Expansion microscopy for the analysis of centrioles and cilia.

Centrioles are vital cellular structures that organise centrosomes and cilia. Due to their subresolutional size, centriole ultrastructural features have been traditionally analysed by electron microscopy. Here we present an adaptation of magnified analysis of the proteome expansion microscopy method, to be used for a robust analysis of centriole number, duplication status, length, structural abnormalities and ciliation by conventional optical microscopes. The method allows the analysis of centriole's structural features from large populations of adherent and nonadherent cells and multiciliated cultures. We validate the method using EM and superresolution microscopy and show that it can be used as an affordable and reliable alternative to electron microscopy in the analysis of centrioles and cilia in various cell cultures. LAY DESCRIPTION: Centrioles are microtubule-based structures organised as ninefold symmetrical cylinders which are, in human cells, ∼500 nm long and ∼230 nm wide. Centrioles assemble dozens of proteins around them forming centrosomes, which nucleate microtubules and organise spindle poles in mitosis. Centrioles, in addition, assemble cilia and flagella, two critically important organelles for signalling and motility. Due to centriole small size, electron microscopy has been a major imaging technique for the analysis of their ultrastructural features. However, being technically demanding, electron microscopy it is not easily available to the researchers and it is rarely used to collect large datasets. Expansion microscopy is an emerging approach in which biological specimens are embedded in a swellable polymer and isotopically expanded several fold. Physical separation of cellular structures allows the analysis of, otherwise unresolvable, structures by conventional optical microscopes. We present an adaptation of expansion microscopy approach, specifically developed for a robust analysis of centrioles and cilia. Our protocol can be used for the analysis of centriole number, duplication status, length, localisation of various centrosomal components and ciliation from large populations of cultured adherent and nonadherent cells and multiciliated cultures. We validate the method against electron microscopy and superresolution microscopy and demonstrate that it can be used as an accessible and reliable alternative to electron microscopy.

The KASH-containing isoform of Nesprin1 giant associates with ciliary rootlets of ependymal cells.

Biallelic nonsense mutations of SYNE1 underlie a variable array of cerebellar and non-cerebellar pathologies of unknown molecular etiology. SYNE1 encodes multiple isoforms of Nesprin1 that associate with the nuclear envelope, with large cerebellar synapses and with ciliary rootlets of photoreceptors. Using two novel mouse models, we determined the expression pattern of Nesprin1 isoforms in the cerebellum whose integrity and functions are invariably affected by SYNE1 mutations. We further show that a giant isoform of Nesprin1 associates with the ciliary rootlets of ependymal cells that line brain ventricles and establish that this giant ciliary isoform of Nesprin1 harbors a KASH domain. Whereas cerebellar phenotypes are not recapitulated in Nes1gSTOP/STOP mice, these mice display a significant increase of ventricular volume. Together, these data fuel novel hypotheses about the molecular pathogenesis of SYNE1 mutations and support that KASH proteins may localize beyond the nuclear envelope in vivo.

Multifractal anisotropic swimming: the optimal foraging behaviour of grouper larvae.

It was hypothesized that the Malabar grouper Ephinephelus malabaricus larvae have developed search patterns adapted to the distribution of their prey to maximise their net energy intake per unit time. Analysis of the swimming behaviour of E. malabaricus larvae in both the presence and absence of Artemia sp. nauplii is presented to test this hypothesis. A method derived from turbulence studies (the moment function of the displacements) was used to characterize the behaviour. The results revealed that larval swimming pattern was multifractal (intermittent and long-range-correlated) and isotropic (i.e. uniform in all directions) in the presence of prey, but multifractal and anisotropic (i.e. more frequent long displacement on the vertical axis) in the absence of prey. It is suggested that the search behaviour observed in the absence of prey is an adaptive response to prey distribution pattern, which is often characterised by multifractality and anisotropy (i.e. larger patches on the horizontal axes). In the presence of prey, E. malabaricus shifted to intensive search behaviour. Other possible contributors to the observed patterns are discussed. It is concluded that multifractality and anisotropy of swimming patterns observed in the experiment are mainly explained in an optimal foraging theory framework.

[What are the predictive factors of the pain during the treatment of kidney stones by extra-corporeal shockwave lithotripsy?]. / Quels sont les facteurs prédictifs de la douleur lors du traitement des calculs rénaux par lithotritie extracorporelle ?

PURPOSE: Identify predictors for selecting patients who requires analgesia during lithotripsy. METHODS: This is a prospective study over a period of 13 months, 100 patients with kidney stones treated by an electromagnetic lithotripter (siemens; lithoskop) were selected. For the study of subjective pain caused by the ESWL at different times of the session, a visual analog scale (VAS) was used at different times (T) of the session (T0 before shots, T1 at 500 shots, T2 at 1500 shots, T end of treatment). A session was considered painless if VAS≤3. To identify predictors, were investigated association between pain and the different characteristics of patients, kidney stones and the shock wave specifications. RESULTS: The analytical study showed that pain was correlated with female gender, anxiety score, skin distance stones, parietal distance and the energy of the shock wave. While age, waist circumference, the circumstance found, the projection of stones on the rib and the number of shots had no impact on the level of pain. CONCLUSION: Our study showed that even with an electromagnetic lithotripter third generation; ESWL is still painful leading to the interruption of the session in 29% of cases. Four major predictors of pain leading to the use of sedo-analgesia early in the session were identified. LEVEL OF EVIDENCE: 3.

[Epidemiology of high risk pregnancy in Sousse, Tunisia]. / Profil épidémiologique des grossesses à risque à Sousse (Tunisie).

This descriptive study assessed the frequency of selected risk factors for high risk pregnancy (HRP) among women in Sousse region. All pregnant women (beyond 28 weeks gestation) giving birth in 4 public maternity hospitals between 15 February 2005 and 15 August 2005 and who had at least 1 risk factorwere enrolled. Data were collected within 24 hours of birth from obstetric and antenatal records and by interview. Of 4660 pregnant women, 1194 (25.6%) had at least 1 risk factor and were considered at-risk pregnancies. Mean age of the at-risk women was 31.3 (SD 5.4) years, 73% were urban residents, 38% had secondary education or higher and 75% were housewives. The mean number of risk factors was 1.5 per woman. The majority of women (59.3%) had 1 risk factor and 30.4% had 2. The mean number of prenatal visits was 4, 68.6% were seen by an obstetrician and 43% used the private sector. Certain factors were inadequately screened: age >35 years, parity >4, previous low birth weight and neonatal death, excessive fundal height and anaemia.

Discovery of dual-target natural antimalarial agents against DHODH and PMT of Plasmodium falciparum: pharmacophore modelling, molecular docking, quantum mechanics, and molecular dynamics simulations.

Malaria is a lethal disease that claims thousands of lives worldwide annually. The objective of this study was to identify new natural compounds that can target two P. falciparum enzymes; P. falciparum Dihydroorotate dehydrogenase (PfDHODH) and P. falciparum phosphoethanolamine methyltransferase (PfPMT). To accomplish this, e-pharmacophore modelling and molecular docking were employed against PfDHODH. Following this, 1201 natural compounds with docking scores of ≤ -7 kcal/mol were docked into the active site of the second enzyme PMT. The top nine compounds were subjected to further investigation using MM-GBSA free binding energy calculations and ADME analysis. The results revealed favourable free binding energy values better than the references, as well as acceptable pharmacokinetic properties. Compounds ZINC000013377887, ZINC000015113777, and ZINC000085595753 were scrutinized to assess their interaction stability with the PfDHODH enzyme, and chemical stability reactivity using molecular dynamics (MD) simulation and density functional theory (DFT) calculations. These findings indicate that the three natural compounds are potential candidates for dual PfDHODH and PfPMT inhibitors for malaria treatment.

Why subchondral bone in osteoarthritis? The importance of the cartilage bone interface in osteoarthritis.

Osteoarthritis is a whole joint disease characterised by the disappearance of the cartilage associated with subchondral bone sclerosis, formation of osteophytes and a mild inflammation of the synovial membrane. Although all these events have been independently studied, functional interactions between these different joint tissues should exist, especially between subchondral bone and cartilage. Moreover, recent studies show that cartilage and subchondral bone act as a single functional unit. This review highlights this novel concept.

Chemical composition and antimicrobial activity of volatile compounds of Tamarix boveana (Tamaricaceae).

The chemical composition of the Tamarix boveana volatile oils obtained from the whole aerial part, flowers, leaves and stems by steam distillation was analysed using gas chromatograph (GC)-flame ionization detectors (FID) and GC-MS. Sixty-two components were identified. Hexadecanoic acid (18.14%), docosane (13.34%), germacrene D (7.68%), fenchyl acetate (7.34%), Benzyl benzoate (4.11%) were found to be the major components in the whole aerial parts. This composition differed according to the tested part: 2.4 Nonadienal was the main compound in the flowers (12.13%) while germacrene D was the major component in leaves (31.43%) and hexadecanoic acid in the stems (13.94%). To evaluate in vitro antimicrobial activity, all volatile oils were tested against six Gram-positive and Gram-negative bacteria and four fungi. The T. boveana volatile oils exhibited an interesting antibacterial activity against all strains tested except Pseudomonas aeruginosa but no antifungal activity was detected.

Molecular characterization of Echinococcus granulosus in Tunisia and Mauritania by mitochondrial rrnS gene sequencing.

Cystic hydatid disease is a zoonotic parasitic disease caused by the cestode Echinococcus granulosus and represents a major public health problem in many countries around the world, including North Africa. E. granulosus exists as a series of genetic variants or strains which differ in a wide variety of criteria that impact on the epidemiology, pathology and control of cystic hydatid disease. Nucleotide sequencing of the mitochondrial rrnS gene was here used to characterize 38 E. granulosus isolates collected from different regions and hosts in Tunisia and Mauritania. The results obtained reveal a significant genetic differentiation between E. granulosus hydatid cysts identified as belonging to the G1 genotype and to the G6/G7 cluster using the rrnS gene as marker, and indicate the circulation of the common sheep strain (G1) in all host species from Tunisia and the camel/pig strain cluster (G6/G7) in camel from Mauritania. Other investigations, using this method, are necessary for further genetic analysis of a wider range of isolates from different host species in order to more fully understand the genetic structure of E. granulosus populations and their transmission dynamics in this and neighbouring African countries.

First Report of Gummy Stem Blight Caused by Didymella bryoniae on Grafted Watermelon in Tunisia.

During the summer of 2006, severe losses were observed in grafted watermelons in the Testour Region in northern Tunisia. Disease symptoms included stem cankers and necrosis and rot of the grafting area that extended a few centimeters along watermelon vines with the production of a brown gummy exudation. Lesions were not observed on leaves or nongrafted plants. Affected plants wilted and eventually died. The presence of small pseudothecia as black specks was observed embedded in the cankers. Isolations from the stems and crown of symptomatic plants onto potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate consistently yielded cultures of a fungal agent. These isolates were transferred to PDA and V8 juice agar and incubated at 23°C for 1 month with a 12-h photoperiod. On PDA, they produced numerous pycnidia with hyaline, cylindrical, one-septate conidia, with mean dimensions of 6.7 × 2.5 µm. On V8 juice agar, they produced sparse ostiolate pseudothecia with bitunicate asci and hyaline, oval, one-septate ascospores, with mean dimensions of 13.7 × 5.1 µm. On the basis of these characters, the isolates were identified as Didymella bryoniae (anamorph Phoma cucurbitacearum) (1,2). To further confirm this identification, the complete internal transcribed spacer regions 1 and 2, including the 5.8S ribosomal DNA, of isolates Di-3 and Di-4 were sequenced (GenBank Accession Nos. EF107641 and EF 107642). These sequences were identical to sequences in GenBank from isolates of D. bryoniae (Accession Nos. AF297228 and AF495850). Pathogenicity tests were conducted on watermelon seedlings cv. Giza and Cucurbita hybrid rootstock seedlings cv. Strong Toza using two isolates, Di-3 and Di-4. Seedlings were inoculated at the two- to three-leaf stage. A 5-mm diameter agar disc, cut from the margin of an 8-day-old culture growing on PDA, was inserted in a basal stem wound made with a sterile scalpel at 2 cm above ground level and sealed with Parafilm. Controls were inoculated with sterile PDA discs. There were 10 replicates for each isolate and host with an equal number of uninoculated plants. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 10 to 15 days after inoculation, symptoms developed as water-soaked lesions followed by necrosis and finally wilting. The fungus was reisolated from the stems of all inoculated plants, completing Koch's postulates. To our knowledge, this is the first report of D. bryoniae in Tunisia. References: (1) A. P. Keinath et al. Phytopathology 85:364, 1995. (2) E. Punithalingam and P. Holliday. No. 332 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1972.

[Physiopathology of osteoarthritis. From normal cartilage to osteoarthritic cartilage: risk factors and inflammatory mechanisms]. / Physiopathologie de l'arthrose. Du cartilage normal au cartilage arthrosique: facteurs de prédisposition et mécanismes inflammatoires.

INTRODUCTION: osteoarthritis is the most prevalent form of arthritis in the world. With the progressive ageing of the population, it becomes a major problem of public health. Osteoarthritis is a degenerative affection characterized by many disorders leading to a structural and functional defect of one or several joints. CURRENT KNOWLEDGE AND KEY POINTS: In this review, we focus on the main inflammatory mechanisms occurring in cartilage during primary osteoarthritis. We also describe some well established risk factors involved in the pathogenesis of this disease such as age, overload and genetic factors. Indeed, osteoarthritis is the result of an imbalance between the processes of degradation and the attempts of repair by the chondrocyte which is the exclusive cell type in cartilage. Degradation is induced by several chemical substances such as proteolytic enzymes (metalloproteinases) and pro-inflammatory cytokines especially interleukin 1beta. To face these events, the chondrocyte starts attempts of repair by secreting growth factors (Transforming Growth Factor and Insulin Growth Factor) or anti-inflammatory cytokines (interleukins 4 and 10) unsuccessfully. All these events will lead to the structural modifications observed in the osteoarthritic cartilage. PROSPECTS: A better comprehension of the physiopathology of osteoarthritis will allow an improvement of therapeutic strategies of this common and invalidating disease.

[Evaluation of quality of life (QOL) of Tunisians patients with COPD]. / Évaluation de la qualité de vie (QDV) des patients tunisiens atteints de bronchopneumopathie chronique obstructive (BPCO).

AIM: To test the Arabic version of the St. George's Hospital Respiratory Questionnaire (SGRQ) in stable Tunisian COPD patients. HYPOTHESES: A correlation coefficient between the post-bronchodilator FEV1 and the "Total" score of SGRQ higher than "-0.40", and QOL scores lower in COPD with "mild to moderate airway obstruction" than in those with "severe to very severe airway obstruction" will be in favor of a possible application of the Arabic version of the SGRQ in Tunisians COPD patients. METHODS: This is a prospective cross-sectional study including 50 clinically stable COPD patients. The Arabic version of the SGRQ was used. Four scores were calculated for the "Symptoms", "Activities", "Impacts" and "Total" components. Patients were divided into two groups according to the severity of their airway obstruction: "mild to moderate, n=30" and "severe to very severe, n=20". The correlation between the QOL "Total" score and postBD FEV1 was evaluated. RESULTS: The mean±SD of age, cigarette smoking and post-bronchodilator FEV1 were, respectively, 60±10, 61±36 pack-years and 55±20 %. The correlation between the post-bronchodilator FEV1 and QOL "Total" score was significant at "-0.65". "Symptoms", "Activities", "Impacts" and "Total" scores were significantly less altered in patients with "mild to moderate airway obstruction" than among those with "severe to very severe airway obstruction" (respectively, 52±24 vs. 77±17, 66±28 vs. 87±18, 48±25 vs. 70±23 and 54±22 vs. 76±17). CONCLUSION: The application of the Arabic version of the SGRQ in stable COPD patients gives reliable results.

Chemical composition and in vitro antifungal and antioxidant activity of the essential oil and methanolic extract of Teucrium sauvagei Le Houerou.

The chemical composition and the in vitro antifungal and antioxidant activity of the essential oil and the methanolic leaf extracts of Teucrium sauvagei Le Houerou, an endemic medicinal plant growing in Tunisia, have been studied. More than 35 constituents having an abundance >or=0.2% were identified in the oil. beta-Eudesmol, T-cadinol, alpha-thujene, gamma-cadinene, and sabinene were the prevalent constituents. Results of the antifungal activity tests indicated that the methanolic extract inhibited the in vitro growth of seven dermatophytes, whereas the essential oil showed average inhibition against only three dermatophytes. In vitro antioxidant properties of the essential oil and the methanolic extract were determined by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)) assays and compared to those of the synthetic antioxidant Trolox. Due to their antifungal and antioxidant properties, the essential oil and the methanolic extract of T. sauvagei may be of use as natural preservative ingredients in food and/or pharmaceutical industries.

Involvement of the interdomain hydrophobic linker and the C-terminal helices in self-association of the molecular chaperone HSC70.

HSP70 from bacteria to man are known to self-associate to form multiple species suggesting that self-association is related to function. In order to determine the structural basis of HSP70 oligomerization, deletion mutants in the C-terminal domain of HSC70, a constitutive member of the HSP70 family, have been constructed and analyzed for their self-association properties by gel electrophoresis, size-exclusion chromatography and analytical ultracentrifugation. The results of this investigation indicate that, whereas deletion of the GGMP rich C-terminal extremity of HSC70, containing EEVD motif stabilizes the oligomeric species, deletions of either the aD-aE C-terminal helices or the inter-domain hydrophobic linker contribute to the stabilization of the monomeric form. Thus, two non-contiguous regions, located at both ends of the C-terminal domain of the protein, appear to form the contact interface in the oligomers and may interact in a dynamic fashion leading to the formation of several coexisting species.

Bio-insecticidal activities of halophytic plant extracts against Tribolium confusum (Coleoptera, Tenebrionidae).

Salt marsh plants were tested for their insecticidal activities against larvae of the confused flour beetle Tribolium confusum (Tenebrionidae). 16 aerial part extracts were obtained using organic solvents of increasing polarity and tested for their anti-feedant and toxicity effects. Responses varied with plant material and extract type. Ethyl acetate extracts of F. laevis, S. echioides and. T. boveana and petroleum ether extract of F. laevis presented, anti-feedant property. However, S. fructicosa seemed to be attractive to the tested flour beetle. Mortalities of 97, 87, 97 and 80% were observed by using respectively ethyl acetate extracts of F. laevis, S. echioides and T. boveana and petroleum ether extract of F. laevis, when applied at a dose of 1%, mixed with the insect diet. This preliminary study showed that F. laevis, S. echioides and T. boveana presented potential bio-insecticidal activity with ethyl acetate extracts, similar result was found with petroleum ether extract of F. laevis. More complementary studies are needed for the use of these extracts to control T. confusum.