Dissecting the energy metabolism in Mycoplasma pneumoniae through genome‐scale metabolic modeling

Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint‐based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time‐dependent changes, albeit using a static model. By performing an in silico knock‐out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.     

Requirements for a minimum standard of care for phenylketonuria: the patients’ perspective

Phenylketonuria (PKU, ORPHA716) is an inherited disorder that affects about one in every 10,000 children born in Europe. Early and continuous application of a modified diet is largely successful in preventing the devastating brain damage associated with untreated PKU. The management of PKU is inconsistent: there are few national guidelines, and these tend to be incomplete and implemented sporadically. In this article, the first-ever pan- European patient/carer perspective on optimal PKU care, the European Society for Phenylketonuria and Allied Disorders (E.S.PKU) proposes recommendations for a minimum standard of care for PKU, to underpin the development of new pan-European guideline for the management of PKU. New standards of best practice should guarantee equal access to screening, treatment and monitoring throughout Europe. Screening protocols and interpretation of screening results should be standardised. Experienced Centres of Expertise are required, in line with current European Union policy, to guarantee a defined standard of multidisciplinary treatment and care for all medical and social aspects of PKU. Women of childbearing age require especially intensive management, due to the risk of severe risks to the foetus conferred by uncontrolled PKU. All aspects of treatment should be reimbursed to ensure uniform access across Europe to guideline-driven, evidence-based care. The E.S.PKU urges PKU healthcare professionals caring for people with PKU to take the lead in developing evidence based guidelines on PKU, while continuing to play an active role in serving as the voice of patients and their families, whose lives are affected by the condition.     

Darkfield Adapter for Whole Slide Imaging: Adapting a Darkfield Internal Reflection Illumination System to Extend WSI Applications

We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.     

Overcoming Electrode‐Induced Losses in Organic Solar Cells by Tailoring a Quasi‐Ohmic Contact to Fullerenes via Solution‐Processed Alkali Hydroxide Layers

It is shown that the performance of inverted organic solar cells can be significantly improved by facilitating the formation of a quasi‐ohmic contact via solution‐processed alkali hydroxide (AOH) interlayers on top of n‐type metal oxide (aluminum zinc oxide, AZO, and zinc oxide, ZnO) layers. AOHs significantly reduce the work function of metal oxides, and are further proven to effectively passivate defect states in these metal oxides. The interfacial energetics of these electron collecting contacts with a prototypical electron acceptor (C₆₀) are investigated to reveal the presence of a large interface dipole and a new interface state between the Fermi energy and the C₆₀ highest occupied molecular orbital for AOH‐modified AZO contacts. These novel interfacial gap states are a result of ground‐state electron transfer from the metal hydroxide‐functionalized AZO contact to the adsorbed molecules, which are hypothesized to be electronically hybridized with the contact. These interface states tail all the way to the Fermi energy, providing for a highly n‐doped (metal‐like) interfacial molecular layer. Furthermore, the strong “light‐soaking” effect is no longer observed in devices with a AOH interface.     

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.     

Reasons for Journal Impact Factor Changes: Influence of Changing Source Items

Both the concept and the application of the impact factor (IF) have been subject to widespread critique, including concerns over its potential manipulation. This study provides a systematic analysis of significant journal Impact Factor changes, based on the relative contribution of either one or both variables of the IF equation (i.e. citations / articles as the numerator / denominator of the quotient). A cohort of JCR-listed journals which faced the most dramatic absolute IF changes between 2013 and 2014 (ΔIF ≥ 3.0, n = 49) was analyzed for the causes resulting in IF changes that theses journals have experienced in the last five years. Along with the variation by number of articles and citations, this analysis includes the relative change of both variables compared to each other and offers a classification of `valid`and `invalid`scenarios of IF variation in terms of the intended goal of the IF to measure journal quality. The sample cohort features a considerable incidence of IF increases (18%) which are qualified as `invalid`according to this classification because the IF increase is merely based on a favorably changing number of articles (denominator). The results of this analysis point out the potentially delusive effect of IF increases gained through effective shrinkage of publication output. Therefore, careful consideration of the details of the IF equation and possible implementation of control mechanisms versus the volatile factor of number of articles may help to improve the expressiveness of this metric.     

Clinical Evaluation of an Affordable Qualitative Viral Failure Assay for HIV Using Dried Blood Spots in Uganda

Background

WHO recommends regular viral load (VL) monitoring of patients on antiretroviral therapy (ART) for timely detection of virological failure, prevention of acquired HIV drug resistance (HIVDR) and avoiding unnecessary switching to second-line ART. However, the cost and complexity of routine VL testing remains prohibitive in most resource limited settings (RLS). We evaluated a simple, low–cost, qualitative viral–failure assay (VFA) on dried blood spots (DBS) in three clinical settings in Uganda.

Methods

We conducted a cross–sectional diagnostic accuracy study in three HIV/AIDS treatment centres at the Joint Clinical Research Centre in Uganda. The VFA employs semi-quantitative detection of HIV–1 RNA amplified from the LTR gene. We used paired dry blood spot (DBS) and plasma with the COBASAmpliPrep/COBASTaqMan, Roche version 2 (VL_ref) as the reference assay. We used the VFA at two thresholds of viral load, (>5,000 or >1,000 copies/ml).

Results

496 paired VFA and VL_ref results were available for comparative analysis. Overall, VFA demonstrated 78.4% sensitivity, (95% CI: 69.7%–87.1%), 93% specificity (95% CI: 89.7%–96.4%), 89.3% accuracy (95% CI: 85%–92%) and an agreement kappa = 0.72 as compared to the VL_ref. The predictive values of positivity and negativity among patients on ART for >12 months were 72.7% and 99.3%, respectively.

Conclusions

VFA allowed 89% of correct classification of VF. Only 11% of the patients were misclassified with the potential of unnecessary or late switch to second–line ART. Our findings present an opportunity to roll out simple and affordable VL monitoring for HIV–1 treatment in RLS.