Semi-continuous scale-down models for clone and operating parameter screening in perfusion bioreactors

Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCD_max (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPR_min (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.     

Chemoattractant action and molecular signaling pathways of Kit ligand on mouse primordial germ cells

Using a Transwell chamber as migration assay for mouse primordial germ cells (PGCs), we show here that these cells posses directional migration in the absence of somatic cell and defined matrix support and in response to a Kit ligand (KL) gradient or medium conditioned by Aorta/Gonad/Mesonephros and gonadal ridges. Other putative PGC chemoattractants such as SDF1 and TGFbeta did not exert any attractive action on PGCs. The chemoattractant activity of KL and conditioned medium was also evidenced by their ability to stimulate actin reorganization in PGCs. In the aim to identify downstream signaling pathways governing KL chemoattraction on PGCs, we demonstrated that in such cells KL rapidly (5 min) increased autophosphorylation of its receptor c-Kit and caused phosphorylation of the serine-threonine kinase AKT through the action of PI3K. 740Y-P peptide, a direct activator of PI3 kinase, stimulated PGC migration at levels similar to those elicited by KL. LY294002 (a specific inhibitor of PI3K) abolished KL-dependent PGC migration or the chemoattractant activity of the conditioned medium and inhibited AKT phosphorylation; Src kinase inhibitors PP2 and SU6656, caused significant reduction of the KL-dependent PGC migration and AKT phosphorylation, while U0126, a selective inhibitor of the MEK/ERK protein kinase cascade, reduced PGC migration and AKT phosphorylation at lesser extent. SU6656 completely abolished the chemoattractant activity of the conditioned medium. Finally, SB202190 (a p38 inhibitor) and rapamycin (mTOR inhibitor) did not affect PGC migration. In addition, to demonstrate that somatic cells are not essential for PGC motility and directional migration, we evidenced a novel role for KL as PGC chemoattractant and for PI3K/AKT and Src kinase, as players involved in the activation of the PGC migratory machinery and likely important for their directional movement towards the gonadal ridges.     

A dense single-nucleotide polymorphism-based genetic linkage map of grapevine (Vitis vinifera L.) anchoring Pinot Noir bacterial artificial chromosome contigs

The construction of a dense genetic map for Vitis vinifera and its anchoring to a BAC-based physical map is described: it includes 994 loci mapped onto 19 linkage groups, corresponding to the basic chromosome number of Vitis. Spanning 1245 cM with an average distance of 1.3 cM between adjacent markers, the map was generated from the segregation of 483 single-nucleotide polymorphism (SNP)-based genetic markers, 132 simple sequence repeats (SSRs), and 379 AFLP markers in a mapping population of 94 F(1) individuals derived from a V. vinifera cross of the cultivars Syrah and Pinot Noir. Of these markers, 623 were anchored to 367 contigs that are included in a physical map produced from the same clone of Pinot Noir and covering 352 Mbp. On the basis of contigs containing two or more genetically mapped markers, region-dependent estimations of physical and recombinational distances are presented. The markers used in this study include 118 SSRs common to an integrated map derived from five segregating populations of V. vinifera. The positions of these SSR markers in the two maps are conserved across all Vitis linkage groups. The addition of SNP-based markers introduces polymorphisms that are easy to database, are useful for evolutionary studies, and significantly increase the density of the map. The map provides the most comprehensive view of the Vitis genome reported to date and will be relevant for future studies on structural and functional genomics and genetic improvement.     

The effect of obesity management on body image in patients seeking treatment at medical centers

Objective: Body image dissatisfaction is common in treatment‐seeking patients with obesity. We aimed to investigate the effects of obesity management on body image in patients with obesity attending Italian medical centers for weight loss programs. Research Methods and Procedures: A total of 473 obese patients seeking treatment in 13 Italian medical centers (80% females; age, 45.9 ± standard deviation 11.0 years; BMI, 36.8 ± 5.7 kg/m²) were evaluated at baseline and after a 6‐month weight loss treatment. Body uneasiness, psychiatric distress, and binge eating were tested by Body Uneasiness Test (BUT, Part A), Symptom CheckList‐90 (SCL‐90), and Binge Eating Scale (BES), respectively. Results: At 6‐month follow‐up, the percentage weight loss was significantly higher in men (9.0 ± 6.3%) than in women (6.8 ± 7.3%; p = 0.010). Both men and women had a significant improvement in BUT Global Severity Index and in all of the BUT subscales with the exception of the Compulsive Self‐Monitoring subscale. Linear regression analysis selected baseline psychological and behavioral measures (global score of BUT and SCL‐90) and improved psychiatric distress and binge eating as independent predictors of changes in basal body dissatisfaction in females, whereas in males, changes were associated only with baseline BUT‐Global Severity Index score, binge eating, and its treatment‐associated improvement. Pre‐treatment BMI and BMI changes did not enter the regression. Discussion: Obesity treatment, even with a modest degree of weight loss, is associated with a significant improvement of body image, in both females and males. This effect depends mainly on psychological factors, not on the amount of weight loss.     

Ethnobotanical notes about some uses of medicinal plants in Alto Tirreno Cosentino area (Calabria, Southern Italy)

The present zootherapeutic study describes the traditional knowledge related to the use of different animals and animal-derived products as medicines by the Saharia tribe reside in the Shahabad and Kishanganj Panchayat Samiti's of Baran district of Rajasthan, India. A field survey was conducted from April to June 2006 by performing interview through structured questionnaire with 21 selected respondents, who provided information regarding use of animals and their products in folk medicine. A total of 15 animal species were recorded and they are used for different ethnomedical purposes, including cough, asthma, tuberculosis, paralysis, earache, herpes, weakness, muscular pain etc. The zootherapeutic knowledge was mostly based on domestic animals, but some protected species like the peacock (Pavo cristatus,), hard shelled turtle (Kachuga tentoria), sambhar (Cervus unicolor) were also mentioned as medicinal resources. We would suggest that this kind of neglected traditional knowledge should be included into the strategies of conservation and management of faunistic resources. Further studies are required for experimental validation to confirm the presence of bioactive compounds in these traditional remedies and also to emphasize more sustainable use of these resources.     

Urine profiling by SELDI-TOF/MS: monitoring of the critical steps in sample collection, handling and analysis

The topic of this study is the impact of several pre-analytical and analytical variables on proteomic profiling of human urine by surface enhanced laser desorption/ionization time of flight-mass spectrometry (SELDI-TOF-MS) in healthy subjects. Urine storage at room temperature caused a progressive degradation of proteins, which was prevented by the addition of protease inhibitors only up to 2 h from the collection. The timing of collection over the day had only a minor impact on protein profile, although influencing the intensity of peaks. Repeated freeze/thaw cycles (up to five) did not affect either the number or the intensity of the peaks. A comparison of the protein profile from eight different healthy individuals showed fairly consistent inter-subject similarities, along with between-subject differences, which were markedly dependent on the sex and the type of ProteinChip array used. The addition of a variety of denaturing agents improved the quality of the spectra with all the chips tested (CM10, Q10 and H50), but not with the copper-coated IMAC-30 chip. Finally, SPA matrix allowed to achieve a better performance of SELDI-TOF/MS spectrum, as compared with CHCA, regardless of the ProteinChip array used and even in the low m/z range (2500-10,000). In conclusion, we suggest that a careful choice of a number of pre-analytical and analytical conditions is required to accomplish and define a unifying protocol for the analysis of human urine by SELDI-TOF/MS, in physiological and in pathological states.