Testing metabolic cold adaptation and the climatic variability hypothesis in two latitudinally distant populations of a supratidal water beetle.

Temperature significantly impacts ectotherm physiology, with thermal tolerance and metabolic traits typically varying with latitude across species ranges. The drivers of this variation remain unclear, however, despite obvious consequences for population persistence and conservation in the face of ongoing global change. This study explored local adaptation and phenotypic plasticity of metabolic rates and thermal limits in the supratidal rockpool beetle Ochthebius lejolisii. Using populations from localities at different ends of the species range that experience contrasting thermal variability, we simultaneously tested two of the major paradigms of spatial physiological ecology: metabolic cold adaptation (MCA) and the climatic variability hypothesis (CVH). Reciprocal acclimation was conducted under spring temperature regimes of both localities, incorporating local diurnal variation. Metabolic rates were measured by closed respirometry, and thermal tolerance limits estimated through thermography. In line with MCA, the higher-latitude population (colder climate) showed higher metabolic rates and temperature coefficients (Q10s) at lower temperatures than the lower-latitude population. As predicted by the CVH, the lower-latitude population (more variable climate) showed higher upper thermal tolerance but only the higher-latitude population was able to acclimate upper thermal limits. This result suggests trade-offs between physiological thermal limits and thermal plasticity in this species. A limited acclimation capacity could make populations on Mediterranean coasts especially vulnerable in the face of projected increases in extreme temperatures under ongoing climate change.

Mean structure of the supercritical turbulent spiral in Taylor-Couette flow.

The large-scale laminar/turbulent spiral patterns that appear in the linearly unstable regime of counter-rotating Taylor-Couette flow are investigated from a statistical perspective by means of direct numerical simulation. Unlike the vast majority of previous numerical studies, we analyse the flow in periodic parallelogram-annular domains, following a coordinate change that aligns one of the parallelogram sides with the spiral pattern. The domain size, shape and spatial resolution have been varied and the results compared with those in a sufficiently large computational orthogonal domain with natural axial and azimuthal periodicity. We find that a minimal parallelogram of the right tilt significantly reduces the computational cost without notably compromising the statistical properties of the supercritical turbulent spiral. Its mean structure, obtained from extremely long time integrations in a co-rotating reference frame using the method of slices, bears remarkable similarity with the turbulent stripes observed in plane Couette flow, the centrifugal instability playing only a secondary role. This article is part of the theme issue 'Taylor-Couette and related flows on the centennial of Taylor's seminal Philosophical transactions paper (Part 2)'.

Prevalence of hypertrophic cardiomyopathy in a large sample of the Spanish working population. / Prevalencia de la miocardiopatía hipertrófica en una amplia muestra de la población laboral española.

BACKGROUND AND OBJECTIVES: To date, in Spain, there are no studies that have evaluated the prevalence of hypertrophic cardiomyopathy in the general population. The aim of this study was to assess the prevalence of hypertrophic cardiomyopathy in a large sample of the working population of Spain. MATERIALS AND METHODS: The study included 13,179 workers (73% men; mean age, 40 years) from 5 regions of Spain who, between May 2008 and November 2010, had a medical examination with an electrocardiogram. The workers with suggestive abnormalities in the electrocardiogram or a predisposing medical history (exertional syncope or sudden death of a family member younger than 50 years) were referred for an echocardiographic evaluation. We defined hypertrophic cardiomyopathy as a parietal thickness ≥13mm in any segment of the left ventricle. We estimated the prevalence of hypertrophic cardiomyopathy in the entire sample and in the workers without hypertension. RESULTS: A total of 1008 workers were selected for the echocardiogram, although only 496 (49.2% of those selected) of these attended the appointment. After the echocardiogram, we detected 16 cases of hypertrophic cardiomyopathy, estimating a prevalence of 0.24% for the entire sample. In the subgroup of workers with no hypertension, we observed 10 cases of hypertrophic cardiomyopathy, which corresponds to an estimated prevalence of 0.19%. CONCLUSIONS: In our sample of the working population in Spain, the estimated prevalence of hypertrophic cardiomyopathy was 0.24%. In the subgroup of patients with no hypertension, the estimated prevalence was 0.19%.

Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods.

BACKGROUND: Molecular and cellular pathophysiological events occurring in the majority of rare kidney diseases remain to be elucidated. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disorder caused by mutations in either CLDN16 or CLDN19 genes. This disease is characterized by massive urinary wasting of magnesium and calcium, osmosis deregulation and polyuria. Patients with p.G20D homozygous mutation in CLDN19 gene exhibit different progression to kidney failure suggesting that beyond the pathogenic mutation itself, other molecular events are favoring disease progression. Due to the fact that biopsy is not clinically indicated in these patients, urinary exosome-like vesicles (uEVs) can be envisioned as a valuable non-invasive source of information of events occurring in the kidney. Exosome research has increased notably to identify novel disease biomarkers but there is no consensus standardized protocols for uEVs isolation in patients with polyuria. For this reason, this work was aimed to evaluate and refine different uEVs isolation methods based on differential centrifugation, the gold standard method. RESULTS: Characterization by NTA, cryo-TEM and immunoblotting techniques identified the most appropriate protocol to obtain the highest yield and purest uEVs enriched fraction possible from urine control samples and FHHNC patients. Moreover, we tested five different RNA extraction methods and evaluated the miRNA expression pattern by qRT-PCR. CONCLUSIONS: In summary, we have standardized the conditions to proceed with the identification of differentially expressed miRNAs in uEVs of FHHNC patients, or other renal diseases characterized by polyuria.

Buchnera has changed flatmate but the repeated replacement of co-obligate symbionts is not associated with the ecological expansions of their aphid hosts.

Symbiotic associations with bacteria have facilitated important evolutionary transitions in insects and resulted in long-term obligate interactions. Recent evidence suggests that these associations are not always evolutionarily stable and that symbiont replacement, and/or supplementation of an obligate symbiosis by an additional bacterium, has occurred during the history of many insect groups. Yet, the factors favouring one symbiont over another in this evolutionary dynamic are not well understood; progress has been hindered by our incomplete understanding of the distribution of symbionts across phylogenetic and ecological contexts. While many aphids are engaged into an obligate symbiosis with a single Gammaproteobacterium, Buchnera aphidicola, in species of the Lachninae subfamily, this relationship has evolved into a 'ménage à trois', in which Buchnera is complemented by a cosymbiont, usually Serratia symbiotica. Using deep sequencing of 16S rRNA bacterial genes from 128 species of Cinara (the most diverse Lachninae genus), we reveal a highly dynamic dual symbiotic system in this aphid lineage. Most species host both Serratia and Buchnera but, in several clades, endosymbionts related to Sodalis, Erwinia or an unnamed member of the Enterobacteriaceae have replaced Serratia. Endosymbiont genome sequences from four aphid species confirm that these coresident symbionts fulfil essential metabolic functions not ensured by Buchnera. We further demonstrate through comparative phylogenetic analyses that cosymbiont replacement is not associated with the adaptation of aphids to new ecological conditions. We propose that symbiont succession was driven by factors intrinsic to the phenomenon of endosymbiosis, such as rapid genome deterioration or competitive interactions between bacteria with similar metabolic capabilities.

Root caries analysis in working population of 35-44 years of age (Spain).

BACKGROUND: The aim of this study was to analyse the influence of socio-demographic variables, toothbrushing frequency, frequency of snacking between meals, and tobacco and alcohol consumption, in root caries in the Spanish working population of Valencia and Murcia regions. MATERIAL AND METHODS: Cross sectional study of 458 workers 35-44 years of age, who underwent a routine work-related check-up, from June 2009 to April 2010, and were also examined, following the WHO methodology, by a calibrated dentist. Stratified random sampling. Participants fulfilled a questionnaire comprising demographic data, toothbrushing frequency, snacking frequency and tobacco and alcohol consumption. RESULTS: The DFS index (root caries) in the employed population of 35-44 years was 0.45 ± 1.3, with a root caries prevalence of 18.6% and an active root caries prevalence of 13.5%. Higher root caries prevalence and active root caries prevalence were associated with male gender, manual occupations, foreign country of origin, lower levels of education and income, lower brushing frequency and higher frequency of snacking between meals. The DFS index was associated with all studied socio-demographic variables, but gender, and it was also associated with brushing frequency. The mean number of root decayed teeth was associated with all socio-demographic variables, but country of origin, and it was also associated with brushing frequency. CONCLUSIONS: Adult workers 35-44 years of age showed worse root condition in regard to caries than general population of this age cohort. In this study, the frequency of toothbrushing and snacking between meals were the variables that influenced more in root caries.

A form of apolipoprotein a-I is found specifically in relapses of focal segmental glomerulosclerosis following transplantation.

Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) following kidney transplantation occurs in a large percentage of patients. Accurate prediction of recurrence and elucidation of its pathogenesis are major therapeutic goals. To detect differential proteins related to FSGS recurrence, proteomic analysis was performed on plasma and urine samples from 35 transplanted idiopathic FSGS patients, divided into relapsing and nonrelapsing. Several proteins were detected increased in urine of relapsing FSGS patients, including a high molecular weight form of apolipoprotein A-I, named ApoA-Ib, found exclusively in relapsing patients. This finding was verified by Western blot individually in the 35 patients and validated in an independent group of 40 patients with relapsing or nonrelapsing FSGS, plus two additional groups: FSGS-unrelated patients showing different proteinuria levels (n = 30), and familial FSGS transplanted patients (n = 14). In the total of 119 patients studied, the ApoA-Ib form was detected in 13 of the 14 relapsing FSGS patients, and in one of the 61 nonrelapsing patients. Only one of the 30 patients with FSGS-unrelated proteinuria tested positive for ApoA-Ib, and was not detected in familial patients. Urinary ApoA-Ib is associated with relapses in idiopathic FSGS and warrants additional investigation to determine its usefulness as biomarker of relapse following transplantation.

STAT3 phosphorylation at serine 727 activates specific genetic programs and promotes clear cell renal cell carcinoma (ccRCC) aggressiveness.

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor mainly activated by phosphorylation in either tyrosine 705 (Y705) or serine 727 (S727) residues that regulates essential processes such as cell differentiation, apoptosis inhibition, or cell survival. Aberrant activation of STAT3 has been related to development of nearly 50% of human cancers including clear cell renal cell carcinoma (ccRCC). In fact, phosho-S727 (pS727) levels correlate with overall survival of ccRCC patients. With the aim to elucidate the contribution of STAT3 phosphorylation in ccRCC development and progression, we have generated human-derived ccRCC cell lines carrying STAT3 Y705 and S727 phosphomutants. Our data show that the phosphomimetic substitution Ser727Asp facilitates a pro-tumoral phenotype in vitro, in a Y705-phosphorylation-independent manner. Moreover, we describe that STAT3 phosphorylation state determines the expression of different subsets of target genes associated with distinct biological processes, being pS727-dependent genes the most related to cellular hallmarks of cancer. In summary, the present study constitutes the first analysis on the role of overall STAT3 phosphorylation state in ccRCC and demonstrates that pS727 promotes the expression of a specific subset of target genes that might be clinically relevant as novel biomarkers and potential therapeutic targets for ccRCC.

[Postoperative unilateral acute glaucoma after abdominal surgery]. / Glaucoma agudo unilateral en el postoperatorio de cirugía abdominal.

An early and correct diagnosis substantially improves the post-operative prognosis of acute angle closure glaucoma (AACG). A 90 year-old woman was operated on for a right colon tumour by laparotomy, under combined anaesthesia without any adverse events. Twelve hours after the operation, the patient described recurrent periorbital pain in her right eye, with ocular hyperaemia, blurred vision, and unresponsive mydriasis. A diagnosis of AACG was made, but although conservative treatment was started YAG laser iridotomies were required to reduce the intraocular pressure. In the AACG postoperative period, as well as with an eye with several predisposed local factors including genetic predisposition, female gender, hypermetropia, increased lens thickness and small corneal diameter, can be added a pupillary block induced by adrenergic and anticholinergic drugs used in anaesthetic procedures. An acute and intensive periorbital or ocular pain, with or without visual disturbance, must aware the doctor. A differential diagnosis with other postoperative ocular diseases and cranial pain causes must be done.

High cardiovascular risk in Spanish workers.

BACKGROUND AND AIMS: To investigate the prevalence of high cardiovascular risk in the Spanish working population, and its distribution among different occupations and gender. METHODS AND RESULTS: Cross-sectional study of 309,955 workers (72.6% males, mean age 36.5 years, range 16-74 years), who underwent a routine medical check-up. Workers were classified as high, intermediate or low cardiovascular risk, according to the SCORE system. Workers with a relative risk greater than 4 were also considered as high-risk. The prevalence of high cardiovascular risk was 7.6% (95% CI 7.5-7.7) in males and 1.7% (95% CI 1.6-1.8) in females. After adjusting for age and gender, the prevalence of high cardiovascular risk was greater in workers from the Agriculture and Construction sectors than in those from Industry and Service sectors. The prevalence of high cardiovascular risk was higher in blue-collar than in white-collar occupations. CONCLUSIONS: A sizeable proportion of workers, especially blue-collar males, are at high cardiovascular risk. Knowledge of this risk for certain workers may serve as a basis for preventive strategies.

Prevalence of hypertrophic cardiomyopathy in a large sample of the Spanish working population.

BACKGROUND AND OBJECTIVES: To date, in Spain, there are no studies that have evaluated the prevalence of hypertrophic cardiomyopathy in the general population. The aim of this study was to assess the prevalence of hypertrophic cardiomyopathy in a large sample of the working population of Spain. MATERIALS AND METHODS: The study included 13,179 workers (73% men; mean age: 40 years) from 5 regions of Spain who, between May 2008 and November 2010, had a medical examination with an electrocardiogram. The workers with suggestive abnormalities in the electrocardiogram or a predisposing medical history (exertional syncope or sudden death of a family member younger than 50 years) were referred for an echocardiographic evaluation. We defined hypertrophic cardiomyopathy as a parietal thickness ≥13mm in any segment of the left ventricle. We estimated the prevalence of hypertrophic cardiomyopathy in the entire sample and in the workers without hypertension. RESULTS: A total of 1008 workers were selected for the echocardiogram, although only 496 (49.2% of those selected) of these attended the appointment. After the echocardiogram, we detected 16 cases of hypertrophic cardiomyopathy, estimating a prevalence of 0.24% for the entire sample. In the subgroup of workers with no hypertension, we observed 10 cases of hypertrophic cardiomyopathy, which corresponds to an estimated prevalence of 0.19%. CONCLUSIONS: In our sample of the working population in Spain, the estimated prevalence of hypertrophic cardiomyopathy was 0.24%. In the subgroup of patients with no hypertension, the estimated prevalence was 0.19%.

Kidney androgen-regulated protein transgenic mice show hypertension and renal alterations mediated by oxidative stress.

BACKGROUND: Kidney androgen-regulated protein (KAP), a proximal tubule androgen-regulated gene, codes for a protein of unknown function. METHODS AND RESULTS: To investigate the consequences of KAP overexpression in kidney, we produced KAP transgenic mice and performed microarray expression analyses in kidneys of control and transgenic males. Downregulation of the androgen-sensitive Cyp4A14 monooxygenase gene in KAP transgenic mice prompted us to analyze blood pressure levels, and we observed that transgenic mice were hypertensive. Inhibition of 20-hydroxyeicosatetraenoic acid synthesis by N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) reduced the increased 20-hydroxyeicosatetraenoic acid levels in urine and normalized arterial pressure in transgenic mice, as did the NADPH oxidase inhibitor apocynin. Increased oxidative stress in transgenic mice was demonstrated by (1) enhanced excretion of urinary markers of oxidative stress, 8-iso-prostaglandin F2alpha, 8-hydroxydeoxyguanosine, and thiobarbituric acid-reacting substances; (2) augmented mitochondrial DNA damage and malondialdehyde levels in kidneys; and (3) diminished catalase and glutathione peroxidase activity in transgenic kidneys. Mice exhibited renal defects that included focal segmental glomerulosclerosis, proteinuria, glycosuria, and fibrosis. CONCLUSIONS: Taken together, these results indicate that KAP expression is critical for cardiovascular-renal homeostasis maintenance and that hypertension is associated with increased oxidative stress. This is the first report showing that overexpression of an androgen-regulated, proximal tubule-specific gene induces hypertension. These observations may shed light on the molecular pathophysiology of gender differences in the prevalence and severity of hypertension and chronic renal disease.

Analysis of the nuclear expression of pSer727-STAT3 as a prognostic factor in patients with clear cell renal carcinoma. / Análisis de la expresión nuclear de pSer727-STAT3 como factor pronóstico en pacientes con carcinoma renal de células claras.

INTRODUCTION: Currently, clear cell renal carcinoma (CCRCC) has no prognostic markers. STAT3 protein (Signal Transducer and Activator of Transcription 3) is involved in the carcinogenesis of CCRCC. Its activation is produced by phosphorylation of the serine 727 residue, translocating to the nucleus where it is involved in carcinogenesis and tumor progression. The primary objective of the study was to evaluate cancer-specific survival rates in a series of 166 patients with CCRCC, and its subsequent correlation with the expression of pSer727-STAT3 as a prognostic marker of CCRCC. MATERIAL AND METHODS: We conducted a retrospective study on 166 patients with CCRCC undergoing partial or radical nephrectomy between 2000 and 2010. A tumor tissue microarray was constructed for immunohistochemical analysis of pSer727-STAT3 expression. The main variable of the study was cancer-specific survival. RESULTS: Patients were classified according to the UICC risk groups as follows: low in 78 patients (47%), intermediate in 52 (31.3%) and high 36 (21.7%); 11 patients (6.7%) were diagnosed with metastatic disease. During a mean follow-up of 97.2 months (1-208), 37 patients (22.3%) developed local and/or distant recurrence. Cancer-specific and overall mortality rates were 28.3% and 67.5%, respectively. The mean expression of pSer727-STAT3 was 92.9 (95% CI: 84.6-101.1) without showing any relationship with risk groups or other prognostic factors. In a Cox logistic regression analysis, pSer727-STAT3 did not behave as an independent predictor of cancer-specific mortality. However, in high-risk and metastatic patients, cancer-specific survival was significantly higher when the expression of pSer727-STAT3 was lower than 110, HR: 5.4 (96% CI: 1.8-16.4) and HR: 2.3 (95% CI: 1.1-4.6) respectively, P<.001. CONCLUSIONS: pSer727-STAT3 is not a survival marker in patients with CCRCC. However, it is a cancer-specific survival marker in high-risk patients, even in metastatic patients undergoing treatment with antiangiogenic agents.

The role of STAT3 protein as a prognostic factor in the clear cell renal carcinoma. Systematic review. / Valor de la proteína STAT3 como factor pronóstico en el carcinoma renal de célula clara. Revisión sistemática.

CONTEXT AND OBJECTIVE: There have been significant advances in the knowledge of renal carcinogenesis n the last years. Nowadays, renal tumors are classified according to their genetic profile and specific treatments based on the identification of therapeutic targets have also been developed. However, no prognostic markers have yet been identified. The aim of this review is to analyse literature that has evaluated the expression of the STAT3 protein as a molecular marker in clear cell renal carcinoma (ccRCC). EVIDENCE ACQUISITION: In January 2018 a systematic review was conducted in Pubmed, Cochrane library and Sciencedirect databases, from papers published from 1990. Search terms were"renal cell carcinoma"and"STAT3"or"STAT-3"and"prognostic factor. Following the principles of the PRISMA declaration and the PICO selection strategy, original articles with series of patients diagnosed with localized or metastatic ccRCC, and where the activity of STAT3 is analysed as a prognostic marker, were selected. A total of 132 publications were identified, of which 10 were finally revised, for they met the inclusion criteria. EVIDENCE SYNTHESIS: STAT3 activation (phosphorylation) through Ser727 is important during ccRCC development and progression. PSTAT3 expression seems to be a prognostic marker and an antiangiogenic-resistance marker in metastatic patients. There is little evidence as prognostic marker in patients with localized disease. CONCLUSIONS: STAT3 (Ser 727) expression in the nucleus of the ccRCC cells can be a prognostic marker and an antiangiogenic-resistance marker. Current scientific evidence is limited and more studies are needed to demonstrate its usefulness.

CCAAT/enhancer binding protein-mediated role of thyroid hormone in the developmental expression of the kidney androgen-regulated protein gene in proximal convoluted tubules.

The kidney androgen-regulated protein (KAP) gene is exclusively expressed in proximal tubules of mouse kidney and in the uterus of pregnant females before they give birth. It displays an exquisite and differential regulation of expression by steroid and thyroid hormones (THs) in different proximal tubule segments. Whereas the pars recta (PR cells) responds to thyroid and sexual hormones, the pars convoluta (PCT cells) represents a truly androgen-dependent compartment because expression occurs only in the presence of androgens and functional androgen receptors. Nevertheless, different hypothyroidism models have indicated that TH might also contribute to the androgenic response in PCT cells. In the present study, we aimed to determine the molecular mechanisms that ultimately control KAP expression in these cells. Using several genetically deficient mouse models and different pharmacologic and hormonal treatments, we determined that thyroid and GH modulate CCAAT/enhancer binding protein alpha and beta levels that, in turn, control KAP expression in PCT cells in a developmentally dependent manner. We demonstrated that these factors bind to sites in the proximal KAP promoter, thereby collaborating with androgens for full KAP expression. Finally, we propose that TH and GH, acting through CCAAT/enhancer binding protein, may constitute a general regulatory mechanism of androgen-dependent genes in mouse kidney.

Systematic review of renal carcinoma prognostic factors. / Revisión sistemática de los factores pronósticos del carcinoma renal.

CONTEXT AND OBJECTIVES: The natural history of renal cell carcinoma is heterogeneous. Some scenarios can be found in terms of clinical presentation, clinical evolution or type of recurrence (local/metastatic). The aim of this publication is to analyze the most important prognostic factors published in the literature. EVIDENCE ACQUISITION: A literature review ob published papers was performed using the Pubmed, from first Motzer's classification published in 1999 to 2015, according to PRISMA declaration. Search was done using the following keywords: kidney neoplasm, kidney cancer, renal cell carcinoma, prognostic factors, mortality, survival and disease progression. Papers were classified according to level of evidence, the number of patients included and the type of study performed. EVIDENCE SYNTHESIS: The evolution in the knowledge of molecular pathways related to renal oncogenesis and the new targeted therapies has left to remain obsolete the old prognostic models. It's necessary to perform a continuous review to actualize nomograms and to adapt them to the new scenarios. CONCLUSIONS: Is necessary to perform a proper external validation of existing prognostic factors using prospective and multicentric studies to add them into the daily urologist clinical practice.

Metabolic syndrome or glucose challenge in first episode of psychosis?

Patients with schizophrenia exhibit a reduced life expectancy. Although unhealthy lifestyle or suicide risk plays a role, the main causes are diverse medical conditions such as cardiovascular diseases, type 2 diabetes mellitus and metabolic syndrome. Albeit pharmacological secondary side effects might also trigger previous conditions, studies in naïve patients reflect diverse anomalies at the onset. Patients with a first episode of psychosis, display a wide scope of metabolic abnormalities, ranging from normality till pathological values depending on the parameters studied. We attempted to evaluate the metabolic syndrome and glycemic homeostasis in a subset of antipsychotic-naïve patients with a first episode of non-affective psychosis. Patients (n=84) showed a similar prevalence of metabolic syndrome compared with a matched control sample (n=98) (6% vs 4%, P=0.562), while glucose homeostasis values differed significantly (14% vs. 5%, P=0.034). Our results suggest that metabolic syndrome is not a useful clinical condition to be evaluated in patients before pharmacological treatment. Abnormal glycemic homeostasis at the onset of the disease requires specific diagnostic tools and preventive measures in order to avoid future cardiovascular events. New strategies must be implemented in order to evaluate the cardiovascular risk and subsequent morbidity in patients at the onset of the disease.

Cell surface antigens of chemically induced fibrosarcomas: detection by a monoclonal antibody of a tumor-restricted Mr 12,000 protein gag antigen encoded by a dual-tropic murine leukemia virus.

Fusion products of spleen cells of W/FuDp rats immunized with a methylcholanthrene-induced BALB/c sarcoma, CA-2, and mouse myeloma cells were screened in an attempt to identify a monoclonal antibody defining the individually distinct tumor-specific transplantation antigen of CA-2. A hybridoma, MP/69/04, was isolated which produces an IgG2a monoclonal antibody that recognized a tumor-restricted antigen of CA-2. In direct binding assay, MP/69/04 reacted only with 2 of 15 methylcholanthrene induced BALB/c sarcomas tested. Thymus, spleen, lymph nodes, bone marrow, brain, adult lung fibroblasts, newborn muscle fibroblasts and 3T3 cells were negative. Absorption tests revealed, however, expression of the MP/69/04 determinant on 8 of the 12 murine leukemia virus (MuLV) producer BALB/c sarcoma tested. The antigen was not detected on any of the three non-producer sarcomas tested nor on a wide range of normal tissues and cell lines. An N-dualtropic MuLV was isolated from CA-2, and cell lines susceptible to infection by this virus were shown to express the MP/69/04 epitope. By Western blotting, the MP/69/04 epitope was identified as being expressed on the MuLV structural protein with a molecular weight of 12,000, present in CA-2 cells and in the purified CA-2 MuLV. These results indicate the MP/69/04 antigen is not a unique tumor-specific transplantation antigen but is a gag product of a recombinant retrovirus which is expressed on the cell surface of many MuLV + methylcholanthrene-induced BALB/c fibrosarcomas.

Assessment of a 16S rRNA amplicon Illumina sequencing procedure for studying the microbiome of a symbiont-rich aphid genus.

The bacterial communities inhabiting arthropods are generally dominated by a few endosymbionts that play an important role in the ecology of their hosts. Rather than comparing bacterial species richness across samples, ecological studies on arthropod endosymbionts often seek to identify the main bacterial strains associated with each specimen studied. The filtering out of contaminants from the results and the accurate taxonomic assignment of sequences are therefore crucial in arthropod microbiome studies. We aimed here to validate an Illumina 16S rRNA gene sequencing protocol and analytical pipeline for investigating endosymbiotic bacteria associated with aphids. Using replicate DNA samples from 12 species (Aphididae: Lachninae, Cinara) and several controls, we removed individual sequences not meeting a minimum threshold number of reads in each sample and carried out taxonomic assignment for the remaining sequences. With this approach, we show that (i) contaminants accounted for a negligible proportion of the bacteria identified in our samples; (ii) the taxonomic composition of our samples and the relative abundance of reads assigned to a taxon were very similar across PCR and DNA replicates for each aphid sample; in particular, bacterial DNA concentration had no impact on the results. Furthermore, by analysing the distribution of unique sequences across samples rather than aggregating them into operational taxonomic units (OTUs), we gained insight into the specificity of endosymbionts for their hosts. Our results confirm that Serratia symbiotica is often present in Cinara species, in addition to the primary symbiont, Buchnera aphidicola. Furthermore, our findings reveal new symbiotic associations with Erwinia- and Sodalis-related bacteria. We conclude with suggestions for generating and analysing 16S rRNA gene sequences for arthropod-endosymbiont studies.