Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Evidences of gentamicin resistance amplification in Klebsiella pneumoniae isolated from faeces of hospitalized newborns.

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenotypical gentamicin resistance amplification (frequencies of 10(-3) to 10(-5), compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromosomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.

Trans-kingdom transposition of the Drosophila element mariner within the protozoan Leishmania.

Transposable elements of the mariner/Tc1 family are postulated to have spread by horizontal transfer and be relatively independent of host-specific factors. This was tested by introducing the Drosophila mauritiana element mariner into the human parasite Leishmania major, a trypanosomatid protozoan belonging to one of the most ancient eukaryotic lineages. Transposition in Leishmania was efficient, occurring in more than 20 percent of random transfectants, and proceeded by the same mechanism as in Drosophila. Insertional inactivation of a specific gene was obtained, and a modified mariner element was used to select for gene fusions, establishing mariner as a powerful genetic tool for Leishmania and other organisms. These experiments demonstrate the evolutionary range of mariner transposition in vivo and underscore the ability of this ubiquitous DNA to parasitize the eukaryotic genome.

Selection against the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus as a probe of genetic alterations in Leishmania major.

The genome of the trypanosomatid protozoan genus Leishmania has been shown to undergo a number of changes relevant to drug resistance and virulence, such as gene amplification, chromosomal rearrangement, and variation in ploidy. Experimental approaches to the study of genomic changes have in some cases been limited by the fact that Leishmania cells are asexual diploids, as are some other trypanosomatids, pathogenic fungi, and cultured mammalian cells. Here we report upon a system which permits the measurement of several types of genomic change occurring at the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus. First, we show that DHFR-TS can function as a positive/negative marker. We used selection against DHFR-TS on a heterozygous line (+/HYG) to generate colonies exhibiting both loss of heterozygosity and structural mutations in DHFR-TS, permitting the first measurement of mutation frequencies in this parasite. Loss of heterozygosity occurred at a frequency ranging from 10(-4) to 10(-6) and was elevated 24-fold by treatment with gamma-irradiation, while the frequency of other events was less than 10(-6) and was increased more than 1,000-fold by nitrosoguanidine treatment. The frequency of loss of heterozygosity relative to other processes such as mutation and gene replacement has important implications for genetic variability in natural Leishmania populations and the generation of both targeted and random mutations. We also developed a protocol for null targeting of diploid cells, in which transfection of a DHFR-TS deletion construct into Leishmania cells followed by negative selection yielded parasites lacking DHFR-TS or foreign sequences. The null-targeting method can be applied to any diploid cell, at any locus for which a negative selection exists. Such marker-free auxotrophic Leishmania cells show potential as an attenuated vaccine, and the methods developed here provide a new approach for manipulating and characterizing the plasticity of the Leishmania genome.

Leishmania amazonensis: multidrug resistance in vinblastine-resistant promastigotes is associated with rhodamine 123 efflux, DNA amplification, and RNA overexpression of a Leishmania mdr1 gene.

A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine. This phenotype was partially reverted by the calcium channel antagonist verapamil. Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux. Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells. An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.

Development of a safe live Leishmania vaccine line by gene replacement.

Vaccination with live Leishmania major has been shown to yield effective immunization in humans; however, this has been discontinued because of problems associated with virulence of the available vaccine lines. To circumvent this, we tested the ability of a dhfr-ts- null mutant of L. major, obtained by gene targeting, to infect and then to vaccinate mice against challenge with virulent L. major. Survival and replication of dhfr-ts- in macrophages in vitro were dependent upon thymidine, with parasites differentiating into amastigotes prior to destruction. dhfr-ts- parasites persisted in BALB/c mice for up to 2 months, declining with a half-life of 2-3 days. Nonetheless, dhfr-ts- was incapable of causing disease in both susceptible and immunodeficient (nu/nu) BALB/c mice. Animal infectivity could be partially restored by thymidine supplementation. When inoculated by the i.v., s.c., or i.m. routes into mice, dhfr-ts- could elicit substantial resistance to a subsequent challenge with virulent L. major. Thus, Leishmania bearing auxotrophic gene knockouts can be safe and induce protective immunity. Potentially, dhfr-ts- could be used as a platform for delivery of immunogens relevant to other diseases.

Identification of GTPase genes in the protozoa parasites Trypanosoma cruzi and Leishmania amazonensis.

A PCR based assay was designed in order to amplify putative ras gene sequences of the GTPase superfamily eventually present in Leishmania amazonensis and Trypanosoma cruzi. A set of primers corresponding to the conserved motifs G1 and G3 of the GTP binding proteins was synthesized. Sequencing of six PCR products (three from Leishmania and three from Trypanosoma) identified, however, two other different GTPases. The 270 bp L. amazonensis clone, pLef-11, shared an amino acid identity of around 80% with an eukaryotic elongation factor 1a of protein synthesis. On the other hand, the 168 bp T. cruzi clone, pTCr1, demonstrated over 60% amino acid identity to ras-related proteins of the rab-YPT-SEC4 family involved in control of vesicular traffic. To our knowledge, this is the first report of GTP binding protein genes in trypanosomatids.