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Veterinary drug residues in food-producing animals pose a threat to human health and public safety, and accurate measurement of these drug residues is necessary. However, detecting nitrofuran metabolite residues is challenging because of their low molecular weight and the long derivatisation time required for their indirect detection. Current antibodies produced against these metabolites have limited accuracy and sensitivity owing to the traditional and ineffective hapten design based on the reaction of 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) with nitrobenzaldehyde. In this study, a new antigen strategy was adopted using an original-structure-based hapten design combined with a new carrier protein with immunosynergistic effects to produce a monoclonal antibody that could recognise the metabolite of furaltadone (AMOZ) directly without derivatisation with excellent analytical performance. Finally, a gold-based paper lateral flow immunoassay for AMOZ without derivatisation was established with a visual limit of detection of 5 ng/mL, IC 50 of 0.97 ng/mL and linear range (IC 20 ∼IC 80) of 0.45–2.07 ng/mL. The method was sensitive and accurate, and detection was completed within 10 min. This new antigen design strategy provides a theoretical basis and reference for antibody production of other ultrasmall molecules. [Display omitted] • An antigen strategy of AMOZ was proposed by hapten design and new carrier proteins. • The excellent antibody directly recognizes AMOZ without derivatization. • Effect of EP distribution and spatial structure to antibody production was studied. • This method could be used as a rapid screening method for AMOZ analysis on site. [ABSTRACT FROM AUTHOR]

Abstract: The “Operon paper” by F. Jacob and J. Monod started 50 years of research into understanding how the expression of genes is regulated on a molecular level. Ten years ago, microRNAs (miRNAs) emerged as major regulators of eukaryotic gene expression. Here, I will review the basic principles of gene regulation by miRNAs and how these principles can be linked to insights from the Operon paper. A lot of what is understood about miRNAs required a combination of computational and experimental methods. I will discuss some examples that illustrate the power of this approach. [Copyright &y& Elsevier]

HucR is a MarR family protein of Deinococcus radiodurans , which binds tightly to the intergenic region of HucR and the uricase gene to inhibit their expression. Urate (or uric acid) antagonizes the repressor function of HucR by binding to HucR to impede its association with the cognate DNA. The previously reported crystal structure of HucR was without the bound urate showing significant structural homology to other MarR structures. In this paper, we report the crystal structure of HucR determined with the urate bound. However, despite the fact that the urate is found at a site well-known to harbor ligands in other MarR family proteins, the overall HucR structure indicates that no significant change in structure takes place with the urate bound. Structure analysis further suggests that the urate interaction in HucR is mediated by histidine/glutamate side chains and ordered water molecules stabilized by various residues. Such interaction is quite unique compared to other known structural interactions between urate and its binding proteins. Furthermore, structural comparison of the apo- and the urate bound forms allows us to hypothesize that the Trp20-mediated water network in the apo-form stabilizes the proper HucR fold for cognate DNA binding, and that urate binding, also via Trp20, and the consequent reorganization of water molecules in the binding pocket, likely disrupts the DNA binding configuration to result in the attenuated DNA binding. • The MarR family Deinococcus radiodurans HucR acts as a transcriptional repressor. • However, urate (uric acid) antagonizes the repression by releasing the cognate DNA. • Here, we report the crystal structure of HucR with the bound urate molecule. • HucR's specific urate interaction is not observed in other urate binding proteins. • The structure suggests a mechanism for urate-induced attenuation of DNA binding. [ABSTRACT FROM AUTHOR]

In this paper, we study the nonlinear dynamics of the MARCKS protein between cytosol and cytoplasmic membrane through the modulational instability phenomenon. The reaction–diffusion generic model used here is firstly transformed into a cubic complex Ginzburg–Landau equation. Then, modulational instability (MI) is carried out in order to derive the MI criteria. We find the domains of some parameter space where nonlinear patterns are expected in the model. The analytical results on the MI growth rate predict that phosphorylation and binding rates affect MARCKS dynamics in opposite way: while the phosphorylation rate tends to support highly localized structures of MARCKS, the binding rate in turn tends to slow down such features. On the other hand, self-diffusion process always amplifies the MI phenomenon. These predictions are confirmed by numerical simulations. As a result, the cyclic transport of MARCKS protein from membrane to cytosol may be done by means of multisolitons-like patterns. • A one-dimensional model for MARKS proteins is considered here to analytically and numerically study modulational instability. • The complex cubic Ginzburg–Landau equation is derived from the generic model by means of the multiple sacles method. • Multisolitons-like patterns are found to govern the cyclic dynamics of MARCKS from cytoplasmic membrane to cytosol. [ABSTRACT FROM AUTHOR]

The post-infection of COVID-19 includes a myriad of neurologic symptoms including neurodegeneration. Protein aggregation in brain can be considered as one of the important reasons behind the neurodegeneration. SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds to heparin and heparin binding proteins. Moreover, heparin binding accelerates the aggregation of the pathological amyloid proteins present in the brain. In this paper, we have shown that the SARS-CoV-2 S1 RBD binds to a number of aggregation-prone, heparin binding proteins including Aβ, α-synuclein, tau, prion, and TDP-43 RRM. These interactions suggests that the heparin-binding site on the S1 protein might assist the binding of amyloid proteins to the viral surface and thus could initiate aggregation of these proteins and finally leads to neurodegeneration in brain. The results will help us to prevent future outcomes of neurodegeneration by targeting this binding and aggregation process. • SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds to heparin and heparin binding proteins. • Heparin binding accelerates the aggregation of the pathological amyloid proteins present in the brain. • SARS-CoV-2 S1 RBD binds to a number of aggregation-prone, heparin binding proteins including Aβ, α-synuclein, tau, prion, and TDP-43 RRM. • Heparin-binding site on the S1 might assist the binding of amyloid proteins to the viral surface and thus could leads to neurodegeneration in brain. [ABSTRACT FROM AUTHOR]

Morquio B disease is an attenuated phenotype within the spectrum of beta galactosidase (GLB1) deficiencies. It is characterised by dysostosis multiplex, ligament laxity, mildly coarse facies and heart valve defects due to keratan sulphate accumulation, predominantly in the cartilage. Morquio B patients have normal neurological development, setting them apart from those with the more severe GM1 gangliosidosis. Morquio B disease, with an incidence of 1:250.000 to 1:1.000.000 live births, is very rare. Here we report the clinical-biochemical data of nine patients. High amounts of keratan sulfate were detected using LC-MS/MS in the patients' urinary samples, while electrophoresis, the standard procedure of qualitative glycosaminoglycans analysis, failed to identify this metabolite in any of the patients' samples. We performed molecular analyses at gene, gene expression and protein expression levels, for both isoforms of the GLB1 gene, lysosomal GLB1, and the cell-surface expressed Elastin Binding Protein. We characterised three novel GLB1 mutations [c.75 + 2 T > G, c.575A > G (p.Tyr192Cys) and c.2030 T > G (p.Val677Gly)] identified in three heterozygous patients. We also set up a copy number variation assay by quantitative PCR to evaluate the presence of deletions/ insertions in the GLB1 gene. We propose a diagnostic plan, setting out the specific clinical- biochemical and molecular features of Morquio B, in order to avoid misdiagnoses and improve patients' management. • Morquio B is a very rare disease and we here report nine affected patients. • Clinical- biochemical and molecular aspects of Morquio B condition are detailed. • LC/MS-MS is proven the gold standard for the detection of keratan sulfate in urines. • Both beta- galactosidase and elastin binding proteins were evaluated. • We present a general paper and define an accurate diagnostic plan for Morquio B. [ABSTRACT FROM AUTHOR]

Macrobrachium rosenbergii Taihu virus (MrTV) is a virulent pathogen that mainly threatens M. rosenbergii larvae. Rab proteins, which are essential for controlling intracellular membrane trafficking, are hijacked by multiple viruses to complete their life cycle. In this paper, we studied the function of M. rosenbergii Rab1A (MrRab1A) in the MrTV infection. Upon MrTV infection, the transcription level of MrRab1A was significantly up-regulated, indicating MrRab1A was a MrTV responsive gene and might be important for MrTV infection. Co-IP and co-localization assays revealed that MrRab1A could directly bind with MrTV and its capsid protein VP3. Moreover, the in vivo neutralization assay demonstrated that pre-incubation of MrTV with recombinant MrRab1A could partially block MrTV infection. These findings indicated that MrRab1A functioned as a virus-binding protein involved in MrTV infection, which shed new light on the mechanism of MrTV infection and provided a potential target for developing anti-MrTV therapies. • We identified MrRab1A as a binding protein of MrTV for the first time. • The transcription level of MrRab1A was significantly increased upon MrTV infection. • Co-IP and co-localization assays revealed that MrRab1A could bind with MrTV. • Preincubation of MrTV with rMrRab1A could partially block MrTV infection. [ABSTRACT FROM AUTHOR]

Identification of regulatory elements is essential for understanding the mechanism behind regulating gene expression. These regulatory elements—located in or near gene—bind to proteins called transcription factors to initiate the transcription process. Their occurrences are influenced by the GC-content or nucleotide composition. For generating synthetic coding sequences with pre-specified amino acid sequence and desired GC-content, there exist two stochastic methods, multinomial and maximum entropy. Both methods rely on the probability of choosing the codon synonymous for usage in regard to a specific amino acid. In spite the latter exhibited unbiased manner, the produced sequences are not exactly obeying the GC-content constraint. In this paper, we present an algorithmic solution to produce coding sequences that follow exactly a primary amino acid sequence and a desired GC-content. The proposed tool, namely CodSeqGen, depends on random selection for smaller subsets to be traversed using the backtracking approach. • Generating random coding sequence is computationally expensive. • For proteins of length n , we need to test 6 n coding sequences to find those satisfying GC-content constraints. • CodSeqGen uses smart backtracking, and produces synonymous coding sequences with the exact desired GC-contents. • Experiments show CodSeqGen produces sequences with GC-contents that satisfy the constraints while NullSeq does not. [ABSTRACT FROM AUTHOR]

DDX43 is a cancer/testis antigen and is thought to stimulate oncogenic pathways in cell proliferation, while its specific function in cancer development is largely unexplored. DDX43 is the member of RNA helicase in DEAD-box family, consists of conserved helicase core and a single K-homology (KH) domain in its N-terminus. In this paper, we expressed and purified human DDX43 protein in E. coli and demonstrated that this protein is a homogeneous monomer. To understand the role and explore the substrates preference of DDX43 in vitro , we systematically studied its binding properties. We found that DDX43 prefers single-strand DNA or RNA with length longer than 12 nt and much prefers guanosine than the other three nucleotides. Achievement of the full binding affinity of protein to substrate needs the existence of all domains, and they must be connected. The absence of either of them or the disjunction can result in a decreased binding affinity to substrates, approximately reduced 10-fold. We also found that the unwinding ability of DDX43 in vitro was neither efficient nor sustainable. • DDX43 prefers single strand G-rich substrate. • Both KH domain and helicase core are required in substrates binding. • KH domain and helicase core must be linked for DDX43 high affinity to substrates. [ABSTRACT FROM AUTHOR]

Agaricus bisporus mannose binding protein (Abmb) demonstrates permeability to epithelial monolayer barrier of the intestine, resistance to gastrointestinal tract conditions and to proteolysis therefore it holds potential as a drug carrier for oral route administration. Abmb also display antiproliferative activity to breast cancer cells and stimulation of immune system thus could potentially be also developed for therapeutic purpose. It is not immunogenic or toxic thereby safe for use. In this paper we further provide evidence that Abmb also lacks of agglutinating activity despite sharing high structural homology to lectins. Abmb is thereby the only mannose specific binding protein that is not member of lectin family. This evidence provides further support on the use of Abmb as pharmaceutical or medicinal agent. Its molecular globularity that may contribute to its lack of agglutination capacity was also evaluated. • Abmb shares high structural homology to lectins. • Abmb has no agglutinating activity hence is not lectin. • Abmb is the only mannose binding protein that is not of lectin family. [ABSTRACT FROM AUTHOR]

Abstract Accumulating evidence suggests a new role for cellular heme as a signalling molecule, in which interactions with target proteins are more transient than found with traditionally-defined hemoproteins. To study this role, a precise method is needed for determining the heme-binding affinity (or dissociation constant, K d). Estimates of K d are commonly made following a spectrophotometric titration of an apo -protein with hemin. An impediment to precise determination is, however, the challenge in discriminating between the Soret absorbance for the product (holo -protein) and that for the titrant (hemin). An altogether different approach has been used in this paper to separate contributions made by these components to absorbance values. The pure component spectra and concentration profiles are estimated by a multivariate curve-resolution (MCR) algorithm. This approach has significant advantages over existing methods. First, a more precise determination of K d can be made as concentration profiles for all three components (apo -protein/ holo -protein/hemin) are determined and can be simultaneously fitted to a theoretical-binding model. Second, an absorption spectrum for the holo -protein is calculated. This is a unique advantage of MCR and attractive for investigating proteins in which the nature of heme binding has not, hitherto, been characterised because the holo -protein spectrum provides information on the interaction. Highlights • Method for obtaining heme-binding affinity with advantages over difference absorption. • Multivariate approach for analysis of spectrophotometric titrations. • Distinguishing Soret absorbances for holo -protein and hemin. • Determination of concentration profiles for apo -protein/ holo -protein/hemin. • Calculation of absorption spectra for uncharacterised holo -proteins. [ABSTRACT FROM AUTHOR]

Abstract To optimize the expression conditions for human lactoferrin production, we have constructed the transgenic chlorella with human lactoferrin named as GTD8A1-HLF, the original chlorella was separated from Gurbantunggut Desert in Xinjiang China. To further improve the production of human lactoferrin, a sequential methodology was used to optimize human lactoferrin production by GTD8A1-HLF. First, a screening trial using a Plackett-Burman design was done, and variables with statistically significant effects on human lactoferrin bio-production were identified. These were further optimized by central composite design experiments and response surface methodology. Finally, we found that the maximum human lactoferrin production (52.70 mg/L) was achieved under the following optimized conditions: Initial pH 5.0, NaNO 3 concentration of 0.600 mol/L, FeSO 4 concentration of 0.006 mol/L, and a CuSO 4 concentration of 0.002 mol/L, with the other medium components constituting the basal culture medium BBM. The yield of HLF protein under optimized culture conditions was approximately 4-fold higher than that obtained by using the basal culture medium BBM. The findings are significant for the potential industrial use of GTD8A1-HLF. Highlights • We first successfully obtained the best culture conditions of HLF production in Desert Chlorella, its expression level is 52.70 mg/L under the optimized conditions as initial pH 5.0, NaNO 3 0.600 mol/L, FeSO 4 0.006 mol/L, and a CuSO 4 0.002 mol/L, with the other medium components constituting the basal culture medium BBM. • It was used to analysis and identify results of this paper that Plackett-Burman design, central composite design experiments and response surface methodology, and also help our to predict best component of improved BBM. The yield of HLF protein using the optimized culture medium was approximately 4-fold of that obtained when basal culture medium was used. • The research also provides a new, safe and non-toxic way to produce human lactoferrin, and will hopefully become as commercial production. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A 3D-QSAR model was produced for the FABP4 inhibitors. • A scaffold hopping suggested 3000 potentially active FABP4 inhibitors. • Three synthesized molecules have shown an IC 50 between 3.70 and 5.59 μM. Abstract Following on the recent publication of pharmacologically relevant effects, small molecule inhibitors of adipocyte fatty-acid binding protein 4 (FABP4) have attracted high interest. FABP4 is mainly expressed in macrophages and adipose tissue, where it regulates fatty acid storage and lipolysis, being also an important mediator of inflammation. In this regard, FABP4 recently demonstrated an interesting molecular target for the treatment of type 2 diabetes, other metabolic diseases and some type of cancers. In the past years, hundreds of effective FABP4 inhibitors have been synthesized. In this paper, a quantitative structure-activity relationship (QSAR) model has been produced, in order to predict the bioactivity of FABP4 inhibitors. The methodology has been combined with a scaffold-hopping approach, allowing to identify three new molecules that act as effective inhibitors of this protein. These molecules, synthesized and tested for their FABP4 inhibitor activity, showed IC 50 values between 3.70 and 5.59 μM, with a high level of agreement with the predicted values. [ABSTRACT FROM AUTHOR]

Graphical abstract A series of oxadiazole-based S1P 1 direct-acting agonists, with high affinity for S1P 1 receptor and favorable selectivity against S1P 3 receptor was designed, synthesized and evaluated in vitro and vivo. Highlights • Oxadiazole-based derivatives were designed and progressively modified as S1P 1 agonists. • The agonism activities of compounds for S1P 1 and S1P 3 receptor were evaluated. • Most compounds show excellent activity and selectivity, particularly for 16-3b and 16 - 3g. • In vivo pharmacological, pharmacokinetics and druggability profiles were studied. • The binding mode between 16-3b and the activated S1P 1 model was also reviewed. Abstract Agonism of S1P 1 receptor has been proven to be responsible for peripheral blood lymphopenia and elicts the identification of various S1P 1 modulators. In this paper we described a series of oxadiazole-based S1P 1 direct-acting agonists disubstituted on terminal benzene ring, with high potency for S1P 1 receptor and favorable selectivity against S1P 3 receptor. In addition, two representative agents named 16-3b and 16 - 3g demonstrated impressive efficacy in lymphocyte reduction along with reduced effect on heart rate when orally administered. Furthermore, these compounds have been shown to possess desired pharmacokinetic (PK) and physicochemical profiles. The binding mode between 16 - 3b and the activated S1P 1 model was also studied. [ABSTRACT FROM AUTHOR]

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. [ABSTRACT FROM AUTHOR]

Many proteins exhibit strong binding affinities to surfaces, with binding energies much greater than thermal fluctuations. When modelling these protein-surface systems with classical molecular dynamics (MD) simulations, the large forces that exist at the protein/surface interface generally confine the system to a single free energy minimum. Exploring the full conformational space of the protein, especially finding other stable structures, becomes prohibitively expensive. Coupling MD simulations with metadynamics (enhanced sampling) has fast become a common method for sampling the adsorption of such proteins. In this paper, we compare three different flavors of metadynamics, specifically well-tempered, parallel-bias, and parallel-tempering in the well-tempered ensemble, to exhaustively sample the conformational surface-binding landscape of model peptide GGKGG. We investigate the effect of mobile ions and ion charge, as well as the choice of collective variable (CV), on the binding free energy of the peptide. We make the case for explicitly biasing ions to sample the true binding free energy of biomolecules when the ion concentration is high and the binding free energies of the solute and ions are similar. We also make the case for choosing CVs that apply bias to all atoms of the solute to speed up calculations and obtain the maximum possible amount of information about the system. [ABSTRACT FROM AUTHOR]

Glucose transporters (GLUTs), expressed in all types of human cells, are responsible for the uptake of sugars as the primary energy source for the normal functions of good cells and for the abnormal growth of cancer cells. The E. coli xylose permease (XylE), a homologue of human GLUTs, has been investigated more thoroughly than other major facilitator proteins in the current literature. In this paper, we present a molecular dynamics (MD) study of an all-atom model system to elucidate the atomistic details and the free-energy landscape along the path of binding a xylopyranose (XYP) from the extracellular space to the inside of the transporter protein XylE. From the MD simulations, the Gibbs free energy of binding was found to be − 4.4 kcal/mol in agreement with the experimental value of − 4.7 kcal/mol. The accuracy of our study is further shown in the computed hydration energy of XYP of − 14.6 kcal/mol in comparison with the experimental data of − 15.0 kcal/mol. Along the binding path, the Gibbs free energy of the XYP-XylE complex first rises from zero in the dissociated state to approximately 4 kcal/mol in the transition state (when XylE slightly increases its opening toward the extracellular side to accommodate XYP) before dropping down to − 9.0 kcal/mol in the bound state. These quantitative insights indicate the fast equilibration between the bound and the unbound states of XylE and XYP. They also serve as an atomistic-dynamic corroboration of the experimental conclusion that XylE is a high-affinity sugar transporter. [ABSTRACT FROM AUTHOR]

Abstract: This paper describes the construction of an atlas of the human basal ganglia. The successive steps of the construction were as follows. First a postmortem specimen was subjected to a MRI acquisition prior to extraction of the brain from the skull. The brain was then cryosectioned (70 μm thickness). One section out of ten (80 sections) was Nissl-stained with cresyl violet, another series of 80 sections was immunostained for the calcium binding protein calbindin. Contours of basal ganglia nuclei including their calbindin-stained functional subdivisions, fiber bundles and ventricles (n =80 structures) were traced from histological sections and digitized. A novelty of this atlas is the MRI acquisition, which represents the core data element of the study. MRI was used for the coregistration of the atlas data and permitted, through multimodal (Nissl, calbindin, images of cryosectioning, T1 and T2 MRI) and 3D optimization, the production of anatomically and geometrically consistent 3D surfaces, which can be sliced through any desired orientation. The atlas MRI is also used for its deformation to provide accurate conformation to the MRI of living patients, thus adding information at the histological level to the patient''s MRI volume. This latter aspect will be presented in a forthcoming paper. [Copyright &y& Elsevier]

We developed a cost-effective assay to measure protein arginine N- methyltransferase (PRMT) activity in a medium-throughput manner by combining P81 filter binding and phosphor screening (FBAPS). Recombinantly-expressed PRMT1 and coactivator-associated arginine methyltransferase 1 (CARM1) were used to develop the FBAPS assay using GST fusions of glycine- and arginine-rich (GAR) protein and polyA binding protein 1 (PABP1(437-488)) as substrates, respectively, and radiolabelled S -adenosyl-L-[ methyl -14C]-methionine as cofactor. Methylation reactions were spotted onto P81 filter paper in a dot blot apparatus and radioactive signals were measured both by phosphor imaging and liquid scintillation counting. Kinetic parameters (K M , k cat) for enzymes and substrates were determined, and IC 50 values were obtained for well-characterized inhibitors. FBAPS yielded kinetic parameters with no statistically significant difference to what was obtained using liquid scintillation counting. The IC 50 values obtained by the FBAPS assay for PRMT1 and CARM1 were comparable to values reported in literature. The FBAPS assay is a modification to the P81 filter binding assay with a dot blot apparatus that allows for processing of samples in a multi-well format, moderately increasing throughput. Signal detection by phosphor imaging offers an affordable and quantitative method that can be used to screen several inhibitors simultaneously against PRMT enzymes with high accuracy. [Display omitted] • A cost-effective method for measuring protein arginine methylation was developed. • FBAPS combines P81 filter binding and phosphor screening. • Parallel sample processing in a multi-well format expedites results. • Rapid determination of kinetic and inhibitory parameters for PRMT1 and CARM1 was perfomed. [ABSTRACT FROM AUTHOR]

The effect of N 2 H 4 (hydrazine) on AnAOB (anaerobic ammonia oxidizing bacteria) metabolic pattern is unknown. Therefore, the main purpose of this paper was to explore the effects of exogenous N 2 H 4 on the SAA (specific anammox activity), characteristics and metabolic pathway of AnAOB. The results showed that low N 2 H 4 concentration (1–5 mg/L) continuous dosing can promote SAA. The promoting effect was found to be more obvious within the dosage of 3–5 mg/L N 2 H 4. It was also indicated that high N 2 H 4 concentration dosing (5–10 mg/L) can trigger the self-protection mechanism of AnAOB granular sludge by secreting a large amount of B-PN (binding polymeric protein). Intermittent addition of N 2 H 4 at low concentration is conducive to the long-term stable operation of anammox process. Exogenous N 2 H 4 can be directly oxidized by AnAOB to promote the consumption of NO 2 −-N and NH 4 +-N. In addition, excess electrons can also drive the process of NO 3 −-N reduction and NO 2 −-N disproportionation. Theoretically, these reaction processes need two and ten extra electrons respectively, which is not easy to occur compared with the anammox process. [Display omitted] • High N 2 H 4 concentration triggered excessive secretion of binding polymeric protein. • The oxidation of N 2 H 4 enhanced the nitrite reduction and anammox process. • N 2 H 4 drive the nitrate reduction and nitrite disproportionation process. • Low-frequency supplement of N 2 H 4 was beneficial for anammox stable operation. [ABSTRACT FROM AUTHOR]

The crystal structure of the NTF2-like domain of the human Ras GTPase SH3 Binding Protein (G3BP), isoform 2, was determined at a resolution of 2.75 Å in complex with a peptide containing a FGDF sequence motif. The overall structure of the protein is highly similar to the homodimeric N-terminal domains of the G3BP1 and Rasputin proteins. Recently, a subset of G3BP interacting proteins was recognized to share a common sequence motif, FGDF. The most studied binding partners, USP10 and viral nsP3, interfere with essential G3BP functions related to assembly of cellular stress granules. Reported molecular modeling suggested that FGDF-motif containing peptides bind in an extended conformation into a hydrophobic groove on the surface of the G3BP NTF2-like domain in a manner similar to the known binding of FxFG nucleoporin repeats. The results in this paper provide evidence for a different binding mode. The FGDF peptide binds and changes conformation of the protruding N-terminal residues by providing hydrophobic interactions to a symmetry related molecule that facilitated crystallization of the G3BP2 isoform. [ABSTRACT FROM AUTHOR]

Each phage infects its specific bacterial host strain through highly specific interactions between the baseplate-associated receptor binding protein (RBP) at the tip of the phage tail and the receptor at the host surface. Baseplates incorporate structural core modules, Dit and Tal, largely conserved among phages, and peripheral modules anchoring the RBPs. Exploiting structural information from the HHpred program and EM data from the Bielmann et al. (2015) paper, a molecular model of the A118 phage baseplate was generated from different building blocks. This model implies the occurrence of baseplate module reshuffling and suggests that listerial phage A118 may have been derived from lactococcal phage TP901-1 through host species exchange. With the increase of available viral module structures, modelling phage baseplates will become easier and more reliant, and will provide insightful information on the nature of the phage host receptor and its mode of recognition. [ABSTRACT FROM AUTHOR]

Serum retinol binding protein 4 levels play critical roles in the early diagnosis and therapeutic monitoring of diabetic kidney disease. In this paper, an liquid chromatography-tandem mass spectrometry approach for the absolute quantification of serum RBP4 with high sensitivity and specificity was presented. Following series of procedures including denaturation, reduction, alkylation and trypsin digestion, the signature peptides of RBP4 were separated on the HPLC system by gradient elution. Quantitative analysis was achieved by a triple quadrupole mass spectrometer under multiple reaction monitoring in the positive ion (ESI+) mode. The calibration range was from 6.25 to 125 mg/L (R2 > 0.993), the lower limit of quantification was 2.50 mg/L, and the lower limit of detection reached 0.0150 mg/L. In the study, the accuracy ranged from 94.6% to 107%, and the relative standard deviation of intra-assay and inter-assay imprecision was less than 5%. The method was demonstrated to realize sensitive and reliable absolute quantification of serum RBP4 conforming to guidelines for bioanalytical method validation. Serum sample was first preprocessed to remove high abundance proteins. After series of procedures including denaturation, reduction, alkylation, proteins were disassembled and further digested to produce peptides by trypsin. Subsequently, the sample was subjected to solid phase extraction and internal standard was added. Finally, the sample was submitted to LC-MS/MS analysis. [Display omitted] • RBP4 is a biomarker reflecting renal impairment. • An LC-MS/MS method was developed for absolute quantification of serum RBP4. • The method is more sensitive, accurate and specific than immunoassay. [ABSTRACT FROM AUTHOR]

Cholecystokinin (CCK) and gastrin are regulatory peptides in vertebrates. Their homologues are widely present in metazoan animals, in form of cionin in tunicates, neuropeptide-like protein 12 in nematodes and sulfakinin (SK) in arthropods. CCK(-like) peptides exert diverse physiological effects through binding their corresponding receptors, which are important members of the hormone-binding G-protein-coupled receptors. In this paper, CCK(-like) peptides and receptors are reviewed in a comparative way at levels of molecular structure, physiological functions and phylogeny. CCK signalling system is widely involved in the regulation of satiety, gastric acid secretion, pancreatic secretion, anxiety and memory processes in vertebrates. Its counterpart SK in arthropods is also found with similar functions on regulation of satiety and gastrointestinal motility. Co-evolution of peptide and receptor has been recognized through metazoans. The CCK(-like) receptors seem to be evolved from a common ancestor based on the phylogenetic analysis, with species-specific events in arthropods. In addition, tetraploidization has been brought up to study the evolution of receptors. There are 2 receptors in chordates and nematodes, whereas, the number of sulfakinin receptor varies in arthropods from 0 to 2. We discussed here that the presence or absence of the SK signalling system is likely to be related to feeding behaviour. [ABSTRACT FROM AUTHOR]

Cysteine-rich intestinal protein (CRIP), a Zn 2+ -binding protein, contains a single copy of the highly conserved double-zinc-finger structure known as the LIM (lin-11-isl-1-mec-3) motif. In this paper, a cDNA encoding MmCRIP was isolated from the Asiatic hard clam Meretrix meretrix . The full-length cDNA of MmCRIP consists of a 237-bp open reading frame that encodes a polypeptide of 78 amino acids with a predicted molecular weight (MW) of 8635.8 Da and theoretical isoelectric point (pI) of 9.01. Bioinformatics analysis showed that it belonged to a new member of the CRIP subfamily. Relationship analysis revealed that MmCRIP has high-levels of sequence similarity to many CRIPs reported in other animals, particularly in invertebrates. Real-time PCR analysis showed that the highest level of MmCRIP expression was in hemocyte tissue and at pediveligers stage. To investigate immune function, mature clams were challenged with Aeromonas hydrophila . During A . hydrophila infection, up-regulation of MmCRIP transcript in clam's hemocyte, gill and hepatopancreas was detected. DsRNAi (double-strand RNA interference) approach was employed to study the function of MmCRIP and the data showed that inactivation of the MmCRIP gene blocked larvae development and caused mass mortalities. The probable roles of MmCRIP in clam early development and innate immunity are presented for the first time. [ABSTRACT FROM AUTHOR]

In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and in the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5 M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome. [ABSTRACT FROM AUTHOR]

Abstract: A major structural component of the cell is the actin cytoskeleton, in which actin subunits are polymerised into actin filaments. These networks can be cross-linked by various types of ABPs (Actin Binding Proteins), such as Filamin A. In this paper, the passive response of cross-linked actin filament networks is evaluated, by use of a numerical and continuum network model. For the numerical model, the influence of filament length, statistical dispersion, cross-link compliance (including that representative of Filamin A) and boundary conditions on the mechanical response is evaluated and compared to experimental results. It is found that the introduction of statistical dispersion of filament lengths has a significant influence on the computed results, reducing the network stiffness by several orders of magnitude. Actin networks have previously been shown to have a characteristic transition from an initial bending-dominated to a stretching-dominated regime at larger strains, and the cross-link compliance is shown to shift this transition. The continuum network model, a modified eight-chain polymer model, is evaluated and shown to predict experimental results reasonably well, although a single set of parameters cannot be found to predict the characteristic dependence of filament length for different types of cross-links. Given the vast diversity of cross-linking proteins, the dependence of mechanical response on cross-link compliance signifies the importance of incorporating it properly in models to understand the roles of different types of actin networks and their respective tasks in the cell. [Copyright &y& Elsevier]

Abstract: Among the methods used to detect food adulteration, the amplification of endogenous reference genes is particularly important. Endogenous reference genes for many different species, such as cotton, papaya, maize and others, have been reported, yet an endogenous reference gene for the peach is still lacking. In this paper, the chlorophyll a/b-binding protein (Lhcb2) gene was identified as a species-specific gene for the peach. Lhcb2 was assayed in 4 species of peaches and 8 non-peach species by both qualitative and quantitative PCR. No amplification was observed in other species. The detection limit of quantitative PCR was as low as 5 pg of DNA, equal to 9 copies, and Southern blot analysis confirmed that the Lhcb2 gene was present in a single copy in the peach genome. All of these experiments indicated that the Lhcb2 gene is a useful endogenous reference gene for the detection of peach material via both qualitative and quantitative PCR assays, even in the processed food samples such as juices containing peach. [Copyright &y& Elsevier]

Abstract: Positive-stranded RNA viruses subvert subcellular membranes to built viral replicases complexes (VRCs) in infected cells. Tombusviruses use peroxisomal membranes for the assembly of their VRCs and they can efficiently switch to the endoplasmic reticulum membrane in the absence of peroxisomes. In this paper, we show that the ER-resident Sec39p vesicular transport protein is critical for the formation of active VRCs in yeast model host. Repression of Sec39p expression in yeast or in plants resulted in greatly reduced tombusvirus accumulation. Moreover, the purified tombusvirus replicase from Sec39p-depleted yeast cells showed low in vitro activity. Also, tombusvirus RNA replication was poor in cell-free extracts or in isolated ER membranes from yeast with repressed Sec39p expression. The tombusvirus p33 replication protein was mislocalized to the ER when Sec39p was depleted in yeast. Overall, Sec39p is the first peroxisomal biogenesis protein characterized that is critical for tombusvirus replication in yeast and plants. [Copyright &y& Elsevier]

Efflux proteins are the transport proteins expressed in the plasma membrane, which are involved in the movement of unwanted toxic substances through specific efflux pumps. Several studies based on computational approaches have been proposed to predict transport proteins and thereby to understand the mechanism of the movement of ions across cell membranes. However, few methods were developed to identify efflux proteins. This paper presents an approach based on the contextualized word embeddings from Bidirectional Encoder Representations from Transformers (BERT) with the Support Vector Machine (SVM) classifier. BERT is the most effective pre-trained language model that performs exceptionally well on several Natural Language Processing (NLP) tasks. Therefore, the contextualized representations from BERT were implemented to incorporate multiple interpretations of identical amino acids in the sequence. A dataset of efflux proteins with annotations was first established. The feature vectors were extracted by transferring protein data through the hidden layers of the pre-trained model. Our proposed method was trained on complete training datasets to identify efflux proteins and achieved the accuracies of 94.15% and 87.13% in the independent tests on membrane and transport datasets, respectively. This study opens a research avenue for the implementation of contextualized word embeddings in Bioinformatics and Computational Biology. [Display omitted] • Efflux proteins facilitate the movement of multi-drugs. • Efflux proteins transport harmful substances via particular efflux pumps. • Bidirectional Encoder Representations from Transformers to identify Efflux protein. • Contextual representations from BERT captures amino acids context in a sequence. • Cosine Similarity measures the degree of similarity between two vectors. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: [18F]FEDAA1106 is expected to be used for evaluating the regional density of the peripheral benzodiazepine receptor (also called TSPO) in several neurodegenerative disorders. Regarding the quantification, direct binding potential (BPND) has been reported to be preferable because of the variation of nondisplaceable distribution volume (VND) among individuals. However, the precise calculation of BPND is difficult in small regions or at voxel levels due to noise. Recently, a new graphical analysis (GA) was proposed to estimate VND in a direct way. In this paper, we evaluated two types of GA for reliable quantification of BPND in PET study with [18F]FEDAA1106 using computer simulations and human data. Methods: In the simulations, time–activity curves were generated with various rate constants and noise levels, and the errors of BPND estimated by GA were analyzed by comparing with true values calculated from rate constants given for the simulations. Thereafter, in a human study with [18F]FEDAA1106 for healthy volunteers, BPND was estimated by two types of GA for region-of-interest (ROI) data. Parametric images of BPND were generated by two types of GA with or without wavelet-denoising. Results: Simulations showed that BPND by GA was well correlated with true values, despite an underestimation. GA reduced unreasonable estimates compared with a conventional nonlinear least-square fitting (NLS), although larger variation of BPND estimates was observed. In a ROI-based analysis of data obtained in a human study, BPNDs estimated by GA were well correlated with those generated by NLS, though they were underestimated. Parametric BPND images by GA could be improved with wavelet-denoising. Conclusion: Graphical analysis could provide BPND values with high stability and simple calculation in both ROI-based and voxel-based analyses of [18F]FEDAA1106 data. [Copyright &y& Elsevier]

Abstract: ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the ATP-binding cassette-type periplasmic Zn-binding proteins. The zinc transporter ZnuABC is composed by three proteins: ZnuB, the membrane permease, ZnuC, the ATPase component and ZnuA, the soluble periplasmic metal-binding protein which captures Zn and delivers it to ZnuB. The ZnuA protein contains a charged flexible loop, rich in histidines and acidic residues, showing significant species-specific differences. Various studies have established that this loop contributes to the formation of a secondary zinc binding site, which has been proposed to be important in the acquisition of periplasmic Zn for its delivery to ZnuB or for regulation of zinc uptake. Due to its high mobility the structure of the histidine-rich loop has never been solved by X-ray diffraction studies. In this paper, through a combined use of molecular modeling, mutagenesis and fluorescence spectroscopy, we confirm the presence of two zinc binding sites characterized by different affinities for the metal ion and show that the flexibility of the loop is modulated by the binding of the zinc ions to the protein. The data obtained by fluorescence spectroscopy have then be used to validate a 3D model including the unsolved histidine-rich loop. [Copyright &y& Elsevier]

Abstract: Notwithstanding several neurotransmission systems are frequently related to memory formation; forgetting process and neurotransmission systems or their transporters; the role of γ-aminobutyric acid (GAT1), glutamate (EACC1), dopamine (DAT) and serotonin (SERT) is poorly understood. Hence, in this paper western-blot analysis was used to evaluate expression of GAT1, EAAC1, DAT and SERT during forgetting in trained and untrained rats treated with the selective serotonin transporter inhibitor fluoxetine, the amnesic drug d-methamphetamine (METH) and fluoxetine plus METH. Transporters expression was determined in the hippocampus (HIP), prefrontal cortex (PFC) and striatum (STR). Results indicated that forgetting of Pavlovian/instrumental autoshaping was associated to up-regulation of GAT1 (PFC and HIP) and DAT (PFC) while SERT (HIP) was down-regulated; no-changes were observed in striatum. Methamphetamine administration did not affect forgetting at 216h post-training but up-regulated hippocampal DAT and EACC, prefrontal cortex DAT and striatal GAT1 or EACC1. Fluoxetine alone prevented forgetting, which was associated to striatal GAT1 and hippocampal DAT up-regulation, but prefrontal cortex GAT1 down-regulation. Fluoxetine plus METH administration was also able to prevent forgetting, which was associated to hippocampal DAT, prefrontal cortex SERT and striatal GAT1, DAT or SERT up-regulation, but prefrontal cortex GAT1 down-regulation. Together these data show that forgetting provokes primarily hippocampal and prefrontal cortex transporters changes; forgetting represent a behavioral process hardly modifiable and its prevention could causes different transporters expression patterns. [Copyright &y& Elsevier]

Abstract: In rodents, the transcription factors early growth response 2 (EGR2) and sterol regulatory element binding protein 1a (SREBP1a) regulate transcription of the stearoyl-CoA desaturase 2 (SCD2) gene during peripheral nerve myelination, which may be important for synthesis of the lipid component of myelin. Most non-rodent genomes do not contain the SCD2 gene, but rather express SCD5 in brain and nervous tissues. In this paper, we asked whether bovine SCD5 is regulated in a similar manner to rodent SCD2. Expression of EGR2 did not result in an increase in endogenous SCD5 mRNA expression in JEG3 cells, but did result in activation of truncated bovine SCD5 promoter luciferase reporter constructs. Similar results were obtained with expression of the active form of SREBP1a; however, unlike rodent SCD2, there was no synergistic activation of the bovine SCD5 promoter reporters when EGR2 and SREBP1a were co-expressed. Mutation of the putative EGR2 binding site in the SCD5 promoter abolished activation by SREBP1a, suggesting that EGR2 and SREBP1a bind to the same site in the SCD5 promoter. Finally, we have identified a region of the bovine SCD5 promoter between 505 and 305 base pairs upstream of the transcriptional start site that appears to be important for maintaining basal levels of transcription of this gene. While it appears that there are some differences between the regulation of rodent SCD2 and bovine SCD5, the promoters of both genes can be activated by EGR2 and SREBP1a. This is the first report of potential regulators of SCD5 transcription. [Copyright &y& Elsevier]

Abstract: FK506, a widely used immunosuppressant, is produced by industrial fermentation processes using various Streptomyces species. Independently of the strain, structurally related compound FK520 is co-produced, resulting in complex and costly isolation procedures. In this paper, we report a chemobiosynthetic approach for exclusive biosynthesis of FK506. This approach is based on the Streptomyces tsukubaensis strain with inactivated allR gene, a homologue of crotonyl-CoA carboxylase/reductase, encoded in the FK506 biosynthetic cluster. This strain produces neither FK506 nor FK520; however, if allylmalonyl-S-N-acetylcysteamine precursor is added to cultivation broth, the production of FK506 is reestablished without FK506-related by-products. Using a combination of metabolic engineering and chemobiosynthetic approach, we achieved exclusive production of FK506, representing a significant step towards development of an advanced industrial bioprocess. [Copyright &y& Elsevier]

Abstract: Here we introduce plusTipTracker, a Matlab-based open source software package that combines automated tracking, data analysis, and visualization tools for movies of fluorescently-labeled microtubule (MT) plus end binding proteins (+TIPs). Although +TIPs mark only phases of MT growth, the plusTipTracker software allows inference of additional MT dynamics, including phases of pause and shrinkage, by linking collinear, sequential growth tracks. The algorithm underlying the reconstruction of full MT trajectories relies on the spatially and temporally global tracking framework described in . Post-processing of track populations yields a wealth of quantitative phenotypic information about MT network architecture that can be explored using several visualization modalities and bioinformatics tools included in plusTipTracker. Graphical user interfaces enable novice Matlab users to track thousands of MTs in minutes. In this paper, we describe the algorithms used by plusTipTracker and show how the package can be used to study regional differences in the relative proportion of MT subpopulations within a single cell. The strategy of grouping +TIP growth tracks for the analysis of MT dynamics has been introduced before (). The numerical methods and analytical functionality incorporated in plusTipTracker substantially advance this previous work in terms of flexibility and robustness. To illustrate the enhanced performance of the new software we thus compare computer-assembled +TIP-marked trajectories to manually-traced MT trajectories from the same movie used in . [Copyright &y& Elsevier]

Abstract: Chemokines are a superfamily of structurally homologous heparin-binding proteins that influence tumor growth and metastasis. Several members of the CXC and CC chemokine families are potent inducers of neovascularization, whereas a subset of the CXC chemokines are potent inhibitors. In this paper, we review the current literature regarding the role of chemokines as mediators of tumor angiogenesis and neovascularization. [Copyright &y& Elsevier]

Abstract: Guanine nucleotides are key signaling molecules and many members of the G-protein family bind and hydrolyze nucleotides, particularly GTP, and regulate intracellular level of GTP and GDP. EngA is one of the members of these universally conserved GTPases. Amino acid sequence alignment of EngA of Mycobacterium tuberculosis H37Rv with other homologous bacterial proteins have shown that EngA of M. tuberculosis H37Rv has significant homology with EngA of other bacteria. EngA protein has shown GTP-binding and GTP hydrolysis activities as intrinsic biochemical properties of protein and this serves as a base to further investigate the physiological significance of this protein in the pathogenesis mechanism of M. tuberculosis H37Rv. In this paper for the first time EngA GTP-binding protein of M. tuberculosis H37Rv was functionally characterized for its GTPase and GTP-hydrolyzing activity. GTPases such as era, obg, lepA, and FtsZ are vital for growth and development and specifically cellular functions of bacteria, in view of these observations it can be concluded that EngA GTPase can be further utilized for the study of its functional role in the pathogenesis of M. tuberculosis H37Rv. [Copyright &y& Elsevier]

Abstract: Fad104 (factor for adipocyte differentiation 104) is a novel gene expressed temporarily in the early stages of adipocyte differentiation. Previously, we showed that fad104 promotes adipocyte differentiation in mouse 3T3-L1 cells and mouse embryonic fibroblasts (MEFs). Furthermore, we reported that implanted wild-type MEFs could develop into adipocytes, whereas fad104-deficient MEFs could not. Interestingly, bone-like tissues were only observed in the implants derived from fad104-deficient MEFs. This result implies that fad104 is involved in osteoblast differentiation. However, the functions of fad104 during osteogenesis are unknown. In this paper, we show that fad104 negatively regulates osteoblast differentiation. During the differentiation process, the level of fad104 expression decreased. Deletion of fad104 facilitated osteoblast differentiation in MEFs, and elevated the level of runx2, a master regulator of osteoblast differentiation. Disruption of fad104 suppressed BMP-2-mediated adipocyte differentiation in MEFs. In conclusion, we demonstrate that fad104 reciprocally regulates differentiation of adipocytes and osteoblast; functions as a positive regulator in adipocyte differentiation and as a negative regulator in osteoblast differentiation. [Copyright &y& Elsevier]

Abstract: Tropomyosin is a two-chained α-helical coiled coil that binds along the length of the actin filament and regulates its function. The paper addresses the question of how a “simple” coiled-coil sequence encodes the information for binding and regulating the actin filament, its universal target. Determination of the tropomyosin sequence confirmed Crick’s predicted heptapeptide repeat of hydrophobic interface residues and revealed additional features that have been shown to be important for its function: a 7-fold periodicity predicted to correspond to actin binding sites and interruptions of the canonical interface with destabilizing residues, such as Ala. Evidence from published work is summarized, leading to the proposal of a paradigm that binding of tropomyosin to the actin filament requires local instability as well as regions of flexibility. The flexibility derives from bends and local unfolding at regions with a destabilized coiled-coil interface, as well as from the dynamic end-to-end complex. The features are required for tropomyosin to assume the form of the helical actin filament, and to bind to actin monomers along its length. The requirement of instability/flexibility for binding may be generalized to the binding of other coiled coils to their targets. [Copyright &y& Elsevier]

Abstract: In this paper, we report the cloning and characterization of the STAT6 gene from the pufferfish, Tetraodon nigroviridis. The TnSTAT6 gene is composed of 20 exons and 19 introns. The exon–intron organization of this gene is similar to that of HsSTAT6 except for the exons encoding the C-terminal transactivation domain. The full-length complementary (c)DNA of TnSTAT6 encodes a 794-amino acid protein that is 31% identical to human STAT6. We generated a constitutively active TnSTAT6-JH1 by fusing the kinase domain of carp JAK1 to the C-terminal end of TnSTAT6 and demonstrated that the fusion protein has specific DNA-binding ability and can activate a reporter construct carrying multiple copies of mammalian IL-4-response elements. Interestingly, TnSTAT6-JH1 associated with and phosphorylated TnSTAT6 on Tyr661. Mutation of this residue, Y661W, in TnSTAT6 abolished its association with TnSTAT6-JH1. This is consistent with the importance of the corresponding Tyr641 of HsSTAT6 in tyrosine phosphorylation and dimer formation. On the other hand, treatment of mammalian IL-4 did not induce tyrosine phosphorylation of wild-type TnSTAT6, suggesting that both the divergent N-terminal domain and coiled-coiled domain of TnSTAT6 may affect the interaction of TnSTAT6 with mammalian IL-4 receptor complexes. [Copyright &y& Elsevier]

Abstract: This paper presents examples of techniques based on the principle of random sampling that allows acquisition of NMR spectra featuring extraordinary resolution. This is due to increased dimensionality and maximum evolution time reached. The acquired spectra of CsPin protein and maltose binding protein were analyzed statistically with the aim to evaluate each technique. The results presented include exemplary spectral cross-sections. The spectral data provided by the proposed techniques allow easy assignment of backbone and side-chain resonances. [Copyright &y& Elsevier]

Abstract: The nuclear membrane has an important role for the dynamic regulation of the genome, besides the well-established cytoskeletal function. The nuclear lamina is emerging as an important player in the organization of the position and functional state of interphase chromosomes. Epigenetic modifications such as DNA methylation and histone modifications are required for genome reprogramming during development, tissue-specific gene expression and global gene silencing. The Methyl-CpG binding protein MeCP2 binds methyl-CpG dinucleotides in the mammalian genome and functions as a transcriptional repressor in vivo by interacting with Sin3A, thereby recruiting histone deacetylases (HDAC). MeCP2 also mediates the formation of higher-order chromatin structures contributing to determine the architectural organization of the nucleus. In this paper, we show that MeCP2 interacts in vitro and in vivo with the inner nuclear membrane protein LBR and that the unstructured aminoacidic sequence linking the MBD and TRD domains of MeCP2 is responsible for this association. The formation of an LBR–MeCP2 protein complex might help providing a molecular explanation to the distribution of part of the heterochromatin at the nuclear periphery linked to inner membrane. [Copyright &y& Elsevier]

Abstract: Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins’ application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense. [Copyright &y& Elsevier]

Abstract: Pleckstrin-2 (PLEK2) has been implicated to be regulated by phosphatidylinositol (PI) 3-kinase, while pleckstrin1 (PLEK1) has been suggested to be a major PKC substrate in platelets. In this paper, we confirmed that PLEK2 specifically bound to the PI 3-kinase products in vitro and explored its behavior. PLEK2 was found to be expressed in various adherent cell lines, while PLEK1 expression was restricted to non-adherent cells in the protein level. Expression of PLEK2 in COS1 cells induced formation of protrusive F-actin structure and enhanced the actin rearrangements induced on collagen- or fibronectin-coated plates. A PLEK2 mutant incapable of binding to the PI 3-kinase products did not show any effect on actin rearrangement. Knockdown of PLEK2 by shRNA inhibited spreading of HCC2998 adenocarcinoma cells. PLEK2 colocalized with Rac and was suggested to be oligomerized. These results suggest that PLEK2 is involved in actin rearrangement in a PI 3-kinase dependent manner. [Copyright &y& Elsevier]

Abstract: In this paper, we report on the isolation, purification and identification of two Toxoplasma gondii membrane proteins binding human lactoferrin. Parasite membrane proteins were isolated using the commercial Mem-PER Eukaryotic Membrane Protein Extraction System. After purification by lactoferrin affinity chromatography, three protein bands were detected with the molecular mass of 74, 63 and 58kDa, two of which (63 and 58kDa) specifically bound biotin labeled human lactoferrin as examined by competitive inhibition. Further identification of latter proteins by ESI/MS/MS amino acid sequencing technique revealed those proteins as Toxoplasma ROP4 (band 63kDa) and ROP2 (band 58kDa) antigens known to be involved in many mechanisms essential for the parasite pathogenicity, including host lactoferrin acquisition as determined in this study. [Copyright &y& Elsevier]

Abstract: Calmodulin (CaM) is a highly versatile Ca2+ signaling transducer known to regulate over a hundred proteins. In this paper, we further demonstrate the versatility of CaM binding by showing that it binds to a synthetic peptide (revCKKp) made by reversing the amino acid sequence of the CaM-binding peptide (CKKp) from CaM-dependent protein kinase kinase (CaMKK) (residues 438–463). Sequence comparison between revCKKp and other CaM-binding peptides (CBPs) from the CaM target databank showed that revCKKp does not resemble any existing classes of CBPs, except CKKp [M. Zhang, T. Yuan, Molecular mechanisms of calmodulin’s functional versatility, Biochem. Cell Biol. 76 (1998) 313–323; S.W. Vetter, E. Leclerc, Novel aspects of calmodulin target recognition and activation, Eur. J. Biochem. 270 (2003) 404–414]. Furthermore, computational modeling showed that revCKKp could bind CaM in a similar manner to CKKp. Lastly, we experimentally showed that our synthetic revCKKp binds to CaM in a reversible Ca2+-dependent manner. [Copyright &y& Elsevier]

Abstract: A rapid method to screen and identify unknown bound proteins to specific nucleic acid probes anchored on ProteinChip array surfaces from crude biological samples has been developed in this paper. It was demonstrated with screening specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts by direct coupling of thioaptamer XBY-S2 to the pre-activated ProteinChip array surfaces. With pre-fractionation of crude nuclear extracts by ion exchange method, specific “on-chip” captured proteins have been obtained that were pure enough to do “on-chip” digestion and the subsequent identification of the “on-chip” bound proteins by microsequencing of the trypsin digested peptide fragments through tandem MS. Five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B1, A3, A/B, and D0 were identified. To verify those bound hnRNPs, a novel thioaptamer/antibody sandwich assay provides highly sensitive and selective identification of proteins on ProteinChip arrays. [Copyright &y& Elsevier]

Abstract: There are currently at least 53 structures of components of nuclear transport in the Protein Databank. In addition to providing critical insights into molecular mechanisms of nuclear transport, these atomic resolution structures provide a large body of information that could guide biochemical and cell biological analyses involving nuclear transport proteins. This paper catalogs 53 crystal and NMR structures of nuclear transport proteins, with the emphasis on providing information useful for mutagenesis and overexpression of recombinant proteins. [Copyright &y& Elsevier]

Abstract: Outer arm dynein is a molecular motor that is positioned at 24nm intervals on outer doublet microtubules in cilia and flagella. In the present paper, we report identification of a 58kDa novel protein with a tetratricopeptide repeat (TPR), referred to as ap58 (for 58kDa axonemal protein) in sea urchin sperm axonemes. Ap58 is extracted along with the outer arm dynein by a high salt solution from axonemes. Sucrose density gradient centrifugation or gel filtration of the extract separates the outer arm dynein core from ap58. Most ap58 sediments to the lower density fraction or elutes in fractions of smaller molecules. However, immunogold localization reveals that ap58 is distributed at ∼25nm intervals on doublet microtubules, suggesting that in situ it is associated with the outer dynein arm. Thus, ap58 with the TPR motif is a new member of outer dynein arm-binding proteins distinct from the outer dynein arm-docking complex. [Copyright &y& Elsevier]