Developing mitoribosomal profiling to investigate quality control of human mitrochondrial protein synthesis

PhD ThesisMitochondria are essential organelles of nucleated cells that have the ability to conduct intra-organellar protein synthesis. Many aspects of this process are poorly understood especially the quality control and the rescue of stalled mitoribosomes. My project was focused on investigating potential candidates involved in quality control and ribosome rescue, including the release factor protein C12orf65. The importance of this protein in mt-translation was confirmed in a patient harbouring a mutation in C12orf65 gene, which displayed a general decrease in de novo mt-translation with subsequent disruption in assembly of OXPHOS complexes I, IV and V. I investigated the consequences on mitochondrial homeostasis in such a cell line. However, for a molecular understanding of the mechanism of this protein’s function a different, more focused approach was needed. To this end I applied ribosome profiling to mitochondria. Ribosome profiling provides a genome-wide analysis of protein synthesis by deep-sequencing of the mRNA fragments protected by ribosomes. It allows monitoring of progression of translation in vivo and can be used to identify contributions by regulating factors. I optimized the protocol for use in human mitochondria, initially on a cell line with a mutation in mt-tRNAVal. The decrease in stability of the uncharged tRNAVal resulted in an increase in mitoribosomal density over the valine codons, consistent with ribosomal stalling. I then used the final protocol, proven effective to study defects in mitoribosome progression, on cells with siRNA depleted C12orf65. Reducing transcript levels to 30% of control gave modest differences in mitoribosomal profiles. The control profiles, however, allowed normal features of mitochondrial translation to be identified. Although the exact function of C12orf65 remains unknown its involvement in mitochondrial protein translation is clear. Further applications of mitoribosome profiling to depleted or mutant cell lines should elucidate mechanisms that rescue stalled mitoribosomes and the potential role of C12orf65

New Achievements in High-Pressure Processing to Preserve Human Milk Bioactivity

High-pressure processing (HPP) is a non-thermal technology that is being increasingly applied in food industries worldwide. It was proposed that this method could be used as an alternative to holder pasteurization (HoP; 62.5°C, 30 min) in milk banks but its impact on the immunologic, enzymatic and hormonal components of human milk has not yet been evaluated in detail. The aim of our study was to compare the effects of HPP in variants: (1) 600 MPa, 10 min (2) 100 MPa, 10 min, interval 10 min, 600 MPa, 10 min (3) 200 MPa, 10 min, interval 10 min, 400 MPa, 10 min (4) 200 MPa, 10 min, interval 10 min, 600 MPa, 10 min in temperature range 19–21°C and HoP on the leptin, adiponectin, insulin, hepatocyte growth factor (HGF), lactoferrin and IgG contents in human milk. HoP was done at the Regional Human Milk Bank in Warsaw at the Holy Family Hospital on S90 Eco pasteurizer (Sterifeed, Medicare Colgate Ltd). Apparatus U4000/65 (Unipress Equipment, Poland) was used for pascalization. Milk samples were obtained from women during 2–6 weeks of lactation. Post-treatment culture showed no endogenous bacterial contamination in any tested option. Concentrations of selected components were determined using ELISA tests. The level of all analyzed components were significantly decreased by HoP: leptin 77.86%, adiponectin 32.79%, insulin 32.40%, HGF 88.72%, lactoferrin 60.31@.%, IgG 49.04%. All HPP variants caused an increase in leptin concentration, respectively (1) 81.79% (2) 90.01% (3) 86.12% (4) 47.96%. Retention of insulin after HPP was (1) 88.20% (2) 81.98% (3) 94.76% (4) 90.31% HGF (1) 36.15% (2) 38.81% 97.15% (3) 97.15% (4) 43.02%, lactoferrin (1) 55.78% (2) 57.63% (3) 78.77% (4) 64.75%. Moreover, HPP variant as 200 + 400 MPa preserved IgG (82.24%) better than HoP and resulted not statistically significant change of adiponectin level (38.55%) compare to raw milk. Our results showed that HPP leads to preservation of adipokines, growth factor, and lactoferrin, IgG much better or comparable with HoP

Barley malt-based composition as a galactagogue — a randomized, controlled trial in preterm mothers

Objectives: Delayed or insufficient breast milk production, as well as low milk supply, is still a challenging problem toovercome, particularly in the case of preterm delivery. Herbal galactagogues might be a good way to increase milk supply,however, there is a lack of clinical studies confirming their efficacy and safety.The aim of this study was to verify the safety and effectiveness as a galactagogue of the unique galactagogue compositionbased on barley malt with β -glucan and lemon balm.Material and methods: The study included 117 mothers of preterm infants randomly divided into the GalactagogueGroup given galactagogue and the Placebo Group. A complete data set was obtained for 80 participants, divided equallybetween two groups.Volume of milk expressed by mothers during the first two weeks after delivery was the primary outcome and safetyof the product was the secondary outcome.Results: Volume of milk recorded on participants’ last visit in the Galactagogue Group was significantly higher than in thePlacebo Group (95 mL vs 62.5 mL, p = 0.049). The total expressed milk volume during the study was 4209 ± 335 mLin the Placebo Group vs 6036 ± 498 mL (p = 0.003) in the Galactagogue Group.Conclusions: Supplementation with unique Galactagogue composition was safe and increased milk output which allowedachieving target minimal volume of 500 mL per day in first week of lactation in preterm mothers

Fortification of Human Milk for Preterm Infants: Update and Recommendations of the European Milk Bank Association (EMBA) Working Group on Human Milk Fortification

Evidence indicates that human milk (HM) is the best form of nutrition uniquely suited not only to term but also to preterm infants conferring health benefits in both the short and long-term. However, HM does not provide sufficient nutrition for the very low birth weight (VLBW) infant when fed at the usual feeding volumes leading to slow growth with the risk of neurocognitive impairment and other poor health outcomes such as retinopathy and bronchopulmonary dysplasia. HM should be supplemented (fortified) with the nutrients in short supply, particularly with protein, calcium, and phosphate to meet the high requirements of this group of babies. In this paper the European Milk Bank Association (EMBA) Working Group on HM Fortification discusses the existing evidence in this field, gives an overview of different fortification approaches and definitions, outlines the gaps in knowledge and gives recommendations for practice and suggestions for future research. EMBA recognizes that “Standard Fortification,” which is currently the most utilized regimen in neonatal intensive care units, still falls short in supplying sufficient protein for some VLBW infants. EMBA encourages the use of “Individualized Fortification” to optimize nutrient intake. “Adjustable Fortification” and “Targeted Fortification” are 2 methods of individualized fortification. The quality and source of human milk fortifiers constitute another important topic. There is work looking at human milk derived fortifiers, but it is still too early to draw precise conclusions about their use. The pros and cons are discussed in this Commentary in addition to the evidence around use of fortifiers post discharge

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression

Evasion of anti-growth signaling: a key step in tumorigenesis and potential target for treatment and prophylaxis by natural compounds

The evasion of anti-growth signaling is an important characteristic of cancer cells. In order to continue to proliferate, cancer cells must somehow uncouple themselves from the many signals that exist to slow down cell growth. Here, we define the anti-growth signaling process, and review several important pathways involved in growth signaling: p53, phosphatase and tensin homolog (PTEN), retinoblastoma protein (Rb), Hippo, growth differentiation factor 15 (GDF15), AT-rich interactive domain 1A (ARID1A), Notch, insulin-like growth factor (IGF), and Krüppel-like factor 5 (KLF5) pathways. Aberrations in these processes in cancer cells involve mutations and thus the suppression of genes that prevent growth, as well as mutation and activation of genes involved in driving cell growth. Using these pathways as examples, we prioritize molecular targets that might be leveraged to promote anti-growth signaling in cancer cells. Interestingly, naturally-occurring phytochemicals found in human diets (either singly or as mixtures) may promote anti-growth signaling, and do so without the potentially adverse effects associated with synthetic chemicals. We review examples of naturally-occurring phytochemicals that may be applied to prevent cancer by antagonizing growth signaling, and propose one phytochemical for each pathway. These are: epigallocatechin-3-gallate (EGCG) for the Rb pathway, luteolin for p53, curcumin for PTEN, porphyrins for Hippo, genistein for GDF15, resveratrol for ARID1A, withaferin A for Notch and diguelin for the IGF1-receptor pathway. The coordination of anti-growth signaling and natural compound studies will provide insight into the future application of these compounds in the clinical setting

A multi-targeted approach to suppress tumor-promoting inflammation

Cancers harbor significant genetic heterogeneity and patterns of relapse following many therapies are due to evolved resistance to treatment. While efforts have been made to combine targeted therapies, significant levels of toxicity have stymied efforts to effectively treat cancer with multi-drug combinations using currently approved therapeutics. We discuss the relationship between tumor-promoting inflammation and cancer as part of a larger effort to develop a broad-spectrum therapeutic approach aimed at a wide range of targets to address this heterogeneity. Specifically, macrophage migration inhibitory factor, cyclooxygenase-2, transcription factor nuclear factor-κB, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase B, and CXC chemokines are reviewed as important antiinflammatory targets while curcumin, resveratrol, epigallocatechin gallate, genistein, lycopene, and anthocyanins are reviewed as low-cost, low toxicity means by which these targets might all be reached simultaneously. Future translational work will need to assess the resulting synergies of rationally designed antiinflammatory mixtures (employing low-toxicity constituents), and then combine this with similar approaches targeting the most important pathways across the range of cancer hallmark phenotypes