Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.     

用于可视化光治疗的Mn3O4@CuS纳米粒的制备研究

摘要 目的：本研究旨在制备用于肿瘤可视化光治疗的多功能Mn₃O₄@CuS核壳型纳米粒,在磁共振成像的引导下,使用近红外光定点辐照,实现局部光热消融治疗。方法：（1）采用高温热解法制备油胺稳定的Mn₃O₄纳米粒,在其表面构建CuS壳层,并进行聚乙二醇修饰,得到分散于水相中的Mn₃O₄@CuS核壳型纳米粒。（2）通过透射电镜、紫外可见近红外吸收光谱等方法对该纳米粒进行理化性质表征,并研究其体外磁共振成像、光热升温等性能。结果：制备的水相分散的Mn₃O₄@CuS纳米粒,粒径均一且分散性较好,形态为近圆形,粒径为9.30±2.29 nm;紫外可见近红外吸收光谱图表明Mn₃O₄@CuS纳米粒在近红外区有较强吸收,最大吸收峰位于1100~1200 nm范围;磁共振成像分析结果可计算出Mn₃O₄@CuS纳米粒的纵向弛豫率r1为1.662 mM^-1s^-1,表明其具有较好的磁共振增强造影效果;光热升温曲线显示Mn₃O₄@CuS纳米粒可在785 nm近红外激光下升温至73.5 ℃,具备较好的光热治疗潜力。结论：本文成功制备出水相分散的Mn₃O₄@CuS核壳型纳米粒,具有良好的磁共振造影成像性能和光热升温效应,有望应用于磁共振成像引导下的肿瘤可视化光治疗。     

影像学方法在淋巴瘤疗效评估中的应用

淋巴瘤通过放化疗可获得很高的治愈率。治疗过程中,疗效评估对于评价或调整治疗方案至关重要。基于病灶形态学改变的传统影像学技术如计算机断层扫描(computed tomography,CT)与磁共振成像(magnetic resonance imaging,MRI)技术在评估淋巴瘤疗效时存在一定的局限性;磁共振加权成像(diffusion-weighted imaging,DWI)结合水分子表观弥散系数(apparent diffusion coefficient,ADC)从分子水平反映疾病的病理生理状态,是一种功能成像新技术;而正电子发射断层显像/X线计算机体层成像(Positron emission tomography/computed tomography,PET/CT)将肿瘤的代谢与形态相结合,是一种新型的功能成像技术,已广泛应用于淋巴瘤患者的分期、疗效监测和预后评估中,可较其他影像学技术更准确的评估疗效;PET/MRI技术在淋巴瘤方面的临床应用研究目前尚处于初步阶段,其临床价值尚需进一步探讨。     

早期糖尿病肾病大鼠模型磁共振弥散加权成像的初步研究

目的:探讨早期糖尿病肾病(Diabetic nephropathy,DN)模型大鼠磁共振弥散加权成像(Diffusion Weight Imaging,DWI)肾实质ADC值变化规律。方法:将20只清洁级雄性SD大鼠随机分成两组,糖尿病肾病组(DN组)12只,正常对照组(NC组)8只;DN组给予60 mg/kg链尿佐菌素腹腔注射诱导糖尿病肾病模型,NC组按照相同方法、相同剂量柠檬酸缓冲液腹腔注射;并对最终糖尿病模型造模成功并且存活的8只DN大鼠、8只NC大鼠进行MRI扫描,包括常规轴位T1WI、T2WI扫描及DWI扫描;扫描结束后收集血液送血肌酐及双肾组织进行病理检查。并测量每只大鼠双肾皮、髓质的ADC值。结果:造模后,DN组大鼠血糖明显升高、尿量明显增加、体重明显减低,DN组大鼠肾脏出现不同程度病理损伤,符合早期DN病理改变。DN组大鼠肾脏皮、髓质ADC值分别为1.522±0.913×10^-3 mm^2/s、1.268±0.388×10^-3 mm^2/s,较NC组肾脏皮、髓质ADC值1.276±0.341×10^-3 mm^2/s、1.011±0.217×10^-3 mm^2/s增高,两组比较有统计学意义(P<0.05)。结论:DWI成像ADC值可能反映早期糖尿病肾病肾脏功能的变化。     

不孕症女性三维子宫输卵管造影联合阴道二维超声的诊断意义

摘要 目的：探讨不孕症女性三维子宫输卵管造影联合阴道二维超声的诊断意义。方法：2019年3月至2020年10月选择在西安医学院第二附属医院和陕西省人民医院诊治的不孕症女性患者90例,所有患者都给予三维子宫输卵管造影联合阴道二维超声检查,记录成像质量与疼痛情况。以X线子宫输卵管造影为金标准,判断诊断价值。结果：检查过程中三维超声造影患者的疼痛评分高于二维超声,对比差异无统计学意义(P>0.05)。三维超声造影的成像质量优良率为100.0 %（90/90）,高于二维超声的93.3 %（84/90）,对比差异有统计学意义(P<0.05)。在90例患者中,三维超声造影判断为卵巢周围组织弥散1级59例,2级16例,3级10例,4级5例。三维子宫输卵管造影联合阴道二维超声判断为输卵管通畅55例,通而不畅25例,阻塞10例。X线子宫输卵管造影判断为输卵管通畅53例,通而不畅26例,阻塞11例,三维子宫输卵管造影联合阴道二维超声诊断的准确性为96.7 %(87/90)。结论：三维子宫输卵管造影联合阴道二维超声在不孕症女性的应用并不会增加患者疼痛,且能提高成像质量,也有利于合理评价与判断患者的输卵管通畅情况。     

一种基于chirp编码的高帧速率超声成像方法

在医学超声成像系统中,帧速率由每帧图像的扫查发射次数所决定.同时发射多条波束可以提高图像的帧速率,但是这会带来不同波束间相互干扰的问题,形成伪像.本文基于编码激励的原理,提出了一种新的高帧速率成像方法.该方法通过发射一组线性频率调制编码信号,有效的降低了波束间的互扰.可以在不影响图像质量的情况下,成倍的提高图像的帧速率.     

Three decades of listeriology through the prism of technological advances

Decades of breakthroughs resulting from cross feeding of microbiological research and technological innovation have promoted Listeria monocytogenes to the rank of model microorganism to study host–pathogen interactions. The extraordinary capacity of this bacterium to interfere with a vast array of host cellular processes uncovered new concepts in microbiology, cell biology and infection biology. Here, we review technological advances that revealed how bacteria and host interact in space and time at the molecular, cellular, tissue and whole body scales, ultimately revolutionising our understanding of Listeria pathogenesis. With the current bloom of multidisciplinary integrative approaches, Listeria entered a new microbiology era.     

Intravital lipid droplet labeling and imaging reveals the phenotypes and functions of individual macrophages in vivo

Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.     

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species'' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.