The effects of washing a collagen sample prior to TEM examination

Transmission electron microscopy (TEM) is an important analysis technique to visualize (bio)macromolecules and their assemblies, including collagen fibers. Many protocols for TEM sample preparation of collagen involve one or more washing steps to remove excess salts from the dispersion that could hamper analysis when dried on a TEM grid. Such protocols are not standardized and washing times as well as washing solvents vary from procedure to procedure, with each research group typically having their own protocol. Here, we investigate the influence of washing with water, ethanol, but also methanol and 2-propanol, for both mineralized and unmineralized collagen samples via a protocol based on centrifugation. Washing with water maintains the hydrated collagen structure and the characteristic banding pattern can be clearly observed. Conversely, washing with ethanol results in dehydration of the fibrils, often leading to aggregation of the fibers and a less obvious banding pattern, already within 1 min of ethanol exposure. As we show, this process is fully reversible. Similar observations were made for methanol and propanol. Based on these results, a standardized washing protocol for collagenous samples is proposed.     

Characteristic tryptic peptides and gelling properties of porcine skin gelatin affected by thermal action

Thermal action in extraction process had effects on characteristic tryptic peptides identification and gelling properties of porcine gelatin. SDS-PAGE, HPLC-LTQ/Orbitrap high-resolution mass spectrometry, texture analyser and rheometer were used to evaluate collagen depolymerisation degree, characteristic tryptic peptides and gelling properties of gelatins prepared in various thermal actions. Results showed that with increasing temperature and time, depolymerisation degree enlarged, while gel strength, gelling and melting temperature decreased. Mass spectra showed that 47 and 49 common characteristic tryptic peptides were identified in gelatins extracted at 50 °C and 100 °C with various times, respectively. Moreover, 34 common characteristic tryptic peptides were identified in all gelatin samples. Further comparison between this work and our previous investigations yielded 20 common characteristic tryptic peptides, which stably exist in various thermal actions. These common characteristic tryptic peptides may be very helpful for the accurate authentication of porcine gelatin.     

Structural Aspects and Prediction of Calmodulin-Binding Proteins

Calmodulin (CaM) is an important intracellular protein that binds Ca²⁺ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM’s ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca²⁺ concentration through interactions with CaM.     

微波辅助孵育法富集麦胚多肽的工艺优化

目的 麦胚孵育过程中蛋白酶将蛋白质水解成肽和氨基酸，多肽具有明确的生理活性和营养调节作用。本研究以麦胚为试验原料，采用微波辅助预处理的方法，研究孵育的温度、时间、pH及料液比4种因素对孵育后的蛋白酶活力及多肽含量的影响。 方法 通过单因素和响应曲面试验进行工艺条件优化。 结果 与未进行微波辅助预处理的样品相比，微波辅助处理（600 W, 10 s）能显著提高孵育后样品中的蛋白酶酶活力（约9.4倍）和肽含量（约3.1倍）。经过微波辅助处理后，孵育温度为51.5℃、pH为4.0、时间为6.33 h、料液比为1:7时，蛋白酶活力达最高为3826.24 U/g；孵育温度为45.0 ℃、pH为4.8、时间为8 h、料液比为1:7时，肽含量达最高为262.63 mg/g。 结论 微波辅助处理能有效的激活麦胚孵育液中的内源性蛋白酶活性，促进蛋白水解反应，显著提高孵育后的肽含量。该研究结果为麦胚多肽的制备新工艺开发提供了研究基础。     

Interleukin-1β Modulation of the Mechanobiology of Primary Human Pulmonary Fibroblasts: Potential Implications in Lung Repair

Pro-inflammatory cytokines like interleukin-1β (IL-1β) are upregulated during early responses to tissue damage and are expected to transiently compromise the mechanical microenvironment. Fibroblasts are key regulators of tissue mechanics in the lungs and other organs. However, the effects of IL-1β on fibroblast mechanics and functions remain unclear. Here we treated human pulmonary fibroblasts from control donors with IL-1β and used Atomic Force Microscopy to unveil that IL-1β significantly reduces the stiffness of fibroblasts concomitantly with a downregulation of filamentous actin (F-actin) and alpha-smooth muscle (α-SMA). Likewise, COL1A1 mRNA was reduced, whereas that of collagenases MMP1 and MMP2 were upregulated, favoring a reduction of type-I collagen. These mechanobiology changes were functionally associated with reduced proliferation and enhanced migration upon IL-1β stimulation, which could facilitate lung repair by drawing fibroblasts to sites of tissue damage. Our observations reveal that IL-1β may reduce local tissue rigidity by acting both intracellularly and extracellularly through the downregulation of fibroblast contractility and type I collagen deposition, respectively. These IL-1β-dependent mechanical effects may enhance lung repair further by locally increasing pulmonary tissue compliance to preserve normal lung distension and function. Moreover, our results support that IL-1β provides innate anti-fibrotic protection that may be relevant during the early stages of lung repair.     

Sequence-Dependent Nanofiber Structures of Phenylalanine and Isoleucine Tripeptides

The molecular design of short peptides to achieve a tailor-made functional architecture has attracted attention during the past decade but remains challenging as a result of insufficient understanding of the relationship between peptide sequence and assembled supramolecular structures. We report a hybrid-resolution model to computationally explore the sequence–structure relationship of self-assembly for tripeptides containing only phenylalanine and isoleucine. We found that all these tripeptides have a tendency to assemble into nanofibers composed of laterally associated filaments. Molecular arrangements within the assemblies are diverse and vary depending on the sequences. This structural diversity originates from (1) distinct conformations of peptide building blocks that lead to different surface geometries of the filaments and (2) unique sidechain arrangements at the filament interfaces for each sequence. Many conformations are available for tripeptides in solution, but only an extended β-strand and another resembling a right-handed turn are observed in assemblies. It was found that the sequence dependence of these conformations and the packing of resulting filaments are determined by multiple competing noncovalent forces, with hydrophobic interactions involving Phe being particularly important. The sequence pattern for each type of assembly conformation and packing has been identified. These results highlight the importance of the interplay between conformation, molecular packing, and sequences for determining detailed nanostructures of peptides and provide a detailed insight to support a more precise design of peptide-based nanomaterials.     

氦氖激光对人胚腱细胞生长分泌的影响

目的 阐明氦氖激光促进人胚腱细胞增殖及分泌胶原的机制。方法 将传代培养的人胚腱细胞分为对照组、照射1日组、照射3日组及照射5日组。在氦氖激光照射的不同时相点，分别测定细胞DNA含量，cAMP水平及胶原分泌量。结果 在氦氖激光作用早期（1－3日），细胞cAMP水平与DNA合成均呈明显升高（P＜0.05)，细胞内信使物质合成及细胞增殖加快，但胶原分泌无变化，而在氦氖激光作用晚期（3－5日），当cAMP水平及DNA合成都不再改变时，腱细胞分泌胶原量显著增加（P＜0.01)。结论 氦氖激光可能通过作用于cAMP蛋白激酶A信号转导系统，从而调控人胚腱细胞增殖及胶原分泌。提示临床应用氛氖激光促进肌腱愈合宜从早期开始，并且需要照射足够长的时间。     

N_2S_2或N_3S型配体的合成、~(99)Tc~m标记及生物分布研究

以MAG3为基本分子骨架，根据构效关系，分别引入合适的天然氨基酸，设计合成了4种N2S2或N3S型小分子多肽新配体，并通过了IR，^1H NMR,^13C NMR,MS谱学鉴定和元素分析表征。采用葡庚糖酸钙（GH）交换法对4个配体进行了^99Tc^m标记，研究了配合物在小鼠体内的生物分布特征。结果表明，^99Tc^m-MVGG肾摄取较高，滞留时间较长，血清除快，且肾与其它组织的活度比值高，具备成为肾功能显像剂的条件；^99Tc^m-MPGG肾初始摄取较高，R（肾／血）活度比值高，但肾清除较快，R（肾／肝）活度比值较低；^99Tc^m-MVTC和^99Tc^m－MPTC心肌初始摄取均较高，但在心肌和血中的清除速度较快。     

胶原改性促进高吸收铬鞣

介绍了胶原改性的几种方法及其所使用的材料 ,并讨论了其对皮革高吸收铬鞣产生的影响     

新生牛肝活性肽的制备及其毒性检测

目的制备新生牛肝活性肽并评价其安全性。方法采用膜法分离制备新生牛肝活性肽。将3批制品于37～40℃,75%相对湿度条件下存放3个月,以多肽含量为指标观察其稳定性,并进行急性毒性试验、小鼠骨髓细胞微核试验、Ames试验、小鼠精子畸形试验和大鼠喂养试验。结果制备的3批新生牛肝活性肽存放3个月后,多肽含量无明显下降,质量稳定。小鼠和大鼠经口灌人大于20.0g/kg体重的新生牛肝活性肽,均无急性毒性。3种致突变试验均未显示出致突变性,大鼠喂养试验各项指标均未见明显毒性。结论新生牛肝活性肽未表现出明显毒性。