Intramembrane cleavage of microneme proteins at the surface of the apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites actively secrete proteins at their apical pole as part of the host cell invasion process. The adhesive micronemal proteins are involved in the recognition of host cell receptors. Redistribution of these receptor-ligand complexes toward the posterior pole of the parasites is powered by the actomyosin system of the parasite and is presumed to drive parasite gliding motility and host cell penetration. The microneme protein protease termed MPP1 is responsible for the removal of the C-terminal domain of TgMIC2 and for shedding of the protein during invasion. In this study, we used site-specific mutagenesis to determine the amino acids essential for this cleavage to occur. Mapping of the cleavage site on TgMIC6 established that this processing occurs within the membrane-spanning domain, at a site that is conserved throughout all apicomplexan microneme proteins. The fusion of the surface antigen SAG1 with these transmembrane domains excluded any significant role for the ectodomain in the cleavage site recognition and provided evidence that MPP1 is constitutively active at the surface of the parasites, ready to sustain invasion at any time.     

Carnitine: an osmolyte that plays a metabolic role

Carnitine, gamma-trimethyl-beta-hydroxybutyrobetaine, is a small molecule widely present in all cells from prokaryotic to eukaryotic ones. It is the sole source of carbon and nitrogen in some bacteria; it serves as osmoprotectant in others. It is a carrier of acyl moieties, and exclusively of long-chain fatty acids for mitochondrial beta-oxidation in mammals. The conspicuously similar composition of the intracellular milieu among widely different species in relation to organic osmolyte systems involves the methylamine family to which carnitine belongs. This prompted us to examine the osmolytic properties of carnitine in an attempt to clarify the metabolic functions carnitine has acquired during evolution. An understanding of the metabolic functions of this organic compatible solute impinge on research involving this compound.     

Hot-spot consensus of fluoroquinolone-mediated DNA cleavage by Gram-negative and Gram-positive type II DNA topoisomerases

Bacterial DNA gyrase and topoisomerase IV are selective targets of fluoroquinolones. Topoisomerase IV versus gyrase and Gram-positive versus Gram-negative behavior was studied based on the different recognition of DNA sequences by topoisomerase–quinolone complexes. A careful statistical analysis of preferred bases was performed on a large number (>400) of cleavage sites. We found discrete preferred sequences that were similar when using different enzymes (i.e. gyrase and topoisomerase IV) from the same bacterial source, but in part diverse when employing enzymes from different origins (i.e. Escherichia coli and Streptococcus pneumoniae). Subsequent analysis on the wild-type and mutated consensus sequences showed that: (i) Gn/Cn-rich sequences at and around the cleavage site are hot spots for quinolone-mediated strand breaks, especially for E. coli topoisomerases: we elucidated positions required for quinolone and enzyme recognition; (ii) for S. pneumoniae enzymes only, A and T at positions −2 and +6 are discriminating cleavage determinants; (iii) symmetry of the target sequence is a key trait to promote cleavage and (iv) the consensus sequence adopts a heteronomous A/B conformation, which may trigger DNA processing by the enzyme–drug complex.     

Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells

Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process.     

The perception of Obstructive Sleep Apnoea/Hypopnoea Syndrome (OSAHS) among Italian general practitioners

Background

Obstructive Sleep Apnoea/Hypopnoea Syndrome (OSAHS) is a common disorder in the general population but often underestimated and underdiagnosed.

Methods

This questionnaire-based study evaluated the overall level of knowledge about OSAHS among Italian General Practitioners (GPs), who are frequently involved in the management of this complex disease. This represents an interesting aspect, because GPs intercept many of the patients with OSAHS, in which C-PAP could be potentially indicated. Randomly-selected GPs were provided with questionnaires, which were then returned anonymously.

Results

80 questionnaires have been validated; the participants in the sample examined were represented by 43 females and 37 males; the average age of participants was 51 years. The general knowledge on OSAHS is overall satisfactory among GPs; it is recognized by most of the GPs interviewed as pathology in constant increase, and associated with predisposing factors such as obesity. High blood pressure is perceived as an independent cardiovascular risk factor in patients with OSAHS, in line with the majority of international studies. The C-PAP has been identified as the care gold standard in patients with OSAHS, despite the lack of patient compliance in relation to this procedure, while polysomnography was found to be the main instrumental procedure used in the diagnostic workup of OSAS. The pulmonologist and a multidisciplinary team have been identified as the specialist figures of reference to which to direct the patient through the diagnostic workup. Respiratory therapists and nurses represent the role of educator in the proper management of the C-PAP in the opinion of 62% of respondents, while only 34% think that this role should be played by the GPs and/or other specialists.

Conclusions

In conclusion, this survey about the perception of OSAHS among GPs in Italy highlighted a satisfactory overall knowledge of OSAHS and only few weak points.     

Tri-, tetra- and heptacyclic perylene analogues as new potential antineoplastic agents based on DNA telomerase inhibition

A recent approach in anticancer chemotherapy envisages telomerase as a potentially useful target. An attractive strategy deals with the development of compounds able to stabilize telomeric DNA in the G-quadruplex folded structure and, among them, a prominent position is found in the perylenes. With the aim to further investigate the role of drug structure, in view of possible pharmaceutical applications, we synthesized a series of compounds related to PIPER, a well-known perylene-based telomerase inhibitor. We modified the number of condensed aromatic rings and introduced different side chains to modulate drug protonation state and extent of self-aggregation. Effective telomerase inhibition was induced by heptacyclic analogues only, some showing a remarkably wide selectivity index with reference to inhibition of Taq polymerase. G-quadruplex stabilization was monitored by circular dichroism and melting experiments. Cell cytotoxicity measurements indicated a poor short-term cell killing ability for the best G-quartet binders. Besides the presence of a planar seven-condensed ring system, the introduction of a cyclic amine in the side chains critically affects the selectivity window.     

Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation,Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukaemia

IGF1R is emerging as an important gene in the pathogenesis of many solid and haematological cancers and its over-expression has been reported as frequently associated with aggressive disease and chemotherapy resistance. In this study we performed an investigation of the role of IGF1R expression in a large and representative prospective series of 217 chronic lymphocytic leukaemia (CLL) patients enrolled in the multicentre O-CLL1 protocol (clinicaltrial.gov #NCT00917540). High IGF1R gene expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high CD38 expression (p<0.0001), trisomy 12 (p<0.0001), and del(11)(q23) (p=0.014). Interestingly, higher IGF1R expression (p=0.002) characterized patients with NOTCH1 mutation (c.7541_7542delCT), identified in 15.5% of cases of our series by next generation sequencing and ARMS-PCR. Furthermore, IGF1R expression has been proven as an independent prognostic factor associated with time to first treatment in our CLL prospective cohort. These data suggest that IGF1R may play an important role in CLL biology, in particular in aggressive CLL clones characterized by IGHV-UM, trisomy 12 and NOTCH1 mutation.     

Effects of assessing the productivity of faculty in academic medical centres: a systematic review

Background:

Many academic medical centres have introduced strategies to assess the productivity of faculty as part of compensation schemes. We conducted a systematic review of the effects of such strategies on faculty productivity.

Methods:

We searched the MEDLINE, Healthstar, Embase and PsycInfo databases from their date of inception up to October 2011. We included studies that assessed academic productivity in clinical, research, teaching and administrative activities, as well as compensation, promotion processes and satisfaction.

Results:

Of 531 full-text articles assessed for eligibility, we included 9 articles reporting on eight studies. The introduction of strategies for assessing academic productivity as part of compensation schemes resulted in increases in clinical productivity (in six of six studies) in terms of clinical revenue, the work component of relative-value units (these units are nonmonetary standard units of measure used to indicate the value of services provided), patient satisfaction and other departmentally used standards. Increases in research productivity were noted (in five of six studies) in terms of funding and publications. There was no change in teaching productivity (in two of five studies) in terms of educational output. Such strategies also resulted in increases in compensation at both individual and group levels (in three studies), with two studies reporting a change in distribution of compensation in favour of junior faculty. None of the studies assessed effects on administrative productivity or promotion processes. The overall quality of evidence was low.

Interpretation:

Strategies introduced to assess productivity as part of a compensation scheme appeared to improve productivity in research activities and possibly improved clinical productivity, but they had no effect in the area of teaching. Compensation increased at both group and individual levels, particularly among junior faculty. Higher quality evidence about the benefits and harms of such assessment strategies is needed.Academic productivity can be defined as a measurable output of a faculty member related to clinical, research, education or administrative activities. Achieving the best possible academic productivity is essential for academic medical centres to maintain or nurture recognition and good reputation.¹ Furthermore, clinical productivity is essential for the survival of academic departments given the economic realities in medicine.²Strategies for productivity assessment help in identifying highly productive faculty, determining areas for faculty and departmental improvement,³ and implementing processes for promotion and tenure.⁴ When coupled with reward schemes, these strategies may improve productivity and compensation at both individual and departmental levels. In the long-term, they may enhance the ability to recruit and retain high-quality faculty and achieve the academic mission of the department. However, these strategies may have some unintended effects such as using time dedicated to education to do more clinical work.³ In addition, they may be challenging to implement.³We conducted a systematic review of the effects of strategies introduced in academic medical centres to assess faculty productivity, compensation, promotion processes and satisfaction.     

Relevance of stereotyped B-cell receptors in the context of the molecular, cytogenetic and clinical features of chronic lymphocytic leukemia

Highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), are expressed in a recurrent fraction of patients affected by chronic lymphocytic leukemia (CLL). We investigated the IGHV status of 1131 productive IG rearrangements from a panel of 1126 CLL patients from a multicenter Italian study group, and correlated the presence and class of HCDR3 stereotyped subsets with the major cytogenetic alterations evaluated by FISH, molecular prognostic factors, and the time to first treatment (TTFT) of patients with early stage disease (Binet A). Stereotyped HCDR3 sequences were found in 357 cases (31.7%), 231 of which (64.7%) were unmutated. In addition to the previously described subsets, 31 new putative stereotypes subsets were identified. Significant associations between different stereotyped HCDR3 sequences and molecular prognostic factors, such as CD38 and ZAP-70 expression, IGHV mutational status and genomic abnormalities were found. In particular, deletion of 17p13 was significantly represented in stereotype subset #1. Notably, subset #1 was significantly correlated with a substantially reduced TTFT compared to other CLL groups showing unmutated IGHV, ZAP-70 or CD38 positivity and unfavorable cytogenetic lesions including del(17)(p13). Moreover, subset #2 was strongly associated with deletion of 13q14, subsets #8 and #10 with trisomy 12, whereas subset #4 was characterized by the prevalent absence of the common cytogenetic abnormalities. Our data from a large and representative panel of CLL patients indicate that particular stereotyped HCDR3 sequences are associated with specific cytogenetic lesions and a distinct clinical outcome.