Direct and label‐free detection of the human growth hormone in urine by an ultrasensitive bimodal waveguide biosensor

A label‐free interferometric transducer showing a theoretical detection limit for homogeneous sensing of 5 × 10^–8 RIU, being equivalent to a protein mass coverage resolution of 2.8 fg mm^–2, is used to develop a high sensitive biosensor for protein detection. The extreme sensitivity of this transducer combined with a selective bioreceptor layer enables the direct evaluation of the human growth hormone (hGH) in undiluted urine matrix in the 10 pg mL^–1 range.

     

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins.     

Bioclimatic transect networks: Powerful observatories of ecological change

Transects that traverse substantial climate gradients are important tools for climate change research and allow questions on the extent to which phenotypic variation associates with climate, the link between climate and species distributions, and variation in sensitivity to climate change among biomes to be addressed. However, the potential limitations of individual transect studies have recently been highlighted. Here, we argue that replicating and networking transects, along with the introduction of experimental treatments, addresses these concerns. Transect networks provide cost‐effective and robust insights into ecological and evolutionary adaptation and improve forecasting of ecosystem change. We draw on the experience and research facilitated by the Australian Transect Network to demonstrate our case, with examples, to clarify how population‐ and community‐level studies can be integrated with observations from multiple transects, manipulative experiments, genomics, and ecological modeling to gain novel insights into how species and systems respond to climate change. This integration can provide a spatiotemporal understanding of past and future climate‐induced changes, which will inform effective management actions for promoting biodiversity resilience.     

Truffle biogeography—A case study revealing ecological niche separation of different Tuber species

Ecology of hypogeic mycorrhizal fungi, such as truffles, remains largely unknown, both in terms of their geographical distribution and their environmental niches. Occurrence of true truffles (Tuber spp.) was therefore screened using specific polymerase chain reaction (PCR) assays and subsequent PCR amplicon sequencing in tree roots collected at 322 field sites across the Czech Republic. These sites spanned a wide range of climatic and soil conditions. The sampling was a priori restricted to areas thought to be suitable for Tuber spp. inasmuch as they were characterized by weakly acidic to alkaline soils, warmer climate, and with tree species previously known to host true truffles. Eight operational taxonomic units (OTUs) corresponding to Tuber aestivum, T. borchii, T. foetidum, T. rufum, T. indicum, T. huidongense, T. dryophilum, and T. oligospermum were detected. Among these, T. borchii was the OTU encountered most frequently. It was detected at nearly 19% of the sites. Soil pH was the most important predictor of Tuber spp. distribution. Tuber borchii preferred weakly acidic soils, T. foetidum and T. rufum were most abundant in neutral soils, and T. huidongense was restricted to alkaline soils. Distribution of T. aestivum was mainly dictated by climate, with its range restricted to the warmest sites. Host preferences of the individual Tuber spp. were weak compared to soil and climatic predictors, with the notable exception that T. foetidum appeared to avoid oak trees. Our results open the way to better understanding truffle ecology and, through this new knowledge, also to better‐informed trufficulture.     

A practical approach in bioreactor scale‐up and process transfer using a combination of constant P/V and vvm as the criterion

Bioreactor scale‐up is a critical step in the production of therapeutic proteins such as monoclonal antibodies (MAbs). With the scale‐up criterion such as similar power input per volume or O₂ volumetric mass transfer coefficient ( ), adequate oxygen supply and cell growth can be largely achieved. However, CO₂ stripping in the growth phase is often inadequate. This could cascade down to increased base addition and osmolality, as well as residual lactate increase and compromised production and product quality. Here we describe a practical approach in bioreactor scale‐up and process transfer, where bioreactor information may be limited. We evaluated the sparger and (CO₂ volumetric mass transfer coefficient) from a range of bioreactor scales (3–2,000 L) with different spargers. Results demonstrated that for oxygen is not an issue when scaling from small‐scale to large‐scale bioreactors at the same gas flow rate per reactor volume (vvm). Results also showed that sparging CO₂ stripping, , is dominated by the gas throughput. As a result, a combination of a minimum constant vvm air or N₂ flow with a similar specific power was used as the general scale‐up criterion. An equation was developed to determine the minimum vvm required for removing CO₂ produced from cell respiration. We demonstrated the effectiveness of using such scale‐up criterion with five MAb projects exhibiting different cell growth and metabolic characteristics, scaled from 3 to 2,000 L bioreactors across four sites. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1146–1159, 2017     

Comparison of spectroscopy technologies for improved monitoring of cell culture processes in miniature bioreactors

Cell culture process development requires the screening of large numbers of cell lines and process conditions. The development of miniature bioreactor systems has increased the throughput of such studies; however, there are limitations with their use. One important constraint is the limited number of offline samples that can be taken compared to those taken for monitoring cultures in large‐scale bioreactors. The small volume of miniature bioreactor cultures (15 mL) is incompatible with the large sample volume (600 µL) required for bioanalysers routinely used. Spectroscopy technologies may be used to resolve this limitation. The purpose of this study was to compare the use of NIR, Raman, and 2D‐fluorescence to measure multiple analytes simultaneously in volumes suitable for daily monitoring of a miniature bioreactor system. A novel design‐of‐experiment approach is described that utilizes previously analyzed cell culture supernatant to assess metabolite concentrations under various conditions while providing optimal coverage of the desired design space. Multivariate data analysis techniques were used to develop predictive models. Model performance was compared to determine which technology is more suitable for this application. 2D‐fluorescence could more accurately measure ammonium concentration (RMSE_CV 0.031 g L^?1) than Raman and NIR. Raman spectroscopy, however, was more robust at measuring lactate and glucose concentrations (RMSE_CV 1.11 and 0.92 g L^?1, respectively) than the other two techniques. The findings suggest that Raman spectroscopy is more suited for this application than NIR and 2D‐fluorescence. The implementation of Raman spectroscopy increases at‐line measuring capabilities, enabling daily monitoring of key cell culture components within miniature bioreactor cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:337–346, 2017     

讲座细胞药物的制备工艺——细胞药物连载之二

细胞药物制备的质量直接关系到细胞治疗的效果。由于细胞治疗所用细胞是具有生物学效应的,细胞药物的制备技术和应用方案具有多样性、复杂性和特殊性,不像一般生物药物那样有统一的制作标准。细胞药物的制备过程主要包括供者筛查、供者检测、采集、加工、分离纯化、储存等,以造血干细胞、间充质干细胞、肝细胞及树突状细胞为例对其进行简要介绍。     

应用活细胞RNA检测纳米技术检测乳腺上皮细胞MCF-10A及乳腺癌细胞MCF-7中miR-142-3p的表达

传统的核酸检测技术如放射性核素、荧光、化学修饰的探针以及核酸扩增等技术无法检测活细胞中核酸的表达量。而活细胞RNA纳米检测技术和传统的检测技术相比,利用纳米金颗粒为探针能对活细胞进行检测,实验步骤更为简单,可以在自然的、无扩增的条件下观察RNA,这可真实地反应基因表达与表型之间的关系。miRNA是一类非编码RNA,其长度为20～24个碱基,在生命活动中起重要的作用。本文应用活细胞RNA检测纳米技术结合荧光定量PCR分别检测正常的乳腺上皮细胞系及乳腺上皮癌细胞系中内源性miR-142-3p的表达,发现乳腺癌细胞系内源性miR-142-3p的表达显著高于正常乳腺上皮细胞系中miR-142-3p的表达,结果提示miR-142-3p可能在乳腺癌细胞发生发展中起到调控作用。     

芒果畸形病病原菌Fusarium mangiferae的快速分子检测

芒果畸形病是芒果上的重要病害之一,由镰孢菌侵染引起,其中以Fusarium mangiferae为主要致病菌.该病害诊断困难,且难于有效控制,因此,一旦发生则对芒果生产造成严重威胁.研究基于ISSR分子标记技术,从50条已知引物中筛选得到一条目的引物UBC 888,该引物可稳定扩增出大小为479bp的F. mangiferae特异性条带(GenBank Accession No. KJ526382).根据获得的特异性片段序列设计引物,成功地将ISSR标记转化为SCAR标记,并获得一对SCAR特异性引物(W342,W1772)和一段大小为1 376bp的特异性扩增片段(GenBank Accession No. KJ526383).通过优化特异性引物扩增条件,获得最适退火温度,构建芒果畸形病病原菌F. mangiferae的快速分子检测技术.此技术操作简单,特异性强,可检测真菌DNA的含量最低为10pg,适用于F. mangiferae和田间带菌芒果组织高灵敏度快速检测,为芒果畸形病的早期诊断和及时预防提供可靠理论依据和技术方法.     

融合蛋白GGH粉针剂制备工艺研究

目的：建立高效液相色谱法测定融合蛋白GGH原料和注射用粉针剂含量的方法,筛选制备粉针剂的辅料和pH,进行6个月的长期稳定性试验。方法：高效液相色谱采用Waters DELTA PAK C18色谱柱,流动相为乙腈和水,梯度洗脱20min,检测波长280nm。根据粉针剂的外形、复溶性、稳定性和活性保留率筛选填充剂及pH。结果：GGH在0.2~1.6mg/ml范围内线性关系良好,可用此方法进行制剂的含量检测;选用4%的甘露醇作为填充剂并调节pH为5.5,可以制备性质较为稳定的GGH冻干粉针剂。结论：可采用含量和纯度检测结合外观性状观察法用于粉针剂的制备工艺筛选,为申报新药提供参考。