Expression, purification, and characterization of the human receptor activator of NF-kappaB ligand (RANKL) extracellular domain

Receptor activator of NF-kappaB ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprotegerin (OPG). We designed a construct encoding the extracellular domain of human RANKL that conformed to reports of native processing. To encourage folding and posttranslational modification of a normally membrane-inserted moiety, we expressed the RANKL truncate as a secreted protein using the signal sequence from OPG in a Trichoplusia ni cell line using a baculovirus expression vector. RANKL was purified by a three-step process including an OPG-Fc affinity column. SDS-PAGE and mass spectral analysis indicated that the protein was >99% pure and glycosylated. Circular dichroism spectra revealed that the protein exhibited structural elements similar to tumor necrosis factor-alpha. By BIAcore analysis, RANKL bound to OPG with an affinity of 6.7 nM. Sedimentation equilibrium analytical ultracentrifugation analyses established that our protein existed as a trimer. We conclude that our expressed human RANKL truncate is folded, is functional, and exhibits self-association consistent with other family members.     

Effects of low-pass filter combinations on lower extremity joint moments in distance running

Inverse dynamics is a standard tool in biomechanics, which requires low-pass filtering of external force and kinematic signals. Unmatched filtering procedures are reported to affect joint moment amplitudes in high impact movements, like landing or cutting, but are also common in the analysis of distance running. We analyzed the effects of cut-off frequencies in 94 rearfoot runners at a speed of 3.5 m/s. Additionally, we investigated whether the evaluation of footwear interventions is affected by the choice of cut-off frequencies. We performed 3D inverse dynamics for the hip, knee and ankle joints using different low-pass filter cut-off frequency combinations for a recursive fourth-order Butterworth filter. We observed fluctuations of joint moment curves in the first half of stance, which were most pronounced for the most unmatched cut-off frequency combination (kinematics: 10 Hz; ground reaction forces (GRFs): 100 Hz) and for more proximal joints. Peak sagittal plane hip joint moments were altered by 94% on average. We observed a change in the ranking of subjects based on joint moment amplitude. We found significant (p < 0.001) footwear by cut-off frequency combination interaction effects for most peak joint moments. These findings highlight the importance of cut-off frequency choice in the analysis of joint moments and the assessment of footwear interventions in distance running. Based on our results, we propose to use matched cut-off frequencies around 20 Hz in order to avoid large artificial fluctuations in joint moment curves while at the same time avoiding a severe removal of physiological high-frequency signal content from the GRF signals.     

Proteins searching for their target on DNA by one-dimensional diffusion: overcoming the “speed-stability” paradox

The sequence dependence of DNA-protein interactions that allows proteins to find the correct reaction site also slows down the 1D diffusion of the protein along the DNA molecule, leading to the so-called “speed-stability paradox,” wherein fast diffusion along the DNA molecule is seemingly incompatible with stable targeting of the reaction site. Here, we develop diffusion-reaction models that use discrete and continuous Gaussian random 1D diffusion landscapes with or without a high-energy cut-off, and two-state models with a transition to and from a “searching” mode in which the protein diffuses rapidly without recognizing the target. We show the conditions under which such considerations lead to a predicted speed-up of the targeting process, and under which the presence of a “searching” mode in a two-state model is nearly equivalent to the existence of a high-energy cut-off in a one-state model. We also determine the conditions under which the search is either diffusion-limited or reaction-limited, and develop quantitative expressions for the rate of successful targeting as a function of the site-specific reaction rate, the roughness of the DNA-protein interaction potential, and the presence of a “searching” mode. In general, we find that a rough landscape is compatible with a fast search if the highest energy barriers can be avoided by “hopping” or by the protein transitioning to a lower-energy “searching” mode. We validate these predictions with the results of Brownian dynamics, kinetic Metropolis, and kinetic Monte Carlo simulations of the diffusion and targeting process, and apply these concepts to the case of T7 RNA polymerase searching for its target site on T7 DNA.     

Four applications of the anesthetic cut-off effect

This article presents and analyses several cases in which the use of the cut-off effect is useful. It starts from the fact that an anesthetic effect of homologous agents is always expressible as a function of their chain lengths and that the cut-off point is a point at which the function vanishes. We then investigate four categories of results: (i) whole body effects. (ii) Cases in which the anesthetics affect the Hodgkin-Huxley parameters of a nerve. (iii) Molecular mechanisms of anesthetic action. (iv) The physical chemistry of the anesthetic process. Our discussion shows that it is possible to incorporate these apparently remote results into one framework. It also shows how to compare results that were gathered by independent measuring methods. In some instances we suggest an interpretation, in others we suggest a further gathering of experimental data. One of the deductions indicates that a weakness exists in the lipid theories of anesthesia.     

应用实验种群生命表评价加州新小绥螨对土耳其斯坦叶螨和截形叶螨的控制能力

【目的】为了解加州新小绥螨Neoseiulus Californicus(McGregor)对土耳其斯坦叶螨Tetranychus turkestani(Ugarov etNikolski)和截形叶螨Tetranychus truncate(Ehara)的控制能力。【方法】采用实验种群生命表方法进行评价。【结果】以土耳其斯坦叶螨为食时加州新小绥螨的净增值率(R_0=33.236)、内禀增长率(r_m=0.218),大于以截形叶螨为食时的值(R_0=32.458,r_m=0.207),而世代平均周期(T=16.340)和种群倍增时间(t=3.173)小于以截形叶螨为食时的值(T=16.849,t=3.355)。【结论】以土耳其斯坦叶螨为食有利于加州新小绥螨种群的发育和繁殖。     

Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.     

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

Background

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.

Results

We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9?=?CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.

Conclusions

Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).

     

The Truncate Mutation of Notch2 Enhances Cell Proliferation through Activating the NF-κB Signal Pathway in the Diffuse Large B-Cell Lymphomas

The Notch2 is a critical membrane receptor for B-cell functions, and also displays various biological roles in lymphoma pathogenesis. In this article, we reported that 3 of 69 (4.3%) diffuse large B-cell lymphomas (DLBCLs) exhibited a truncate NOTCH2 mutation at the nucleotide 7605 (G/A) in the cDNA sequence, which led to partial deletion of the C-terminal of PEST (proline-, glutamic acid-, serine- and threonine-rich) domain. The truncate Notch2 activated both the Notch2 and the NF-κB signals and promoted the proliferation of B-cell lymphoma cell lines, including DLBCL and Burkitt''s lymphoma cell lines. Moreover, the ectopic proliferation was completely inhibited by ammonium pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor. Simultaneously, PDTC also reduced the expression level of Notch2. Based on these results, we conclude that the Notch2 receptor with PEST domain truncation enhances cell proliferation which may be associated with the activation of the Notch2 and the NF-κB signaling. Our results are expected to provide a possible target for new DLBCL therapies by suppressing the Notch2 and the NF-κB signaling.     

Tic40 is important for reinsertion of proteins from the chloroplast stroma into the inner membrane

Chloroplast inner-membrane proteins Tic40 and Tic110 are first imported from the cytosol into the chloroplast stroma, and subsequently reinserted from the stroma into the inner membrane. However, the mechanism of reinsertion remains unclear. Here we show that Tic40 itself is involved in this reinsertion process. When precursors of either Tic40 or a Tic110 C-terminal truncate, tpTic110-Tic110N, were imported into chloroplasts isolated from a tic40-null mutant, soluble Tic40 and Tic110N intermediates accumulated in the stroma of tic40-mutant chloroplasts, due to a slower rate of reinsertion. We further show that a larger quantity of soluble Tic21 intermediates also accumulated in the stroma of tic40-mutant chloroplasts. In contrast, inner-membrane insertion of the triose-phosphate/phosphate translocator was not affected by the tic40 mutation. Our data suggest that multiple pathways exist for the insertion of chloroplast inner-membrane proteins.     

Neonatal screening for congenital adrenal hyperplasia in North-Eastern Italy: a report three years into the program

AIMS: To evaluate the incidence of congenital adrenal hyperplasia (CAH) in the Northern Italian population and the efficiency of the North-Eastern Italy screening program. To adjust cut-off levels for 17-hydroxyprogesterone (17-OHP) in relation to gestational age and birth weight, comparing the benefits in terms of reduction of recall rates with the two approaches and ultimately choosing the better of the two. SUBJECTS AND METHODS: Since September 2001, blood samples from neonates born in North-Eastern Italy have been screened with a fluoroimmunoassay method for 17-OHP determination (DELFIA). A preliminary cut-off level of > or = 30 nmol/l was set both for term and preterm newborns. The values of 17-OHP were analysed using statistical methods in relation to gestational age and birth weight in order to modify the cut-off on the basis of our data. RESULTS: After 33 months of screening we screened 128,282 newborns and detected 6 affected babies. During the first 8 months of screening among the recalled babies, 89.6 and 78.1% were preterm and low-birth-weight newborns, respectively, with a recall rate of 2.59% for premature neonates and of 4.94% for babies with birth weights < 2,500 g. We chose a new cut-off value of 50 nmol/l for preterm newborns only and, after 4 months, the recall rate was reduced to 0.83% for these infants and to 1.83% for low-birth-weight infants. CONCLUSION: After 33 months of screening for CAH in North-Eastern Italy, we report an incidence of 1:21,380. In 5 out of 6 affected babies, the diagnosis was established only after a positive screening test, which prevented a severe salt-wasting crisis in these babies. The cut-off level related to gestational age led to a significant reduction in the number of false-positives among preterm babies.We therefore intend to continue with the screening program for CAH in North-Eastern Italy, keeping a gestational-age-related cut-off in the hope that our data may encourage a national screening program for CAH.     

Bacillus subtilis SepF Binds to the C-Terminus of FtsZ

Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.     

Isolating a trimer intermediate in the self-assembly of E2 protein cage

Understanding the self-assembly mechanism of caged proteins provides clues to develop their potential applications in nanotechnology, such as a nanoscale drug delivery system. The E2 protein from Bacillus stearothermophilus , with a virus-like caged structure, has drawn much attention for its potential application as a nanocapsule. To investigate its self-assembly process from subunits to a spherical protein cage, we truncate the C-terminus of the E2 subunit. The redesigned protein subunit shows dynamic transition between monomer and trimer, but not the integrate 60-mer. The results indicate the role of the trimer as the intermediate and building block during the self-assembly of the E2 protein cage. In combination with the molecular dynamics simulations results, we conclude that the C-terminus modulates the self-assembly of the E2 protein cage from trimer to 60-mer. This investigation elucidates the role of the intersubunit interactions in engineering other functionalities in other caged structure proteins.     

Intron 5 alpha of the COXI gene of yeast mitochondrial DNA is a mobile group I intron.

We have found that intron 5 alpha of the COXI gene (al5 alpha) of yeast mtDNA is a mobile group I intron in crosses between strains having or lacking the intron. We have demonstrated the following hallmarks of that process: 1) co-conversion of flanking optional intron markers; 2) mutations that truncate the intron open reading frame block intron mobility; and 3) the intron open reading frame encodes an endonuclease activity that is required for intron movement. The endonuclease activity, termed I-Sce IV, cleaves the COXI allele lacking al5 alpha near the site of intron insertion, making a four-base staggered cut with 3' OH overhangs. Three cloned DNAs derived from different forms of the COXI gene, which differ in primary sequence at up to seven nucleotides around the cleavage site, are all good substrates for in vitro I-Sce IV cleavage activity. Two of the strains from which these substrates were derived were tested in crosses and are comparably efficient as al5 alpha recipients. When compared with omega mobility occurring simultaneously in one cross, al5 alpha is less efficient as a mobile element.     

The effect of interactions on the cellular uptake of nanoparticles

We present a simple two-state model to understand the size-dependent endocytosis of nanoparticles. Using this model, we elucidate the relevant energy terms required to understand the size-dependent uptake mechanism and verify it by correctly predicting the behavior at large and small particle sizes. In the absence of interactions between the nanoparticles, we observe an asymmetric distribution of sizes with maximum uptake at intermediate sizes and a minimum size cut-off below which there can be no endocytosis. Including the effect of interactions in our model has remarkable effects on the uptake characteristics. Attractive interactions shift the minimum size cut-off and increase the optimal uptake while repulsive interactions make the distribution more symmetric lowering the optimal uptake.     

Human mutations in NDE1 cause extreme microcephaly with lissencephaly [corrected

Genes disrupted in human microcephaly (meaning "small brain") define key regulators of neural progenitor proliferation and cell-fate specification. In comparison, genes mutated in human lissencephaly (lissos means smooth and cephalos means brain) highlight critical regulators of neuronal migration. Here, we report two families with extreme microcephaly and grossly simplified cortical gyral structure, a condition referred to as microlissencephaly, and show that they carry homozygous frameshift mutations in NDE1, which encodes a multidomain protein that localizes to the centrosome and mitotic spindle poles. Both human mutations in NDE1 truncate the C-terminal NDE1domains, which are essential for interactions with cytoplasmic dynein and thus for regulation of cytoskeletal dynamics in mitosis and for cell-cycle-dependent phosphorylation of NDE1 by Cdk1. We show that the patient NDE1 proteins are unstable, cannot bind cytoplasmic dynein, and do not localize properly to the centrosome. Additionally, we show that CDK1 phosphorylation at T246, which is within the C-terminal region disrupted by the mutations, is required for cell-cycle progression from the G2 to the M phase. The role of NDE1 in cell-cycle progression probably contributes to the profound neuronal proliferation defects evident in Nde1-null mice and patients with NDE1 mutations, demonstrating the essential role of NDE1 in human cerebral cortical neurogenesis.     

The role of the notochord for epaxial myotome formation in the mouse.

The vertebrate somite is the source of all trunk skeletal muscles. Myogenesis in avian embryos is thought to depend on signals from notochord and neural tube for the epaxial muscles, and signals from lateral mesoderm and surface ectoderm for the hypaxial muscles. However, this hypothesis has to be tested because in mouse mutants lacking a notochord the presence of a fused myotome beneath the neural tube has been reported. We have compared the expression pattern of myogenic markers and markers for the hypaxial muscle precursors in the mutants Brachyury curtailed, truncate, Danforth's short tail and Pintail. In regions lacking notochord and sclerotome, we found small, ventrally located domains of Myf5 and MyoD expression, concomitant with ventrally expanded Pax3 signals and upregulated expression of the hypaxial marker Lbx1, suggesting that only the hypaxial program is active. We therefore hypothesise that in mammals, as in birds, the formation of the epaxial musculature depends on the presence of notochord derived signals.     

Optimal cut-off score for diagnosing depression with the Patient Health Questionnaire (PHQ-9): a meta-analysis

Background:

The brief Patient Health Questionnaire (PHQ-9) is commonly used to screen for depression with 10 often recommended as the cut-off score. We summarized the psychometric properties of the PHQ-9 across a range of studies and cut-off scores to select the optimal cut-off for detecting depression.

Methods:

We searched Embase, MEDLINE and PsycINFO from 1999 to August 2010 for studies that reported the diagnostic accuracy of PHQ-9 to diagnose major depressive disorders. We calculated summary sensitivity, specificity, likelihood ratios and diagnostic odds ratios for detecting major depressive disorder at different cut-off scores and in different settings. We used random-effects bivariate meta-analysis at cutoff points between 7 and 15 to produce summary receiver operating characteristic curves.

Results:

We identified 18 validation studies (n = 7180) conducted in various clinical settings. Eleven studies provided details about the diagnostic properties of the questionnaire at more than one cut-off score (including 10), four studies reported a cut-off score of 10, and three studies reported cut-off scores other than 10. The pooled specificity results ranged from 0.73 (95% confidence interval [CI] 0.63–0.82) for a cut-off score of 7 to 0.96 (95% CI 0.94–0.97) for a cut-off score of 15. There was major variability in sensitivity for cut-off scores between 7 and 15. There were no substantial differences in the pooled sensitivity and specificity for a range of cut-off scores (8–11).

Interpretation:

The PHQ-9 was found to have acceptable diagnostic properties for detecting major depressive disorder for cut-off scores between 8 and 11. Authors of future validation studies should consistently report the outcomes for different cut-off scores.Depressive disorders are still under-recognized in medical settings despite major associated disability and costs. The use of short screening questionnaires may improve the recognition of depression in different medical settings.¹ The depression module of the Patient Health Questionnaire (PHQ-9) has become increasingly popular in research and practice over the past decade.² In its initial validation study, a score of 10 or higher had a sensitivity of 88% and a specificity of 88% for detecting major depressive disorders. Thus, a score of 10 has been recommended as the cut-off score for diagnosing this condition.³In a recent review of the PHQ-9, Kroenke and colleagues argued against inflexible adherence to a single cut-off score.² A recent analysis of the management of depression in general practice in the United Kingdom showed that the accuracy of predicting major depressive disorder could be improved by using 12 as the cut-off score.⁴Given the widespread use of PHQ-9 in screening for depression and that certain cut-off scores are being recommended as part of national strategies to screen for depression (based on initial validation studies, which might not be generalizable),^4,5 we attempted to determine whether the cut-off of 10 is optimum for screening for depression. This question could not be answered by two previous systematic reviews^6,7 because of the small number of primary studies available at the time. We also aimed to provide greater clarity about the proper use of PHQ-9 given the many settings in which it is used.     

Effect of dsDNA binding to SmD-derived peptides on clinical accuracy in the diagnosis of systemic lupus erythematosus

Systemic lupus erythematosus is characterized by antibodies to a variety of intracellular self-antigens, such as dsDNA and Sm, and these serve as hallmarks in the diagnosis of systemic autoimmune diseases. Several studies have shown that SmD1 and SmD3 synthetic peptides represent highly functional antigens for autoantibody detection and thus for diagnostic applications. The present study analysed the technical and clinical accuracy of an anti-SmD1 (amino acids 83–119) and an anti-SmD3 (amino acids 108–122) ELISA for the detection of anti-Sm antibodies. Depending on the cut-off value of the SmD1 ELISA, we found a high degree of concordance between the two tests. At an optimized cut-off value of 100 units for SmD1 we found the same clinical sensitivity (12.5%) and specificity (100%) in a group of systemic lupus erythematosus patients (n = 48) and in controls (n = 99). The concordance at this cut-off value was 100% (P < 0.0001; χ² = 127.61). Using a second panel of sera (n = 65) preselected based on positive anti-Sm results, we confirmed the high degree of concordance between the two assays. Using dsDNA-coated ELISA plates and biotinylated peptides we confirmed the high dsDNA binding properties for SmD1, which were significantly higher than the SmD3-derived peptide. However, no cross-linking of anti-dsDNA antibodies to SmD1 was observed after adding increasing amounts of dsDNA to anti-dsDNA positive, anti-SmD1 negative serum. We therefore conclude that the reported difference in the sensitivity is related to the different cut-off levels and not to the detection of anti-dsDNA antibodies bridged via dsDNA to the SmD1 peptide. Moreover, we found that a subpopulation of anti-Sm antibodies cross-reacted with SmD1 and SmD3. Taken together, the data indicate that both SmD peptide ELISAs represent accurate assays and may be used as important standards for the detection of anti-Sm antibodies.