The impact of endogenous content,replicates and pooling on genome capture from faecal samples

Target‐capture approach has improved over the past years, proving to be very efficient tool for selectively sequencing genetic regions of interest. These methods have also allowed the use of noninvasive samples such as faeces (characterized by their low quantity and quality of endogenous DNA) to be used in conservation genomic, evolution and population genetic studies. Here we aim to test different protocols and strategies for exome capture using the Roche SeqCap EZ Developer kit (57.5 Mb). First, we captured a complex pool of DNA libraries. Second, we assessed the influence of using more than one faecal sample, extract and/or library from the same individual, to evaluate its effect on the molecular complexity of the experiment. We validated our experiments with 18 chimpanzee faecal samples collected from two field sites as a part of the Pan African Programme: The Cultured Chimpanzee. Those two field sites are in Kibale National Park, Uganda (N = 9) and Loango National Park, Gabon (N = 9). We demonstrate that at least 16 libraries can be pooled, target enriched through hybridization, and sequenced allowing for the genotyping of 951,949 exome markers for population genetic analyses. Further, we observe that molecule richness, and thus, data acquisition, increase when using multiple libraries from the same extract or multiple extracts from the same sample. Finally, repeated captures significantly decrease the proportion of off‐target reads from 34.15% after one capture round to 7.83% after two capture rounds, supporting our conclusion that two rounds of target enrichment are advisable when using complex faecal samples.     

On the effect of sodium dodecyl sulfate on the structure of beta-galactosidase from Escherichia coli. A fluorescence study.

An understanding of the structure-function relationship of proteins under different chemical-physical conditions is of fundamental importance for an understanding of their structure and function in cells. In this paper we report the effects of sodium dodecyl sulfate and temperature on the structure of beta-galactosidase from Escherichia coli, as monitored by fluorescence spectroscopy. The structure of the protein was studied in the temperature range of 10-60 degrees C in the absence and presence of sodium dodecyl sulfate by frequency-domain measurement of the intrinsic fluorescence intensity and anisotropy decays. The time-resolved fluorescence data in the absence of SDS indicated that at 10 degrees C the tryptophanyl emission decays were well described by a three exponential decays model, and that the temperature increase resulted in shortening of the long-lived component with little change in the short- and middle-lived components. The addition of SDS to the protein solution also affected the long-lived component. The effects of the detergent and temperature on the enzyme structure were also investigated by means of quenching experiments and anisotropy decays. The obtained results showed that the presence of SDS confers more flexibility to the protein structure, and suggest a strict relation between enzyme activity and protein flexibility.     

The accumulation of chromosome aberrations and Dlb-1 mutations in mice with highly fractionated exposure to gamma radiation

The dichotomy between the doses at which experimental measurements of genetic effects can be made and the doses to which people are exposed is often different by two or more orders of magnitude. This presents a significant problem when determining the effects of low doses of radiation or chemicals. The solution has usually involved extrapolating the data by curve-fitting or by applying theoretical considerations. Both approaches are unsatisfactory due to uncertainties of the assumptions used in each process. The alternative solution has been to increase the sample size enormously at the lower doses. This is impractical beyond a certain point due to the variation in the spontaneous frequency and the need to quadruple the sample size for a doubling of precision. The development of new methods for measuring stable genetic effects, however, permits a simple and effective approach to this problem: if the genetic events being detected have no effect on survival, i.e., are selectively neutral, then the effects of multiple independent treatments will be additive. If the independent treatments are identical, then the effect of each is easily calculated by dividing the total effect by the number of treatments. Here we report a limited test of this approach using mice. Chromosome aberrations induced in lymphocytes and Dlb-1 mutations induced in the small intestine were measured after daily doses of 0.64, 1.85 or 5.5 cGy ¹³⁷Cs gamma rays administered for 21, 42 or 63 days. The dose response curve for chromosome translocations obtained in this way, combined with the data from single larger acute doses, shows no evidence for a threshold over a 500-fold dose range. Dlb-1 mutations were increased at each dose and time but the results do not permit reliable extrapolations. The results suggest that translocations might be useful for quantifying the effect of doses below 0.05 cGy and that the effect of dose rate and dose fractionation at much lower doses than reported here could be investigated.     

Neo-adjuvant chemo-(immuno-)therapy of advanced squamous-cell head and neck carcinoma: a multicenter, phase III, randomized study comparing cisplatin + 5-fluorouracil (5-FU) with cisplatin + 5-FU + recombinant interleukin 2

We carried out an open, randomized, phase III, multicenter clinical trial to compare, in neo-adjuvant setting, the clinical response and toxicity of the combination chemotherapy cisplatin + 5-FU with the same combination plus s.c. recombinant interleukin-2 (rIL-2) in patients with advanced (stage III–IV) head and neck squamous-cell carcinoma (HNSCC). Regimen A was the classical Al Sarraf treatment: 100 mg/m² cisplatin i.v. on day 1 plus 1000 mg m⁻² day⁻¹ 5-FU on days 1–5 as a continuous infusion. Regimen B was the same as regimen A plus 4.5 MIU/day rIL-2 s.c. on days 8–12 and 15–19. Treatment was repeated every 3 weeks for three cycles. A total of 33 patients were enrolled in the study; 30 were evaluable for toxicity and 28 for response. Seventeen patients were assigned to group A and 16 were assigned to group B. Three patients (20%) of group A and 4 (31%) of group B had a complete response, 9 patients (60%) of group A and 6 (46%) of group B had a partial response, with an overall response rate of 12 patients (80%) for group A and 10 patients (77%) for group B. Two patients (13%) of group A and 3 patients (23%) group B had stable disease; 1 patient (7%) of group A had progressive disease. Thus, there was not a statistically significant difference in response rate between the two groups and therefore there was no benefit from the addition of immunotherapy with rIL-2 to the standard chemotherapy. Both regimens were well tolerated. There were 2 toxic deaths (6.7%), 1 from hematological causes in group A and 1 from cardiac causes in group B. Myelosuppression and gastrointestinal toxicity, mainly nausea/vomiting and stomatitis, were the most frequent toxicities. The calculated number of patients for the sample has not yet been reached; however, the projection of our present results suggests that it is highly improbable that a clinically significant difference between the two treatment groups will be observed even if the calculated patient sample size is achieved. Received: 9 April 1998 / Accepted: 30 June 1998     

Genomic-assisted characterisation of Pseudomonas sp. strain Pf4, a potential biocontrol agent in hydroponics

In an attempt to select potential biocontrol agents against Pythium spp. and Rhizoctonia spp. root pathogens for use in soilless systems, 12 promising bacteria were selected for further investigations. Sequence analysis of the 16S rRNA gene revealed that three strains belonged to the genus Enterobacter, whereas nine strains belonged to the genus Pseudomonas. In in vitro assays, one strain of Pseudomonas sp., Pf4, closely related to Pseudomonas protegens (formerly Pseudomonas fluorescens), showed noteworthy antagonistic activity against two strains of Pythium aphanidermatum and two strains of Rhizoctonia solani AG 1-IB, with average inhibition of mycelial growth >80%. Strain Pf4 was used for in vivo treatments on lamb’s lettuce against R. solani root rot in small-scale hydroponics. Pf4-treated and untreated plants were daily monitored for symptom development and after two weeks of infection, a significant protective effect of Pf4 against root rot was recorded. The survival and population density of Pf4 on roots were also checked, demonstrating a density above the threshold value of 10⁵?CFU?g^?1 of root required for disease suppression. Known loci for the synthesis of antifungal metabolites, detected using PCR, and draft-genome sequencing of Pf4 demonstrated that Pseudomonas sp. Pf4 has the potential to produce an arsenal of secondary metabolites (plt, phl, ofa and fit-rzx gene clusters) very similar to that of the well-known biocontrol P. protegens strain Pf-5.     

Bacterial production and reconstitution in proteoliposomes of <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> CAT2: a transporter of basic amino acids and organic cations

Key message

The vacuolar SlCAT2 was cloned, over-produced in E. coli and reconstituted in proteoliposomes.Arg, Ornithine and Lys were identified as substrates. Unexpectedly, also the organic cationsTetraethylammonium and Acetylcholine were transported indicating involvement of SlCAT2 insignaling.

Abstract

In land plants several transporters are involved in ion and metabolite flux across membranes of cells or intracellular organelles. The vacuolar amino acid transporter CAT2 from Solanum lycopersicum was investigated in this work. SlCAT2 was cloned from tomato flower cDNA, over-produced in Escherichia coli and purified by Nichel-chelating chromatography. For functional studies, the transporter was reconstituted in proteoliposomes. Competence of SlCAT2 for Arg transport was demonstrated measuring uptake of [³H]Arg in proteoliposomes which was trans-stimulated by internal Arg or ornithine. Uptake of [³H]Ornithine and [³H]Lys was also detected at lower efficiency with respect to [³H]Arg. Transport was activated by the presence of intraliposomal ATP suggesting regulation by the nucleotide. The prototype for organic cations tetraethylammonium (TEA) was also transported by SlCAT2. However, scarce reciprocal inhibition between TEA and Arg was found, while the biguanide metformin was able to strongly inhibit uptake of both substrates. These findings suggest that amino acids and organic cations may interact with the transporter through different functional groups some of which are common for the two types of substrates. Interestingly, reconstituted SlCAT2 showed competence for acetylcholine transport, which was also inhibited by metformin. Kinetics of Arg and Ach transport were performed from which Km values of 0.29 and 0.79 mM were derived, respectively.

     

Interaction of mildronate with the mitochondrial carnitine/acylcarnitine transport protein

The interaction of mildronate [3-(2,2,2-trimethylhydrazine) propionate] with the purified mitochondrial carnitine/acylcarnitine transporter reconstituted in liposomes has been studied. Mildronate, externally added to the proteoliposomes, strongly inhibited the carnitine/carnitine antiport catalyzed by the reconstituted transporter with an IC(50) of 560 muM. A kinetic analysis revealed that the inhibition is completely competitive, that is, mildronate interacts with the substrate-binding site. The half-saturation constant of the transporter for external mildronate (K(i)) is 530 muM. Carnitine/mildronate antiport has been measured as [(3)H]carnitine uptake into proteoliposomes containing internal mildronate or as [(3)H]carnitine efflux from proteoliposomes in the presence of external mildronate, indicating that mildronate is transported by the carnitine/acylcarnitine transporter and that the inhibition observed was due to the transport of mildronate in the place of carnitine. The intraliposomal half-saturation constant for mildronate transport (K(m)) has been determined. Its value, 18 mM, is much higher than the external half-saturation constant (K(i)) in agreement with the asymmetric properties of the transporter. In vivo, the antiport reaction between cytosolic (administered) mildronate and matrix carnitine may cause intramitochondrial carnitine depletion. This effect, together with the inhibition of the physiological transport, will lead to impairment of fatty acid utilization.     

Tetanus, botulinum and snake presynaptic neurotoxins

Tetanus and botulinum neurotoxins, produced by anaerobic bacteria of the genus Clostridium, are the most toxic proteins known and are solely responsible for the pathogenesis of tetanus and botulism. They are metallo-proteases that enter nerve terminals and cleave proteins of the neuroexocytosis apparatus causing a persistent, but reversible, inhibition of neurotransmitter release. Botulinum neurotoxins are used in the therapy of many human syndromes caused by hyperactive nerve terminals. Snake presynaptic PLA2 neurotoxins block nerve terminals by binding to the nerve membrane and catalyzing phospholipid hydrolysis with production of lysophospholipids and fatty acids. These compounds change the membrane conformation causing enhanced fusion of synaptic vesicle via hemifusion intermediate with release of neurotransmitter and, at the same time, inhibition of vesicle fission and recycling. It is possible to envisage clinical applications of the lysophospholipid/fatty acid mixture to inhibit hyperactive superficial nerve terminals.

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The Potential Role of Pharmacogenomic and Genomic in the Adjuvant Treatment of Early Stage Non Small Cell Lung Cancer

Although notable progress has been made in the treatment of non-small-cell lung cancer (NSCLC) in recent years, this disease is still associated with a poor prognosis. Despite early-stage NSCLC is considered a potentially curable disease following complete resection, the majority of patients relapse and eventually die after surgery. Adjuvant chemotherapy prolongs survival, altough the absolute improvement in 5-year overall survival is only approximately 5%.Trying to understand the role of genes which could affect drug activity and response to treatment is a major challenge for establishing an individualised chemotherapy according to the specific genetic profile of each patient. Among genes involved in the DNA repair system, the excision repair cross-complementing 1 (ERCC1) is a useful markers of clinical resistance to platinum-based chemotherapy. In the International Lung Cancer Trial (IALT) adjuvant chemotherapy significantly prolonged survival among patients with ERCC1 negative tumors but not among ERCC1-positive patients. BRCA1 and ribonucleotide reductase M1 (RRM1), two other key enzymes in DNA synthesis and repair, appear to be modulators of drug sensitivity and may provide additional information for customizing adjuvant chemotherapy.Several clinical trials suggest that overexpression of class III β-tubulin is an adverse prognostic factor in cancer since it could be responsible for resistance to anti-tubulin agents. A retrospective analysis of NCIC JBR.10 trial showed that high tubulin III expression is associated with a higher risk of relapse following surgery alone but also with a higher probability of benefit from adjuvant cisplatin plus vinorelbine chemotherapy.Finally, the use of gene expression patterns such as the lung metagene model could provide a potential mechanism to refine the estimation of a patient’s risk of disease recurrence and could affect treatment decision in the management of early stage of NSCLC.In this review we will discuss the potential role of pharmacogenomic approaches to guide the medical treatment of early stage NSCLC.Key Words: NSCLC, adjuvant treatment, molecular markers, ERCC1, RRM1, β-tubulin, EGFR.