Disclosure of a putative biosignature for respiratory chain disorders through a metabolomics approach

The diagnosis of respiratory chain deficiencies (RCDs) is complicated and the need for a diagnostic biomarker or biosignature has been widely expressed. In this study, the metabolic profile of a selected group of 29 RCD patients, with a predominantly muscle disease phenotype, and 22 controls were investigated using targeted and untargeted analyses of three sub-sections of the human metabolome, including urinary organic acids and amino acids [measured by gas chromatography–mass spectrometry (GC–MS)], as well as acylcarnitines (measured by electrospray ionization tandem MS). Although MS technologies are highly sensitive and selective, they are restrictive by being applied only to sub-sections of the metabolome; an untargeted nuclear magnetic resonance (NMR) spectroscopy approach was therefore also included. After data reduction and pre-treatment, a biosignature comprising six organic acids (lactic, succinic, 2-hydroxyglutaric, 3-hydroxyisobutyric, 3-hydroxyisovaleric and 3-hydroxy-3-methylglutaric acids), six amino acids (alanine, glycine, glutamic acid, serine, tyrosine and α-aminoadipic acid) and creatine, was constructed from uni- and multivariate statistical analyses and verified by cross-validation. The results presented here provide the first proof-of-concept that the metabolomics approach is capable of defining a biosignature for RCDs. We postulate that the composite of organic acids ≈ amino acids > creatine > betaine > carnitines represents the basic biosignature for RCDs. Validated through a prospective study, this could offer an improved ability to assign individual patients to a group with defined RCD characteristics and improve case selection for biopsy procedures, especially in infants and children.     

Estimates of Excess Medically Attended Acute Respiratory Infections in Periods of Seasonal and Pandemic Influenza in Germany from 2001/02 to 2010/11

Background

The number of patients seeking health care is a central indicator that may serve several different purposes: (1) as a proxy for the impact on the burden of the primary care system; (2) as a starting point to estimate the number of persons ill with influenza; (3) as the denominator data for the calculation of case fatality rate and the proportion hospitalized (severity indicators); (4) for economic calculations. In addition, reliable estimates of burden of disease and on the health care system are essential to communicate the impact of influenza to health care professionals, public health professionals and to the public.

Methodology/Principal Findings

Using German syndromic surveillance data, we have developed a novel approach to describe the seasonal variation of medically attended acute respiratory infections (MAARI) and estimate the excess MAARI attributable to influenza. The weekly excess inside a period of influenza circulation is estimated as the difference between the actual MAARI and a MAARI-baseline, which is established using a cyclic regression model for counts. As a result, we estimated the highest ARI burden within the last 10 years for the influenza season 2004/05 with an excess of 7.5 million outpatient visits (CI95% 6.8–8.0). In contrast, the pandemic wave 2009 accounted for one third of this burden with an excess of 2.4 million (CI95% 1.9–2.8). Estimates can be produced for different age groups, different geographic regions in Germany and also in real time during the influenza waves.     

A dynamic model linking cell growth to intracellular metabolism and extracellular by-product accumulation

Mathematical modeling of animal cell growth and metabolism is essential for the understanding and improvement of the production of biopharmaceuticals. Models can explain the dynamic behavior of cell growth and product formation, support the identification of the most relevant parameters for process design, and significantly reduce the number of experiments to be performed for process optimization. Few dynamic models have been established that describe both extracellular and intracellular dynamics of growth and metabolism of animal cells. In this study, a model was developed, which comprises a set of 33 ordinary differential equations to describe batch cultivations of suspension AGE1.HN.AAT cells considered for the production of α1-antitrypsin. This model combines a segregated cell growth model with a structured model of intracellular metabolism. Overall, it considers the viable cell concentration, mean cell diameter, viable cell volume, concentration of extracellular substrates, and intracellular concentrations of key metabolites from the central carbon metabolism. Furthermore, the release of metabolic by-products such as lactate and ammonium was estimated directly from the intracellular reactions. Based on the same set of parameters, this model simulates well the dynamics of four independent batch cultivations. Analysis of the simulated intracellular rates revealed at least two distinct cellular physiological states. The first physiological state was characterized by a high glycolytic rate and high lactate production. Whereas the second state was characterized by efficient adenosine triphosphate production, a low glycolytic rate, and reactions of the TCA cycle running in the reverse direction from α-ketoglutarate to citrate. Finally, we show possible applications of the model for cell line engineering and media optimization with two case studies.     

Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS

This study aims at substituting the essential functions of photoreceptors in patients who are blind owing to untreatable forms of hereditary retinal degenerations. A microelectronic neuroprosthetic device, powered via transdermal inductive transmission, carrying 1500 independent microphotodiode-amplifier-electrode elements on a 9 mm² chip, was subretinally implanted in nine blind patients. Light perception (8/9), light localization (7/9), motion detection (5/9, angular speed up to 35 deg s⁻¹), grating acuity measurement (6/9, up to 3.3 cycles per degree) and visual acuity measurement with Landolt C-rings (2/9) up to Snellen visual acuity of 20/546 (corresponding to decimal 0.037 or corresponding to 1.43 logMAR (minimum angle of resolution)) were restored via the subretinal implant. Additionally, the identification, localization and discrimination of objects improved significantly (n = 8; p < 0.05 for each subtest) in repeated tests over a nine-month period. Three subjects were able to read letters spontaneously and one subject was able to read letters after training in an alternative-force choice test. Five subjects reported implant-mediated visual perceptions in daily life within a field of 15° of visual angle. Control tests were performed each time with the implant''s power source switched off. These data show that subretinal implants can restore visual functions that are useful for daily life.     

A Powerful Molecular Engineering Tool Provided Efficient Chlamydomonas Mutants as Bio-Sensing Elements for Herbicides Detection

This study was prompted by increasing concerns about ecological damage and human health threats derived by persistent contamination of water and soil with herbicides, and emerging of bio-sensing technology as powerful, fast and efficient tool for the identification of such hazards. This work is aimed at overcoming principal limitations negatively affecting the whole-cell-based biosensors performance due to inadequate stability and sensitivity of the bio-recognition element. The novel bio-sensing elements for the detection of herbicides were generated exploiting the power of molecular engineering in order to improve the performance of photosynthetic complexes. The new phenotypes were produced by an in vitro directed evolution strategy targeted at the photosystem II (PSII) D1 protein of Chlamydomonas reinhardtii, using exposures to radical-generating ionizing radiation as selection pressure. These tools proved successful to identify D1 mutations conferring enhanced stability, tolerance to free-radical-associated stress and competence for herbicide perception. Long-term stability tests of PSII performance revealed the mutants capability to deal with oxidative stress-related conditions. Furthermore, dose-response experiments indicated the strains having increased sensitivity or resistance to triazine and urea type herbicides with I₅₀ values ranging from 6×10⁻⁸ M to 2×10⁻⁶ M. Besides stressing the relevance of several amino acids for PSII photochemistry and herbicide sensing, the possibility to improve the specificity of whole-cell-based biosensors, via coupling herbicide-sensitive with herbicide-resistant strains, was verified.     

Differential Expression of CRH,UCN, CRHR1 and CRHR2 in Eutopic and Ectopic Endometrium of Women with Endometriosis

Endometriosis is considered as a benign aseptic inflammatory disease, characterised by the presence of ectopic endometrium-like tissue. Its symptoms (mostly pain and infertility) are reported as constant stressors. Corticotropin releasing hormone (CRH) and urocortin (UCN) are neuropeptides, strongly related to stress and inflammation. The effects of CRH and UCN are mediated through CRHR1 and CRHR2 receptors which are implicated in several reproductive functions acting as inflammatory components. However, the involvement of these molecules to endometriosis remains unknown. The aim of this study was to examine the expression of CRHR1 and CRHR2 in endometriotic sites and to compare the expression of CRHR1 and CRHR2 in eutopic endometrium of endometriotic women to that of healthy women. We further compared the expression of CRH, UCN, CRHR1 and CRHR2 in ectopic endometrium to that in eutopic endometrium of women with endometriosis. Endometrial biopsy specimens were taken from healthy women (10 patients) and endometrial and endometriotic biopsy specimens were taken from women with endometriosis (16 patients). Τhe expression of CRH, UCN, CRHR1, and CRHR2 was tested via RT-PCR, immunohistochemistry and Western blotting. This study shows for the first time that CRH and UCN receptor subtypes CRHR1β and CRHR2α are expressed in endometriotic sites and that they are more strongly expressed (p<0.01) in eutopic endometrium of women with endometriosis compared to healthy women endometrium at the mRNA and protein level. CRH, UCN, CRHR1 and CRHR2 mRNA were also more highly expressed in ectopic rather than eutopic endometrium (CRH, UCN, CRHR2α: p<0.01, CRHR1β: p<0.05) and protein (CRH and UCN: p<0.05, CRHR1 and CRHR2: p<0.01) in women with endometriosis. These data indicate that CRH and UCN might play an immunoregulatory role in endometriotic sites by affecting reproductive functions such as decidualization and implantation of women with endometriosis.     

Ezrin and HNRNP expression correlate with increased virus release rate and early onset of virus‐induced apoptosis of MDCK suspension cells

With the aim to adapt high‐yield adherent cell lines to suspension growth, Madin Darby canine kidney (MDCK) suspension cells were developed recently that achieved comparable influenza virus yields despite an early induction of apoptosis compared to the parental adherent cell line. For both cell lines, a comprehensive study under comparable infection conditions is performed comprising information on: time course of viral infection, antiviral state of cells, virus‐induced apoptosis, and virus‐induced cellular protein expression for early and late infection with influenza A/PuertoRico/8/34 H1N1. The proteomic analysis is performed with 2D differential gel electrophoreses followed by mass spectrometry. Based on flow cytometric data and on the differential expression of various stress and apoptosis‐related proteins, the earlier onset of virus‐induced apoptosis is confirmed for suspension cells. Surprisingly, the data indicated an increased virus release rate for suspension cells. These observations correlate with an increased expression of the apical marker protein ezrin, known to play a role in influenza‐induced cytoskeletal rearrangement, and the differential expression of heterogeneous nuclear ribonucleoproteins, known to bind actively influenza viral proteins and play a central role in regulating gene expression. Based on these findings, additional studies towards the design of MDCK suspension cells with further increase in influenza virus yields will be performed.     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.     

Breeding habitat and nest‐site selection by an obligatory “nest‐cleptoparasite”, the Amur Falcon Falco amurensis

The selection of a nest site is crucial for successful reproduction of birds. Animals which re‐use or occupy nest sites constructed by other species often have limited choice. Little is known about the criteria of nest‐stealing species to choose suitable nesting sites and habitats. Here, we analyze breeding‐site selection of an obligatory “nest‐cleptoparasite”, the Amur Falcon Falco amurensis. We collected data on nest sites at Muraviovka Park in the Russian Far East, where the species breeds exclusively in nests of the Eurasian Magpie Pica pica. We sampled 117 Eurasian Magpie nests, 38 of which were occupied by Amur Falcons. Nest‐specific variables were assessed, and a recently developed habitat classification map was used to derive landscape metrics. We found that Amur Falcons chose a wide range of nesting sites, but significantly preferred nests with a domed roof. Breeding pairs of Eurasian Hobby Falco subbuteo and Eurasian Magpie were often found to breed near the nest in about the same distance as neighboring Amur Falcon pairs. Additionally, the occurrence of the species was positively associated with bare soil cover, forest cover, and shrub patches within their home range and negatively with the distance to wetlands. Areas of wetlands and fallow land might be used for foraging since Amur Falcons mostly depend on an insect diet. Additionally, we found that rarely burned habitats were preferred. Overall, the effect of landscape variables on the choice of actual nest sites appeared to be rather small. We used different classification methods to predict the probability of occurrence, of which the Random forest method showed the highest accuracy. The areas determined as suitable habitat showed a high concordance with the actual nest locations. We conclude that Amur Falcons prefer to occupy newly built (domed) nests to ensure high nest quality, as well as nests surrounded by available feeding habitats.