Identification of spider-mite species and their endosymbionts using multiplex PCR

Spider mites of the genus Tetranychidae are severe crop pests. In the Mediterranean a few species coexist, but they are difficult to identify based on morphological characters. Additionally, spider mites often harbour several species of endosymbiotic bacteria, which may affect the biology of their hosts. Here, we propose novel, cost-effective, multiplex diagnostic methods allowing a quick identification of spider-mite species as well as of the endosymbionts they carry. First, we developed, and successfully multiplexed in a single PCR, primers to identify Tetranychus urticae, T. evansi and T. ludeni, some of the most common tetranychids found in southwest Europe. Moreover, we demonstrated that this method allows detecting multiple species in a single pool, even at low frequencies (up to 1/100), and can be used on entire mites without DNA extraction. Second, we developed another set of primers to detect spider-mite endosymbionts, namely Wolbachia, Cardinium and Rickettsia in a multiplex PCR, along with a generalist spider-mite primer to control for potential failure of DNA amplification in each PCR. Overall, our method represents a simple, cost-effective and reliable method to identify spider-mite species and their symbionts in natural field populations, as well as to detect contaminations in laboratory rearings. This method may easily be extended to other species.     

Hormonal responses to exercise during moderate cold exposure in young vs. middle-age subjects.

The influence of moderate cold exposure on the hormonal responses of atrial natriuretic factor (ANF), arginine vasopressin (AVP), catecholamines, and plasma renin activity (PRA) after exhaustive exercise was studied in 9 young and 10 middle-aged subjects. Exercise tests were randomly performed in temperate (30 degrees C) and cold (10 degrees C) environments. Heart rate, oxygen consumption, and peripheral arterial blood pressure were measured at regular intervals. Blood samples were collected before and immediately after exercise at 30 or 10 degrees C. Plasma sodium and potassium concentrations as well as hemoglobin and hematocrit were measured, and the change in plasma volume was calculated. At rest and during exercise, oxygen consumption was similar during exposure to both temperate and cold temperatures. During submaximal exercise intensities, the rise in heart rate was blunted while the increase in systolic blood pressure was significantly greater at 10 than at 30 degrees C. The increases in plasma sodium and potassium concentrations after exhaustion were similar between environments, as was the decrease in plasma volume. In both groups, all plasma hormones were significantly elevated postexercise, with the AVP response similar at 10 and 30 degrees C. However, the norepinephrine and ANF responses were significantly greater while the PRA response was significantly reduced at 10 degrees C. In the middle-aged subjects the epinephrine response to exercise was higher at 10 than at 30 degrees C. The greater ANF and reduced PRA responses to exercise in the cold may have resulted from central hemodynamic changes caused by cold-induced cutaneous vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct Merkel Cell Polyomavirus Molecular Features in Tumour and Non Tumour Specimens from Patients with Merkel Cell Carcinoma

Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083). Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease.     

Creating outbred and inbred populations in haplodiploids to measure adaptive responses in the laboratory

Laboratory studies are often criticized for not being representative of processes occurring in natural populations. One reason for this is the fact that laboratory populations generally do not capture enough of the genetic variation of natural populations. This can be mitigated by mixing the genetic background of several field populations when creating laboratory populations. From these outbred populations, it is possible to generate inbred lines, thereby freezing and partitioning part of their variability, allowing each genotype to be characterized independently. Many studies addressing adaptation of organisms to their environment, such as those involving quantitative genetics or experimental evolution, rely on inbred or outbred populations, but the methodology underlying the generation of such biological resources is usually not explicitly documented. Here, we developed different procedures to circumvent common pitfalls of laboratory studies, and illustrate their application using two haplodiploid species, the spider mites Tetranychus urticae and Tetranychus evansi. First, we present a method that increases the chance of capturing high amounts of variability when creating outbred populations, by performing controlled crosses between individuals from different field‐collected populations. Second, we depict the creation of inbred lines derived from such outbred populations, by performing several generations of sib‐mating. Third, we outline an experimental evolution protocol that allows the maintenance of a constant population size at the beginning of each generation, thereby preventing bottlenecks and diminishing extinction risks. Finally, we discuss the advantages of these procedures and emphasize that sharing such biological resources and combining them with available genetic tools will allow consistent and comparable studies that greatly contribute to our understanding of ecological and evolutionary processes.     

Lipid Peroxidation and Total Cholesterol in HAART-Na?ve Patients Infected with Circulating Recombinant Forms of Human Immunodeficiency Virus Type-1 in Cameroon

Background

HIV infection has commonly been found to affect lipid profile and antioxidant defense.

Objectives

To determine the effects of Human Immunodeficiency Virus (HIV) infection and viral subtype on patient’s cholesterol and oxidative stress markers, and determine whether in the absence of Highly Active Antiretroviral Therapy (HAART), these biochemical parameters could be useful in patient’s management and monitoring disease progression in Cameroon. For this purpose, we measured total cholesterol (TC), LDL cholesterol (LDLC), HDL cholesterol (HDLC), total antioxidant ability (TAA), lipid peroxidation indices (LPI), and malondialdehyde (MDA) in HIV negative persons and HIV positive HAART-naïve patients infected with HIV-1 group M subtypes.

Methods

We measured serum TC, LDLC, HDLC, plasma MDA, and TAA concentrations, and calculated LPI indices in 151 HIV-positive HAART-naïve patients and 134 seronegative controls. We also performed gene sequence analysis on samples from 30 patients to determine the effect of viral genotypes on these biochemical parameters. We also determined the correlation between CD4 cell count and the above biochemical parameters.

Results

We obtained the following controls/patients values for TC (1.96±0.54/1. 12±0. 48 g/l), LDLC (0. 67±0. 46/0. 43±0. 36 g/l), HDLC (105. 51±28. 10/46. 54±23. 36 mg/dl) TAA (0. 63±0. 17/0. 16±0. 16 mM), MDA (0. 20±0. 07/0. 41±0. 10 µM) and LPI (0. 34±0. 14/26. 02±74. 40). In each case, the difference between the controls and patients was statistically significant (p<0.05). There was a positive and statistically significant Pearson correlation between CD4 cell count and HDLC (r = +0.272; p<0.01), TAA (r = +0.199; p<0.05) and a negative and statistically significant Pearson correlation between CD4 cell count and LPI (r = −0.166; p<0.05). Pearson correlation between CD4 cell count and TC, CD4cell count and LDLC was positive but not statistically significant while it was negative but not statistically significant with MDA. The different subtypes obtained after sequencing were CRF02_AG (43.3%), CRF01_AE (20%), A1 (23.3%), H (6.7%), and G (6.7%). None of the HIV-1 subtypes significantly influenced the levels of the biochemical parameters, but by grouping them as pure subtypes and circulating recombinant forms (CRFs), the CRF significantly influenced TC levels. TC was significantly lower in patients infected with CRF (0.87±0.27 g/l) compared to patients infected with pure HIV-1 subtypes (1.32±0.68 g/l) (p<0.017). MDA levels were also significantly higher in patients infected with HIV-1CRF01_AE (0.50±0.10 µM), compared to patients infected with CRF02_AG (0. 38±0. 08 µM) (p<0.018).

Conclusion

These results show that HIV infection in Cameroon is associated with significant decrease in TAA, LDLC, HDLC and TC, and increased MDA concentration and LPI indices which seem to be linked to the severity of HIV infection as assessed by CD4 cell count. The data suggests increased oxidative stress and lipid peroxidation in HIV-infected patients in Cameroon, and an influence of CRFs on TC and MDA levels.     

Interaction domains and nuclear targeting signals in subunits of the U2 small nuclear ribonucleoprotein particle-associated splicing factor SF3a

Human splicing factor SF3a is a component of the mature U2 small nuclear ribonucleoprotein particle (snRNP) and its three subunits of 60, 66, and 120 kDa are essential for splicing in vitro and in vivo. The SF3a heterotrimer forms in the cytoplasm and enters the nucleus independently of the U2 snRNP. Here, we have analyzed domains required for in vitro interactions between the SF3a subunits. Our results indicate that the SF3a66-SF3a120 interaction is mediated by a 27-amino acid region in SF3a120 C-terminal to the second suppressor-of-white-apricot and prp21/spp91 domain and amino acids 108-210 of SF3a66. Neither of these sequences contains known structural motifs, suggesting that the interaction domains are novel. Moreover, an ～100-amino acid region, including the SURP2 domain of SF3a120 but extending into neighboring regions, is sufficient for binding to SF3a60. Analysis of determinants for nuclear import of SF3a demonstrates that SF3a120 provides the major nuclear localization signal and SF3a60 contributes to nuclear import.     

Substrate specificity overlap and interaction between adrenoleukodystrophy protein (ALDP/ABCD1) and adrenoleukodystrophy-related protein (ALDRP/ABCD2)

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene, which encodes a peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D called ALDP. ALDP is supposed to function as a homodimer allowing the entry of CoA-esters of very-long chain fatty acids (VLCFA) into the peroxisome, the unique site of their β-oxidation. ALDP deficiency can be corrected by overexpression of ALDRP, its closest homolog. However, the exact nature of the substrates transported by ALDRP and its relationships with ALDP still remain unclear. To gain insight into the function of ALDRP, we used cell models allowing the induction in a dose-dependent manner of a wild type or a mutated non-functional ALDRP-EGFP fusion protein. We explored the consequences of the changes of ALDRP expression levels on the fatty acid content (saturated, monounsaturated, and polyunsaturated fatty acids) in phospholipids as well as on the levels of β-oxidation of 3 suspected substrates: C26:0, C24:0, and C22:6n-3 (DHA). We found an inverse correlation between the fatty acid content of saturated (C26:0, C24:0) and monounsaturated (C26:1, C24:1) VLCFA and the expression level of ALDRP. Interestingly, we obtained a transdominant-negative effect of the inactive ALDRP-EGFP on ALDP function. This effect is due to a physical interaction between ALDRP and ALDP that we evidenced by proximity ligation assays and coimmunoprecipitation. Finally, the β-oxidation assays demonstrate a role of ALDRP in the metabolism of saturated VLCFA (redundant with that of ALDP) but also a specific involvement of ALDRP in the metabolism of DHA.     

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.     

Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila

In Drosophila, intestinal stem cells (ISCs) respond to oxidative challenges and inflammation by increasing proliferation rates. This phenotype is part of a regenerative response, but can lead to hyperproliferation and epithelial degeneration in the aging animal. Here we show that Nrf2, a master regulator of the cellular redox state, specifically controls the proliferative activity of ISCs, promoting intestinal homeostasis. We find that Nrf2 is constitutively active in ISCs and that repression of Nrf2 by its negative regulator Keap1 is required for ISC proliferation. We further show that Nrf2 and Keap1 exert this function in ISCs by regulating the intracellular redox balance. Accordingly, loss of Nrf2 in ISCs causes accumulation of reactive oxygen species and accelerates age-related degeneration of the intestinal epithelium. Our findings establish Keap1 and Nrf2 as a critical redox management system that regulates stem cell function in high-turnover tissues.     

Notch-Mediated Suppression of TSC2 Expression Regulates Cell Differentiation in the Drosophila Intestinal Stem Cell Lineage

Epithelial homeostasis in the posterior midgut of Drosophila is maintained by multipotent intestinal stem cells (ISCs). ISCs self-renew and produce enteroblasts (EBs) that differentiate into either enterocytes (ECs) or enteroendocrine cells (EEs) in response to differential Notch (N) activation. Various environmental and growth signals dynamically regulate ISC activity, but their integration with differentiation cues in the ISC lineage remains unclear. Here we identify Notch-mediated repression of Tuberous Sclerosis Complex 2 (TSC2) in EBs as a required step in the commitment of EBs into the EC fate. The TSC1/2 complex inhibits TOR signaling, acting as a tumor suppressor in vertebrates and regulating cell growth. We find that TSC2 is expressed highly in ISCs, where it maintains stem cell identity, and that N-mediated repression of TSC2 in EBs is required and sufficient to promote EC differentiation. Regulation of TSC/TOR activity by N signaling thus emerges as critical for maintenance and differentiation in somatic stem cell lineages.