the extent to which detection of M. gallisepticum DNA or antibodies against the bacteria in a non-fringillid bird species is over- or underestimated using black-capped chickadees Poecile atricapillus, a species in which antibodies against M. gallisepticum are frequently detected in free-living individuals. After keeping black-capped chickadees in captivity for 12 weeks, during which period the birds remained negative for M. gallisepticum, four were inoculated with M. gallisepticum and four were sham inoculated in both eyes to serve as negative controls. Simultaneously we inoculated six house finches with the same isolate of M. gallisepticum as a positive control. All inoculated birds of both species developed infections detectable by qPCR in the conjunctiva. For the 6 weeks following inoculation we detected antibodies in all M. gallisepticum-inoculated house finches but in only three of the four M. gallisepticum-inoculated black-capped chickadees. All house finches developed severe eye lesions but none of the black-capped chickadees did. Modeling the Rapid Plate Agglutination test results of black-capped chickadees shows that the rate of false-positive tests would be not more than 3.2%, while the estimated rate of false negatives is 55%. We conclude that the proportion of wild-caught individuals in which we detect M. gallisepticum-specific antibodies using Rapid Plate Agglutination is, if anything, substantially underestimated.     

Status and needs for online control of tertiary ozone-based water treatment: use of surrogate correlation models for removal of trace organic contaminants

The number of studies dealing with tertiary ozonation to remove trace organic contaminants (TrOCs) in effluents originating from wastewater treatment plants (WWTPs) is increasing due to the need for upgrading the WWTPs overall performance. To follow-up TrOCs removal in real-time during ozone-based treatment, online surrogate measurements are necessary, of which mainly spectroscopic surrogates (i.e. UV–VIS absorbance and fluorescence) are the emerging techniques in literature. This paper summarizes and reflects on the state-of-the-art as retrieved from more than 100 peer-reviewed studies published between January 2007 and December 2020 and dealing with (1) surrogate correlation models for the prediction of TrOCs removal in secondary effluent and (2) control strategies to adjust the ozone dose during (full-scale) operation. Next to the flow and load proportional ozone dosing strategies, controlling the ozone dose solely based on the characteristics of the effluent entering the ozonation unit, also a differential control strategy based on the change in characteristics due to ozonation of the WWTP effluent is highlighted. The latter seems the best option as flow and load proportional ozone dosing do not consider the amount and/or reactivity of the matrix constituents. The presence of organic and inorganic scavengers of ozone and radicals in the effluent matrix has a significant impact on the TrOCs removal efficiency. This effluent quality can differ in time and between WWTPs, hence the surrogate correlation models should be widely applicable. At the end of the review, recommendations are made for future research and implementation of an effective control strategy for (full-scale) applications.

     

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background

A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.

Methodology/Findings

An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10⁻³ par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories.

Conclusion/Significance

This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.     

Auxin and Plant-Microbe Interactions

Microbial synthesis of the phytohormone auxin has been known for a long time. This property is best documented for bacteria that interact with plants because bacterial auxin can cause interference with the many plant developmental processes regulated by auxin. Auxin biosynthesis in bacteria can occur via multiple pathways as has been observed in plants. There is also increasing evidence that indole-3-acetic acid (IAA), the major naturally occurring auxin, is a signaling molecule in microorganisms because IAA affects gene expression in some microorganisms. Therefore, IAA can act as a reciprocal signaling molecule in microbe-plant interactions. Interest in microbial synthesis of auxin is also increasing in yet another recently discovered property of auxin in Arabidopsis. Down-regulation of auxin signaling is part of the plant defense system against phytopathogenic bacteria. Exogenous application of auxin, e.g., produced by the pathogen, enhances susceptibility to the bacterial pathogen.The phytohormone auxin (from the Greek “auxein,” meaning to grow) regulates a whole repertoire of plant developmental processes, as documented in previous articles on this topic. Perhaps less well known is the fact that some microorganisms also produce auxin (Costacurta and Vanderleyden 1995; Patten and Glick 1996). In their interaction with plants, these microorganisms can interfere with plant development by disturbing the auxin balance in plants. This is best documented for phytopathogenic bacteria like Agrobacterium spp. and Pseudomonas savastanoi pv. savastanoi, causing tumors and galls, respectively (Jameson 2000; Mole et al. 2007), and plant growth promoting rhizobacteria (PGPR) such as Azospirillum spp. that impact on plant root development (Persello-Cartieaux et al. 2003; Spaepen et al. 2007a). The term rhizobacteria refers to the fact that their numbers are highly enriched in the rhizosphere, i.e., the narrow band of soil that surrounds the root (Hiltner 1904; Smalla et al. 2006; van Loon 2007). Of more recent date is the observation that auxin (indole-3-acetic acid or IAA) is a signaling molecule in some microorganisms (Spaepen et al. 2007a). Bringing these data together, it follows that auxin can have a major impact in microorganism-plant interactions. This is the main theme addressed in this article. Finally, the recent finding that auxin signaling in plants is also part of the Arabidopsis defense response against a leaf pathogen (Navarro et al. 2006) is discussed in relation to bacterial IAA synthesis.     

Efficacy study of novel diamidine compounds in a Trypanosoma evansi goat model

Three diamidines (DB 75, DB 867 and DB 1192) were selected and their ability to cure T. evansi experimentally infected goats was investigated. A toxicity assessment and pharmacokinetic analysis of these compounds were additionally carried out. Goats demonstrated no signs of acute toxicity, when treated with four doses of 1 mg/kg/day (total dose 4 mg/kg). Complete curative efficacy of experimentally infected goats was seen in the positive control group treated with diminazene at 5 mg/kg and in the DB 75 and DB 867 groups treated at 2.5 mg/kg. Drug treatment was administered once every second day for a total of seven days. Complete cure was also seen in the group of goats treated with DB 75 at 1.25 mg/kg. DB 1192 was incapable of curing goats at either four-times 2.5 mg/kg or 1.25 mg/kg. Pharmacokinetic analysis clearly demonstrated that the treatment failures of DB 1192 were due to sub-therapeutic compound levels in goat plasma, whilst compound levels for DB 75 and DB 867 remained well within the therapeutic window. In conclusion, two diamidine compounds (DB 75 and DB 867) presented comparable efficacy at lower doses than the standard drug diminazene and could be considered as potential clinical candidates against T. evansi infection.