Profiles of lymphomas consequent to MLV integration into the genome of pre-B cells in the bone marrow.

<p>+, ++: Five- and ten-fold increase in expression level, respectively, relative to normal control pre-B cells in the bone marrow, according to a quantitative RT-PCR assay for <i>Stat5a</i> and flow cytometry for IgM, Lambda5, and Vpreb.</p

<i>In vitro</i> MLV-LTR integration into <i>Stat5a</i>.

<p>The vertical axis to the left represents the number of integrations into each nucleotide in M0 (native <i>Stat5a</i>), modified sequences M1-M4, and control same length random sequences R1-R5. These sequences are 400-bp in length, portions of which are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031533#pone-0031533-g001" target="_blank">Figure 1</a>. The horizontal axis represents the bases 1105–1153 in the <i>Stat5a</i> gene. The sequences shown are the junction of the target sequence and 5′- MLV LTR when the MLV is inserted at nucleotide 1130. (A) Integration sites identified with the <i>in vitro</i> assay using sequences M0-M4. Black circles (L) represent the number of mice suffering from lymphomas resulting from MLV integration into the individual nucleotides shown. The number of integrations into nucleotide 1130 per 2000 integrations was significantly greater with sequences M0 and M1 than with sequences M2-M5 (*<i>P</i><0.05). (B) Integration sites identified with the <i>in vitro</i> assay using the 5 random sequences (R1-R5) inserted into the plasmid DNA.</p

<i>In vitro</i> integration using the <i>c-myc</i> promoter sequence and buffers containing variable concentrations of MgCl₂.

<p>(A) Thermodynamic analysis of the presumed cruciform structure from the <i>c-myc</i> promoter sequence DNA (GENEBANK M12345, No. 711-980) in the presence of 60 mM MgCl₂. Black arrows indicate the previously reported integration sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031533#pone.0031533-Nielsen1" target="_blank">[15]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031533#pone.0031533-Hansen1" target="_blank">[19]</a>. Red arrows indicate representative <i>in vitro</i> integration sites. A box indicates the hot spot segment. (B) Graph showing the relative area of supercoiled plasmid DNA with (target +) or without target <i>c-myc</i> DNA (target −) at 60 min after incubation in buffer containing various concentrations of MgCl₂ (unit mM, n = 6; mean ± s.d.). (C) Graph showing the total number of integration sites within the target <i>c-myc</i> DNA and the number of integration into the hot spot at 60 min after incubation in buffer containing various concentrations of MgCl₂ (unit mM, n = 6; mean ± s.d.).</p

Target sequence length and integration.

<p>(A) Presumed secondary structure of the top strand generating a cruciform in the presence of 60 mM of MgCl₂, as predicted using the M-fold program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031533#pone.0031533-Zuker1" target="_blank">[20]</a>. (B) Absolute value of the Gibbs' free energy change during DNA folding and generation of a cruciform by the top strand. Arrows represent the marginal points in which the lengths are threshold values of the free energy change. (C) Number of integrations into nucleotide 1130 in the top strand cruciform. Arrows represent the marginal points in which the lengths are the threshold value. Arrows correspond well to the positions of those in (B) (n = 6; mean ± s.d.). The square of the correlation coefficient for the absolute energy value shown in (B) and the number of integrations shown in (C) was 0.838.</p

<i>In vitro</i> integration in buffers of varying MgCl₂ concentration.

<p>(A) Electrophoresis of plasmid DNA with (upper) or without (lower) the target sequence DNA. Supercoiled DNA was electrophoresed at 0 min, at 30 min, and at 60 min after incubation. Arrows and arrowheads indicate fragments corresponding to peaks shown in (B). (B) Electropherogram of the plasmid with the target sequence and the plasmid alone at incubation for 0 min and 30 min. (C) Graph showing the relative area of the electrophoretic signal of supercoiled plasmid DNA with (red) or without (blue) target <i>Stat5a</i> DNA at 60 min after incubation in buffer containing various concentrations of MgCl₂ (unit mM, n = 6; mean ± s.d.). (D) Graph showing the total number of integration sites within the target <i>Stat5a</i> DNA (red) and the number of integrations at nucleotide 1130 (blue) at 60 min after incubation in buffer containing various concentrations of MgCl₂ (unit mM, n = 6; mean ± s.d.). (E) Sample photo of a secondary structure when using a buffer containing 60 mM and 30 mM of manganese dichloride (mM). Supercoiled DNA (in the upper photo) and globular DNA (in the lower photo) are displayed.</p

<i>In vitro</i> integration using retroviral LTRs.

<p>(A) The 5′ and 3′ LTR of MLV proviral DNA (red line) was used after removal of other elements encoding <i>gag</i>, <i>pol</i>, <i>pro</i> and <i>env</i>. The sequence shown displays the MLV LTR in the form integrated into the host DNA. The target DNA (grey line) was ligated into the pCR2.1 TOPO plasmid vector (black line). Arrowheads next to the proviral DNA sequence represent the processed ends. After incubation of retroviral and target DNA with integrase, proviral DNA was integrated into the target sequence or plasmid. The integration site was then sequenced. (B) The MLV integration site hot spot in the lymphoma genome of SL/Kh mice is represented by the red square. The utilized target sequences M0-M4 are shown below. M0 is identical to the native <i>Stat5a</i> sequence. Red letters in the sequence indicate the most frequent sites of integration in hematopoietic tumors as previously reported by us <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031533#pone.0031533-Tsuruyama2" target="_blank">[14]</a>.</p

DataSheet_1_Multisite chronic pain and the risk of autoimmune diseases: A Mendelian randomization study.docx

BackgroundAccumulating evidence has demonstrated that an association between chronic pain and autoimmune diseases (AIDs). Nevertheless, it is unclear whether these associations refer to a causal relationship. We used a two-sample Mendelian randomization (MR) method to determine the causal relationship between chronic pain and AIDs.MethodsWe assessed genome-wide association study (GWAS) summary statistics for chronic pain [multisite chronic pain (MCP) and chronic widespread pain (CWP)], and eight common AIDs, namely, amyotrophic lateral sclerosis (ALS), celiac disease (CeD), inflammatory bowel disease (IBD), multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus Erythematosus (SLE), type 1 diabetes (T1D) and psoriasis. Summary statistics data were from publicly available and relatively large-scale GWAS meta-analyses to date. The two-sample MR analyses were first performed to identify the causal effect of chronic pain on AIDs. The two-step MR and multivariable MR were used to determine if mediators (BMI and smoking) causally mediated any connection and to estimate the proportion of the association mediated by these factors combined.ResultsWith the utilization of MR analysis, multisite chronic pain was associated with a higher risk of MS [odds ratio (OR) = 1.59, 95% confidence interval (CI) = 1.01-2.49, P = 0.044] and RA (OR = 1.72, 95% CI = 1.06-2.77, P = 0.028). However, multisite chronic pain had no significant effect on ALS (OR = 1.26, 95% CI = 0.92-1.71, P = 0.150), CeD (OR = 0.24, 95% CI = 0.02-3.64, P = 0.303), IBD (OR = 0.46, 95% CI = 0.09-2.27, P = 0.338), SLE (OR = 1.78, 95% CI = 0.82-3.88, P = 0.144), T1D (OR = 1.15, 95% CI = 0.65-2.02, P = 0.627) or Psoriasis (OR = 1.59, 95% CI = 0.22-11.26, P = 0.644). We also found positive causal effects of MCP on BMI and causal effects of BMI on MS and RA. Moreover, there were no causal connections between genetically predicted chronic widespread pain and the risk of most types of AIDs disease.ConclusionOur MR analysis implied a causal relationship between MCP and MS/RA, and the effect of MCP on MS and RA may be partially mediated by BMI.</p

sj-docx-1-tpp-10.1177_20451253241243260 – Supplemental material for Attentional bias modification and attention control training in PTSD: a systematic review and meta-analysis

Supplemental material, sj-docx-1-tpp-10.1177_20451253241243260 for Attentional bias modification and attention control training in PTSD: a systematic review and meta-analysis by Fan Zhang, Chenwei Huang, Wenjie Yan, Hui Ouyang and Weizhi Liu in Therapeutic Advances in Psychopharmacology</p

High-Performance Smart Hydrogels with Redox-Responsive Properties Inspired by Scallop Byssus

Smart hydrogels with versatile properties, including a tunable gelation time, nonswelling attributes, and biocompatibility, are in great need in the biomedical field. To meet this urgent demand, we explored novel biomaterials with the desired properties from sessile marine organisms. To this end, a novel protein, Sbp9, derived from scallop byssus was extensively investigated, which features typical epidermal growth factor-like (EGFL) multiple repetitive motifs. Our current work demonstrated that the key fragment of Sbp9 (calcium-binding domain (CBD) and 4 EGFL repeats (CE4)) was able to form a smart hydrogel driven by noncovalent interactions and facilitated by disulfide bonds. More importantly, this smart hydrogel demonstrates several desirable and beneficial features, which could offset the drawbacks of typical protein-based hydrogels, including (1) a redox-responsive gelation time (from <1 to 60 min); (2) tunable mechanical properties, nonswelling abilities, and an appropriate microstructure; and (3) good biocompatibility and degradability. Furthermore, proof-of-concept demonstrations showed that the newly discovered hydrogel could be used for anticancer drug delivery and cell encapsulation. Taken together, a smart hydrogel inspired by marine sessile organisms with desirable properties was generated and characterized and demonstrated to have extensive applicability potential in biomedical applications, including tissue engineering and drug release

Discovery and characterization of tyrosinases from sea anemone pedal disc

The formation of underwater adhesion is a complicated physiological process and many different types of enzymes are found to be essential apart from structural proteins. Previous studies have shown that various tyrosinases were present in marine adhesives, but little information is available about the over-expression and enzymatic characterization of these enzymes. Specifically, this study first identified four significantly up-regulated tyrosinases in the pedal disc of Haliplanella luciae by means of multi-omics technology, and made preliminary bioinformatics predictions. Sequence alignment showed that the Tyr1_Hl contained six conserved His residues that bind to copper ions, of which a tyrosinase with diphenolase activity named as Tyr1_HlΔ, was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Enzymatic characterization showed that the activity of Tyr1_HlΔ was Cu2+ dependent and maximum catalytic activities were in 20 mM Tris–HCl (pH 8.0) at 37 °C. In summary, we identified novel tyrosinases in the pedal disks of sea anemone for the first time and the Tyr1_HlΔ was successfully recombinant expressed. Our study will provide basis for future exploration of bio-adhesion mechanism and design of bio-adhesives derived from sea anemones.</p