Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required

Effective gauge group of pure loop quantum gravity is SO(3): New estimate of the Immirzi parameter

We argue that the effective gauge group for {\it pure} four-dimensional loop quantum gravity(LQG) is SO(3) (or $SO(3,C)$) instead of SU(2) (or $SL(2,C)$). As a result, links with half-integer spins in spin network states are not realized for {\it pure} LQG, implying a modification of the spectra of area and volume operators. Our observations imply a new value of $\gamma \approx 0.170$ for the Immirzi parameter which is obtained from matching the Bekenstein-Hawking entropy to the number of states from LQG calculations. Moreover, even if the dominant contribution to the entropy is not assumed to come from configurations with the minimum spins, the results of both pure LQG and the supersymmetric extension of LQG can be made compatible when only integer spins are realized for the former, while the latter also contains half-integer spins, together with an Immirzi parameter for the supersymmetric case which is twice the value of the SO(3) theory. We also verify that the $-{1/2}$ coefficient of logarithmic correction to the Bekenstein-Hawking entropy formula is robust, independent of whether only integer, or also half-integer spins, are realized.Comment: new value of Immirzi parameter is ~0.170; dominance of miminum spin configurations is not assumed in comparing with Bekenstein-Hawking formula; typos corrected. Version pressed in PL

Projecting the 10-year costs of care and mortality burden of depression until 2032: a Markov modelling study developed from real-world data

Background Based on real-world data, we developed a 10-year prediction model to estimate the burden among patients with depression from the public healthcare system payer's perspective to inform early resource planning in Hong Kong. Methods We developed a Markov cohort model with yearly cycles specifically capturing the pathway of treatment-resistant depression (TRD) and comorbidity development along the disease course. Projected from 2023 to 2032, primary outcomes included costs of all-cause and psychiatric care, and secondary outcomes were all-cause deaths, years of life lived, and quality-adjusted life-years. Using the territory-wide electronic medical records, we identified 25,190 patients aged ≥10 years with newly diagnosed depression from 2014 to 2016 with follow-up until 2020 to observe the real-world time-to-event pattern, based on which costs and time-varying transition inputs were derived using negative binomial modelling and parametric survival analysis. We applied the model as both closed cohort, which studied a fixed cohort of incident patients in 2023, and open cohort, which introduced incident patients by year from 2014 to 2032. Utilities and annual new patients were from published sources. Findings With 9217 new patients in 2023, our closed cohort model projected the 10-year cumulative costs of all-cause and psychiatric care to reach US$309.0 million and US$58.3 million, respectively, with 899 deaths (case fatality rate: 9.8%) by 2032. In our open cohort model, 55,849–57,896 active prevalent cases would cost more than US$322.3 million and US$60.7 million, respectively, with more than 943 deaths annually from 2023 to 2032. Fewer than 20% of cases would live with TRD or comorbidities but contribute 31–54% of the costs. The greatest collective burden would occur in women aged above 40, but men aged above 65 and below 25 with medical history would have the highest costs per patient-year. The key cost drivers were relevant to the early disease stages. Interpretation A limited proportion of patients would develop TRD and comorbidities but contribute to a high proportion of costs, which necessitates appropriate attention and resource allocation. Our projection also demonstrates the application of real-world data to model long-term costs and mortality, which aid policymakers anticipate foreseeable burden and undertake budget planning to prepare for the care need in alternative scenarios

Elevated adipogenesis of marrow mesenchymal stem cells during early steroid-associated osteonecrosis development

<p>Abstract</p> <p>Background</p> <p>Increased bone marrow lipid deposition in steroid-associated osteonecrosis (ON) implies that abnormalities in fat metabolism play an important role in ON development. The increase in lipid deposition might be explained by elevated adipogenesis of marrow mesenchymal stem cells (MSCs). However, it remains unclear whether there is a close association between elevated adipogenesis and steroid-associated ON development.</p> <p>Objective</p> <p>The present study was designed to test the hypothesis that there might be a close association between elevated adipogenesis and steroid-associated ON development.</p> <p>Methods</p> <p>ON rabbit model was induced based on our established protocol. Dynamic-MRI was employed for local intra-osseous perfusion evaluation in bilateral femora. Two weeks after induction, bone marrow was harvested for evaluating the ability of adipogenic differentiation of marrow MSCs at both cellular and mRNA level involving adipogenesis-related gene peroxisome proliferator-activated receptor gamma2 (PPARγ2). The bilateral femora were dissected for examining marrow lipid deposition by quantifying fat cell number, fat cell size, lipid deposition area and ON lesions. For investigating association among adipogenesis, lipid deposition and perfusion function with regard to ON occurrence, the rabbits were divided into ON⁺(with at least one ON lesion) group and ON^-(without ON lesion) group. For investigating association among adipogenesis, lipid deposition and perfusion function with regard to ON extension, the ON⁺rabbits were further divided into sub-single-lesion group (SON group: with one ON lesion) and sub-multiple-lesion group (MON group: with more than one ON lesion).</p> <p>Results</p> <p>Local intra-osseous perfusion index was found lower in either ON⁺or MON group when compared to either ON^-or SON group, whereas the marrow fat cells number and area were much larger in either ON⁺or MON group as compared with ON^-and SON group. The adipogenic differentiation ability of MSCs and PPARγ2 expression in either ON⁺or MON group were elevated significantly as compared with either ON^-or SON group.</p> <p>Conclusion</p> <p>These findings support our hypothesis that there is a close association between elevated adipogenesis and steroid-associated osteonecrosis development.</p

Comparative proteomic analysis of malformed umbilical cords from somatic cell nuclear transfer-derived piglets: implications for early postnatal death

Background: Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets. Results: Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC. Conclusion: These results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development

Could social robots facilitate children with autism spectrum disorders in learning distrust and deception?

Social robots have been increasingly involved in our daily lives and provide a new environment for children\u27s growth. The current study aimed to examine how children with and without Autism Spectrum Disorders (ASD)learned complex social rules from a social robot through distrust and deception games. Twenty children with ASD between the ages of 5–8 and 20 typically-developing (TD)peers whose age and IQ were matched participated in distrust and deception tasks along with an interview about their perception of the human-likeness of the robot. The results demonstrated that: 1)children with ASD were slower to learn to and less likely to distrust and deceive a social robot than TD children and 2)children with ASD who perceived the robot to appear more human-like had more difficulty in learning to distrust the robot. Besides, by comparing to a previous study the results showed that children with ASD appeared to have more difficulty in learning to distrust a human compared to a robot, particularly in the early phase of learning. Overall, our study verified that social robots could facilitate children with ASD\u27s learning of some social rules and showed that children\u27s perception of the robot plays an important role in their social learning, which provides insights on robot design and its clinical applications in ASD intervention

A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein

10.1371/journal.pone.0033451PLoS ONE74

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely