Cover Picture: Biotechnology Journal 5/2016

This regular issue of BTJ includes articles on biocatalysis, biomaterials, and industrial biotechnology. The cover picture shows several aspects of bacteriophage therapy, including phages adsorbing to bacterial cell surfaces, commercial preparations Pyo‐ and Intesi‐phages that are currently produced and used in Georgia, and administration of phages by medical staff to a patient. Image is provided by Aleksandre Ujmajuridze, Jon Caplin, Nino Chanishvili, and Aidan Coffey authors of ”Silk route to the acceptance and re‐implementation of bacteriophage therapy“ ( http://dx.doi.org/10.1002/biot.201600023 ).     

Cover Picture: Biotechnology Journal 11/2012

This “regular” issue of Biotechnology Journal gathers the state‐of‐the‐art in biotechnology, with articles on bioenergy, biofuels, medical biotechnology, biomaterials, etc. Image: © Sergej Khackimullin – Fotolia.com.     

Urinary biomarkers for renal tract malformations

Introduction: Renal tract malformations (RTMs) are congenital anomalies of the kidneys and urinary tract, which are the major cause of end-stage renal disease in children. Using immunoassay-based approaches (ELISA, western blot), individual urinary proteins including transforming growth factor β, tumor necrosis factor and monocyte attractant proteins 1 were found to be associated to RTMs. However, only mass spectrometry (MS) based methods leading to the identification of panels of protein-based markers composed of fragments of the extracellular matrix allowed the prediction of progression of RTMs and its complications.

Areas covered: In this review, we summarized relevant studies identified in “Pubmed” using the keywords “urinary biomarkers” and “proteomics” and “renal tract malformations” or “hydronephrosis” or “ureteropelvic junction obstruction” or “posterior urethral valves” or “vesicoureteral reflux”. These publications represent studies on potential protein-based biomarkers, either individually or combined in panels, of RTMs in human and animal models.

Expert commentary: Successful use in the clinic of these protein-based biomarkers will need to involve larger scale studies to reach sufficient power. Improved performance will potentially come from combining immunoassay- and MS-based markers.     

Letter to the Editor

Regarding Paper “Stratified Fisher's exact test and its sample size calculation” by Sin‐Ho Jung Biometrical Journal (2014) 56 (1): 129–140 Article: http://dx.doi.org/10.1002/bimj.201300048     

The phosphoproteome of Aspergillus nidulans reveals functional association with cellular processes involved in morphology and secretion

We describe the first phosphoproteome of the model filamentous fungus Aspergillus nidulans. Phosphopeptides were enriched using titanium dioxide, separated using a convenient ultra‐long reverse phase gradient, and identified using a “high‐high” strategy (high mass accuracy on the parent and fragment ions) with higher‐energy collisional dissociation. Using this approach 1801 phosphosites, from 1637 unique phosphopeptides, were identified. Functional classification revealed phosphoproteins were overrepresented under GO categories related to fungal morphogenesis: “sites of polar growth,” “vesicle mediated transport,” and “cytoskeleton organization.” In these same GO categories, kinase‐substrate analysis of phosphoproteins revealed the majority were target substrates of CDK and CK2 kinase families, indicating these kinase families play a prominent role in fungal morphogenesis. Kinase‐substrate analysis also identified 57 substrates for kinases known to regulate secretion of hydrolytic enzymes (e.g. PkaA, SchA, and An‐Snf1). Altogether this data will serve as a benchmark that can be used to elucidate regulatory networks functionally associated with fungal morphogenesis and secretion. All MS data have been deposited in the ProteomeXchange with identifier PXD000715 ( http://proteomecentral.proteomexchange.org/dataset/PXD000715 ).     

Transcriptome analysis reveals the difference between “healthy” and “common” aging and their connection with age‐related diseases

A key goal of aging research was to understand mechanisms underlying healthy aging and develop methods to promote the human healthspan. One approach is to identify gene regulations unique to healthy aging compared with aging in the general population (i.e., “common” aging). Here, we leveraged Genotype‐Tissue Expression (GTEx) project data to investigate “healthy” and “common” aging gene expression regulations at a tissue level in humans and their interconnection with diseases. Using GTEx donors' disease annotations, we defined a “healthy” aging cohort for each tissue. We then compared the age‐associated genes derived from this cohort with age‐associated genes from the “common” aging cohort which included all GTEx donors; we also compared the “healthy” and “common” aging gene expressions with various disease‐associated gene expressions to elucidate the relationships among “healthy,” “common” aging and disease. Our analyses showed that 1. GTEx “healthy” and “common” aging shared a large number of gene regulations; 2. Despite the substantial commonality, “healthy” and “common” aging genes also showed distinct function enrichment, and “common” aging genes had a higher enrichment for disease genes; 3. Disease‐associated gene regulations were overall different from aging gene regulations. However, for genes regulated by both, their regulation directions were largely consistent, implying some aging processes could increase the susceptibility to disease development; and 4. Possible protective mechanisms were associated with some “healthy” aging gene regulations. In summary, our work highlights several unique features of GTEx “healthy” aging program. This new knowledge could potentially be used to develop interventions to promote the human healthspan.     

Glucosyltransferase‐dependent and ‐independent effects of TcdB on the proteome of HEp‐2 cells

Toxin B (TcdB) of the nosocomial pathogen C. difficile has been reported to exhibit a glucosyltransferase‐dependent and ‐independent effect on treated HEp‐2 cells at toxin concentration above 0.3 nM. In order to investigate and further characterize both effects epithelial cells were treated with wild type TcdB and glucosyltransferase‐deficient TcdB_NXN and their proteomes were analyzed by LC‐MS. Triplex SILAC labeling was used for quantification. Identification of 5212 and quantification of 4712 protein groups was achieved. Out of these 257 were affected by TcdB treatment, 92 by TcdB_NXN treatment and 49 by both. TcdB mainly led to changes in proteins that are related to “GTPase mediated signaling” and the “cytoskeleton” while “chromatin” and “cell cycle” related proteins were altered by both, TcdB and TcdB_NXN. The obtained dataset of HEp‐2 cell proteome helps us to better understand glucosyltransferase‐dependent and ‐independent mechanisms of TcdB and TcdB_NXN, particularly those involved in pyknotic cell death. All proteomics data have been deposited in the ProteomeXchange with the dataset identifier PXD006658 ( https://proteomecentral.proteomexchange.org/dataset/PXD006658 ).     

CYP2E1 Rsa Ι/Pst Ι polymorphism and lung cancer susceptibility: A meta‐analysis involving 10,947 subjects

The above article, published in the Journal of Cellular and Molecular Medicine on 14 September 2016 in Wiley Online Library (wileyonlinelibrary.com), and in Volume 19, pp. 2136‐2142, has been retracted by agreement between the authors, the journal Editor in Chief, Stefan Constantinescu, and John Wiley & Sons Ltd. The retraction has been agreed due to unattributed overlap of the language used in the “Materials and method” and “Discussion” sections of this study and the following article published in Lung Cancer: “CYP2E1 Rsa I/Pst I polymorphism is associated with lung cancer risk among Asians” by Ping Zhan, Jing Wang, Yu Zhang, Li‐Xin Qiu, Su‐feng Zhao, Qian Qian, Shu‐Zhen Wei, Li‐Ke Yu and Yong Song, Volume 69, 2010, pages 19‐25. REFERENCE Shen Z‐T, Wu X‐H, Li B, Shen J‐S, Wang Z, Li J, Zhu X‐X. CYP2E1 Rsa Ι/Pst Ι polymorphism and lung cancer susceptibility: a meta‐analysis involving 10,947 subjects. J Cell Mol Med. 2015;19:2136‐2142. https://doi.org/10.1111/jcmm.12579     

Letter to the Editor

Regarding Paper “Sample size determination in clinical trials with multiple co‐primary endpoints including mixed continuous and binary variables” by T. Sozu , T. Sugimoto , and T. Hamasaki Biometrical Journal (2012) 54 (5): 716–729 Article: http://dx.doi.org/10.1002/bimj.201100221 Authors' Reply: http://dx.doi.org/10.1002/bimj.201300032 This paper recently introduced a methodology for calculating the sample size in clinical trials with multiple mixed binary and continuous co‐primary endpoints modeled by the so‐called conditional grouped continuous model (CGCM). The purpose of this note is to clarify certain aspects of the methodology and propose an alternative approach based on latent means tests for the binary endpoints. We demonstrate that our approach is more powerful, yielding smaller sample sizes at powers comparable to those used in the paper.     

Cover Picture: Biotechnology Journal 5/2017

The cover shows a cartoon of wave motion that provides an ideal hydrodynamic environment to induce cell‐cell collision and subsequent aggregation of human mesenchymal stem cells with controlled aggregate size in suspension. The cover is prepared by Ang‐Chen Tsai, Yijun Liu, Xuegang Yuan, Ravindran Chella and Teng Ma authors of the article ”Aggregation kinetics of human mesenchymal stem cells under wave motion“ ( https://doi.org/10.1002/biot.201600448 ).     

A conserved threonine spring‐loads precursor for intein splicing

Protein splicing is an autocatalytic process where an “intein” self‐cleaves from a precursor and ligates the flanking N‐ and C‐“extein” polypeptides. Inteins occur in all domains of life and have myriad uses in biotechnology. Although the reaction steps of protein splicing are known, mechanistic details remain incomplete, particularly the initial peptide rearrangement at the N‐terminal extein/intein junction. Recently, we proposed that this transformation, an N‐S acyl shift, is accelerated by a localized conformational strain, between the intein's catalytic cysteine (Cys1) and the neighboring glycine (Gly‐1) in the N‐extein. That proposal was based on the crystal structure of a catalytically competent trapped precursor. Here, we define the structural origins and mechanistic relevance of the conformational strain using a combination of quantum mechanical simulations, mutational analysis, and X‐ray crystallography. Our results implicate a conserved, but largely unstudied, threonine residue of the Ssp DnaE intein (Thr69) as the mediator of conformational strain through hydrogen bonding. Further, the strain imposed by this residue is shown to position the splice junction in a manner that enhances the rate of the N‐S acyl shift substantially. Taken together, our results not only provide fundamental understanding of the control of the first step of protein splicing but also have important implications in various biotechnological applications that require precursor manipulation.     

Cover Picture: Biotechnology Journal 8/2017

Thaw what is frozen: it is generally accepted that the initial genetic code evolved from an ambiguous to a well‐defined (”frozen“) code with the repertoire of 20 (+2) canonical amino acids. If code is perfectly optimized by evolution, is it then possible to change it experimentally? If so, what are the barriers to overcome? Kubyshkin and Budisa have tried to answer this question by building a model that predicts ”entry points“ for invading the code with noncanonic amino acids. These experiments should lead to a chemical alienation of cells which represent an important step towards the creation of artificial life. The cover is prepared by Vladimir Kubyshkin and Nediljko Budisa authors of the article ”Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?” ( https://doi.org/10.1002/biot.201600097 ).     

Recent advances in exploiting ionic liquids for biomolecules: Solubility,stability and applications

The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) – potentially attractive ”green“ and ”designer“ solvents – rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule‐based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein‐, enzyme‐, and DNA‐based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule‐friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL‐based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule‐based applications.     

Progress in scaffold‐free bioprinting for cardiovascular medicine

Biofabrication of tissue analogues is aspiring to become a disruptive technology capable to solve standing biomedical problems, from generation of improved tissue models for drug testing to alleviation of the shortage of organs for transplantation. Arguably, the most powerful tool of this revolution is bioprinting, understood as the assembling of cells with biomaterials in three‐dimensional structures. It is less appreciated, however, that bioprinting is not a uniform methodology, but comprises a variety of approaches. These can be broadly classified in two categories, based on the use or not of supporting biomaterials (known as “scaffolds,” usually printable hydrogels also called “bioinks”). Importantly, several limitations of scaffold‐dependent bioprinting can be avoided by the “scaffold‐free” methods. In this overview, we comparatively present these approaches and highlight the rapidly evolving scaffold‐free bioprinting, as applied to cardiovascular tissue engineering.     

Cover Picture: Biotechnology Journal 10/2016

This regular issue of BTJ includes articles on analytical biotechnology, industrial biotechnology and metabolic engineering. The cover is inspired by the article "Chlorella species as hosts for genetic engineering and expression of heterologous proteins: Progress, challenge and perspective" by Bo Yang, Jin Liu, Yue Jiang and Feng Chen which is describing the heterologous proteins expressed in unicellular Chlorella species combined with the production of fine value‐added bioactives ( http://dx.doi.org/10.1002/biot.201500617 ).     

Cover Picture: Biotechnology Journal 4/2017

Poly(ethylene glycol)‐based polymers are used as carbon sources to re‐program the skin microbiome via specifically induction of fermentation of skin commensal Staphylococcus epidermidis bacteria. The production of short‐chain fatty acids by this fermentation diminishes the colonization of methicillin‐resistant Staphylococcus aureus. The cover is prepared by Ming‐Shan Kao, Stephen Huang, Wei‐Lin Chang, Ming‐Fa Hsieh, Chun‐Jen Huang, Richard L. Gallo and Chun‐Ming Huang authors of the article ”Microbiome precision editing: Using PEG as a selective fermentation initiator against methicillin‐resistant Staphylococcus aureus“ ( https://doi.org/10.1002/biot.201600399 ).     

Protective Cellular Mechanism of Estrogen Against Kidney Stone Formation: A Proteomics Approach and Functional Validation

Females have less incidence/prevalence of kidney stone disease than males. Estrogen thus may serve as the protective factor but with unclear mechanism. This study explores cellular mechanism underlying such stone preventive mechanism of estrogen. Madin darby canine kidney (MDCK) renal tubular cells are incubated with or without 20 nm 17β‐estradiol for 7 days. Comparative proteomics reveals 58 differentially expressed proteins in estrogen‐treated versus control cells that are successfully identified by nanoLC–ESI–Q‐TOF‐MS/MS. Interestingly, these altered proteins are involved mainly in “binding and receptor,” “metabolic process,” and “migration and healing” networks. Functional investigations demonstrate reduction of calcium oxalate (CaOx) crystal‐binding capability of the estrogen‐treated cells consistent with the decreased levels of annexin A1 and α‐enolase (the known CaOx crystal‐binding receptors) on the cell surface. High‐calcium and high‐oxalate challenge initially enhances surface expression of annexin A1 and α‐enolase, respectively, both of which return to their basal levels by estrogen. Additionally, estrogen reduces intracellular ATP level and promotes cell migration and tissue healing. Taken together, estrogen causes changes in cellular proteome of renal tubular cells that lead to decreased surface expression of CaOx crystal receptors, reduced intracellular metabolism, and enhanced cell proliferation and tissue healing, all of which may contribute, at least in part, to stone prevention.     

A critique of the “ultra‐high risk” and “transition” paradigm

The transdiagnostic expression of psychotic experiences in common mental disorder (anxiety/depression/substance use disorder) is associated with a poorer prognosis, and a small minority of people may indeed develop a clinical picture that meets criteria for schizophrenia. However, it appears neither useful nor valid to observe early states of multidimensional psychopathology in young people through the “schizo”‐prism, and apply misleadingly simple, unnecessary and inefficient binary concepts of “risk” and “transition”. A review of the “ultra‐high risk” (UHR) or “clinical high risk” (CHR) literature indicates that UHR/CHR samples are highly heterogeneous and represent individuals diagnosed with common mental disorder (anxiety/depression/substance use disorder) and a degree of psychotic experiences. Epidemiological research has shown that psychotic experiences are a (possibly non‐causal) marker of the severity of multidimensional psychopathology, driving poor outcome, yet notions of “risk” and “transition” in UHR/CHR research are restrictively defined on the basis of positive psychotic phenomena alone, ignoring how baseline differences in multidimensional psychopathology may differentially impact course and outcome. The concepts of “risk” and “transition” in UHR/CHR research are measured on the same dimensional scale, yet are used to produce artificial diagnostic shifts. In fact, “transition” in UHR/CHR research occurs mainly as a function of variable sample enrichment strategies rather than the UHR/CHR “criteria” themselves. Furthermore, transition rates in UHR/CHR research are inflated as they do not exclude false positives associated with the natural fluctuation of dimensional expression of psychosis. Biological associations with “transition” thus likely represent false positive findings, as was the initial claim of strong effects of omega‐3 polyunsatured fatty acids in UHR samples. A large body of UHR/CHR intervention research has focused on the questionable outcome of “transition”, which shows lack of correlation with functional outcome. It may be more productive to consider the full range of person‐specific psychopathology in all young individuals who seek help for mental health problems, instead of “policing” youngsters for the transdiagnostic dimension of psychosis. Instead of the relatively inefficient medical high‐risk approach, a public health perspective, focusing on improved access to a low‐stigma, high‐hope, small scale and youth‐specific environment with acceptable language and interventions may represent a more useful and efficient strategy.