Simultaneous analysis of coccidiostats and sulphonamides in non-target feed by HPLC-MS/MS and validation following the Commission Decision 2002/657/EC

Taking into consideration the maximum level (ML) for coccidiostats included in the European Regulation 574/2011 and the fact that the presence of residues of sulphonamides in non-target feed is forbidden, the aim of this article is to present an analytical method based on HPLC-MS/MS for the identification and quantification of sulphonamides and coccidiostats in non-target feeds. The method was validated following Commission Decision 2002/657/EC, and recovery, repeatability and reproducibility were within the limits established in the Decision. For coccidiostats, the decision limit and detection capability were calculated for the different species taking into account the ML allowed in Regulation 574/2011. The applicability of the method was investigated in 50 feed samples collected from dairy farms, 50 obtained from feed mills and 10 interlaboratory feed samples.     

Estradiol-17β Regulates Expression of Luteal DNA Methyltransferases and Genes Involved in the Porcine Corpus Luteum Function In Vivo

The corpus luteum (CL) is a temporary endocrine gland vital for pregnancy establishment and maintenance. Estradiol-17β (E2) is the major embryonic signal in pigs supporting the CL’s function. The mechanisms of the luteoprotective action of E2 are still unclear. The present study aimed to determine the effect of E2 on luteal expression of factors involved in CL function. An in vivo model of intrauterine E2 infusions was applied. Gilts on day 12 of pregnancy and the estrous cycle were used as referential groups. Concentrations of E2 and progesterone were elevated in CLs of gilts receiving E2 infusions, compared to placebo-treated gilts. Estradiol-17β stimulated luteal expression of DNA-methyltransferase 1 (DNMT1), but decreased expression of DNMT3B gene and protein, as well as DNMT3A protein. Similar results for DNMT3A and 3B were observed in CLs on day 12 of pregnancy compared to day 12 of the estrous cycle. Intrauterine infusions of E2 altered luteal expression of the genes involved in CL function: PTGFR, PTGES, STAR, HSD17B1, CYP19A1, and PGRMC1. Our findings indicate a role for E2 in expression regulation of factors related to CL function and a novel potential for E2 to regulate DNA methylation as putative physiological mechanisms controlling luteal gene expression.     

LATS1 Is a Mediator of Melanogenesis in Response to Oxidative Stress and Regulator of Melanoma Growth

The LATS1 kinase has been described as a tumor suppressor in various cancers. However, its role in melanoma has not been fully elucidated. There are several processes involved in tumorigenesis, including melanin production. Melanin content positively correlates with the level of reactive oxygen species (ROS) inside the cell. Accordingly, the purpose of the study was to assess the role of LATS1 in melanogenesis and oxidative stress and its influence on tumor growth. We have knocked down LATS1 in primary melanocytes and melanoma cells and found that its expression is crucial for melanin synthesis, ROS production, and oxidative stress response. We showed that LATS1 ablation significantly decreased the melanogenesis markers’ expression and melanin synthesis in melanocyte and melanoma cell lines. Moreover, silencing LATS1 resulted in enhanced oxidative stress. Reduced melanin content in LATS1 knocked down tumors was associated with increased tumor growth, pointing to melanin’s protective role in this process. The study demonstrated that LATS1 is highly engaged in melanogenesis and oxidative stress control and affects melanoma growth. Our results may find the implications in the diagnosis and treatment of pigmentation disorders, including melanoma.     

Symposium review: Genomic investigations of flavor formation by dairy microbiota

Flavor is one of the most important attributes of any fermented dairy product. Dairy consumers are known to be willing to experiment with different flavors; thus, many companies producing fermented dairy products have looked at culture manipulation as a tool for flavor diversification. The development of flavor is a complex process, originating from a combination of microbiological, biochemical, and technological aspects. A key driver of flavor is the enzymatic activities of the deliberately inoculated starter cultures, in addition to the environmental or “nonstarter” microbiota. The contribution of microbial metabolism to flavor development in fermented dairy products has been exploited for thousands of years, but the availability of the whole genome sequences of the bacteria and yeasts involved in the fermentation process and the possibilities now offered by next-generation sequencing and downstream “omics” technologies is stimulating a more knowledge-based approach to the selection of desirable cultures for flavor development. By linking genomic traits to phenotypic outputs, it is now possible to mine the metabolic diversity of starter cultures, analyze the metabolic routes to flavor compound formation, identify those strains with flavor-forming potential, and select them for possible commercial application. This approach also allows for the identification of species and strains not previously considered as potential flavor-formers, the blending of strains with complementary metabolic pathways, and the potential improvement of key technological characteristics in existing strains, strains that are at the core of the dairy industry. An in-depth knowledge of the metabolic pathways of individual strains and their interactions in mixed culture fermentations can allow starter blends to be custom-made to suit industry needs. Applying this knowledge to starter culture research programs is enabling research and development scientists to develop superior starters, expand flavor profiles, and potentially develop new products for future market expansion.     

Feeding strategy impacts heterologous protein production in Yarrowia lipolytica fed-batch cultures—Insight into the role of osmolarity

Fed-batch cultivation is the preferred bioprocessing strategy applied in microbial production of proteins. Feeding strategy is crucial parameters to be optimized upon development of a fed-batch process. In this study, we investigated impact of different feeding strategies on production of recombinant enzymatic protein in Yarrowia lipolytica cultures. From amongst tested strategies, comprising intermittent and continuous feedings, also in cascade with respiratory factors, intermittent feeding executed after complete exhaustion of glycerol from the medium, with moderate amplitude of osmolarity, was the most beneficial in terms of the secretory enzyme amount, its volumetric productivity and specific activity. Because adopted feeding strategies strongly modulated osmolarity of the cultures, the effect of osmotic pressure on production of the target heterologous protein was investigated in a series of batch cultivations with addition of osmoactive compounds (NaCl, sorbitol, sucrose, and glycerol) at different concentrations. Although obvious promoting effect of the osmoactive substances on the enzyme production was clear, no straightforward correlation between the medium osmolarity and the target enzyme's specific activity could be observed. These results suggest that not only the level of osmolarity but also chemical character of the osmoactive compound have both important impact on the production of secretory proteins in Y. lipolytica cultures.     

Determination of Total Concentrations and Chemical and Physical Fractionation Forms of Manganese in Infusions of Ground Coffees

A simple and fast method of the determination of total Mn in infusions of ground coffees was proposed and included the acidification of infusions with HNO₃ followed by the analysis by flame atomic absorption spectrometry. The method provided the precision and the accuracy better than 3 %. In addition, chemical and physical fractionation patterns of Mn in coffee infusions were assessed using solid phase extraction and ultrafiltration-based procedures, respectively. It was found that Mn in infusions of ground coffees is predominantly present in the form of cationic (64–81 % of the total content) low molecular weight (61–68 % of the total content) species. This points out that this metal is highly bioaccessible from the coffee brew.     

Immunological Prognostic Factors in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell neoplasm characterized by an abnormal proliferation of clonal, terminally differentiated B lymphocytes. Current approaches for the treatment of MM focus on developing new diagnostic techniques; however, the search for prognostic markers is also crucial. This enables the classification of patients into risk groups and, thus, the selection of the most optimal treatment method. Particular attention should be paid to the possible use of immune factors, as the immune system plays a key role in the formation and course of MM. In this review, we focus on characterizing the components of the immune system that are of prognostic value in MM patients, in order to facilitate the development of new diagnostic and therapeutic directions.     

Evaluation of heterologous α‐amylase production in two expression platforms dedicated for Yarrowia lipolytica: commercial Po1g–pYLSC (php4d) and custom‐made A18–pYLTEF (pTEF)

In view of the constantly increasing demand for cost‐effective, low‐energy and environmentally friendly industrial processes and household care products, enzyme production occupies an essential place in the field of biotechnology. Along with increasing demand for industrial and household care enzymes, the demand for heterologous expression platforms has also increased. Apart from the conventional hosts, e.g. Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris, routinely used in heterologous protein expression, the non‐conventional ones have become more and more exploited in this field. Among the available yeast host systems, Yarrowia lipolytica appears to be an attractive alternative. The aim of this study was to compare efficiency of two Yarrowia‐based expression platforms, commercial Po1g–pYLSC and custom‐made A18‐pYLTEF, in expression of an insect‐derived, raw‐starch‐digesting α‐amylase, to select the ‘champion’ system for further studies on this valuable enzyme. Both expression platforms were compared with respect to copy number of the integrated expression cassette/transformed genome, and the recombinant strains performance (Po1g–pYLSC‐derived 4.29 strain, and A18‐pYLTEF‐derived B9 strain) during batch bioreactor cultures. Our results demonstrate that the average number of integration events into the recipient's genome was comparable for both expression systems under investigation, but with varying distribution of the multicopy integrants; and the number of the recombinant gene copies was highly correlated with the acquired amylolytic activity of the strains. Due to severe susceptibility of the recombinant AMY1 polypeptide to native proteases of the custom‐made expression system, the final yield of the enzyme was substantially lower when compared to the commercial Po1g–pYLSC (reaching a maximum level of 142.84 AU/l). Copyright © 2015 John Wiley & Sons, Ltd.     

Structural anomaly and electrical relaxation in (Na2/3Pb1/3)(Mn1/2Nb1/2)O3 ceramics

The X-ray diffraction patterns of (Na_2/3Pb_1/3)(Mn_1/2Nb_1/2)O₃ ceramics were measured within 15–850 K temperature range. The anomaly in the thermal expansion temperature dependence occurred in 250–365 K range. The generalised Cole–Cole model was proposed to describe the measured effective electric permittivity influenced by high electric conduction and the coexistence of two contributions ?*(T,f) = ?*_lattice + ?*_carriers was considered. The analysis of the electric permittivity and conduction exhibited two relaxation processes. The electric conduction relaxation characteristic time values indicated the small polaron mechanism with τ₀ ≈ 10⁻¹³ s occurring in 240–345 K range and the ionic mechanism with τ₀ ≈ 10⁻¹¹ s involved in the other relaxation occurring in the 320–510 K range. The ionic relaxation process was ascribed to a subsystem of defects, which was weakly interrelated to the anomaly in thermal expansion of the (Na_2/3Pb_1/3)(Mn_1/2Nb_1/2)O₃ ceramics. The Gate model was proposed to describe the ionic relaxation mechanism.     

Culturomics Approach to Identify Diabetic Foot Infection Bacteria

The main goal of the study was to evaluate the usefulness of the culturomics approach in the reflection of diabetic foot infections (DFIs) microbial compositions in Poland. Superficial swab samples of 16 diabetic foot infection patients (Provincial Polyclinical Hospital in Toruń, Poland) were subjected to culturing using 10 different types of media followed by the identification via the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Biotyper platform. Identified 204 bacterial isolates representing 18 different species—mostly Enterococcus faecalis (63%) and Staphylococcus aureus (44%). Most of the infections (81%) demonstrated a polymicrobial character. Great differences in the species coverage, the number of isolated Gram-positive and Gram-negative bacteria, and the efficiency of the microbial composition reflection between the investigated media were revealed. The use of commonly recommended blood agar allowed to reveal only 53% of the entire microbial composition of the diabetic foot infection samples, which considerably improved when the chromagar orientation and vancomycin-resistant enterococi agar were applied. In general, efficiency increased in the following order: selective < universal < enriched < differential media. Performed analysis also revealed the impact of the culture media composition on the molecular profiles of some bacterial species, such as Corynebacterium striatum, Proteus mirabilis or Morganella morganii that contributed to the differences in the identification quality. Our results indicated that the culturomics approach can significantly improve the accuracy of the reflection of the diabetic foot infections microbial compositions as long as an appropriate media set is selected. The chromagar orientation and vancomycin-resistant enterococi agar media which were used for the first time to study diabetic foot infection microbial profiles demonstrate the highest utility in the culturomics approach and should be included in further studies directed to find a faster and more reliable diabetic foot infection diagnostic tool.