Absolute quantification of biological samples provides precise numerical expression levels, enhancing accuracy, and performance for rare templates. Current methodologies, however, face challenges-flow cytometers are costly and complex, whereas fluorescence imaging, relying on software or manual counting, is time-consuming and error-prone. It is presented that Deep-qGFP, a deep learning-aided pipeline for the automated detection and classification of green fluorescent protein (GFP) labeled microreactors, enables real-time absolute quantification. This approach achieves an accuracy of 96.23% and accurately measures the sizes and occupancy status of microreactors using standard laboratory fluorescence microscopes, providing precise template concentrations. Deep-qGFP demonstrates remarkable speed, quantifying over 2000 microreactors across ten images in just 2.5 seconds, with a dynamic range of 56.52-1569.43 copies µL-1 . The method demonstrates impressive generalization capabilities, successfully applied to various GFP-labeling scenarios, including droplet-based, microwell-based, and agarose-based applications. Notably, Deep-qGFP is the first all-in-one image analysis algorithm successfully implemented in droplet digital polymerase chain reaction (PCR), microwell digital PCR, droplet single-cell sequencing, agarose digital PCR, and bacterial quantification, without requiring transfer learning, modifications, or retraining. This makes Deep-qGFP readily applicable in biomedical laboratories and holds potential for broader clinical applications.

Osteoarthritis (OA) is a whole joint disorder that is characterized by cartilage damage and abnormal remodeling of subchondral bone. Injecting adipose-derived stem cells (ASCs) into the knee joint cavity can assist in repairing osteoarthritic joints, but their ability to migrate to the damaged site is limited. Our previous studies have shown that knee loading can improve the symptoms of OA, but the effect and mechanism of knee loading on the migration of ASCs in OA remain unclear. We employed a mouse model of OA in the knee and applied knee loading (1 N at 5 Hz for 6 min/day for 2 weeks) after the intra-articular injection of ASCs. The cartilage and subchondral bone repair were assessed by histopathological analysis. Immunofluorescence assays were also used to analyze the migration of ASCs. Using cell cultures, we evaluated the migration of ASCs using the transwell migration and wound healing assays. In vivo experiments showed that knee loading promoted the migration of ASCs, increased the local SDF-1 level, and accelerated the repair of the OA-damaged sites. Mechanistically, the observed effects were blocked by the SDF-1/CXCR4 inhibitor. The in vitro results further revealed that knee loading promoted the migration of ASCs and the inhibition of SDF-1/CXCR4 significantly suppressed the beneficial loading effect. The results herein suggested that the migration of ASCs was enhanced by knee loading through the SDF-1/CXCR4 regulatory axis, and mechanical loading promoted the joint-protective effect of ASCs.

Difficulty in preventing crops from plant viruses urges to discover novel efficient antiviral chemicals, which is sped up by precise screening methods. Fluorescence-based methods have recently been applied as innovative and rapid tools for visually monitoring the replication of viruses and screening of antivirals, whereas the quantification of fluorescence signals mainly depends on manually calculating the fluorescent spots, which is time-consuming and imprecise. In the present work, the fluorescence spots were automatically identified, and the fluorescence area was directly quantified by a program developed in our group, which avoided subjective errors from the operators. We further employed this digital and visual screening assay to identify antivirals using the tobacco mosaic virus-green fluorescence protein (TMV-GFP) construct, in which the expression of GFP intuitively reflected the efficacy of antivirals. The accuracy of this assay was validated by quantifying the activities of the commercial antiviral inhibitors ribavirin and ningnanmycin and then was applied to evaluate the subtle activity differences of a series of newly synthesized carbazole and β-carboline alkaloid derivatives. Among them, compounds 5 (76%) and 11 (63%) exhibited anti-TMV activities comparable to that of ningnanmycin (65%) at 50 μM, and they delayed the multiplication of TMV in the early stage of infection without phytotoxicity. Taken together, these findings demonstrated that the digital and visual TMV-GFP screening method was competent to test the antiviral activities of compounds with subtle modifications and facilitated the discovery of novel antivirals.

Cotton encounters long-term drought stress problems resulting in major yield losses. Transcription factors (TFs) plays an important role in response to biotic and abiotic stresses. The coexpression patterns of gene networks associated with drought stress tolerance were investigated using transcriptome profiles. Applying a weighted gene coexpression network analysis, we discovered a salmon module with 144 genes strongly linked to drought stress tolerance. Based on coexpression and RT-qPCR analysis GH_D01G0514 was selected as the candidate gene, as it was also identified as a hub gene in both roots and leaves with a consistent expression in response to drought stress in both tissues. For validation of GH_D01G0514, Virus Induced Gene Silencing was performed and VIGS plants showed significantly higher excised leaf water loss and ion leakage, while lower relative water and chlorophyll contents as compared to WT (Wild type) and positive control plants. Furthermore, the WT and positive control seedlings showed higher CAT and SOD activities, and lower activities of hydrogen peroxide and MDA enzymes as compared to the VIGS plants. Gh_D01G0514 (GhNAC072) was localized in the nucleus and cytoplasm. Y2H assay demonstrates that Gh_D01G0514 has a potential of auto activation. It was observed that the Gh_D01G0514 was highly upregulated in both tissues based on RNA Seq and RT-qPCR analysis. Thus, we inferred that, this candidate gene might be responsible for drought stress tolerance in cotton. This finding adds significantly to the existing knowledge of drought stress tolerance in cotton and deep molecular analysis are required to understand the molecular mechanisms underlying drought stress tolerance in cotton.

Eukaryotic filamentous yellow-green algae from the Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptⅡ plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase Ⅱ-encoding G418-resistant gene (nptⅡ) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub- nptⅡ were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement.

Sugars containing cationic polymers are potential carriers for in vitro and in vivo nucleic acid delivery. Monosaccharides such as glucose and galactose have been chemically conjugated to various materials of synergistic poly-lysine dendrimer systems for efficient and biocompatible delivery of short interfering RNA (siRNA). The synergistic dendrimers, which contain lipid conjugated glucose terminalized lysine dendrimers, have significantly lower adverse impact on cells while maintaining efficient cellular entry. Moreover, the synergistic dendrimers complexed to siRNA induced RNA interference (RNAi) in the cells and profoundly knocked down green fluorescence protein (GFP) as well as the endogenously expressing disease related gene Plk1. The new synergic dendrimers may be promising system for biocompatible and efficient siRNA delivery.

Understanding the long-term fate and potential mechanisms of mesenchymal stem cells (MSCs) after transplantation is essential for improving functional benefits of stem cell-based stroke treatment. Magnetic resonance imaging (MRI) is considered an attractive and clinically translatable tool for longitudinal tracking of stem cells, but certain controversies have arisen in this regard. In this study, we used SPION-loaded cationic polymersomes to label green fluorescent protein (GFP)-expressing MSCs to determine whether MRI can accurately reflect survival, long-term fate, and potential mechanisms of MSCs in ischemic stroke therapy. Our results showed that MSCs could improve the functional outcome and reduce the infarct volume of stroke in the brain. In vivo MRI can verify the biodistribution and migration of grafted cells when pre-labeled with SPION-loaded polymersome. The dynamic change of low signal volume on MRI can reflect the tendency of cell survival and apoptosis, but may overestimate long-term survival owing to the presence of iron-laden macrophages around cell graft. Only a small fraction of grafted cells survived up to 8 weeks after transplantation. A minority of these surviving cells were differentiated into astrocytes, but not into neurons. MSCs might exert their therapeutic effect via secreting paracrine factors rather than directing cell replacement through differentiation into neuronal and/or glial phenotypes.

The methods of the quantitative assay of the antiviral activity of interferons (IFNs) (type I, II or III) are very important during carrying out of the research of them, since they were found. Here a recombinant vesicular stomatitis virus expressing green fluorescent protein (GFP) (VSV/GFP) and MDBK cells were used to develop an antiviral assay (AVA) for IFNs. This method was carried out on a 96-well cell culture plate, and the half reduction of virus replication was quantified by assaying GFP. To quantify GFP, cell lysis buffer was directly added to the wells infected with VSV/GFP to lyse cells, the VSV/GFP was then inactivated, and relative fluorescence unit (RFU) of GFP was measured and used to calculate the antiviral activity. This method needed only one step instead of three steps in the staining method with naphthol blue black, medium with phenol red can be used, and it had good reproducibility. The GFP-containing samples could be stored at 4°C in a wet box for at least 1 week without affecting the assay results. In addition, the results obtained with this method were similar to those obtained with the staining method. In conclusion, a safe, rapid, convenient and low-cost AVA of IFN based on recombinant VSV/GFP was established.

OBJECTIVE: To construct a low-pH-sensing system in Streptococcus mutans (S. mutans) and to visually detect the pH in situ.
METHODS: Promoter of ureaseⅠ(PureⅠ) and green fluorescence protein (gfp) DNA fragments were amplified by polymerase chain reaction (PCR) from the genome of Streptococcus salivarius 57.I and S. mutans containing the gfp fragment. The two amplified DNA fragments were ligated together and further integrated into pDL278 to construct the recombinant plasmid pDL278-pureⅠ-gfp. This recombinant plasmid was then transformed into S. mutans UA159 cells. Subsequently, the intensity of the optical density per unit area of the low-pH-sensing system was measured and compared under different pH conditions and different processing times.
RESULTS: PureⅠ and gfp DNA fragments were amplified successfully with the correct molecule sizes (450 and 717 bp, respectively). The recombinant plasmid pDL278-pureⅠ-gfp was constructed and further verified by PCR and sequencing. The intensity of the optical density per unit area of the low-pH-sensing system increased with decreasing pH and increasing processing time.
CONCLUSIONS: A low-pH-sensing system was constructed successfully in S. mutans. Our research verified that pureⅠ of Streptococcus salivarius can function well in S. mutans as an acid induced promoter, and provided a new method of detecting the pH of plaque biofilms in situ.
目的 构建变异链球菌低pH感应系统，原位可视地检测其所处环境的pH。方法 通过聚合酶链反应（PCR）和基因重组技术，克隆得到唾液链球菌57.I的ureⅠ基因启动子片段（pureⅠ）及绿色荧光蛋白（GFP）的编码基因gfp，经过酶切后将两个基因片段连接融合，然后再经双酶切将融合片段与大肠杆菌-变异链球菌穿梭载体pDL278连接，构建出重组质粒pDL278-pureⅠ-gfp。将重组质粒转化到变异链球菌UA159中，使用荧光显微镜观察其在不同pH条件和不同处理时间的单位面积荧光强度。结果 目的基因pureⅠ和gfp扩增片段大小分别为450 bp和717 bp，与预期大小相符。构建的重组质粒pDL278-pureⅠ-gfp测序结果与数据库比对完全一致。重组变异链球菌低pH报告菌株PCR扩增片段与预期结果相符。在一定范围内，变异链球菌低pH感应系统单位面积荧光强度随pH值的降低和处理时间的延长而增强。结论 本研究成功构建了变异链球菌低pH感应系统，同时验证了唾液链球菌酸诱导启动子pureⅠ能在变异链球菌中正常发挥功能，为今后研究菌斑生物膜中原位pH的动态变化提供了新方法。.

Cell-based therapy with mesenchymal stem cells (MSCs) is a promising strategy for acute ischemic stroke. In vivo tracking of therapeutic stem cells with magnetic resonance imaging (MRI) is imperative for better understanding cellular survival and migrational dynamics over time. In this study, we develop a novel biocompatible nanocomplex (ASP-SPIONs) based on cationic amylose, by introducing spermine and the image label, ultrasmall superparamagnetic iron oxide nanoparticles (SPIONs), to label MSCs. The capacity, efficiency, and cytotoxicity of the nanocomplex in transferring SPIONs into green fluorescence protein-modified MSCs were tested; and the performance of in vivo MRI tracking of the transplanted cells in acute ischemic stroke was determined. The results demonstrated that the new class of SPIONs-complexed nanoparticles based on biodegradable amylose can serve as a highly effective and safe carrier to transfer magnetic label into stem cells. A reliable tracking of transplanted stem cells in stroke was achieved by MRI up to 6 weeks, with the desirable therapeutic benefit of stem cells on stroke retained. With the advantages of a relatively low SPIONs concentration and a short labeling period, the biocompatible complex of cationic amylose with SPIONs is highly translatable for clinical application. It holds great promise in efficient, rapid, and safe labeling of stem cells for subsequent cellular MRI tracking in regenerative medicine.