Development and validation of a triplex real-time qPCR for sensitive detection and quantification of major rat bite fever pathogen Streptobacillus moniliformis.

Streptobacillus (S.) moniliformis is the most important pathogen causing rat bite fever (RBF) worldwide. This zoonotic pathogen is understudied mainly due to difficulties in culturing S. moniliformis as a fastidious microorganism. Therefore, advances in molecular detection techniques are highly needed, especially with regard to the widespread availability of real-time quantitative (q) PCR in laboratories. In this study, we aimed to develop a qPCR for the identification of Streptobacillus species and quantification of S. moniliformis in clinical samples, especially those derived from tissue samples of animal origin. We optimized a previously described PCR protocol in order to develop a qPCR, which can detect different Streptobacillus species with high specificity and is simultaneously able to quantitate S. moniliformis in different clinical matrices. The qPCR exhibited a limit of detection (LOD) of 21 copies/reaction representing ~4-5 streptobacilli, while the limit of quantification (LOQ) was 2.1 × 103 copies/reaction. It was also more sensitive than conventional PCR by two orders of magnitude and proved to have a substantial agreement (Kappa 0.74) compared to it with a superior detection rate in 374 samples from wild rats, laboratory rats and animals from holdings of wild-trapped rats. To conclude, the qPCR described in this study is an important molecular tool that is able to quantify S. moniliformis in tissue samples of animal origin. It represents a suitable tool for future establishment and evaluation of other molecular assays that are highly needed for a better understanding of epidemiology and pathophysiology of RBF. In experimental studies, it will also be useful for titration purposes since the quantification of the organism using classical plate counting technique is problematic and inaccurate.

Clinical infection in house rats (Rattus rattus) caused by Streptobacillus notomytis.

Rat bite fever is an under-reported, under-diagnosed emerging zoonosis with worldwide distribution. Besides Spirillum minus, Streptobacillus moniliformis is the major causative microorganism although it usually colonises rats without any clinical signs. A group of house rats (Rattus rattus) kept in a zoo exhibition for educational purposes suffered from neurological signs including disorientation, torticollis, stall walking, ataxia and death. Gross pathological and histo-pathological examinations of the investigated rats revealed high-grade otitis interna et media, from which Streptobacillus notomytis was isolated in pure culture or as the predominant microorganism. This case series underlines a previously expressed hypothesis that R. rattus might be naturally colonised with S. notomytis, whereas the traditional rat bite fever organism, S. moniliformis, might be restricted to the Norway rat (Rattus norvegicus). However, the general paucity of Streptobacillus isolates, especially from their respective animal hosts, precludes definitive proof of these host tropisms. This is the first report of S. notomytis detection outside Asia and Australia and the first evidence for its role as a facultative pathogen in house rats.

Beyond Cuticular Hydrocarbons: Chemically Mediated Mate Recognition in the Subsocial Burying Beetle Nicrophorus vespilloides.

Burying beetles have fascinated scientists for centuries due to their elaborate form of biparental care that includes the burial and defense of a vertebrate carcass, as well as the subsequent feeding of the larvae. However, besides extensive research on burying beetles, one fundamental question has yet to be answered: what cues do males use to discriminate between the sexes? Here, we show in the burying beetle Nicrophorus vespilloides that cuticular lipids trigger male mating behavior. Previous chemical analyses have revealed sex differences in cuticular hydrocarbon (CHC) composition; however, in the current study, fractionated-guided bioassay showed that cuticular lipids, other than CHCs, elicit copulation. Chemical analyses of the behaviorally active fraction revealed 17 compounds, mainly aldehydes and fatty acid esters, with small quantitative but no qualitative differences between the sexes. Supplementation of males with hexadecanal, the compound contributing most to the statistical separation of the chemical profiles of males and females, did not trigger copulation attempts by males. Therefore, a possible explanation is that the whole profile of polar lipids mediates sex recognition in N. vespilloides.

USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B-cell lymphoma.

The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC-induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X-linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B-cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event-free survival in patients treated with spindle poison-containing chemotherapy. Accordingly, aggressive B-cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine Eµ-Myc lymphoma model. Together, we specify the USP9X-XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B-cell lymphoma.

Changes in Nutrition-Related Behaviors in Alcohol-Dependent Patients After Outpatient Detoxification: The Role of Chocolate.

BACKGROUND: Previous studies have reported changes in nutrition-related behaviors in alcohol-dependent patients after alcohol detoxification, but prospective studies assessing the effects of these changes on maintaining abstinence are lacking. OBJECTIVES: To assess changes in craving and consumption of chocolate and other sweets over time up to six months after outpatient alcohol detoxification treatment and to detect differences in abstinent versus nonabstinent patients. METHODS: One hundred and fifty alcohol-dependent patients were included in this prospective observational study. Participants completed self-report questionnaires on nutrition-related behaviors and craving before detoxification treatment (baseline, t1), one week (t2), one month (t3), and six months later (t4). RESULTS: Significant changes in craving for and consumption of chocolate as well as in craving for other sweets were observed over time. Increases were most prominent within the first month. Patients who remained abstinent until t3 consumed three times more chocolate than nonabstainers. One quarter of the patients switched from being rare (t1) to frequent (t3) chocolate eaters, and 84% of these remained abstinent until t3. No significant correlations were found between craving for alcohol and craving for or consumption of chocolate or other sweets. CONCLUSIONS/IMPORTANCE: In the first month after outpatient alcohol detoxification treatment, significant changes in nutrition-related behaviors were observed. These changes were not associated with alcohol craving. For a subgroup, increasing the frequency of chocolate consumption might be a temporary protective factor with respect to alcohol relapse.

A hormone-related female anti-aphrodisiac signals temporary infertility and causes sexual abstinence to synchronize parental care.

The high energetic demand of parental care requires parents to direct their resources towards the support of existing offspring rather than investing into the production of additional young. However, how such a resource flow is channelled appropriately is poorly understood. In this study, we provide the first comprehensive analysis of the physiological mechanisms coordinating parental and mating effort in an insect exhibiting biparental care. We show a hormone-mediated infertility in female burying beetles during the time the current offspring is needy and report that this temporary infertility is communicated via a pheromone to the male partner, where it inhibits copulation. A shared pathway of hormone and pheromone system ensures the reliability of the anti-aphrodisiac. Female infertility and male sexual abstinence provide for the concerted investment of parental resources into the existing developing young. Our study thus contributes to our deeper understanding of the mechanisms underlying adaptive parental decisions.

The Role of Psychological Distress in Relapse Prevention of Alcohol Addiction. Can High Scores on the SCL-90-R Predict Alcohol Relapse?

OBJECTIVE: The aim of this study was to identify if psychological distress may contribute to treatment outcome in alcohol-addicted patients during a follow-up period of 5 months after detoxification. METHODS: As part of a prospective, multicenter, randomized study in relapse prevention, patients' levels of psychological distress were assessed using the Symptome Checklist (SCL-90-R). At study inclusion, all patients were detoxified and showed no more withdrawal symptoms. The patients who relapsed during the 5-month follow-up period were compared with those who remained abstinent. Predictors for relapse were investigated in a logistic regression. RESULTS: First, a significant difference in initial psychological distress between patients who stayed abstinent and patients who relapsed was found: following detoxification, patients who relapsed scored significantly higher on the SCL-90-R at study inclusion. In addition, psychological distress differed over time in both groups. Second, patients without relapse showed a larger decrease in some SCL-90-R scales between the beginning and the end of the observation period than patients who relapsed. Third, the logistic regression analyses showed that high scores on the overall score GSI (Global Severity Index) of the SCL-90-R can be seen as a predictor for future relapse. CONCLUSION: The SCL-90-R may be a useful instrument to predict relapse. As our study indicates that high levels of psychological distress increases the risk of relapse, specific interventions may be targeted at this risk factor.

Size Exclusion High Performance Liquid Chromatography: Re-Discovery of a Rapid and Versatile Method for Clean-Up and Fractionation in Chemical Ecology.

Solvent extraction of bioactive molecules from glands, tissues, or whole organisms is a common first step in chemoecological studies. Co-extraction of a surplus of high boiling materials such as triacylglycerides (TAGs) and other lipids with higher molecular weight might hamper the identification of volatile or medium-volatile semiochemicals by high resolution chromatographic and spectroscopic techniques. Therefore, effective clean-up procedures are needed to separate potential semiochemicals from the accompanying materials. Size exclusion high performance liquid chromatography (SE-HPLC), a technique often disregarded by chemoecologists, has proved to be a rapid and efficient clean-up method for complex crude extracts. We demonstrated that TAGs can be baseline separated from typical semiochemicals within less than 10 min on a porous gel stationary phase based on highly cross-linked polystyrene/divinylbenzene. We applied the method as a rapid one-step clean-up procedure for the analysis of juvenile hormone III in insect hemolymph by gas chromatography-mass spectrometry. We furthermore introduced some recent application examples on insect pheromones to demonstrate that SE-HPLC is not only an effective method for the purification of crude extracts, but can as well be used as a first fractionation step for the bioassay-guided identification of behavior modifying natural products. SE-HPLC can be well operated with low-boiling solvents such as dichloromethane, and results in fraction volumes of typically less than one ml, which decreases the danger of losing volatile analytes during subsequent concentration steps.

Acceptance threshold theory can explain occurrence of homosexual behaviour.

Same-sex sexual behaviour (SSB) has been documented in a wide range of animals, but its evolutionary causes are not well understood. Here, we investigated SSB in the light of Reeve's acceptance threshold theory. When recognition is not error-proof, the acceptance threshold used by males to recognize potential mating partners should be flexibly adjusted to maximize the fitness pay-off between the costs of erroneously accepting males and the benefits of accepting females. By manipulating male burying beetles' search time for females and their reproductive potential, we influenced their perceived costs of making an acceptance or rejection error. As predicted, when the costs of rejecting females increased, males exhibited more permissive discrimination decisions and showed high levels of SSB; when the costs of accepting males increased, males were more restrictive and showed low levels of SSB. Our results support the idea that in animal species, in which the recognition cues of females and males overlap to a certain degree, SSB is a consequence of an adaptive discrimination strategy to avoid the costs of making rejection errors.

Disruption of the PRKCD-FBXO25-HAX-1 axis attenuates the apoptotic response and drives lymphomagenesis.

We searched for genetic alterations in human B cell lymphoma that affect the ubiquitin-proteasome system. This approach identified FBXO25 within a minimal common region of frequent deletion in mantle cell lymphoma (MCL). FBXO25 encodes an orphan F-box protein that determines the substrate specificity of the SCF (SKP1-CUL1-F-box)(FBXO25) ubiquitin ligase complex. An unbiased screen uncovered the prosurvival protein HCLS1-associated protein X-1 (HAX-1) as the bona fide substrate of FBXO25 that is targeted after apoptotic stresses. Protein kinase Cδ (PRKCD) initiates this process by phosphorylating FBXO25 and HAX-1, thereby spatially directing nuclear FBXO25 to mitochondrial HAX-1. Our analyses in primary human MCL identify monoallelic loss of FBXO25 and stabilizing HAX1 phosphodegron mutations. Accordingly, FBXO25 re-expression in FBXO25-deleted MCL cells promotes cell death, whereas expression of the HAX-1 phosphodegron mutant inhibits apoptosis. In addition, knockdown of FBXO25 significantly accelerated lymphoma development in Eµ-Myc mice and in a human MCL xenotransplant model. Together we identify a PRKCD-dependent proapoptotic mechanism controlling HAX-1 stability, and we propose that FBXO25 functions as a haploinsufficient tumor suppressor and that HAX1 is a proto-oncogene in MCL.

An adult patient with Nijmegen Breakage Syndrome and Hodgkin's Lymphoma.

BACKGROUND: Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive DNA repair disorder characterized by immune deficiency, microcephaly, mental retardation and a disposition for the development of hematological malignancies. So far, mostly pediatric patients have been described, since the underlying condition is often fatal before adulthood. Many patients diagnosed with Hodgkin lymphoma (HL) due to this DNA repair defect receive reduced treatment followed by early progression and fatal outcome. CASE PRESENTATION: We describe here a 26-year old male caucasian patient with NBS who presented with multi organ failure due to HL. Immediate intensive chemotherapy lead to complete remission and reversed organ failure. CONCLUSION: We show that application of standard chemotherapy can lead to long-term disease free survival in patients with a DNA repair disorder. Furthermore, we describe here, to the best of our knowledge, the first adult patient with NBS and HL.

Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in-vitro enzyme activity with clinical phenotype in argininosuccinic aciduria.

BACKGROUND: The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme. METHODS: Bacterial in-vitro expression studies allowed the enzyme analysis of purified mutant ASL proteins p.I100T (c.299 T > C), p.V178M (c.532 G > A), p.E189G (c.566A > G), p.Q286R (c.857A > G), p.K315E (c.943A > G), p.R379C (c.1135 C > T) and p.R385C (c.1153 C > T) in comparison to the wildtype protein. RESULTS: In the bacterial in-vitro expression system, ASL wild-type protein was successfully expressed. The known classical p.Q286R, the novel classical p.K315E and the known mutations p.I100T, p.E189G and p.R385C, which all have been linked to a mild phenotype, showed no significant residual activity. There was some enzyme activity detected with the p.V178M (5 % of wild-type) and p.R379C (10 % of wild-type) mutations in which K(m) values for argininosuccinic acid differed significantly from the wild-type ASL protein. CONCLUSION: The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.

Integrating biological invasions, climate change and phenotypic plasticity.

Invasive species frequently change the ecosystems where they are introduced, e.g., by affecting species interactions and population densities of native species. We outline the connectedness of biological invasions, climate change and the phenomenon of phenotypic plasticity. Integrating these hot topics is important for understanding the biology of many species, their information transfer and general interactions with other organisms. One example where this is particularly true is the zooplankton species Daphnia lumholtzi, which has successfully invaded North America. The combination of a high thermal tolerance and a phenotypically plastic defense in D. lumholtzi might be responsible for its invasion success. Its morphological defense consists of rigid spines and is formed after sensory detecting the presence of native fish predators. The integration of biological invasions, climate change and phenotypic plasticity is an important goal for integrative biology.

Inducible defences as key adaptations for the successful invasion of Daphnia lumholtzi in North America?

The mechanisms underlying successful biological invasions often remain unclear. In the case of the tropical water flea Daphnia lumholtzi, which invaded North America, it has been suggested that this species possesses a high thermal tolerance, which in the course of global climate change promotes its establishment and rapid spread. However, D. lumholtzi has an additional remarkable feature: it is the only water flea that forms rigid head spines in response to chemicals released in the presence of fishes. These morphologically (phenotypically) plastic traits serve as an inducible defence against these predators. Here, we show in controlled mesocosm experiments that the native North American species Daphnia pulicaria is competitively superior to D. lumholtzi in the absence of predators. However, in the presence of fish predation the invasive species formed its defences and became dominant. This observation of a predator-mediated switch in dominance suggests that the inducible defence against fish predation may represent a key adaptation for the invasion success of D. lumholtzi.

Mutations and polymorphisms in the human argininosuccinate synthetase (ASS1) gene.

Citrullinemia type I is an autosomal recessive disorder that is caused by a deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS1). Deficiency of ASS1 shows various clinical manifestations encompassing severely affected patients with fatal neonatal hyperammonemia as well as asymptomatic individuals with only a biochemical phenotype. This is a comprehensive report of all 87 mutations found to date in the ASS1 gene on chromosome 9q34.1. A large proportion of the mutations (n=27) are described here for the first time. Mutations are distributed throughout exons 3 to 15, most of them being identified in exons 5, 12, 13, and 14. The mutation G390R in exon 15 is the single most common mutation in patients with the classical phenotype. Certain mutations clearly link to specific clinical courses but the clinical phenotype cannot be anticipated in all patients. This update presents a survey of the correlation between mutations in the ASS1 gene and the respective clinical courses as described so far. It also sheds light on the geographic incidence of the mutations. Enzymatic studies have been done in bacterial and human cell systems. However, the prognostic value of genetic aberrations with respect to their effect on protein function and clinical manifestation remains uncertain.

Analysis of mRNA transcripts improves the success rate of molecular genetic testing in OTC deficiency.

BACKGROUND: Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of urea metabolism that can lead to hyperammonemic crises and orotic aciduria. To date, a total of 341 causative mutations within the OTC gene have been described. However, in about 20% of the patients with enzymatically confirmed OTC deficiency no mutation can be detected when sequencing of genomic DNA analyzing exons and adjacent intronic segments of the OTC gene is performed. METHODS: Standard genomic DNA analysis of the OTC gene in five consecutive patients from five families revealed no mutation. Hence, liver tissue was obtained by needle sampling or open biopsy and RNA extracted from liver was analyzed. RESULTS: Complex rearrangements of the OTC transcript (three insertions and two deletions) were found in all five patients. CONCLUSION: In patients with a strong suspicion of OTC deficiency despite normal results of sequencing exonic regions of the OTC gene, characterization of liver OTC mRNA is highly effective in resolving the genotype. Liver tissue sampling by needle aspiration allows for both enzymatic analysis and RNA based diagnostics of OTC deficiency.

2-Methylbutyryl-coenzyme A dehydrogenase deficiency: functional and molecular studies on a defect in isoleucine catabolism.

2-Methylbutyryl-CoA dehydrogenase (MBD; coded by the ACADSB gene) catalyzes the step in isoleucine metabolism that corresponds to the isovaleryl-CoA dehydrogenase reaction in the degradation of leucine. Deficiencies of both enzymes may be detected by expanded neonatal screening with tandem-mass spectrometry due to elevated pentanoylcarnitine (C5 acylcarnitine) in blood, but little information is available on the clinical relevance of MBD deficiency. We biochemically and genetically characterize six individuals with MBD deficiency from four families of different ethnic backgrounds. None of the six individuals showed clinical symptoms attributable to MBD deficiency although the defect in isoleucine catabolism was demonstrated both in vivo and in vitro. Several mutations in the ACADSB gene were identified, including a novel one. MBD deficiency may be a harmless metabolic variant although significant impairment of valproic acid metabolism cannot be excluded and further study is required to assess the long-term outcome of individuals with this condition. The relatively high prevalence of ACADSB gene mutations in control subjects suggests that MBD deficiency may be more common than previously thought but is not detected because of its usually benign nature.