Laparoscopic versus open appendectomy for complicated appendicitis in high risk patients.

INTRODUCTION: Laparoscopic appendectomy is widely used for the treatment of complicated appendicitis. Its use in patients with high operative risk is still on debate. The aim of the presented study was to investigate the benefits of laparoscopic appendectomy in patients with high peri- and postoperative risk factors. METHODS: We performed a retrospective analysis of all patients who underwent appendectomy in our center between 2006 and 2013. Patients were classified according to their preoperative risk (classification of the American Society of Anesthesia--ASA score). Only patients with ASA 3 and 4 were included and were divided into two groups--open appendectomy (OA group) and laparoscopic appendectomy (LA group). RESULTS: The operation time was slightly longer in the LA group (p = 0.05), but hospital stay was shorter (p = 0.05). Complications graded according to the Clavien Dindo classification were slightly more frequent in patients after LA, whereas severe complications occurred more frequently in patients after OA (p = 0.01). The postoperative WBC decreased steadily and significantly in patients after OA, whereas the decrease in patients after LA was delayed (p = 0.03). CRP slightly increased after OA and decreased thereafter, whereas it steadily decreased after LA (p = 0.05). CONCLUSION: Laparoscopic appendectomy can be recommended for patients with complicated appendicitis even with higher risk categories.

The genome of the simian and human malaria parasite Plasmodium knowlesi.

Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells.

Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreER(T2), and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreER(T2). The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts.

Toxin-antitoxin based transgene expression in mammalian cells.

Long-term, recombinant gene expression in mammalian cells depends on the nature of the transgene integration site and its inherent properties to modulate transcription (epigenetic effects). Here we describe a method by which high transgene expression is achieved and stabilized in extensively proliferating cultures. The method is based on strict co-expression of the transgene with an antitoxin in cells that express the respective toxin. Since the strength of antitoxin expression correlates with an advantage for cell growth, the cells with strong antitoxin expression are enriched over time in cultures of heterogeneous cells. This principle was applied to CHO cell lines that conditionally express the toxin kid and that are transduced to co-express the antitoxin kis together with different transgenes of interest. Cultivation of pools of transfectants that express the toxin steadily increase their transgene expression within several weeks to reach a maximum that is up to 120-fold over the initial status. In contrast, average transgene expression drops in the absence of toxin expression. Together, we show that cells conditionally expressing kid can be employed to create overexpressing cells by a simple coupling of kis to the transgene of interest, without further manipulation and in absence of selectable drugs.

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration.

The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN gamma-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinase-mediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN gamma-retroviral vectors for clinical trials.

GARP: a key receptor controlling FOXP3 in human regulatory T cells.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4(+)CD25(hi) T (T(reg)) cells. Based on transcriptional profiling of ex vivo activated T(reg) and helper CD4(+)CD25(-) T (T(h)) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human T(reg) cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific T(h) cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other T(reg)-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in T(reg) cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-beta induced T(reg) cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in T(reg) cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

A giant ubiquitin-conjugating enzyme related to IAP apoptosis inhibitors.

Ubiquitin-conjugating enzymes (UBC) catalyze the covalent attachment of ubiquitin to target proteins and are distinguished by the presence of a UBC domain required for catalysis. Previously identified members of this enzyme family are small proteins and function primarily in selective proteolysis pathways. Here we describe BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), a giant (528-kD) ubiquitin-conjugating enzyme from mice. BRUCE is membrane associated and localizes to the Golgi compartment and the vesicular system. Remarkably, in addition to being an active ubiquitin-conjugating enzyme, BRUCE bears a baculovirus inhibitor of apoptosis repeat (BIR) motif, which to this date has been exclusively found in apoptosis inhibitors of the IAP-related protein family. The BIR motifs of IAP proteins are indispensable for their anti-cell death activity and are thought to function through protein-protein interaction. This suggests that BRUCE may combine properties of IAP-like proteins and ubiquitin-conjugating enzymes and indicates that the family of IAP-like proteins is structurally and functionally more diverse than previously expected.

Contiguity to males in utero affects avoidance responding in adult female mice.

Female mice that had been situated in utero between two female fetuses displayed higher levels of active avoidance responding in adult life than females that had been located between two male fetuses and males for whom uterine position was without effect. Uterine position, therefore, influences acquired as well as species-typical behaviors.

Suprachiasmatic nuclear lesions do not abolish food-shifted circadian adrenal and temperature rhythmicity.

Daytime restriction of food and water availability in nocturnal animals phase shifts the circadian periodicity of plasma corticosteroid concentrations and body temperature. These shifted rhythms persist in animals with lesions of the suprachiasmatic nuclei who are arrhythmic under normal conditions. These findings suggest the existence of an additional "clock" that may be involved in the generation of the rhythm.

[CT coronary angiogram: state of the art in 2009]. / Coronarographie par CT-scan en 2009.

Thanks to 64 multi-detector CT-scan it is nowadays possible to visualise non invasively the coronary arteries: the recently published series have shown excellent sensitivity and specificity in the detection of coronary artery disease. Actually, the principal interest of the technique is the excellent negative predictive value which is very useful to rule out a significant coronary artery stenosis. For the time being, the angio CT is recommended for symptomatic patients with low to intermediate probability of coronary artery disease with inconclusive functional test. Despite some technical ameliorations, the irradiation doses delivered by multi-detector CT are significant and should restrict its indications specifically in women and young patients, in whom a late radio-induced cancer may be a concern.

A soft matter computer for soft robots.

Despite the growing interest in soft robotics, little attention has been paid to the development of soft matter computational mechanisms. Embedding computation directly into soft materials is not only necessary for the next generation of fully soft robots but also for smart materials to move beyond stimulus-response relationships and toward the intelligent behaviors seen in biological systems. This article describes soft matter computers (SMCs), low-cost, and easily fabricated computational mechanisms for soft robots. The building block of an SMC is a conductive fluid receptor (CFR), which maps a fluidic input signal to an electrical output signal via electrodes embedded into a soft tube. SMCs could perform both analog and digital computation. The potential of SMCs is demonstrated by integrating them into three soft robots: (i) a Softworm robot was controlled by an SMC that generated the control signals necessary for three distinct gaits; (ii) a soft gripper was given a set of reflexes that could be programmed by adjusting the parameters of the CFR; and (iii) a two-degree of freedom bending actuator was switched between three distinct behaviors by varying only one input parameter. SMCs are a low-cost way to integrate computation directly into soft materials and an important step toward entirely soft autonomous robots.

Translation of NRF mRNA is mediated by highly efficient internal ribosome entry.

The ubiquitous transcription factor NRF (NF-kappaB repressing factor) is a constitutive transcriptional silencer of the multifunctional cytokine interferon-beta. NRF mRNA contains a long 5' untranslated region (5'UTR) predicted to fold into a strong secondary structure. The presence of stable hairpins is known to be incompatible with efficient translation by ribosomal scanning. Using dicistronic reporter gene constructs, we show that the NRF 5'UTR acts as an internal ribosome entry site (IRES) which directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES in various cell lines is at least 30-fold higher than that of picornaviral IRESs. The NRF 5'UTR also functions as a translational enhancer in the context of monocistronic mRNAs. Our results indicate that the NRF 5'UTR contains a highly potent IRES, which may allow for an alternate mode of translation under physiological conditions in which cap-dependent translation is inhibited.

The actin-related protein Act3p of Saccharomyces cerevisiae is located in the nucleus.

Actin-related proteins, a group of protein families that exhibit about 50% sequence identity among each other and to conventional actin, have been found in a variety of eukaryotic organisms. In the budding yeast Saccharomyces cerevisiae, genes for one conventional actin (ACT1) and for three actin-related proteins (ACT2, ACT3, and ACT5) are known. ACT3, which we recently discovered, is an essential gene coding for a polypeptide of 489 amino acids (Act3p), with a calculated molecular mass of 54.8 kDa. Besides its homology to conventional actin, Act3p possesses a domain exhibiting weak similarity to the chromosomal protein HMG-14 as well as a potential nuclear localization signal. An antiserum prepared against a specific segment of the ACT3 gene product recognizes a polypeptide band of approximately 55 kDa in yeast extract. Indirect immunofluorescence experiments with this antiserum revealed that Act3p is located in the nucleus. Nuclear staining was observed in all cells regardless of the stage of the cell cycle. Independently, immunoblotting experiments with subcellular fractions showed that Act3p is indeed highly enriched in the nuclear fraction. We suggest that Act3p is an essential constituent of yeast chromatin.

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange.

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.

Recognition of 5'-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2.

TAR, a 59 nt 5'-terminal hairpin in human immuno-deficiency virus 1 (HIV-1) mRNA, binds viral Tat and several cellular proteins. We report that eukaryotic translation initiation factor 2 (eIF2) recognizes TAR. TAR and the AUG initiation codon domain, located well downstream from TAR, both contribute to the affinity of HIV-1 mRNA for eIF2. The affinity of TAR for eIF2 was insensitive to lower stem mutations that modify sequence and structure or to sequence changes throughout the remainder that leave the TAR secondary structure intact. Hence, eIF2 recognizes structure rather than sequence in TAR. The affinity for eIF2 was severely reduced by a 3 nt change that converts the single A bulge into a 7 nt internal loop. T1 footprinting showed that eIF2 protects nucleotides in the loop as well as in the strand opposite the A bulge. Thus, eIF2 recognizes the TAR loop and lower part of the sub-apical stem. Though not contiguous, these regions are brought into proximity in TAR by a bend in the helical structure induced by the UCU bulge; binding of eIF2 opens up the bulge context and apical stem. The ability to bind eIF2 suggests a function for TAR in HIV-1 mRNA translation. Indeed, the 3 nt change that reduces the affinity of TAR for eIF2 impairs the ability of reporter mRNA to compete in translation. Interaction of TAR with eIF2 thus allows HIV-1 mRNA to compete more effectively during protein synthesis.

Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs.

In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.

Growth suppression of the hepatocellular carcinoma cell line Hepa1-6 by an activatable interferon regulatory factor-1 in mice.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis and few therapeutic options. The aim of the study was to evaluate the potential of IFN regulatory factor-1 (IRF-1) for cytokine gene therapy of HCC using an IRF-1/human estrogen receptor fusion protein (IRF-1hER), which is reversibly activatable by beta-estradiol (E2). IRF-1hER stably expressing murine Hepa1-6 HCC cells (HepaIRF-1hER) were characterized by lowMHC 1, highCD54, and lack of MHC II, CD80, and CD86 expression. Activation of HepaIRF-1hER cells induced a highMHC I, lowMHC II, and highCD54 phenotype. Furthermore, they were characterized by IFN-beta secretion, decreased anchorage-independent growth in a soft agar assay, and diminished cell growth. Tumor growth in E2-treated syngeneic C57L/J mice, but not in E2-untreated mice, was suppressed. These E2-treated mice were protected against rechallenge with HepaIRF-1hER and wild-type Hepa1-6 tumors even in the absence of E2, suggesting induction of tumor specific immunity. In fact, significant CTL activity against Hepa1-6 tumors and the endogenously expressed HCC-specific self antigen alpha-fetoprotein was observed. Antitumoral effects, however, were only partially dependent on both CD4+ and CD8+ T cells. IRF-1 treatment of mice bearing HepaIRF-1hER tumors resulted in growth arrest of tumors, and a significant survival benefit was observed in comparison to E2-untreated mice. In conclusion, our data demonstrate that IRF-1 suppresses HCC growth through both a direct antitumor growth effect and enhanced immune cell recognition of the tumor and is a promising candidate for gene therapy of HCC.