Facile tethering of stable and unstable proteins for optical tweezers experiments

Single-molecule force spectroscopy with optical tweezers has emerged as a powerful tool for dissecting protein folding. The requirement to stably attach “molecular handles” to specific points in the protein of interest by preparative biochemical techniques is a limiting factor in applying this methodology, especially for large or unstable proteins that are difficult to produce and isolate. Here, we present a streamlined approach for creating stable and specific attachments using autocatalytic covalent tethering. The high specificity of coupling allowed us to tether ribosome-nascent chain complexes, demonstrating its suitability for investigating complex macromolecular assemblies. We combined this approach with cell-free protein synthesis, providing a facile means of preparing samples for single-molecule force spectroscopy. The workflow eliminates the need for biochemical protein purification during sample preparation for single-molecule measurements, making structurally unstable proteins amenable to investigation by this powerful single-molecule technique. We demonstrate the capabilities of this approach by carrying out pulling experiments with an unstructured domain of elongation factor G that had previously been refractory to analysis. Our approach expands the pool of proteins amenable to folding studies, which should help to reduce existing biases in the currently available set of protein folding models.     

Intraocular iron injection induces oxidative stress followed by elements of geographic atrophy and sympathetic ophthalmia

Iron has been implicated in the pathogenesis of age‐related retinal diseases, including age‐related macular degeneration (AMD). Previous work showed that intravitreal (IVT) injection of iron induces acute photoreceptor death, lipid peroxidation, and autofluorescence (AF). Herein, we extend this work, finding surprising chronic features of the model: geographic atrophy and sympathetic ophthalmia. We provide new mechanistic insights derived from focal AF in the photoreceptors, quantification of bisretinoids, and localization of carboxyethyl pyrrole, an oxidized adduct of docosahexaenoic acid associated with AMD. In mice given IVT ferric ammonium citrate (FAC), RPE died in patches that slowly expanded at their borders, like human geographic atrophy. There was green AF in the photoreceptor ellipsoid, a mitochondria‐rich region, 4 h after injection, followed later by gold AF in rod outer segments, RPE and subretinal myeloid cells. The green AF signature is consistent with flavin adenine dinucleotide, while measured increases in the bisretinoid all‐trans‐retinal dimer are consistent with the gold AF. FAC induced formation carboxyethyl pyrrole accumulation first in photoreceptors, then in RPE and myeloid cells. Quantitative PCR on neural retina and RPE indicated antioxidant upregulation and inflammation. Unexpectedly, reminiscent of sympathetic ophthalmia, autofluorescent myeloid cells containing abundant iron infiltrated the saline‐injected fellow eyes only if the contralateral eye had received IVT FAC. These findings provide mechanistic insights into the potential toxicity caused by AMD‐associated retinal iron accumulation. The mouse model will be useful for testing antioxidants, iron chelators, ferroptosis inhibitors, anti‐inflammatory medications, and choroidal neovascularization inhibitors.     

Rare Fungal Keratitis Caused by Coprinellus Radians

Mycopathologia - A case of fungal keratitis due to Coprinellus radians is reported. To our knowledge, fungal keratitis caused by this species was rare. Fungal hyphae were detected in corneal...     

A doubly auxotrophic CHO-K1 cell line for the production of recombinant monoclonal antibodies

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO-K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR-Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively). Survival of these doubly auxotrophic cells depends on the provision of sources of purines and pyrimidines or on the transfection and integration of open reading frames encoding these two enzymes. We successfully used one such double auxotroph (UA10) to select for stable transfectants carrying (a) the recombinant tumor necrosis factor-α receptor fusion protein etanercept and (b) the heavy and light chains of the anti-Her2 monoclonal antibody trastuzumab. Transfectant clones produced these recombinant proteins in a stable manner and in substantial amounts. The availability of this double auxotroph provides a rapid and efficient selection method for the serial or simultaneous transfer of genes for multiple polypeptides of choice into CHO cells using readily available purine- and pyrimidine-free commercial media.     

金钟藤入侵群落的种间联结及生态位特征

金钟藤(Decalobanthus boisianus)是林业有害植物, 其暴发生长和扩散对森林生态系统造成了严重破坏。本文以海南岛48个金钟藤典型分布群落为研究对象, 用方差比率法和贡献定律法探究群落的稳定性; 用χ ²统计量、联结系数(AC)、共同出现百分率(PC)、Ochiai指数和Dice指数分析金钟藤与伴生物种的种间联结关系; 用生态位宽度、生态位相似性系数和生态位重叠指数研究群落中各物种的生态位特征, 以期为金钟藤生物防治的植物物种筛选提供借鉴。结果表明: (1)金钟藤所在48个群落共有156种伴生植物, 其中大戟科、茜草科、桑科、无患子科和樟科植物占优势; (2)群落中优势物种呈正联结关系, 植物种类累积倒数百分比与累积相对频度交点坐标为(44.53, 55.47), 远离稳定交点坐标(20, 80), 说明群落处于不稳定状态; (3)金钟藤与芳槁润楠(Machilus suaveolens)、黄椿木姜子(Litsea variabilis)、岭南山竹子(Garcinia oblongifolia)、显脉杜英(Elaeocarpus dubius)、鸭脚木(Schefflera octophylla)和银柴(Aporusa dioica)都紧密关联, 说明金钟藤与这些物种的资源利用方式较相似; (4)金钟藤的生态位宽度最大, 与伴生物种间的生态位重叠度较高, 但伴生物种间的生态位重叠度较低。金钟藤的入侵导致群落处于不稳定状态, 并与伴生物种间存在激烈的竞争关系。因此, 建议在金钟藤已入侵的群落中大量栽种芳槁润楠、黄椿木姜子、显脉杜英、鸭脚木和银柴, 以遏制其蔓延; 大量栽种翻白叶树(Pterospermum heterophyllum)、海南菜豆树(Radermachera hainanensis)、九节(Psychotria rubra)和肉实树(Sarcosperma laurinum)用于金钟藤入侵群落的植被恢复。     

AZD8835 inhibits osteoclastogenesis and periodontitis-induced alveolar bone loss in rats

Chronic periodontitis (CP) is one of the most common oral diseases, which is characterized by the loss of connective tissue and alveolar bone in adults. AZD8835, a novel dual phosphoinositide-3-kinase (PI3K) inhibitor, is currently in phase 1 clinical evaluation to treat breast cancer. However, whether AZD8835 has any effect on teeth and alveolar bone health remains unclear. In the current study, we aimed to investigate the potential effect of AZD8835 in treating CP in vitro and in vivo. We found that AZD8835 could inhibit osteoclast differentiation, bone resorption, and downregulate the expression of osteoclast marker genes, such as tartrate-resistant acid phosphatase (Trap), cathepsin K (Ctsk), V-ATPase d2 (Atp6v0d2), and calcitonin receptor (Ctr). In addition, AZD8835 suppressed osteoclastogenesis by inhibiting receptor activator of nuclear factor kappa B ligand (RANKL)-induced PI3K/protein kinase B (AKT), extracellular signal-regulated kinase, and nuclear factor-κB signaling in BMMs. In vivo, AZD8835 greatly ameliorated alveolar bone (ABL) loss in rats with CP. Meanwhile, histological examination showed fewer osteoclasts in the treatment group. In conclusion, these results indicated that AZD8835 is a promising agent to reduce ABL in CP.     

结直肠癌组织RPN11与Ki67的表达及其临床病理学意义

目的观察去泛素化酶RPN11和增殖相关核标记物Ki67在结直肠癌组织中的表达,研究其与结直肠癌肿瘤细胞增殖的相关性及与结直肠癌临床病理特征的关系。方法采用免疫组织化学SABC法检测56例结直癌组织及20例癌旁正常组织中的RPN11和Ki67表达,结合临床病理学资料进行统计分析。结果免疫组织化学染色显示:RPN11及Ki67在结直肠癌组织的阳性表达率明显高于正常结直肠组织;RPN11和Ki67的表达均与肿瘤分化程度、TNM分期、转移有关,而与性别、年龄无明显相关;RPN11与Ki67的表达呈正相关。结论RPN11和Ki67可能共同参与结直肠癌肿瘤细胞的增殖调控,并促进结直肠癌的发生发展以及浸润转移。     

A Water‐Soluble Cu Complex as Molecular Catalyst for Electrocatalytic CO2 Reduction on Graphene‐Based Electrodes

A structurally simple molecular 1,10‐phenanthroline‐Cu complex on a mesostructured graphene matrix that can be active and selective toward CO₂ reduction over H₂ evolution in an aqueous solution is reported. The active sites consist of Cu(I) center in a distorted trigonal bipyramidal geometry, which enables the adsorption of CO₂ with η¹‐COO‐like configuration to commence the catalysis, with a turnover frequency of ≈45 s^?1 at ?1 V versus reversible hydrogen electrode. Using in situ infrared spectroelectrochemical investigation, it is demonstrated that the Cu complex can be reversibly heterogenized near the graphene surface via potential control. An increase of electron density in the complex is observed as a result of the interaction from the electric field, which further tunes the electron distribution in the neighboring CO₂. It is also found that the mesostructure of graphene matrix favored CO₂ reduction on the Cu center over hydrogen evolution by limiting mass transport from the bulk solution to the electrode surface.     

Ursolic acid prevents doxorubicin‐induced cardiac toxicity in mice through eNOS activation and inhibition of eNOS uncoupling

In addition to the known antitumour effects of ursolic acid (UA), increasing evidence indicates that this molecule plays a role in cardiac protection. In this study, the effects of ursolic acid on the heart in mice treated with doxorubicin (DOX) were assessed. The results showed that ursolic acid improved left ventrical fractional shortening (LVFS) and left ventrical ejection fraction (LVEF) of the heart, increased nitrogen oxide (NO) levels, inhibited reactive oxygen species (ROS) production and decreased cardiac apoptosis in mice treated with doxorubicin. Mechanistically, ursolic acid increased AKT and endothelial nitric‐oxide synthase (eNOS) phosphorylation levels, and enhanced eNOS expression, while inhibiting doxorubicin induced eNOS uncoupling through NADPH oxidase 4 (NOX4) down‐regulation. These effects of ursolic acid resulted in heart protection from doxorubicin‐induced injury. Therefore, ursolic acid may be considered a potential therapeutic agent for doxorubicin‐associated cardiac toxicity in clinical practice.