Predictive microbiology for cosmetics based on physicals, chemicals and concentration parameters.

OBJECTIVE: Challenge test (CT) is essential to assure the efficiency of the preservative system in products. A previous study realized by our staff in 2012, carried out to evaluate the influence of three parameters (ethanol, pH and water) on the microbiological cosmetics products conservation. Following this work, a correlation between aw (based on the glycerine concentration) and the selected parameter has been demonstrated. In the present study, smaller limits of ethanol, pH and glycerine were applied to determinate CT necessity. METHODS: Sixteen stables O/W cosmetics creams with different concentration of ethanol (1-19%), glycerine (3-16%) and different pH (6-11) were formulated. To evaluate the efficiency of the different formulations, CTs were performed according to the International Standard ISO 11930:2012. To determine the influence of the parameters, a D-optimal plan generated by Design Expert(®) was applied. Design of Experiments software offers to plan, estimate and control the statistics and models for factorial and no-factorial designs. RESULTS: Challenge tests results show that 10 formula passed criteria A, two passed criteria B and four are not conform. Mostly, an ethanol concentration higher than 16% exempts products of CT. It has been shown that an ethanol concentration between 10.5% and 16%, and an glycerine concentration >10%; or if the ethanol concentration is between 5% and 10.5%, glycerine is >6% and pH is ≥10, the CT is not required. Ethanol has a significant impact on conservation and especially when it is correlated with glycerine and pH. Finally, a glycerine concentration higher than 16% exempts products of CT. CONCLUSION: Following the analysis of the different concentration, a correlation between glycerine and ethanol that directly influence microbiological protection of cosmetics products has been established. Indeed, by controlling ethanol, pH and glycerine, many products may be exempted from the CT.

Determination of the influence of factors (ethanol, pH and a(w) ) on the preservation of cosmetics using experimental design.

OBJECTIVE: Ethanol, pH and water activity are three well-known parameters that can influence the preservation of cosmetic products. With the new constraints regarding the antimicrobial effectiveness and the restrictive use of preservatives, a D-optimal design was set up to evaluate the influence of these three parameters on the microbiological conservation. METHODS: To monitor the effectiveness of the different combination of these set parameters, a challenge test in compliance with the International standard ISO 11930: 2012 was implemented. The formulations established in our study could support wide variations of ethanol concentration, pH values and glycerin concentration without noticeable effects on the stability of the products. RESULTS: In the conditions of the study, determining the value of a single parameter, with the tested concentration, could not guarantee microbiological conservation. However, a high concentration of ethanol associated with an extreme pH could inhibit bacteria growth from the first day (D0). Besides, it appears that despite an aw above 0.6 (even 0.8) and without any preservatives incorporated in formulas, it was possible to guarantee the microbiological stability of the cosmetic product when maintaining the right combination of the selected parameters. CONCLUSION: Following the analysis of the different values obtained during the experimentation, there seems to be a correlation between the aw and the selected parameters aforementioned. An application of this relationship could be to define the aw of cosmetic products by using the formula, thus avoiding the evaluation of this parameter with a measuring device.

Uniquely conserved non-translated regions are involved in generation of the two major transcripts of protein phosphatase 2Cbeta.

Partial cDNAs of different isoforms of protein phosphatase 2Cbeta (PP2Cbeta or PPM1B) have been characterized in mammals. We disclose here the full cDNAs of two major PP2Cbeta isoforms from human, rat and mouse. These cDNAs (2.6 and 3.3 kb) are able to encode 53 kDa (PP2Cbetal) and 43 kDa (PP2Cbetas) polypeptides, respectively. The isoforms are co-expressed ubiquitously with the highest level in skeletal muscle, as assessed by Northern-blot analysis. Western and in situ analyses using monoclonal antibodies against PP2Cbeta confirmed the existence of two isoforms in the cytoplasm. Comparative sequence analysis revealed that both cDNAs consist of six exons with an alternate usage of the 3' exons that underlies the differences between them. The genomic structure of PP2Cbeta is similar to that of other PP2C paralogs and includes a non-coding first exon followed by a large intron and a large second exon that encoded most of the catalytic domain. Both variants of the ending exon include large non-coding regions. All non-translated regions (NTRs) are highly conserved between the orthologous genes, indicating their regulatory function. The 5'-NTR is long (379 bp), includes upstream start codons and is predicted to contain stable secondary structures. Such features inhibit translation initiation by the scanning mechanism. Introduction of this NTR element into a bi-luciferase expression-cassette enabled expression of the second cistron, suggesting that it might serve as an internal ribosome entry site, or it contains a cryptic promoter. Overexpression of PP2Cbeta under CMV-promoter in 293 cells led to cell-growth arrest or cell death.

Differentiation-induced internal translation of c-sis mRNA: analysis of the cis elements and their differentiation-linked binding to the hnRNP C protein.

In previous reports we showed that the long 5' untranslated region (5' UTR) of c-sis, the gene encoding the B chain of platelet-derived growth factor, has translational modulating activity due to its differentiation-activated internal ribosomal entry site (D-IRES). Here we show that the 5' UTR contains three regions with a computer-predicted Y-shaped structure upstream of an AUG codon, each of which can confer some degree of internal translation by itself. In nondifferentiated cells, the entire 5' UTR is required for maximal basal IRES activity. The elements required for the differentiation-sensing ability (i.e., D-IRES) were mapped to a 630-nucleotide fragment within the central portion of the 5' UTR. Even though the region responsible for IRES activation is smaller, the full-length 5' UTR is capable of mediating the maximal translation efficiency in differentiated cells, since only the entire 5' UTR is able to confer the maximal basal IRES activity. Interestingly, a 43-kDa protein, identified as hnRNP C, binds in a differentiation-induced manner to the differentiation-sensing region. Using UV cross-linking experiments, we show that while hnRNP C is mainly a nuclear protein, its binding activity to the D-IRES is mostly nuclear in nondifferentiated cells, whereas in differentiated cells such binding activity is associated with the ribosomal fraction. Since the c-sis 5' UTR is a translational modulator in response to cellular changes, it seems that the large number of cross-talking structural entities and the interactions with regulated trans-acting factors are important for the strength of modulation in response to cellular changes. These characteristics may constitute the major difference between strong IRESs, such as those seen in some viruses, and IRESs that serve as translational modulators in response to developmental signals, such as that of c-sis.

PDGF2/c-sis mRNA leader contains a differentiation-linked internal ribosomal entry site (D-IRES).

It has become clear that a given cell type can qualitatively and quantitatively affect the expression of the platelet-derived growth factor B (PDGF2/c-sis) gene at multiple levels. In a previous report, we showed that PDGF2/c-sis 5'-untranslated region has a translational modulating activity during megakaryocytic differentiation of K562 cells. This study points to the mechanism used for this translational modulation. The unusual mRNA leader, which imposes a major barrier to conventional ribosomal scanning, was found to contain an internal ribosomal entry site that becomes more potent in differentiating cells and was termed differentiation-linked internal ribosomal entry site (D-IRES). The D-IRES element defines a functional role for the cumbersome 1022-nucleotide-long mRNA leader and accounts for its uncommon, evolutionary conserved architecture. The differentiation-linked enhancement of internal translation, which provides an additional step to the fine tuning of PDGF2/c-sis gene expression, might be employed by numerous critical regulatory genes with unusual mRNA leaders and might have widespread implications for cellular growth and development.