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.     

Discovery and Structure–Activity Relationships of N-Aryl 6-Aminoquinoxalines as Potent PFKFB3 Kinase Inhibitors

Energy and biomass production in cancer cells are largely supported by aerobic glycolysis in what is called the Warburg effect. The process is regulated by key enzymes, among which phosphofructokinase PFK-2 plays a significant role by producing fructose-2,6-biphosphate; the most potent activator of the glycolysis rate-limiting step performed by phosphofructokinase PFK-1. Herein, the synthesis, biological evaluation and structure–activity relationship of novel inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which is the ubiquitous and hypoxia-induced isoform of PFK-2, are reported. X-ray crystallography and docking were instrumental in the design and optimisation of a series of N-aryl 6-aminoquinoxalines. The most potent representative, N-(4-methanesulfonylpyridin-3-yl)-8-(3-methyl-1-benzothiophen-5-yl)quinoxalin-6-amine, displayed an IC₅₀ of 14 nm for the target and an IC₅₀ of 0.49 μm for fructose-2,6-biphosphate production in human colon carcinoma HCT116 cells. This work provides a new entry in the field of PFKFB3 inhibitors with potential for development in oncology.     

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.     

Diabetes and Cardiovascular Risk in Renal Transplant Patients

End-stage kidney disease (ESKD) is a main public health problem, the prevalence of which is continuously increasing worldwide. Due to adverse effects of renal replacement therapies, kidney transplantation seems to be the optimal form of therapy with significantly improved survival, quality of life and diminished overall costs compared with dialysis. However, post-transplant patients frequently suffer from post-transplant diabetes mellitus (PTDM) which an important risk factor for cardiovascular and cardiovascular-related deaths after transplantation. The management of post-transplant diabetes resembles that of diabetes in the general population as it is based on strict glycemic control as well as screening and treatment of common complications. Lifestyle interventions accompanied by the tailoring of immunosuppressive regimen may be of key importance to mitigate PTDM-associated complications in kidney transplant patients. More transplant-specific approach can include the exchange of tacrolimus with an alternative immunosuppressant (cyclosporine or mammalian target of rapamycin (mTOR) inhibitor), the decrease or cessation of corticosteroid therapy and caution in the prescribing of diuretics since they are independently connected with post-transplant diabetes. Early identification of high-risk patients for cardiovascular diseases enables timely introduction of appropriate therapeutic strategy and results in higher survival rates for patients with a transplanted kidney.     

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.     

Enhanced adsorption of two azo dyes produced by carbon modification of TiO2

The process of removal of two azo dyes (Reactive Red 198 and Direct Green 99) from water was investigated. The adsorption of azo dyes onto surfaces of pristine TiO₂, P25 and carbon-modified TiO₂ (at 120 °C for 24 h) was presented. The Freundlich model of adsorption isotherm was found for pristine TiO₂ and TiO₂-P25. Modification of TiO₂ by carbon lead to the change from the Freundlich model to the Langmuir model of adsorption isotherm. For the TiO₂-C photocatalyst the adsorption capacity was determined, which was almost two times higher for Direct Green 99 than Reactive Red 198 dyes. As a result we observed the increase of photocatalytic activity of carbon-modified TiO₂ photocatalyst.