SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.

Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency.

BACKGROUND: Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVES: This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS: Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. RESULTS: A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSIONS: This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.

Safety and immunogenicity of an adjuvanted recombinant spike protein-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine, SpikeVet™, in selected Carnivora, Primates and Artiodactyla in Australian zoos.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect a broad range of animal species and has been associated with severe disease in some taxa. Few studies have evaluated optimal strategies to mitigate the risk to susceptible zoo animals. This study evaluated the safety and immunogenicity of a protein-based veterinary SARS-CoV-2 vaccine (SpikeVet™) in zoo animals. Two to three doses of SpikeVet™ were administered intramuscularly or subcutaneously 3-4 weeks apart to 354 zoo animals representing 38 species. SpikeVet™ was very well tolerated across all species. Minor adverse effects were observed in 1.69% of animals vaccinated, or 1.04% of vaccine doses administered. Preliminary immunogenicity analyses in representative carnivores (meerkats, lions) and an artiodactylid (domestic goat) showed SpikeVet™-immunized animals developed serum antibodies able to neutralize a range of SARS-CoV-2 variants, including the vaccine-homologous Wuhan and Mu variants, as well as vaccine-heterologous Omicron BA.2 and XBB.1 strains. Prior to vaccination, all eight lions were seropositive for Wuhan strain by surrogate viral neutralization testing, suggesting past infection with SARS-CoV-2 or cross-reactive antibodies generated by another closely related coronavirus. These results from a range of zoo species support the ongoing development of SpikeVet™ as a safe and effective veterinary SARS-CoV-2 vaccine.

Immunologic and Genetic Contributors to CD46-Dependent Immune Dysregulation.

Mutations in CD46 predispose to atypical hemolytic uremic syndrome (aHUS) with low penetrance. Factors driving immune-dysregulatory disease in individual mutation carriers have remained ill-understood. In addition to its role as a negative regulator of the complement system, CD46 modifies T cell-intrinsic metabolic adaptation and cytokine production. Comparative immunologic analysis of diseased vs. healthy CD46 mutation carriers has not been performed in detail yet. In this study, we comprehensively analyzed clinical, molecular, immune-phenotypic, cytokine secretion, immune-metabolic, and genetic profiles in healthy vs. diseased individuals carrying a rare, heterozygous CD46 mutation identified within a large single family. Five out of six studied individuals carried a CD46 gene splice-site mutation causing an in-frame deletion of 21 base pairs. One child suffered from aHUS and his paternal uncle manifested with adult-onset systemic lupus erythematosus (SLE). Three mutation carriers had no clinical evidence of CD46-related disease to date. CD4+ T cell-intrinsic CD46 expression was uniformly 50%-reduced but was comparable in diseased vs. healthy mutation carriers. Reconstitution experiments defined the 21-base pair-deleted CD46 variant as intracellularly-but not surface-expressed and haploinsufficient. Both healthy and diseased mutation carriers displayed reduced CD46-dependent T cell mitochondrial adaptation. Diseased mutation carriers had lower peripheral regulatory T cell (Treg) frequencies and carried potentially epistatic, private rare variants in other inborn errors of immunity (IEI)-associated proinflammatory genes, not found in healthy mutation carriers. In conclusion, low Treg and rare non-CD46 immune-gene variants may contribute to clinically manifest CD46 haploinsufficiency-associated immune-dysregulation.

Influence of inter-aquifer leakage on well-injection capacity: Theory and aquifer-scale mapping for artificial recharge.

Aquifer storage and recovery (ASR) is an important water resources management technique that involves the injection of a large volume of water underground. For the successful implementation of an ASR project, a target aquifer should have a sufficient injection capacity, which is the maximum volume of water that can be safely injected. In nature, no aquitard is perfectly impermeable, and inter-aquifer leakage may have a major impact on injection capacity. Despite the importance of determining the injection capacity for ASR planning, there is no quantitative methodology that estimates the injection capacity of leaky aquifers. In this study, we first develop a solution for injection capacity with inter-aquifer leakage based on the Hantush - Jacob solution, and conduct a comprehensive sensitivity analysis to elucidate the influence of inter-aquifer leakage on injection capacity. From the sensitivity analysis, we show that inter-aquifer leakage can impact injection capacity by more than one order of magnitude, depending on the hydrogeological and operational parameters. We then develop a practical mapping methodology that estimates the injection capacity of leaky aquifers. We demonstrate the proposed methodology by applying it to a potential ASR site in Minnesota, USA, where ASR is considered as a solution to alleviate groundwater contamination by PFAS chemicals. The case study results reveal significant spatial variability in injection capacity over the study area and show an average increase in the injection capacity of about 26% compared to that in the nonleaky scenario. We also analyze the uncertainty in the estimated injection capacity due to the variability of aquitard properties and show that the variability of aquitard vertical hydraulic conductivity leads to a larger uncertainty in the estimated injection capacity than does the variability of aquitard thickness. This study elucidates the effects of inter-aquifer leakage on injection capacity and provides a practical methodology for injection capacity mapping.

Plectus of the Prairie: A Case Study of Taxonomic Resolution from a Nematode Biodiversity Survey.

Taxonomic resolution is a critical component of biodiversity assessments. In this case study, we examined a single taxon within a larger study of nematode diversity to evaluate the taxonomic resolution of different diversity assessment methods. The selected taxon was the microbial-feeding genus Plectus, a group considered to include multiple cosmopolitan species. The methods included a morphological evaluation by light microscopy, Sanger sequencing of PCR amplicons of COI and 18S gene regions, and 18S metabarcoding sequencing. The study sites were 15 remnant tallgrass prairie plots in eastern Nebraska. In the morphological analysis, we observed two basic morphotypes, a short-tailed form with a small amphid and a long-tailed form with a large amphid. Sanger sequencing of COI sorted Plectus diversity into six distinct clades. The largest two of these six clades keyed to P. parietinus and P. rhizophilus based on morphology. BLAST analysis with COI revealed no close matches in GenBank. Sanger sequencing of the 18S region did not differentiate the six clades. These results illustrate that the method of diversity assessment strongly influences estimates of biodiversity. An additional 95 Plectus specimens, from outside the remnant sites, added taxonomic breadth to the COI phylogenetic tree. There were no geographically widespread COI haplotypes and no evidence of cosmopolitan Plectus species.

Nematode biodiversity assessments need vouchered databases: A BOLD reference library for plant-parasitic nematodes in the superfamily Criconematoidea.

Nematodes are frequently cited as underrepresented in faunistic surveys using DNA barcoding with COI. This underrepresentation is generally attributed to a limited presence of nematodes in DNA databases which, in turn, is often ascribed to structural variability and high evolutionary rates in nematode mitochondrial genomes. Empirical evidence, however, indicates that many taxa are readily amplified with primer sets specifically targeted to different nematode families. Here we report the development of a COI reference library of 1726 specimens in the terrestrial plant parasitic nematode superfamily Criconematoidea. Specimens collected during an ecoregion survey of North America were individually photographed, measured, and PCR amplified to produce a 721 bp region of COI for taxonomic analysis. A neighbor-joining tree structured the dataset into 179 haplotype groups that generally conformed to morphospecies in traditional analysis or Barcode Index Numbers (BINs) in the BOLD system, although absent formal BIN membership due to insufficient overlap with the Folmer region of COI. Approximately one-third of the haplotype groups could be associated with previously described species. The geographic distribution of criconematid nematode species suggests a structure influenced by the major habitat types in the United States and Canada. All sequences collected in the ecoregion survey are deposited in BOLD.

Spontaneous and Fungicide-Induced Genomic Variation in Sclerotinia sclerotiorum.

Stress from exposure to sublethal fungicide doses may cause genomic instability in fungal plant pathogens, which may accelerate the emergence of fungicide resistance or other adaptive traits. In a previous study, five strains of Sclerotinia sclerotiorum were exposed to sublethal doses of four fungicides with different modes of action, and genotyping showed that such exposure induced mutations. The goal of the present study was to characterize genome-wide mutations in response to sublethal fungicide stress in S. sclerotiorum and study the effect of genomic background on the mutational repertoire. The objectives were to determine the effect of sublethal dose exposure and genomic background on mutation frequency/type, distribution of mutations, and fitness costs. Fifty-five S. sclerotiorum genomes were sequenced and aligned to the reference genome. Variants were called and quality filtered to obtain high confidence calls for single nucleotide polymorphisms (SNPs), insertions/deletions (INDELs), copy number variants, and transposable element (TE) insertions. Results suggest that sublethal fungicide exposure significantly increased the frequency of INDELs in two strains from one genomic background (P value ≤ 0.05), while TE insertions were generally repressed for all genomic backgrounds and under all fungicide exposures. The frequency and/or distribution of SNPs, INDELs, and TE insertions varied with genomic background. A propensity for large duplications on chromosome 7 and aneuploidy of this chromosome were observed in the S. sclerotiorum genome. Mutation accumulation did not significantly affect the overall in planta strain aggressiveness (P value > 0.05). Understanding factors that affect pathogen mutation rates can inform disease management strategies that delay resistance evolution.

Comparison of some cardiopulmonary effects of etorphine and thiafentanil during the chemical immobilization of blesbok (Damaliscus pygargus phillipsi).

OBJECTIVE: To determine the cardiopulmonary effects of etorphine and thiafentanil for immobilization of blesbok. STUDY DESIGN: Blinded, randomized, two-way crossover study. ANIMALS: A group of eight adult female blesbok. METHODS: Animals were immobilized twice, once with etorphine (0.09 mg kg-1) and once with thiafentanil (0.09 mg kg-1) administered intramuscularly by dart. Immobilization quality was assessed and analysed by Wilcoxon signed-rank test. Time to final recumbency was compared between treatments by one-way analysis of variance. Cardiopulmonary effects including respiratory rate (ƒR), arterial blood pressures and arterial blood gases were measured. A linear mixed model was used to assess the effects of drug treatments over the 40 minute immobilization period. Significant differences between treatments, for treatment over time as well as effect of treatment by time on the variables, were analysed (p < 0.05). RESULTS: There was no statistical difference (p = 0.186) between treatments for time to recumbency. The mean ƒR was lower with etorphine (14 breaths minute-1) than with thiafentanil (19 breaths minute-1, p = 0.034). The overall mean PaCO2 was higher with etorphine [45 mmHg (6.0 kPa)] than with thiafentanil [41 mmHg (5.5 kPa), p = 0.025], whereas PaO2 was lower with etorphine [53 mmHg (7.1 kPa)] than with thiafentanil [64 mmHg (8.5 kPa), p < 0.001]. The systolic arterial pressure measured throughout all time points was higher with thiafentanil than with etorphine (p = 0.04). The difference varied from 30 mmHg at 20 minutes after recumbency to 14 mmHg (standard error difference 2.7 mmHg) at 40 minutes after recumbency. Mean and diastolic arterial pressures were significantly higher with thiafentanil at 20 and 25 minute measurement points only (p < 0.001). CONCLUSIONS: Both drugs caused clinically relevant hypoxaemia; however, it was less severe with thiafentanil. Ventilation was adequate. Hypertension was greater and immobilization scores were lower with thiafentanil.

Comparison of cardiopulmonary effects of etorphine and thiafentanil administered as sole agents for immobilization of impala (Aepyceros melampus).

OBJECTIVE: To compare the cardiopulmonary effects of the opioids etorphine and thiafentanil for immobilization of impala. STUDY DESIGN: Two-way crossover, randomized study. ANIMALS: A group of eight adult female impala. METHODS: Impala were given two treatments: 0.09 mg kg-1 etorphine or 0.09 mg kg-1 thiafentanil via remote dart injection. Time to recumbency, quality of immobilization and recovery were assessed. Respiratory rate, heart rate (HR), mean arterial blood pressure (MAP) and arterial blood gases were measured. A linear mixed model was used to analyse the effects of treatments, treatments over time and interactions of treatment and time (p < 0.05). RESULTS: Time to recumbency was significantly faster with thiafentanil (2.0 ± 0.8 minutes) than with etorphine (3.9 ± 1.6 minutes; p = 0.007). Both treatments produced bradypnoea, which was more severe at 5 minutes with thiafentanil (7 ± 4 breaths minute-1) than with etorphine (13 ± 12 breaths minute-1; p = 0.004). HR increased with both treatments but significantly decreased over time when etorphine (132 ± 17 to 82 ± 11 beats minute-1) was compared with thiafentanil (113 ± 22 to 107 ± 36 beats minute-1; p < 0.001). Both treatments caused hypertension which was more profound with thiafentanil (mean overall MAP = 140 ± 14 mmHg; p < 0.001). Hypoxaemia occurred with both treatments but was greater with thiafentanil [PaO2 37 ± 13 mmHg (4.9 kPa)] than with etorphine [45 ± 16 mmHg (6.0 kPa)] 5 minutes after recumbency (p < 0.001). After 30 minutes, PaO2 increased to 59 ± 10 mmHg (7.9 kPa) with both treatments (p < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: The shorter time to recumbency with thiafentanil may allow easier and faster retrieval in the field. However, thiafentanil caused greater hypertension, and ventilatory effects during the first 10 minutes, after administration.

Pratylenchus smoliki, a new nematode species (Pratylenchidae: Tylenchomorpha) from the Great Plains region of North America.

Pratylenchus smoliki is a new species of root-lesion nematode described from corn-soybean production fields in the Central Great Plains of North America. It is characterized by populations with relatively abundant males, two lip annuli, females with a round functional spermatheca and a conoid to subcylindrical tail with a non-crenate, smooth terminus. In host preference tests, corn and wheat produce the largest nematode populations, whereas sorghum and soybeans produce less than 20% the numbers observed on corn. Scanning electron microscopy reveals that the en face patterns compare to those seen in Pratylenchus pseudocoffeae, P. scribneri, P. hexincisus, and P. alleni. The pattern is described as rectangular to trapezoidal subdorsal and subventral lips adjoining oral disc, but with a clear demarcation between the oral disc and the subdorsal and subventral sectors. A Maximum Likelihood COI tree recognizes P. smoliki as a moderately-well-supported clade with several haplotype subgroups. A Maximum Likelihood partial 28S tree provides strong support for the P. smoliki clade and reinforces the close relationships between species with similar en face patterns. Topotype specimens of P. alleni were demonstrably different from P. smoliki using DNA markers. The geographic range of P. smoliki overlaps with the ranges of P. alleni, P. scribneri, P. neglectus, P. hexicisus, and P. dakotaensis. The observed host range (corn, rye, sunflower, and wheat) suggests that P. smoliki may be native to the tallgrass prairie region of the Great Plains.

Subclinical infection of captive Asian elephants (Elephas maximus) in Thailand with elephant endotheliotropic herpesvirus.

Elephant endotheliotropic herpesvirus (EEHV) infection is a conservation threat to the endangered Asian elephant (Elephas maximus), causing fatal hemorrhagic disease in juvenile elephants throughout the world, including Thailand. This study revealed a subclinical EEHV1 infection rate of 5.5% in healthy captive Asian elephants in Thailand (n = 362). The virus was detected in all age classes above one year old, in both sexes, and across the country - even in facilities with no history of hemorrhagic disease (EEHV HD). Subclinical EEHV infection in Thailand urgently requires proper health management.

Streptococcus iniae associated mass marine fish kill off Western Australia.

Streptococcus iniae causes high mortality in cultured and wild fish stocks globally. Since the first report in captive Amazon river dolphins Inia geoffrensis in 1976, it has emerged in finfish across all continents except Antarctica. In March 2016, an estimated 17000 fish were observed dead and dying along a remote 70 km stretch of the Kimberley coastline north of Broome, Western Australia. Affected species included finfish (lionfish Pterois volitans, angelfish Pomacanthus sp., stripey snapper Lutjanus carponotatus, sand bass Psammoperca waigiensis, yellowtail grunter Amniataba caudavittata, damselfish Pomacentridae sp.), flatback sea turtles Natator depressus, and olive (Aipysurus laevis) and black-ringed (Hydrelaps darwiniensis) sea snakes. Moribund fish collected during the event exhibited exophthalmia and abnormal behaviour, such as spiralling on the surface or within the water column. Subsequent histopathological examination of 2 fish species revealed bacterial septicaemia with chains of Gram-positive cocci seen in multiple organs and within brain tissue. S. iniae was isolated and identified by bacterial culture, species-specific PCR, Matrix-Assisted Laser Desorption Ionisation Time-Of-Flight (MALDI-TOF) and biochemical testing. This is the first report of S. iniae associated with a major multi-species wild marine fish kill in Australia. Extreme weather events in the region including a marked decrease in water temperatures, followed by an extended period of above-average coastal water temperatures, were implicated as stressors potentially contributing to this outbreak.

Pharmacokinetics and bioavailability after intramuscular injection of the 5-HT1A serotonin agonist R-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) in domestic goats (Capra aegagrus hircus).

To determine the bioavailability and pharmacokinetic properties of the serotonin 5-HT1A receptor agonist R-8-OH-DPAT in goats, and 0.1 mg kg-1 R-8-OH-DPAT hydrobromide was administered intramuscularly (i.m.) and intravenously (i.v.) to six goats in a two-phase cross-over design experiment. Venous blood samples were collected from the jugular vein 2, 5, 10, 15, 20, 30, 40 and 60 min following treatment and analysed by liquid chromatography tandem mass spectrometry. Bioavailability and pharmacokinetic parameters were determined by a one-compartment analysis. Mean bioavailability of R-8-OH-DPAT when injected i.m. was 66%. The mean volume of distribution in the central compartment was 1.47 L kg-1 . The mean plasma body clearance was 0.056 L kg-1  min-1 . All goats injected i.v. and two of six goats injected i.m. showed signs of serotonin toxicity. In conclusion, R-8-OH-DPAT is well absorbed following i.m. injection and the observed pharmacokinetics suggest that administration via dart is feasible. Administration of R-8-OH-DPAT hydrobromide, at a dosage of 0.1 mg kg-1 , resulted in the observation of clinical signs of serotonin toxicity in the goats. It is suggested that dosages for the clinical use of the compound should be lower in order to achieve the desired clinical effect without causing serotonin toxicity.

Dose-effect study of the serotonin agonist R-8-OH-DPAT on opioid-induced respiratory depression in blesbok (Damaliscus pygargus philipsi) and impala (Aepyceros melampus).

OBJECTIVE: To determine whether the R-enantiomer of 8-hydroxy-2-(di-n-propylamino) tetralin (R-8-OH-DPAT) alleviates respiratory depression in antelope species immobilized with etorphine. The experiment also aimed to establish the most clinically effective dose of this serotonin 5- HT1A receptor agonist. ANIMALS: A group of six female blesbok and six female impala. STUDY DESIGN: Each animal was subjected to four immobilization treatments in a prospective four-way crossover design-control treatment consisting of only etorphine at 0.09 mg kg-1 and three treatments consisting of etorphine at 0.09 mg kg-1 combined with 0.005, 0.02 and 0.07 mg kg-1 of R-8-OH-DPAT, respectively. Induction, quality of immobilization and recovery were monitored in each treatment. Physiological variables including heart rate, respiratory rate, arterial blood pressure and blood gases were measured for 35 minutes during immobilization. A linear mixed model was used to assess the effects of treatments over the recumbency period. RESULTS: R-8-OH-DPAT did not influence induction, immobilization or recovery scores. Respiratory rate in blesbok was increased in the medium- and high-dosage R-8-OH-DPAT treatment group. However, this increased respiratory rate did not translate into improvements of arterial partial pressure of oxygen (PaO2) values in the blesbok. The medium and higher dosages of R-8-OH-DPAT in impala led to an improved PaO2 as well as to decreased opioid-induced tachycardia during the first 10 minutes of immobilization. CONCLUSIONS AND CLINICAL RELEVANCE: Previous reports indicated that the racemic mixture of 8-OH-DPAT injected intravenously had a positive effect on blood-gas values in etorphine-treated hypoxemic goats. In this experiment, similar effects could be seen in impala at the higher dosage rates of R-8-OH-DPAT. However, failure to achieve an improvement of blood-gas values in blesbok was an unexpected result. It could be speculated that the dosage, species-specific differences of serotonin receptors or the use of the R-enantiomer of 8-OH-DPAT might play a role.

DNA barcoding evidence for the North American presence of alfalfa cyst nematode, Heterodera medicaginis.

Specimens of Heterodera have been collected from alfalfa fields in Kearny County, Kansas & Carbon County, Montana. DNA barcoding with the COI mitochondrial gene indicate that the species is not Heterodera glycines, soybean cyst nematode, H. schachtii, sugar beet cyst nematode, or H. trifolii, clover cyst nematode. Maximum likelihood phylogenetic trees show that the alfalfa specimens form a sister clade most closely related to H. glycines, with a 4.7% mean pairwise sequence divergence across the 862 nucleotides of the COI marker. Morphological analyses of juveniles and cysts conform to the measurements of H. medicaginis, the alfalfa cyst nematode originally described from the USSR in 1971. Initial host testing demonstrated that the nematode reproduced on alfalfa, but not on soybeans, tomato, or corn. Collectively, the evidence suggests that this finding represents the first record of H. medicaginis in North America. Definitive confirmation of this diagnosis would require COI sequence of eastern European isolates of this species.Specimens of Heterodera have been collected from alfalfa fields in Kearny County, Kansas & Carbon County, Montana. DNA barcoding with the COI mitochondrial gene indicate that the species is not Heterodera glycines, soybean cyst nematode, H. schachtii, sugar beet cyst nematode, or H. trifolii, clover cyst nematode. Maximum likelihood phylogenetic trees show that the alfalfa specimens form a sister clade most closely related to H. glycines, with a 4.7% mean pairwise sequence divergence across the 862 nucleotides of the COI marker. Morphological analyses of juveniles and cysts conform to the measurements of H. medicaginis, the alfalfa cyst nematode originally described from the USSR in 1971. Initial host testing demonstrated that the nematode reproduced on alfalfa, but not on soybeans, tomato, or corn. Collectively, the evidence suggests that this finding represents the first record of H. medicaginis in North America. Definitive confirmation of this diagnosis would require COI sequence of eastern European isolates of this species.

MAP kinases associate with high molecular weight multiprotein complexes.

Plant responses to the environment and developmental processes are mediated by a complex signaling network. The Arabidopsis thaliana mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 and their orthologs in other plants are shared signal transducers that respond to many developmental and environmental signals and thus represent highly connected hubs in the cellular signaling network. In animals, specific MAPK signaling complexes are assembled which enable input-specific protein-protein interactions and thus specific signaling outcomes. In plants, not much is known about such signaling complexes. Here, we report that MPK3, MPK6, and MPK10 orthologs in tomato, tobacco, and Arabidopsis as well as tomato MAPK kinase 4 (MKK4) associate with high molecular weight (~250-550 kDa) multiprotein complexes. Elicitation by the defense-associated peptides flg22 and systemin resulted in phosphorylation and activation of the monomeric MAPKs, whereas the complex-associated MAPKs remained unphosphorylated and inactive. In contrast, treatment of tomato cells with a phosphatase inhibitor resulted in association of phosphorylated MPK1/2 with the complex. These results demonstrate that plant MAPKs and MAPKKs dynamically assemble into stable multiprotein complexes and this may depend on their phosphorylation status. Identification of the constituents of these multiprotein complexes promises a deeper understanding of signaling dynamics.

Discovery and Identification of Meloidogyne Species Using COI DNA Barcoding.

DNA barcoding with a new cytochrome oxidase c subunit 1 primer set generated a 721 to 724 bp fragment used for the identification of 322 Meloidogyne specimens, including 205 new sequences combined with 117 from GenBank. A maximum likelihood analysis grouped the specimens into 19 well-supported clades and four single-specimen lineages. The "major" tropical apomictic species ( Meloidogyne arenaria , Meloidogyne incognita , Meloidogyne javanica ) were not discriminated by this barcode although some closely related species such as Meloidogyne konaensis were characterized by fixed diagnostic nucleotides. Species that were collected from multiple localities and strongly characterized as discrete lineages or species include Meloidogyne enterolobii , Meloidogyne partityla , Meloidogyne hapla , Meloidogyne graminicola , Meloidogyne naasi , Meloidogyne chitwoodi , and Meloidogyne fallax . Seven unnamed groups illustrate the limitations of DNA barcoding without the benefit of a well-populated reference library. The addition of these DNA sequences to GenBank and the Barcode of Life Database (BOLD) should stimulate and facilitate root-knot nematode identification and provide a first step in new species discovery.

Detection of Sp110 by Flow Cytometry and Application to Screening Patients for Veno-occlusive Disease with Immunodeficiency.

Mutations in Sp110 are the underlying cause of veno-occlusive disease with immunodeficiency (VODI), a combined immunodeficiency that is difficult to treat and often fatal. Because early treatment is critically important for patients with VODI, broadly usable diagnostic tools are needed to detect Sp110 protein deficiency. Several factors make establishing the diagnosis of VODI challenging: (1) Current screening strategies to identify severe combined immunodeficiency are based on measuring T cell receptor excision circles (TREC). This approach will fail to identify VODI patients because the disease is not associated with severe T cell lymphopenia at birth; (2) the SP110 gene contains 17 exons, making it a challenge for Sanger sequencing. The recently developed next-generation sequencing (NGS) platforms that can rapidly determine the sequence of all 17 exons are available in only a few laboratories; (3) there is no standard functional assay to test for the effects of novel mutations in Sp110; and (4) it has been difficult to use flow cytometry to identify patients who lack Sp110 because of the low level of Sp110 protein in peripheral blood lymphocytes. We report here a novel flow cytometric assay that is easily performed in diagnostic laboratories and might thus become a standard assay for the evaluation of patients who may have VODI. In addition, the assay will facilitate investigations directed at understanding the function of Sp110.