bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein Production for Biotechnology
Authors: Korajczyk Peter J , Zerbs Sarah , Collart Frank R 
Published: October 2017

Abstract

Purified proteins are useful as experimental tools, commercial products and therapeutics for the treatment of disease. The benefits of these products are dependent on specific qualities such as protein functionality, yield and purity. To meet these protein production criteria, a vast repertoire of constructs, genetic tools, methods and host organisms has been developed to enable multiple approaches for the production of diverse protein products. Consideration of the advantages and limitations of various heterologous expression systems is necessary to ensure that the resulting proteins will be useful for the specific needs of the intended project. Multiple approaches are often evaluated to accommodate the wide diversity in protein characteristics and functions. A systematic approach is outlined for evaluation of protein properties incorporating the requirements for the intended use of protein production to provide a reliable strategy for the selection of appropriate system(s) for large‐scale protein production.

Key Concepts

Controlled production of purified proteins is necessary for scientific research and has many therapeutic and commercial applications.

The intended use of a protein product strongly influences the methods selected for protein production.

Each biological expression system has unique advantages, disadvantages and requirements for protein production.

Multiple technological approaches and hosts may be screened to meet project requirements.

In vitro
expression methods allow for the systematic development of proteins with unique modifications or properties.

A large resource of technological materials in support of protein production enables the generation of products with substantial economic and scientific interest.
Single‐Step Method of Total
RNA
Isolation by Guanidine–Phenol Extraction
Authors: Chomczynski Piotr , Wilfinger William , Mackey Karol 
Published: October 2017

Abstract

The original single‐step method is the first procedure to isolate purified total ribonucleic acid (RNA) from a variety of sources including tissues and cells from human, animal, plant, yeast, bacterial and viral origins, without the requirement of high‐speed ultracentrifugation. This method is based on liquid‐phase separation resulting in sequestration of pure RNA into the aqueous phase. RNA is precipitated from the aqueous phase, dissolved, reprecipitated and washed with alcohol before the final solubilisation step. The entire procedure can be completed in less than 4 h and it provides RNA that is suitable for many sensitive downstream applications such as RNase protection assays, northern blotting, RNA sequencing studies and reverse transcription‐polymerase chain reaction. This pioneering methodology has served as the impetus for the development of newer and improved RNA extraction methodology that now enables investigators to extract and purify RNA in less than 60 min. Some of these newer methods do not require a halogenated organic solvent, or combine the single‐step method with column purification of the RNA.

Key Concepts

Enzymes within living cells rapidly degrade RNA after a tissue sample is removed from the donor. To prevent RNA degradation, tissue samples that cannot be immediately processed must be rapidly frozen with dry ice or liquid nitrogen and stored frozen at −80°C until the RNA can be extracted.

At the time of RNA extraction, frozen cells or tissues must be immersed in the denaturing solution and rapidly homogenised before the tissue thaws in order to inactivate RNAse and avoid RNA degradation.

The quality of the recovered RNA is dependent on a delicate balance of salt concentration and optimal pH. Overloading the extraction solution with too much tissue or diluting the denaturing solution beyond what is specified in the protocol will impact the quality of the resulting RNA.

The extraction of RNA from samples that have a high buffering capacity, such as blood, plasma or tissue culture medium, requires greater care in order to maintain optimal salt balance and pH control.

Overdrying of the RNA pellets will degrade RNA and impede RNA solubilisation.

RNA should be solubilised at a concentration that will be appropriate for meaningful spectrophotometric quantitation as well as subsequent downstream molecular biology applications.

Enzymes that are involved in RNA degradation are ubiquitous and special care must be taken to avoid RNAse contamination during RNA solubilisation and storage.
Oxidative Phosphorylation System: Nuclear Genes and Genetic Disease
Authors: Sánchez‐Caballero Laura , Baertling Fabian , Nijtmans Leo , Smeitink Jan AM 
Published: October 2017

Abstract

The mitochondrial oxidative phosphorylation (OXPHOS) system is located in the inner mitochondrial membrane, and it is the major energy provider of the cell. It consists of five multiprotein complexes (complex I–V). Defects in one or several of these enzymes cause mitochondrial disorders. Identifying underlying mutations within the nuclear genome has been an arduous task until the widespread use of whole exome sequencing in recent years. Since then, a big effort has been put into deciphering underlying mutations in nuclear genes that directly or indirectly affect the OXPHOS system. This led to the discovery of a great number of new gene defects involved in these multifaceted diseases. These findings not only improved the diagnostic process but also helped to understand the mechanisms behind mitochondrial diseases and laid the groundwork for novel approaches to therapy.

Key Concepts

Mitochondrial diseases are caused by a deficient pyruvate oxidation that involves the Krebs cycle and the oxidative phosphorylation (OXPHOS) system.

The OXPHOS system comprises the four respiratory chain enzymes (complexes I–IV) and complex V, also termed ATP synthase.

Defects in one or several of these enzymes cause mitochondrial disorders and give rise to a broad spectrum of clinical signs and symptoms ranging from mild adult‐onset myopathy to neonatal-onset fatal multisystem disease.

Impairments in OXPHOS functionality can not only be caused by mutations in genes encoding subunits of the respective complex but also by defects in nuclear genes encoding proteins required for OXPHOS complex biogenesis, mitochondrial translation, or mitochondrial redox homeostasis.
Segmental Duplications and Genetic Disease
Authors: Yilmaz Feyza , Shaikh Tamim H , Emanuel Beverly S 
Published: October 2017

Abstract

The human genome contains many different types of repetitive DNA elements that vary by size and copy number. Segmental duplications (SDs) are one such class of repetitive elements that are relatively large in size, have low copy number in the genome and their copies share high levels of sequence identity with each other. These characteristic features of SDs make them excellent substrates for genomic rearrangements, resulting from aberrant recombination between the highly identical copies. Several of the genomic rearrangements mediated by SDs lead to copy number variations of large genomic regions containing many genes. Consequently, SD‐mediated rearrangements are often associated with genetic diseases that manifest as syndromes characterised by multiple congenital anomalies due to dosage imbalance of one or more genes.

Key Concepts

Segmental duplications are repetitive DNA elements in the human genome.

Segmental duplications mediate genomic rearrangements associated with many genomic diseases.

Segmental duplications mediate genomic rearrangements via nonallelic homologous recombination (NAHR), or they can stimulate or promote rearrangements via nonhomologous end‐joining (NHEJ) or replication‐based mechanism (RBM).

Segmental duplications have been associated with both recurrent and nonrecurrent chromosomal rearrangements including microdeletions, microduplications, translocations, inversions and other complex rearrangements.

Segmental duplications have played an important role in the human genome evolution, genetic variation and disease predisposition.
The Genetics of Autism
Authors: Warrier Varun , Baron‐Cohen Simon 
Published: October 2017

Abstract

Autism spectrum conditions (henceforth ‘autism’) refer to a group of neurodevelopmental conditions involving difficulties in social interaction and communication and unusually repetitive and restricted behaviours and interest. Twin and family studies have established a significant heritability for autism. Autism is polygenic with variations across the allele frequency spectrum contributing to risk. Early linkage and candidate gene association studies were statistically underpowered to identify significant loci. Current genome‐wide association studies have identified significant positive genetic correlation between autism and various measures of cognition. The use of genetic microarrays and next‐generation DNA sequencing has identified tens of genes and copy number variants associated with autism. In addition, RNA microarray and sequencing studies of postmortem brain samples have identified transcriptionally altered genes and pathways in autism. Multiple lines of evidence converge on altered glial, synaptic and chromatin pathways as contributing to autism risk.

Key Concepts

Autism spectrum conditions (autism) represent a group of genetically and phenotypically heterogeneous neurodevelopmental conditions.

Autism is highly heritable as evidenced by twin studies, familial recurrence and molecular genetic analyses.

Autism is highly polygenic with common genetic variation, rare genetic variation and structural variation contributing to genetic risk.

Human DNA microarray and next‐generation sequencing of large cohorts have identified tens of associated genes and genetic loci.

Genome‐wide association studies point to significant heritability and genetic correlation with measures of cognition.

RNA sequencing and microarray studies have identified transcriptionally dysregulated genes in the autism postmortem cortex.

Pathway analysis across different genetic data sets identified synaptic and neuronal pathways, chromatin remodelling and a role for microglia and astrocytes.
Biobanking for Genetic Diseases
Authors: Filocamo Mirella , Casareto Lorena , Baldo Chiara 
Published: October 2017

Abstract

Biobanks are bioresources of human samples linked to relevant personal and health data of the participants, which are collected, processed and stored in an organised system. Their primary purpose is to provide biospecimens for researchers to help improve knowledge on human health and diseases, while appropriately protecting the interests, rights and privacy of the participants.
The biobanking community poses a variety of legal, ethical, social and regulatory challenges, particularly involved in genetic diseases, to be overcome because of the sensitive data they store. A crucial avenue for a biobank to build and maintain public trust is therefore through the development of governance mechanisms ensuring that, in pursuing its remit, the biobank acts in compliance with the applicable laws and regulations of the country in which it is located, as well as the international regulations in order to ease the cross‐border sample exchange and the international cooperation of biobanks.

Key Concepts

Biobanks are collections of human samples linked to relevant personal and health data of the participants, organised and managed in a systematic way.

Primary purpose of biobanks is to help improve knowledge on human health and diseases.

Biobanking poses a variety of challenges including ethical, legal and social issues (ELSI).

Because of the preciousness of the samples and the sensitive nature of the data stored in biobanks, proper governance is essential to protecting confidentiality, choices and rights of participants.

As critical components in the field of clinical practice and basic research, biobanks develop standard operating procedures devoted to collect, process, store, retrieve and transfer to researchers a set of biospecimens and associated information.

In the field of rare diseases (RDs), collectively affecting 6–8% of the population, international cooperation among RD‐biobanks is fundamental for sharing and achieving a critical mass of samples and data for basic, translational and clinical research.

To ease an international cooperation, biobanks also need to act in compliance with international regulations.
Vertebrate Embryo: Myogenesis and Muscle Development
Authors: Robson Lesley G 
Published: October 2017

Abstract

The formation and patterning of three types of muscle tissues are found in vertebrates: smooth, cardiac and skeletal. All three muscle types share the property that they contract to move substances around the body (smooth and cardiac) or move and stabilise the skeleton (skeletal). Although there are common signalling pathways utilised by all muscle types, each utilises a unique pathway. The varied origins of myogenic cells and the important role of local signals coordinate the correct migration from their site of origin to final location. The local environmental signals also coordinate proliferation and expansion of myogenic precursors and finally the correct patterning, orientation and differentiation of myogenic cells. It is also evidenced that the environment continues to have an important role in the regenerative and growth of muscle tissue postnatally.

Key Concepts

That often the same signalling pathways are used independent of the phenotype of muscle being produced.

Although many different cell lineages are used to produce smooth muscle, cardiac and skeletal muscles use more specific cell lineages.

The environment that myogenic cells inhabit often helps to specify the early myogenic specification pathway and the later patterning of the differentiating myogenic cells into specific muscle groups and fibre types.

The regenerative capacity in the postnatal muscle tissue is also regulated by the environment.
Alpha‐Synuclein
Authors: Cremades Nunilo 
Published: October 2017

Abstract

α‐Synuclein (αS) is a presynaptic small protein that has attracted much interest because its aggregation and accumulation in the form of amyloid fibrils is the hallmark of a range of neurodegenerative disorders, collectively referred to as synucleinopathies. Despite intense research on this protein since it was first identified two decades ago as the major component of the proteinaceous intracellular inclusion characteristics of Parkinson disease, there is still no consensus on the physiological function of the protein and much remains to be established on the molecular basis of its toxicity. Recently, important steps have been undertaken to identify the different conformational states that this protein is able to adopt and elucidate their role in physiological and pathological conditions.

Key Concepts

The physiological function(s) of α‐synuclein remains controversial, although recent evidences suggest a major regulatory role in synapsis.

At physiological conditions, α‐synuclein is in a dynamic equilibrium between a membrane‐bound α‐helical (likely multimeric) conformation and a cytosolic intrinsically disordered (monomeric) conformation.

α‐Synuclein aggregation and fibril formation likely play a central role in Parkinson disease and other neurodegenerative disorders.

Different strains or fibril polymorphs of α‐synuclein have different degrees of infectivity and might be associated with distinct types of pathologies.

Different mechanisms of formation of α‐synuclein amyloid aggregates have been observed
in vitro
, but their relative relevance
in vivo
remains unknown.

Recent studies support the idea that multiple aggregated species of α‐synuclein can be generated through diverse misfolding pathways during the process of amyloid aggregation and could play distinct roles during the development of disease.

Combined methods to target specifically different α‐synuclein conformations could potentially prevent α‐synuclein‐associated toxicity.
Molecular Genetics of Common Variable Immunodeficiency
Authors: Bogaert Delfien J , Dullaers Melissa , De Baere Elfride , Haerynck Filomeen 
Published: October 2017

Abstract

Common variable immunodeficiency (CVID) is one of the most frequently diagnosed primary immunodeficiencies (PIDs) characterised by antibody deficiency, poor responses to vaccination and an increased susceptibility to infections and complications due to immune dysregulation. The clinical diagnosis of CVID is an umbrella covering an immunologically and genetically heterogeneous group of diseases. To date, 22 disease‐causing genes have been associated with monogenic forms of CVID, encompassing 2–10% of reported cases. However, these genetic subtypes are now considered to account for distinct disease entities and have been removed from under the CVID umbrella. Furthermore, accumulating data suggest that the majority of CVID patients have a complex or multifactorial disorder, in which multiple genetic, epigenetic and/or environmental factors are involved. Genetic testing for monogenic causes should especially be performed in CVID patients with early onset of disease, noninfectious complications, a positive family history and/or consanguineous parents, since this could be important for genetic counselling, follow up and/or treatment decisions.

Key Concepts

CVID is a diagnosis of exclusion, encompassing a clinically, immunologically and genetically heterogeneous group of PID patients.

Monogenic defects have so far been reported in 2–10% of CVID patients.

The majority of CVID patients are believed to have a complex or multifactorial aetiology.

Patients in whom a monogenic cause is identified are no longer classified under CVID, but under separate disease entities (IUIS classification).

A monogenic cause of CVID is more probable in case of early onset of disease, complications of immune dysregulation, a positive family history and/or consanguinity.

Especially in CVID patients with early onset of disease and/or noninfectious complications, genetic workup should be performed to identify or exclude an underlying monogenic cause since this could be important for patient management decisions.
Mechanisms and Roles of Mitophagy
Authors: Vigié Pierre , Bhatia‐Kiššová Ingrid , Camougrand Nadine 
Published: October 2017

Abstract

Mitochondria are highly dynamic organelles that provide essential metabolic functions and represent the major bioenergetic hub of eukaryotic cells. Mitochondrial dysfunctions are implicated in numerous diseases. Therefore, maintenance of a healthy pool of mitochondria is required for cellular function and survival. Mitochondrial quality control is secured by several mechanisms that act at different levels: proteases and chaperones, the ubiquitin‐proteasome system (UPS) and mitophagy. Multiple mitophagy programs either operate independently or undergo crosstalk and require modulated receptor activities in the mitochondrial membranes. In mammals, different mitophagy effectors have been characterised, such as the receptors NIX, BNIP3, FUNDC1, BCL2L13, Prohibitin2, cardiolipin and PINK1/Parkin pathway.

Key Concepts

Mitophagy is a process that degrades mitochondria selectively via autophagy.

Mitophagy operates at the organelle scale.

Proteases, chaperones and ubiquitin‐proteasome system operate at the molecular level.

PINK1/Parkin‐dependent and PINK1/Parkin‐independent pathways regulate mitophagy in mammals.

Receptors localised in mitochondrial membranes play a role in mitophagy.
Genome im Fokus: Entwicklung und Anwendungen von CRISPR‐Cas9‐Bildgebungstechnologien
Authors: Knight Spencer C. , Tjian Robert , Doudna Jennifer A. 
Published: October 2017

AbstractDie Entdeckung der CRISPR‐Cas9‐Endonuklease ermöglicht die einfache Genom‐Editierung von lebenden Zellen und Organismen. Katalytisch deaktivierte Cas9 (dCas9) behält die Fähigkeit, DNA in einer RNA‐abhängigen Weise zu binden, und wurde zusätzlich als ein Werkzeug für die Transkriptionsmodulation, die Epigenom‐Editierung und die genomische Bildgebung entwickelt. Dieser Aufsatz zeigt die jüngsten Fortschritte und Herausforderungen bei der Entwicklung von dCas9 für die Bildgebung genomischer Loci auf. Die Entstehung und Weiterentwicklung dieser Technologie bietet das Potenzial, neue mechanistische Fragen über die Chromosomendynamik und die dreidimensionale Genomorganisation in vivo zu beantworten.
Thioamide‐Directed Cobalt(III)‐Catalyzed Selective Amidation of C(sp3)−H Bonds
Authors: Tan Peng Wen , Mak Adrian M. , Sullivan Michael B. , Dixon Darren J. , Seayad Jayasree 
Published: October 2017

AbstractA mild, oxidant‐free, and selective Cp*CoIII‐catalyzed amidation of thioamides with robust dioxazolone amidating agents via C(sp3)−H bond activation to generate the desired amidated products is reported. The method is efficient and allows for the C−H amidation of a wide range of functionalized thioamides with aryl‐, heteroaryl‐, and alkyl‐substituted dioxazolones under the Cp*CoIII‐catalyzed conditions. The observed regioselectivity towards primary C(sp3)−H activation is supported by computational studies and the cyclometalation is proposed to proceed by means of an external carboxylate‐assisted concerted metalation/deprotonation mechanism. The reported method is a rare example of the use of a directing group other than the commonly used pyridine and quinolone classes for Cp*CoIII‐catalyzed C(sp3)−H functionalization and the first to exploit thioamides.
Identification of key residues modulating the stereoselectivity of nitrile hydratase toward rac‐mandelonitrile by semi‐rational engineering
Authors: Cheng Zhongyi , Peplowski Lukasz , Cui Wenjing , Xia Yuanyuan , Liu Zhongmei , Zhang Jialei , Kobayashi Michihiko , Zhou Zhemin 
Published: October 2017

AbstractOptically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high‐value chiral chemicals is often time‐, money‐, and resource‐intensive; traditional semi‐rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot‐spot residues. In this study, through ligand‐protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity toward rac‐mandelonitrile (MAN). All the four selected residues had numerous collisions with rac‐MAN. Five mutants significantly shifting stereoselectivity towards (S)‐MAN were obtained from site‐saturation mutagenesis, one of them, at position βPhe37, exhibiting efficient production of (S)‐MAN with 96.8% eep, was isolated and further analyzed. The increased pulling force observed during SMD simulation was found to be in good coincidence with the formation of hydrogen bonds between (R)‐MAN and residue βHis37. (R)‐MAN had to break these barriers to enter the active site of nitrile hydratase and S selectivity was thus improved. The results indicated that combining steered molecular dynamics simulation with a traditional semi‐rational design significantly reduced the select range of hot‐spot residues for the evolution of NHase stereoselectivity, which could serve as an alternative for the modulation of enzyme stereoselectivity.