Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability

Bryan S. Der, Ron Jacak, Brian Kuhlman, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of AmericaChristien Kluwe, Aleksandr E. Miklos, Andrew D. Ellington , Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, United States of AmericaChristien Kluwe, Aleksandr E. Miklos, George Georgiou, Andrew D. Ellington, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, United States of AmericaAleksandr E. Miklos, Andrew D. Ellington , Applied Research Laboratories, University of Texas at Austin, Austin, Texas, United States of AmericaSergey Lyskov, Jeffrey J. Gray, Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of AmericaBrian Kuhlman, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of AmericaReengineering protein surfaces to exhibit high net charge, referred to as “supercharging”, can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from −11 to −61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and −30 net charge. Mid-charge variants demonstrated ~3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding.This work was supported by the Defense Advanced Research Projects Agency (HR-0011-10-1-0052 to A.E.) and the Welch Foundation (F-1654 to A.E.), the National Institutes of Health grants GM073960 (B.K.) and R01-GM073151 (J.G. and S.L.), the Rosetta Commons (S.L.), the National Science Foundation graduate research fellowship (2009070950 to B.D.), the UNC Royster Society Pogue fellowship (B.D.), and National Institutes of Health grant T32GM008570 for the UNC Program in Molecular and Cellular Biophysics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Center for Systems and Synthetic BiologyCellular and Molecular BiologyApplied Research LaboratoriesEmail: [email protected]

Non-pharmacological treatment-related changes of molecular biomarkers in major depressive disorder: A systematic review and meta-analysis

Background Major depressive disorder (MDD) is a serious mood disorder and leading cause of disability. Despite treatment advances, approximately 30% of individuals with MDD do not achieve adequate clinical response. Better understanding the biological mechanism(s) underlying clinical response to specific psychopharmacological interventions may help fine tune treatments in order to further modulate their underlying mechanisms of action. However, little is known regarding the effect of non-pharmacological treatments (NPTs) on candidate molecular biomarker levels in MDD. This review aims to identify molecular biomarkers that may elucidate NPT response for MDD. Methods We performed a systematic review and a multilevel linear mixed-effects meta-analyses, and a meta-regression. Searches were performed in PubMed, Scopus, and PsycINFO in October 2020 and July 2021. Results From 1387 retrieved articles, 17 and six studies were included in the systematic review and meta-analyses, respectively. Although there was little consensus associating molecular biomarker levels with symptomology and/or treatment response, brain metabolites accessed via molecular biomarker-focused neuroimaging techniques may provide promising information on whether an individual with MDD would respond positively to NPTs. Furthermore, non-invasive brain stimulation interventions significantly increased the expression of neurotrophic factors (NTFs) compared to sham/placebo, regardless of add-on pharmacological treatment. Conclusions NTFs are candidate biomarkers to fine-tune NIBS for MDD treatment.publishe

Factors Influencing Brazilian sugar and ethanol refineries’ failure

Purpose: This study aims to examine the dimensions of organisational failure in the Brazilian sugarcane and ethanol refineries, as reported in judicial recovery plans. Design/methodology/approach: This paper follows a qualitative, inductive approach that uses content analysis to examine the details of recovery plans. Besides, a cause-and-effect relationship diagram is proposed, making it possible to identify the interconnections between the identified variables. Findings: There is evidence that organisational failures are not a linear outcome. Organisational failures are complex and occur because of several factors, often interdependent and operating at different levels. Research implications: Organisational failures basically have three interrelated levels: the macro-level (external environment); the meso-level (organisational environment); and the micro-level (associated with the decision-maker). The relationship between these levels is not trivial and involves coordinated research efforts. Practical implications: Organisations must consider all types of failure levels when developing business reorganisation plans. Reorganisation plans are more than a formal document to achieve judicial recovery, as they should incorporate strategic factors. Social implications: Failures are a regularity in organisations’ day-to-day. Understanding failure’s sources is vital to design firms’ strategies and public policies. Originality/value: The study of organisational failure involves the analysis of complex and multidimensional phenomena. Judicial recovery plans are the means for companies to get a second chance. To that end, this article addresses the sources of organisational failures through the lens of judicial plans

Atlas de otorrinolaringologia

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Neurofibromatosis

Neurofibromatosis (NF) is one of the most common genetic disorders. Inherited in an autosomal dominant fashion, this phacomatosis is classified into two genetically distinct subtypes characterized by multiple cutaneous lesions and tumors of the peripheral and central nervous system. Neurofibromatosis type 1 (NF1), also referred to as Recklinghausen's disease, affects about 1 in 3500 individuals and presents with a variety of characteristic abnormalities of the skin and the peripheral nervous system. Neurofibromatosis type 2 (NF2), previously termed central neurofibromatosis, is much more rare occurring in less than 1 in 25 000 individuals. Often first clinical signs of NF2 become apparent in the late teens with a sudden loss of hearing due to the development of bi-or unilateral vestibular schwannomas. In addition NF2 patients may suffer from further nervous tissue tumors such as meningiomas or gliomas. This review summarizes the characteristic features of the two forms of NF and outlines commonalities and distinctions between NF1 and NF2

Cancer Stem Cell-Like Cells Derived from Malignant Peripheral Nerve Sheath Tumors

This study aims to examine whether or not cancer stem cells exist in malignant peripheral nerve sheath tumors (MPNST). Cells of established lines, primary cultures and freshly dissected tumors were cultured in serum free conditions supplemented with epidermal and fibroblast growth factors. From one established human MPNST cell line, S462, cells meeting the criteria for cancer stem cells were isolated. Clonal spheres were obtained, which could be passaged multiple times. Enrichment of stem cell-like cells in these spheres was also supported by increased expression of stem cell markers such as CD133, Oct4, Nestin and NGFR, and decreased expression of mature cell markers such as CD90 and NCAM. Furthermore, cells of these clonal S462 spheres differentiated into Schwann cells, smooth muscle/fibroblast and neurons-like cells under specific differentiation-inducing cultural conditions. Finally, subcutaneous injection of the spheres into immunodeficient nude mice led to tumor formation at a higher rate compared to the parental adherent cells (66% versus 10% at 2.5×105). These results provide evidence for the existence of cancer stem cell-like cells in malignant peripheral nerve sheath tumors

Graduates

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DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance

Klinische Bedeutung der Bestimmung der Bindung von Trijodthyronin an Serumproteine mittels Dextran-Gel-Filtration

Neben den bewährten älteren Verfahren zur Bestimmung des proteingebundenen127Jods und des Radiojodumsatzes hat sich die gleichzeitige Bestimmung des sog. freien und des proteingebundenen Anteils an in vitro mit Serum inkubiertem L-Trijodthyronin-131Jod mittels Dextran-Gel-Filtration klinisch zur Differentialdiagnose von Hyperthyreose und Euthyreose bewährt. Bei Ausnützung der Verdrängung von proteingebundenem L-Trijodthyronin-131Jod durch nichtmarkiertes Hormon und bei Variation der Dextran-Gel-Menge in der Säule bietet die Methode gute Differenzierungsmöglichkeiten auch für die Schilddrüsenfunktionszustände Euthyreose und Hypothyreose. Bei dem Verfahren wird der Patient nicht mit radioaktivem Jod belastet, ein für die Kinderklinik wichtiger Gesichtspunkt. Manche Störfaktoren, die den131Jodspeicherungstest und die Bestimmung des proteingebundenen Jods (PB127I) verfälschen, haben keinen Einfluß auf die mit der Dextran-Gel-Filtration untersuchten Proteinbindungsverhältnisse für L-Trijodthyronin-131Jod. So hat sich das Verfahren für die Untersuchung von Patienten mit operativ oder durch131Jodbehandlung verkleinerten Schilddrüsen, mit endokrinem Exophthalmus und in Fällen mit vorausgegangener Jodgabe, z. B. in Form von Kontrastmitteln, besonders bewährt. Mit der Bestimmung des sog. freien L-Trijodthyronin-131Jods wird ein physiologisch und pathogenetisch wichtiger Parameter der Schilddrüsenfunktion ermittelt. Die klinische Bedeutung der Bestimmung der Bindungs-und Transportverhältnisse für Trijodthyronin mittels Dextran-Gel-Filtration wird diskutiert.In addition to conventional methods of assay of protein bound iodine (PB127I) and of131iodine turnover in the thyroid, the simultaneous determination of socalled free and protein bound 1-triiodothyronine-131I, added in vitro to serum, using dextran gel filtration was found to be clinically helpful for diagnosis of euthyroidism and hyperthyroidism. Employing discharge effects of non-labelled triiodothyronine on protein bound 1-triiodothyronine-131I and varying the amount of dextran gel in the columns, the method provides reasonably good differentiation of euthyroid and hypothyroid states. No radioactive iodine is given to patients during this procedure, a fact of importance for pediatriciens. Some factors, that influence131iodine uptake or PB127I levels, do not disturb protein binding of 1-triiodothyronine-131I as determined by dextran gel filtration. The latter method was found to be especially useful for the examination of patients with surgically, or by therapy with131iodine dissected thyroid glands, with endocrine exophthalmos, and in cases of previous iodine administration (e.g. X-ray procedures). Determination of socalled free 1-triiodothyronine-131I provides information about a factor of physiological and pathogenetical significance, its clinical meaning is discussed