Microbiological control in stem cell banks: approaches to standardisation

The transplant of cells of human origin is an increasingly complex sector of medicine which entails great opportunities for the treatment of a range of diseases. Stem cell banks should assure the quality, traceability and safety of cultures for transplantation and must implement an effective programme to prevent contamination of the final product. In donors, the presence of infectious micro-organisms, like human immunodeficiency virus, hepatitis B virus, hepatitis C virus and human T cell lymphotrophic virus, should be evaluated in addition to the possibility of other new infectious agents (e.g. transmissible spongiform encephalopathies and severe acute respiratory syndrome). The introduction of the nucleic acid amplification can avoid the window period of these viral infections. Contamination from the laboratory environment can be achieved by routine screening for bacteria, fungi, yeast and mycoplasma by European pharmacopoeia tests. Fastidious micro-organisms, and an adventitious or endogenous virus, is a well-known fact that will also have to be considered for processes involving in vitro culture of stem cells. It is also a standard part of current good practice in stem cell banks to carry out routine environmental microbiological monitoring of the cleanrooms where the cell cultures and their products are prepared. The risk of viral contamination from products of animal origin, like bovine serum and mouse fibroblasts as a “feeder layer” for the development of embryonic cell lines, should also be considered. Stem cell lines should be tested for prion particles and a virus of animal origin that assure an acceptable quality.     

Enhanced silymarin accumulation is related to calcium deprivation in cell suspension cultures of Silybum marianum (L.) Gaertn

Elimination of calcium ions from the medium of cell cultures of Silybum marianum (L.) Gaertn increased flavonolignan production. Silymarin accumulation was not altered by treatment of cultures with the calcium ionophore A23187. The specific Ca2+ chelator, EGTA, enhanced the silymarin content in cells by 200%, and its secretion by 3-4 times. The inorganic ion La3+, as well as the calcium channel inhibitor verapamil, also stimulated production. Several reagents known to block intracellular calcium movement, such as ruthenium red, thapsigargin and TMB-8 appreciably increased silymarin accumulation. These results suggest that inhibition of external and internal calcium fluxes plays a significant role in flavonolignan metabolism of S. marianum cell cultures.     

Life-long supplementation with a low dosage of coenzyme Q10 in the rat: effects on antioxidant status and DNA damage

Life-long low-dosage supplementation of coenzyme Q(10) (CoQ(10)) is studied in relation to the antioxidant status and DNA damage. Thirty-two male rats were assigned into two experimental groups differing in the supplementation or not with 0.7 mg/kg/day of CoQ(10). Eight rats per group were killed at 6 and 24 months. Plasma retinol, alpha-tocopherol, coenzyme Q, total antioxidant capacity and fatty acids were analysed. DNA strand breaks were studied in peripheral blood lymphocytes. Aging and supplementation led to significantly higher values for CoQ homologues, retinol and alpha-tocopherol. No difference in total antioxidant capacity was detected at 6 months but significantly lower values were found in aged control animals. Similar DNA strand breaks levels were found at 6 months. Aging led to significantly higher DNA strand breaks levels in both groups but animals supplemented with CoQ(10) led to a significantly lower increase in that marker. Aged rats showed significantly higher polyunsaturated fatty acids. This study demonstrates that lifelong intake of a low dosage of CoQ(10) enhances plasma levels of CoQ(9), CoQ(10), alpha-tocopherol and retinol. In addition, CoQ(10) supplementation attenuates the age-related fall in total antioxidant capacity of plasma and the increase in DNA damage in peripheral blood lymphocytes.     

The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes

Classical studies on protist diversity of freshwater environments worldwide have led to the idea that most species of microbial eukaryotes are known. One exemplary case would be constituted by the ciliates, which have been claimed to encompass a few thousands of ubiquitous species, most of them already described. Recently, molecular methods have revealed an unsuspected protist diversity, especially in oceanic as well as some extreme environments, suggesting the occurrence of a hidden diversity of eukaryotic lineages. In order to test if this holds also for freshwater environments, we have carried out a molecular survey of small subunit ribosomal RNA genes in water and sediment samples of two ponds, one oxic and another suboxic, from the same geographic area. Our results show that protist diversity is very high. The majority of phylotypes affiliated within a few well established eukaryotic kingdoms or phyla, including alveolates, cryptophytes, heterokonts, Cercozoa, Centroheliozoa and haptophytes, although a few sequences did not display a clear taxonomic affiliation. The diversity of sequences within groups was very large, particularly that of ciliates, and a number of them were very divergent from known species, which could define new intra-phylum groups. This suggests that, contrary to current ideas, the diversity of freshwater protists is far from being completely described.     

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His₆-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K _m values for nitrite and NADH were 0.5 mM and 20 μM, respectively, at the pH and temperature optima (pH 9 and 30°C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.     

Improving secondary embryogenesis in <Emphasis Type="Italic">Quercus robur</Emphasis>: application of temporary immersion for mass propagation

The effects of the culture system used for embryo proliferation were investigated with the aim of improving multiplication rates and somatic embryo quality in two embryogenic lines of Quercus robur derived from mature trees (B-17 and Sainza). Embryo proliferation medium was defined following comparison of five different semi-solid media, and the highest multiplication rates (based on the total number of embryos and number of cotyledonary-shaped embryos) were achieved with medium supplemented with 0.44 μM benzyladenine for both lines. Embryo proliferation on semi-solid medium was compared with that obtained by a temporary immersion system (TIS), in which four cycles with immersion frequencies of 1 min every 6, 8, 12 or 24 h were tested. TIS promoted a significant increase in proliferated embryo biomass, with the growth index (GI) two and four times higher than in semi-solid medium in B-17 and Sainza genotypes, respectively. An immersion cycle of 1 min every 8 or 12 h produced approximately 700 somatic embryos (B-17) and 1,500 somatic embryos (Sainza) per RITA^® bioreactor, with significant differences in the latter genotype with respect to gelled medium. TIS had also a significant effect on somatic embryo synchronization as it enabled a higher production of cotyledonary embryos (90%), which represents increases of 14% (B-17) and 20% (Sainza) with respect to gelled medium. For germination of embryos proliferated in TIS two maturation systems were applied: (1) culture in semi-solid medium containing 6% sorbitol or (2) culture by TIS (without sorbitol) at a frequency of 1 min immersion every 48 h. Germination ability was higher after maturation on sorbitol medium and plantlet conversion occurred in 48% (B-17) and 13% (Sainza) embryos. TIS produced large numbers of well-developed cotyledonary embryos, hence reduced the cost and labor.     

Role of Kras Status in Patients with Metastatic Colorectal Cancer Receiving First-Line Chemotherapy plus Bevacizumab: A TTD Group Cooperative Study

Background

In the MACRO study, patients with metastatic colorectal cancer (mCRC) were randomised to first-line treatment with 6 cycles of capecitabine and oxaliplatin (XELOX) plus bevacizumab followed by either single-agent bevacizumab or XELOX plus bevacizumab until disease progression. An additional retrospective analysis was performed to define the prognostic value of tumour KRAS status on progression-free survival (PFS), overall survival (OS) and response rates.

Methodology/Principal Findings

KRAS data (tumour KRAS status and type of mutation) were collected by questionnaire from participating centres that performed KRAS analyses. These data were then cross-referenced with efficacy data for relevant patients in the MACRO study database. KRAS status was analysed in 394 of the 480 patients (82.1%) in the MACRO study. Wild-type (WT) KRAS tumours were found in 219 patients (56%) and mutant (MT) KRAS in 175 patients (44%). Median PFS was 10.9 months for patients with WT KRAS and 9.4 months for patients with MT KRAS tumours (p = 0.0038; HR: 1.40; 95% CI:1.12–1.77). The difference in OS was also significant: 26.7 months versus 18.0 months for WT versus MT KRAS, respectively (p = 0.0002; HR: 1.55; 95% CI: 1.23–1.96). Univariate and multivariate analyses showed that KRAS was an independent variable for both PFS and OS. Responses were observed in 126 patients (57.5%) with WT KRAS tumours and 76 patients (43.4%) with MT KRAS tumours (p = 0.0054; OR: 1.77; 95% CI: 1.18–2.64).

Conclusions/Significance

This analysis of the MACRO study suggests a prognostic role for tumour KRAS status in patients with mCRC treated with XELOX plus bevacizumab. For both PFS and OS, KRAS status was an independent factor in univariate and multivariate analyses.     

“The 3/3 Strategy”: A Successful Multifaceted Hospital Wide Hand Hygiene Intervention Based on WHO and Continuous Quality Improvement Methodology

Background

Only multifaceted hospital wide interventions have been successful in achieving sustained improvements in hand hygiene (HH) compliance.

Methodology/Principal Findings

Pre-post intervention study of HH performance at baseline (October 2007– December 2009) and during intervention, which included two phases. Phase 1 (2010) included multimodal WHO approach. Phase 2 (2011) added Continuous Quality Improvement (CQI) tools and was based on: a) Increase of alcohol hand rub (AHR) solution placement (from 0.57 dispensers/bed to 1.56); b) Increase in frequency of audits (three days every three weeks: “3/3 strategy”); c) Implementation of a standardized register form of HH corrective actions; d) Statistical Process Control (SPC) as time series analysis methodology through appropriate control charts. During the intervention period we performed 819 scheduled direct observation audits which provided data from 11,714 HH opportunities. The most remarkable findings were: a) significant improvements in HH compliance with respect to baseline (25% mean increase); b) sustained high level (82%) of HH compliance during intervention; c) significant increase in AHRs consumption over time; c) significant decrease in the rate of healthcare-acquired MRSA; d) small but significant improvements in HH compliance when comparing phase 2 to phase 1 [79.5% (95% CI: 78.2–80.7) vs 84.6% (95% CI:83.8–85.4), p<0.05]; e) successful use of control charts to identify significant negative and positive deviations (special causes) related to the HH compliance process over time (“positive”: 90.1% as highest HH compliance coinciding with the “World hygiene day”; and “negative”:73.7% as lowest HH compliance coinciding with a statutory lay-off proceeding).

Conclusions/Significance

CQI tools may be a key addition to WHO strategy to maintain a good HH performance over time. In addition, SPC has shown to be a powerful methodology to detect special causes in HH performance (positive and negative) and to help establishing adequate feedback to healthcare workers.     

Some common signal transduction events are not necessary for the elicitor-induced accumulation of silymarin in cell cultures of Silybum marianum

A variety of pharmacological effectors of signal transduction pathways were used to investigate the elicitor-activated sequence of cellular responses by which yeast extract (YE) or methyljasmonate (MeJA) enhanced production of silymarin in cell cultures of Silybum marianum. As we recently showed that inhibition of external and internal calcium fluxes significantly increased flavonolignan production in S. marianum cultures, we examined whether calcium mediates signaling events leading to enhancement of silymarin production upon YE or MeJA elicitation. Pre-treatment of cultures with calcium chelators, calcium blockers or intracellular antagonists enhanced the elicitor effect of YE or MeJA. The increase of intracellular-free Ca(2+) level also promoted the elicitor effect, suggesting that an external source of calcium or alterations in internal calcium fluxes were not required for the elicitation to occur. Activation of phosphorylation/dephosphorylation cascades did not appear to mediate the elicitation mechanism; the increase in silymarin induced by elicitation was not suppressed by inhibitors of protein phosphatases or by protein kinase inhibitors. No H(2)O(2) generation was detected at any time after elicitation. Also, diphenyleneiodonium, a potent inhibitor of NAD(P)H-oxidase, did not block silymarin production in elicited cultures. From these results, we conclude that S. marianum cell cultures do not appear to employ conserved signaling components in the transduction of the elicitor signal to downstream responses such as silymarin production.     

Designing healthy,climate friendly and affordable school lunches

Purpose

This study aims to develop a model with which to build diets taking into account nutritional, climate change and economic aspects. A case study is used to test the proposed model, consisting of finding the optimal menus for school children in Spain from combinations of 20 starters, 20 main dishes and 7 desserts for a 20-day planning period.

Methods

An optimizing technique, specifically integer goal programming, is used as a means of designing diets which take into account the aforementioned aspects. Goal programming (GP) is used to design those menus that meet, or nearly meet, all the requirements with respect to caloric content, caloric share among macronutrients, nutrients to encourage and nutrients to limit, while reducing the carbon footprint (CFP) and the lunch budget. In order to have real, acceptable dishes, a school catering company provided information about the typical dishes they serve. The CFP of each dish was assessed, based on literature about life cycle assessment and CFP studies on food products. The nutritional value of each dish was obtained from databases, whereas prices were gathered from a wholesaler.

Results and discussion

After solving the goal programming model for several CFP and budget goals, the results show reductions with respect to the average CFP of between ?13 and ?24 %, and reductions with respect to the average budget between ?10 and ?15 % while maintaining the nutritional aspects similar to the average of the proposed menus. The results show that a wide range of budget is available, maintaining an almost constant CFP and meeting nutritional requirements to a similar degree; therefore, it is possible to avoid trade-offs between the CFP and the budget. The analysis of the dishes selected shows how the optimization model, in general, avoids the dishes which have a high CFP and high price and which are low in iron content, but high in protein and cholesterol.

Conclusions

Goal programming constitutes a suitable tool for designing diets which are economically, environmentally and nutritionally sustainable. Its flexibility enables specific issues to be studied, such as the existence of possible trade-offs between budget and CFP, attained by changing the budget and the CFP goals. By means of an iterative process, new dishes could be introduced or the existing ones could be improved, thus providing catering companies with useful information.