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Choosing an appropriate somatic embryogenesis medium of carrot (Daucus carota L.) by data mining technology. 通过数据挖掘技术选择合适的胡萝卜(Daucus carota L.)体细胞胚胎发生培养基。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-27 DOI: 10.1186/s12896-024-00898-7
Masoumeh Fallah Ziarani, Masoud Tohidfar, Mohsen Hesami

Introduction: Developing somatic embryogenesis is one of the main steps in successful in vitro propagation and gene transformation in the carrot. However, somatic embryogenesis is influenced by different intrinsic (genetics, genotype, and explant) and extrinsic (e.g., plant growth regulators (PGRs), medium composition, and gelling agent) factors which cause challenges in developing the somatic embryogenesis protocol. Therefore, optimizing somatic embryogenesis is a tedious, time-consuming, and costly process. Novel data mining approaches through a hybrid of artificial neural networks (ANNs) and optimization algorithms can facilitate modeling and optimizing in vitro culture processes and thereby reduce large experimental treatments and combinations. Carrot is a model plant in genetic engineering works and recombinant drugs, and therefore it is an important plant in research works. Also, in this research, for the first time, embryogenesis in carrot (Daucus carota L.) using Genetic algorithm (GA) and data mining technology has been reviewed and analyzed.

Materials and methods: In the current study, data mining approach through multilayer perceptron (MLP) and radial basis function (RBF) as two well-known ANNs were employed to model and predict embryogenic callus production in carrot based on eight input variables including carrot cultivars, agar, magnesium sulfate (MgSO4), calcium dichloride (CaCl2), manganese (II) sulfate (MnSO4), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and kinetin (KIN). To confirm the reliability and accuracy of the developed model, the result obtained from RBF-GA model were tested in the laboratory.

Results: The results showed that RBF had better prediction efficiency than MLP. Then, the developed model was linked to a genetic algorithm (GA) to optimize the system. To confirm the reliability and accuracy of the developed model, the result of RBF-GA was experimentally tested in the lab as a validation experiment. The result showed that there was no significant difference between the predicted optimized result and the experimental result.

Conclutions: Generally, the results of this study suggest that data mining through RBF-GA can be considered as a robust approach, besides experimental methods, to model and optimize in vitro culture systems. According to the RBF-GA result, the highest somatic embryogenesis rate (62.5%) can be obtained from Nantes improved cultivar cultured on medium containing 195.23 mg/l MgSO4, 330.07 mg/l CaCl2, 18.3 mg/l MnSO4, 0.46 mg/l 2,4- D, 0.03 mg/l BAP, and 0.88 mg/l KIN. These results were also confirmed in the laboratory.

简介:体细胞胚胎发生是胡萝卜体外繁殖和基因转化成功的主要步骤之一。然而,体细胞胚胎发生受不同内在因素(遗传学、基因型和外植体)和外在因素(如植物生长调节剂、培养基成分和胶凝剂)的影响,这给制定体细胞胚胎发生方案带来了挑战。因此,优化体细胞胚胎发生是一个繁琐、耗时且成本高昂的过程。通过混合使用人工神经网络(ANN)和优化算法的新型数据挖掘方法可促进体外培养过程的建模和优化,从而减少大量的实验处理和组合。胡萝卜是基因工程和重组药物的模式植物,因此是研究工作中的重要植物。本研究首次使用遗传算法(GA)和数据挖掘技术对胡萝卜(Daucus carota L.)的胚胎发生进行了回顾和分析:在本研究中,数据挖掘方法通过多层感知器(MLP)和径向基函数(RBF)这两种著名的方差网络(ANNs)来建模和预测胡萝卜胚胎性胼胝体的产生,这两种方法基于八个输入变量,包括胡萝卜栽培品种、琼脂、硫酸镁(GA)、硼砂(GA)、硼砂(GA)、硼砂(GA)和硼砂(GA)、琼脂、硫酸镁 (MgSO4)、二氯化钙 (CaCl2)、硫酸锰 (MnSO4)、2,4-二氯苯氧乙酸 (2,4-D)、6-苄基氨基嘌呤 (BAP) 和激肽 (KIN)。为了证实所开发模型的可靠性和准确性,在实验室对 RBF-GA 模型得出的结果进行了测试:结果表明,RBF 比 MLP 有更好的预测效率。然后,将所开发的模型与遗传算法(GA)相结合,对系统进行优化。为了证实所开发模型的可靠性和准确性,RBF-GA 的结果作为验证实验在实验室进行了实验测试。结果表明,预测的优化结果与实验结果之间没有明显差异:总体而言,本研究的结果表明,除了实验方法外,通过 RBF-GA 进行数据挖掘可被视为体外培养系统建模和优化的一种稳健方法。根据 RBF-GA 的结果,在含有 195.23 毫克/升 MgSO4、330.07 毫克/升 CaCl2、18.3 毫克/升 MnSO4、0.46 毫克/升 2,4-D、0.03 毫克/升 BAP 和 0.88 毫克/升 KIN 的培养基上培养的南特改良品种体细胞胚胎发生率最高(62.5%)。这些结果也在实验室中得到了证实。
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引用次数: 0
Establishment of a novel cell line for producing replication-competent adenovirus-free adenoviruses. 建立生产无复制能力腺病毒的新型细胞系。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-27 DOI: 10.1186/s12896-024-00894-x
Eun Yeong Han, Yeon-Jeong Kim

Adenoviruses are commonly utilized as viral vectors for gene therapy, genetic vaccines, and recombinant protein expression. To generate replication-defective adenoviruses, E1-complementing cell lines such as HEK293A are utilized; however, limitations remain. Repeated passage of E1-deleted virus in HEK293A cells increases the occurrence of replication-competent adenoviruses (RCAs). In the present study, we developed a novel cell line originating from human primary cells. L132 cells were transduced two times with E1-encoded retrovirus and three times with E1A-encoded retrovirus. Finally, we selected the most productive L132 cell line for generation of RCA-free adenovirus, GT541. GT541 can serve as an alternative cell line to HEK293A and other adenovirus-producing cells.

腺病毒通常被用作基因治疗、基因疫苗和重组蛋白表达的病毒载体。为了产生复制缺陷型腺病毒,需要使用 HEK293A 等 E1 互补细胞系;但这仍然存在局限性。在 HEK293A 细胞中重复通过 E1 缺失病毒会增加复制能力腺病毒(RCA)的出现。在本研究中,我们开发了一种源自人类原代细胞的新型细胞系。我们用 E1 编码的逆转录病毒对 L132 细胞进行了两次转导,用 E1A 编码的逆转录病毒进行了三次转导。最后,我们选择了产量最高的 L132 细胞系来生成不含 RCA 的腺病毒 GT541。GT541 可作为 HEK293A 和其他生产腺病毒细胞的替代细胞系。
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引用次数: 0
Systematic design and evaluation of aptamers for VEGF and PlGF biomarkers of Preeclampsia. 系统设计和评估子痫前期血管内皮生长因子和 PlGF 生物标记物的适配体。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-27 DOI: 10.1186/s12896-024-00891-0
Samavath Mallawarachchi, Rümeysa E Cebecioglu, Majed Althumayri, Levent Beker, Sandun Fernando, Hatice Ceylan Koydemir

Preeclampsia is a potentially life-threatening condition for both mother and baby, characterized by hypertension and potential organ damage. Early diagnosis is crucial to mitigate its adverse health effects. Traditional diagnostic methods, which focus on late-manifesting symptoms like hypertension and proteinuria, underscore the need for molecular diagnostic approaches for timely detection. This study successfully designs and evaluates novel aptamers with high specificity and affinity for Vascular Endothelial Growth Factor (VEGF) and Placental Growth Factor (PlGF), biomarkers closely associated with preeclampsia. Using molecular docking, molecular dynamics simulations, and BioLayer Interferometry (BLI), we identified aptamers that demonstrated strong binding affinities, comparable or superior to traditional antibodies. Our findings suggest that these aptamers have the potential to be integrated into cost-effective, point-of-care diagnostic tools, significantly improving early detection and intervention strategies for preeclampsia. The robust performance of these aptamers marks a pivotal step toward the development of more reliable and accessible diagnostic solutions, with implications for better maternal and fetal health outcomes.

子痫前期是一种可能危及母婴生命的疾病,其特点是高血压和潜在的器官损伤。早期诊断对减轻其对健康的不利影响至关重要。传统的诊断方法主要针对高血压和蛋白尿等晚期症状,因此需要分子诊断方法来及时发现。这项研究成功地设计并评估了对血管内皮生长因子(VEGF)和胎盘生长因子(PlGF)具有高特异性和亲和力的新型适配体,这两种生长因子是与子痫前期密切相关的生物标志物。通过分子对接、分子动力学模拟和生物层干涉测量法(BLI),我们鉴定出了具有强大结合亲和力的适配体,其结合亲和力可与传统抗体相媲美甚至更胜一筹。我们的研究结果表明,这些适配体有可能被整合到具有成本效益的床旁诊断工具中,从而大大改善子痫前期的早期检测和干预策略。这些适配体的强大性能标志着我们朝着开发更可靠、更易获得的诊断解决方案迈出了关键一步,这对改善孕产妇和胎儿的健康状况具有重要意义。
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引用次数: 0
Preparation and preliminary application of fluorescent microsphere test strips for feline parvovirus antibodies. 猫细小病毒抗体荧光微球试纸的制备和初步应用。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-27 DOI: 10.1186/s12896-024-00900-2
Jinyuan Shang, Manping Yan, Xiaohao Zhang, Wei Liu, Shun Wu, Zhenjun Wang, Li Yi, Chunxia Wang, Erkai Feng, Yuening Cheng, Guoliang Luo

This study introduces a novel diagnostic modality for the detection of feline panleukopenia virus (FPV) antibodies in feline serum by using fluorescent microsphere immunochromatographic test strips (FM-ICTS). Leveraging the inherent specificity of antigen-antibody interactions, the FM-ICTS approach demonstrates considerable potential for efficient and accurate FPV antibody detection within a short timeframe. The FM-ICTS method demonstrates strong diagnostic performance, with consistent accuracy and stability over time. PBS buffer dilution enables detection across the range of FPV antibody haemagglutination inhibition (HI) titres in both healthy and immunized or infected cats. A high correlation (R² = 0.9733) between the T/C ratio and FPV antibody titres confirms the method's effectiveness in quantifying these titres. Clinical validation with 84 samples supports its reliability by matching results with HI assays. Additionally, stability tests show that the test strips maintain performance during storage, with a coefficient of variation (CV) below 12% over three months at 25℃. This innovative FM-ICTS framework emerges as a promising avenue for expedient and dependable disease diagnosis within the realm of veterinary science, offering implications for timely disease management and surveillance.

本研究利用荧光微球免疫层析试纸条(FM-ICTS)介绍了一种检测猫血清中猫泛白细胞减少症病毒(FPV)抗体的新型诊断方法。利用抗原-抗体相互作用的固有特异性,FM-ICTS 方法在短时间内高效、准确地检测 FPV 抗体方面具有相当大的潜力。FM-ICTS 方法具有很强的诊断性能,准确性和稳定性始终如一。用 PBS 缓冲液稀释可检测健康猫、免疫猫或感染猫的各种 FPV 抗体血凝抑制滴度(HI)。T/C 比值与 FPV 抗体滴度之间的高度相关性(R² = 0.9733)证实了该方法在量化这些滴度方面的有效性。84 份样本的临床验证结果与 HI 检测结果一致,证明了该方法的可靠性。此外,稳定性测试表明,试纸在 25℃ 下保存三个月后,变异系数(CV)仍能保持在 12% 以下。这种创新的 FM-ICTS 框架是在兽医科学领域内进行快速、可靠的疾病诊断的一条大有可为的途径,对疾病的及时管理和监测具有重要意义。
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引用次数: 0
Unraveling the impact of pH, sodium concentration, and medium osmolality on Vibrio natriegens in batch processes. 揭示批处理过程中 pH 值、钠浓度和培养基渗透压对纳氏弧菌的影响。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-23 DOI: 10.1186/s12896-024-00897-8
Eva Forsten, Steffen Gerdes, René Petri, Jochen Büchs, Jørgen Magnus

Background: Vibrio natriegens, a halophilic marine γ-proteobacterium, holds immense biotechnological potential due to its remarkably short generation time of under ten minutes. However, the highest growth rates have been primarily observed on complex media, which often suffer from batch-to-batch variability affecting process stability and performance. Consistent bioprocesses necessitate the use of chemically defined media, which are usually optimized for fermenters with pH and dissolved oxygen tension (DOT) regulation, both of which are not applied during early-stage cultivations in shake flasks or microtiter plates. Existing studies on V. natriegens' growth on mineral media report partially conflicting results, and a comprehensive study examining the combined effects of pH buffering, sodium concentration, and medium osmolality is lacking.

Results: This study evaluates the influence of sodium concentration, pH buffering, and medium osmolality on the growth of V. natriegens under unregulated small-scale conditions. The maximum growth rate, time of glucose depletion, as well as the onset of stationary phase were observed through online-monitoring the oxygen transfer rate. The results revealed optimal growth conditions at an initial pH of 8.0 with a minimum of 300 mM MOPS buffer for media containing 20 g/L glucose or 180 mM MOPS for media with 10 g/L glucose. Optimal sodium chloride supplementation was found to be between 7.5 and 15 g/L, lower than previously reported ranges. This is advantageous for reducing industrial corrosion issues. Additionally, an osmolality range of 1 to 1.6 Osmol/kg was determined to be optimal for growth. Under these optimized conditions, V. natriegens achieved a growth rate of 1.97 ± 0.13 1/h over a period of 1 h at 37 °C, the highest reported rate for this organism on a mineral medium.

Conclusion: This study provides guidelines for cultivating V. natriegens in early-stage laboratory settings without pH and DOT regulation. The findings suggest a lower optimal sodium chloride range than previously reported and establish an osmolality window for optimal growth, thereby advancing the understanding of V. natriegens' physiology. In addition, this study offers a foundation for future research into the effects of different ions and carbon sources on V. natriegens.

背景:纳氏弧菌(Vibrio natriegens)是一种嗜卤海洋γ-蛋白细菌,由于其生成时间极短,不到十分钟,因此具有巨大的生物技术潜力。然而,最高的生长率主要是在复杂的培养基上观察到的,而这种培养基往往会因批次间的差异而影响工艺的稳定性和性能。稳定的生物工艺需要使用化学定义的培养基,这些培养基通常是为具有 pH 值和溶解氧张力(DOT)调节功能的发酵罐而优化的,而这两种功能在摇瓶或微孔板的早期培养过程中并不适用。关于 V. natriegens 在矿物培养基上生长的现有研究报告的结果部分相互矛盾,目前还缺乏对 pH 缓冲、钠浓度和培养基渗透压综合影响的全面研究:结果:本研究评估了钠浓度、pH 缓冲和培养基渗透压对 V. natriegens 在不受限制的小规模条件下生长的影响。通过在线监测氧转移率,观察了最大生长速率、葡萄糖耗尽时间以及静止期的开始。结果表明,最佳生长条件为初始 pH 值为 8.0,含 20 克/升葡萄糖的培养基至少需要 300 毫摩尔 MOPS 缓冲液,含 10 克/升葡萄糖的培养基至少需要 180 毫摩尔 MOPS 缓冲液。最佳氯化钠补充量为 7.5 至 15 克/升,低于之前报道的范围。这有利于减少工业腐蚀问题。此外,1 至 1.6 Osmol/kg 的渗透压范围被确定为最佳生长条件。在这些优化条件下,V. natriegens 在 37 °C、1 小时内的生长速度达到了 1.97 ± 0.13 1/h,这是该生物在矿物培养基上的最高生长速度:本研究为在没有 pH 值和 DOT 调节的早期实验室环境中培养 V. natriegens 提供了指导。研究结果表明,最佳氯化钠范围比以前报道的要低,并建立了一个最佳生长的渗透压窗口,从而加深了人们对 V. natriegens 生理学的了解。此外,这项研究还为今后研究不同离子和碳源对纳氏酵母菌的影响奠定了基础。
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引用次数: 0
Anti-inflammatory potential of aspergillus unguis SP51-EGY: TLR4-dependent effects & chemical diversity via Q-TOF LC-HRMS unguis SP51-EGY 的抗炎潜力:通过 Q-TOF LC-HRMS 分析 TLR4 依赖性效应和化学多样性
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-18 DOI: 10.1186/s12896-024-00890-1
Soad Nasr, Abdelhameed S. Dawood, Amal Mosad Ibrahim, Mohamed S. Abdel-Aziz, Walid Fayad, Anwar Abdelnaser, Faten K. Abd EL-Hady
Inflammation serves as an intricate defense mechanism for tissue repair. However, overactivation of TLR4-mediated inflammation by lipopolysaccharide (LPS) can lead to detrimental outcomes such as sepsis, acute lung injury, and chronic inflammation, often associated with cancer and autoimmune diseases. This study delves into the anti-inflammatory properties of “Aspergillus unguis isolate SP51-EGY” on LPS-stimulated RAW 264.7 macrophages. Through real-time qPCR, we assessed the expression levels of pivotal inflammatory genes, including iNOS, COX-2, TNF-α, and IL-6. Remarkably, our fungal extracts significantly diminished NO production and showed noteworthy reductions in the mRNA expression levels of the aforementioned genes. Furthermore, while Nrf2 is typically associated with modulating inflammatory responses, our findings indicate that the anti-inflammatory effects of our extracts are not Nrf2-dependent. Moreover, the chemical diversity of the potent extract (B Sh F) was elucidated using Q-TOF LC-HRMS, identifying 54 compounds, some of which played vital roles in suppressing inflammation. Most notably, compounds like granisetron, fenofibrate, and umbelliprenin were found to downregulate TNF-α, IL-1β, and IL-6 through the NF-κB signaling pathway. In conclusion, “Aspergillus unguis isolate SP51-EGY”, isolated from the Red Sea, Egypt, has been unveiled as a promising TLR4 inhibitor with significant anti-inflammatory potentials, presenting novel insights for their potential therapeutic use in inflammation.
炎症是一种复杂的组织修复防御机制。然而,脂多糖(LPS)过度激活 TLR4 介导的炎症可导致败血症、急性肺损伤和慢性炎症等有害结果,而慢性炎症通常与癌症和自身免疫性疾病相关。本研究探讨了" Unguis 曲霉分离物 SP51-EGY "对 LPS 刺激的 RAW 264.7 巨噬细胞的抗炎特性。通过实时 qPCR,我们评估了关键炎症基因的表达水平,包括 iNOS、COX-2、TNF-α 和 IL-6。值得注意的是,我们的真菌提取物明显减少了 NO 的产生,并显著降低了上述基因的 mRNA 表达水平。此外,虽然 Nrf2 通常与调节炎症反应有关,但我们的研究结果表明,我们提取物的抗炎作用并不依赖于 Nrf2。此外,我们还利用 Q-TOF LC-HRMS 对强效提取物(B Sh F)的化学多样性进行了阐释,鉴定出 54 种化合物,其中一些在抑制炎症方面发挥了重要作用。最值得注意的是,研究发现格拉司琼、非诺贝特和脐橙素等化合物可通过 NF-κB 信号通路下调 TNF-α、IL-1β 和 IL-6。总之,从埃及红海分离出的 "unguis曲霉分离物SP51-EGY "是一种很有前途的TLR4抑制剂,具有显著的抗炎潜力,为其在炎症中的潜在治疗用途提供了新的见解。
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引用次数: 0
Synthesis and evaluation of nanosized aluminum MOF encapsulating Umbelliferon: assessing antioxidant, anti-inflammatory, and wound healing potential in an earthworm model 包覆伞形酮的纳米铝 MOF 的合成与评估:在蚯蚓模型中评估抗氧化、抗炎和伤口愈合潜力
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-15 DOI: 10.1186/s12896-024-00889-8
Rabab M. Thabit, Fatma El-Zahraa A. Abd El-Aziz, A. Abu El-Fadl, A. A. Abu-Sehly, Ahmed M. Sayed
Nanoporous aluminum metal–organic framework (Al-MOF) was synthesized via solvothermal methods and employed as a carrier matrix for in vitro drug delivery of Umbelliferon (Um). The encapsulated Um was gradually released over seven days at 37 °C, using simulated body fluid phosphate-buffered saline (PBS) at pH 7.4 as the release medium. The drug release profile suggests the potential of Al-MOF nanoparticles as effective drug delivery carriers. Structural and chemical analyses of Um-loaded Al-MOF nanoparticles (Um-Al MOF) were conducted using Fourier-transform infrared (FTIR) spectroscopy, X-ray diffractometry (XRD), and ultraviolet–visible (UV–Vis) spectroscopy. Thermal gravimetric analysis (TGA) was employed to investigate the thermal stability of the Al-MOF nanoparticles, while Transmission Electron Microscopy (TEM) was utilized to assess their morphological features. Um-Al MOF nanoparticles demonstrated notable antioxidant and anti-inflammatory properties compared to Um and Al-MOF nanoparticles individually. Moreover, they exhibited significant enhancement in wound healing in an earthworm model. These findings underscore the potential of Al-MOF nanoparticles as a promising drug delivery system, necessitating further investigations to explore their clinical applicability.
通过溶热法合成了纳米多孔铝金属有机框架(Al-MOF),并将其作为载体基质用于茵贝酮(Um)的体外给药。以 pH 值为 7.4 的模拟体液磷酸盐缓冲盐水(PBS)为释放介质,在 37 °C、7 天内逐渐释放被包裹的 Um。药物释放曲线表明,Al-MOF 纳米粒子具有作为有效药物递送载体的潜力。利用傅立叶变换红外光谱(FTIR)、X 射线衍射仪(XRD)和紫外可见光谱(UV-Vis)对负载 Um 的 Al-MOF 纳米粒子(Um-Al MOF)进行了结构和化学分析。热重分析(TGA)用于研究 Al-MOF 纳米粒子的热稳定性,而透射电子显微镜(TEM)则用于评估其形态特征。与单独的 Um 和 Al-MOF 纳米粒子相比,Um-Al MOF 纳米粒子具有显著的抗氧化和抗炎特性。此外,它们还在蚯蚓模型中显示出明显的伤口愈合能力。这些发现凸显了 Al-MOF 纳米粒子作为一种前景广阔的给药系统的潜力,因此有必要对其进行进一步研究,以探索其临床适用性。
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引用次数: 0
A marker-free genetic manipulation method for Glaesserella parasuis strains developed by alternately culturing transformants at 37°C and 30°C. 通过在 37°C 和 30°C 温度下交替培养转化株,开发出一种无标记的寄生褐藻菌株遗传操作方法。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-03 DOI: 10.1186/s12896-024-00887-w
Jing Xiao, Yuxin Wang, Dongfang Wu, Yuping Song, Xuwang Cai, Huanchun Chen, Hongbo Zhou, Xiaojuan Xu

Background: Glaesserella parasuis (G. parasuis) is the causative agent of Glässer's disease, which causes significant economic losses in the swine industry. However, research on the pathogenesis of G. parasuis has been hampered by the lack of a simple and efficient marker-free knockout system.

Results: In this study, a marker-free knockout system was developed for G. parasuis using a temperature-sensitive vector. By alternating the incubation of transformants at 30°C and 37°C, we optimized the screening process for this system. The system was successfully applied to knockout the KanR cassette from JS0135ΔnanH::KanR, achieving a knockout efficiency of 90% in the final round of screening. To confirm that temperature variation was a key factor, we proceeded with knocking out the nanH and apd genes in the CF7066 strain. The knockout efficiency reached up to 100%, with the shortest screening time being only four days. The knockout of the nanH gene resulted in a significant reduction in the growth vitality of the strains, while the knockout of the apd gene led to an approximate 56% improvement in the adhesion rate. Additionally, we observed that the expression of recombinant genes in transformants was higher at 30℃ than at 37℃, with the recC gene being upregulated approximately 7-fold. In contrast, there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in the wild-type strains. This discrepancy was likely due to an elevated copy number of target plasmids at 30℃, which may have resulted in the enhanced expression of recombinant genes.

Conclusions: In conclusion, this newly developed gene knockout system for G. parasuis presents a valuable tool for advancing research on this organism.

背景:寄生格氏菌(Glaesserella parasuis,G. parasuis)是格莱塞氏病的病原体,它给养猪业造成了巨大的经济损失。然而,由于缺乏简单高效的无标记基因敲除系统,对寄生璃泽氏菌发病机制的研究一直受到阻碍:结果:本研究利用对温度敏感的载体为寄生虫开发了一种无标记基因剔除系统。通过交替在 30°C 和 37°C 下培养转化子,我们优化了该系统的筛选过程。该系统被成功用于敲除 JS0135ΔnanH::KanR 中的 KanR 盒,在最后一轮筛选中,敲除效率达到了 90%。为了证实温度变化是一个关键因素,我们继续在 CF7066 菌株中敲除 nanH 和 apd 基因。基因敲除效率高达 100%,最短筛选时间仅为四天。敲除 nanH 基因会显著降低菌株的生长活力,而敲除 apd 基因则会使粘附率提高约 56%。此外,我们还观察到,重组基因在转化株中的表达量在 30℃ 比 37℃ 高,其中 recC 基因上调了约 7 倍。相比之下,野生型菌株重组基因的表达在 30℃ 和 37℃ 之间几乎没有差异。这种差异可能是由于 30℃ 时目的质粒的拷贝数增加,从而导致重组基因的表达增强:总之,这种新开发的寄生虫基因敲除系统是推进寄生虫研究的重要工具。
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引用次数: 0
Purification and characterization of soluble recombinant Crimean-Congo hemorrhagic fever virus glycoprotein Gc expressed in mammalian 293F cells. 在哺乳动物 293F 细胞中表达的可溶性重组克里米亚-刚果出血热病毒糖蛋白 Gc 的纯化和表征。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-27 DOI: 10.1186/s12896-024-00885-y
Nigel Aminake Makoah, Matefo Millicent Litabe, Fredy Brice Nemg Simo, Katlego Keith Maboho, Felicity Jane Burt

Background: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies.

Methods: We designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen.

Results: The optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation.

Conclusions: The purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen's suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment.

背景:克里米亚-刚果出血热(CCHF)是一种由蜱虫传播的人畜共患病,表现为严重的出血性症状,致死率很高。病原体克里米亚-刚果出血热病毒(CCHFV)是世界卫生组织确定的高度优先病原体,目前还没有获得批准的疫苗或特效治疗方法。此外,亟需加强诊断工具,以提高公共卫生意识、改进预防措施和疾病控制策略:方法:我们设计了质粒来纯化在哺乳动物 293 F 细胞中表达的可溶性 CCHFV 糖蛋白 Gc,然后使用亲和层析和尺寸排阻层析进行纯化。纯化后的抗原通过 SDS-PAGE 和 Western 印迹法进行分析,以确认其与 CCHF 存活者的抗体的反应性。此外,还使用纯化的 Gc 作为包被抗原开发了一种内部间接 ELISA:结果:经过优化的表达系统在亲和层析后成功产生了可溶性纯Gc抗原。在 Western 印迹分析中,该蛋白与 CCHFV 阳性血清抗体呈特异性反应。间接酶联免疫吸附试验在区分 CCHFV 阳性和阴性血清样本方面表现出很高的效率,表明它有潜力成为一种有价值的诊断工具。尺寸排阻色谱法进一步证实了我们制备的蛋白质中存在聚集体:纯化的 Gc 抗原有望用于开发 CCHFV 的直接诊断测定。结论:纯化的 Gc 抗原有望用于开发 CCHFV 的直接诊断检测,该抗原是否适合亚单位疫苗的开发,以及是否可用作从存活者中分离单克隆抗体的诱饵,还有待于进一步研究。这项工作为今后研究开发用于野外部署的快速诊断测试奠定了基础。
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引用次数: 0
Correction: Comparison of lipidome profiles in serum from lactating dairy cows supplemented with Acremonium terrestris culture based on UPLC-QTRAP-MS/MS. 更正:基于 UPLC-QTRAP-MS/MS 的泌乳奶牛血清脂质体图谱比较。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-22 DOI: 10.1186/s12896-024-00886-x
Chenmiao Zhang, Yiran Zhao, Shijiao Guo, Feifei Li, Xu Gong, Jiarui Gao, Linshu Jiang, Jinjin Tong
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引用次数: 0
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BMC Biotechnology
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