Unbiased and Mobile Gait Analysis Detects Motor Impairment in Parkinson's Disease

Motor impairments are the prerequisite for the diagnosis in Parkinson''s disease (PD). The cardinal symptoms (bradykinesia, rigor, tremor, and postural instability) are used for disease staging and assessment of progression. They serve as primary outcome measures for clinical studies aiming at symptomatic and disease modifying interventions. One major caveat of clinical scores such as the Unified Parkinson Disease Rating Scale (UPDRS) or Hoehn&Yahr (H&Y) staging is its rater and time-of-assessment dependency. Thus, we aimed to objectively and automatically classify specific stages and motor signs in PD using a mobile, biosensor based Embedded Gait Analysis using Intelligent Technology (eGaIT). eGaIT consist of accelerometers and gyroscopes attached to shoes that record motion signals during standardized gait and leg function. From sensor signals 694 features were calculated and pattern recognition algorithms were applied to classify PD, H&Y stages, and motor signs correlating to the UPDRS-III motor score in a training cohort of 50 PD patients and 42 age matched controls. Classification results were confirmed in a second independent validation cohort (42 patients, 39 controls). eGaIT was able to successfully distinguish PD patients from controls with an overall classification rate of 81%. Classification accuracy increased with higher levels of motor impairment (91% for more severely affected patients) or more advanced stages of PD (91% for H&Y III patients compared to controls), supporting the PD-specific type of analysis by eGaIT. In addition, eGaIT was able to classify different H&Y stages, or different levels of motor impairment (UPDRS-III). In conclusion, eGaIT as an unbiased, mobile, and automated assessment tool is able to identify PD patients and characterize their motor impairment. It may serve as a complementary mean for the daily clinical workup and support therapeutic decisions throughout the course of the disease.     

Quantitative Ultrastructural Immunocytochemistry Using a Computerized Image Analysis System

Secretory granules in human pituitary adenoma cells have been examined indirectly for hormone epitopes by immunogold labelling of resin-embedded ultra thin sections. The specific binding of different immunoglobulin-gold complexes to the antigrowth hormone antibodies over the secretory granules was measured using a computerized image analysis system. This facilitated the assessment of the preferential binding to the target granules of gold particles with three different average particle diameters (Au₇, Au₁₁, Au₁₇). The time of pretreatment of sections with H₂0₂ or a buffer was found to influence the staining considerably. The scanning electron microscopic findings of protruded secretory granules with a mountain-like surface might be relevant to the uneven distribution of immunolabels seen over the secretory granules in the adenohypophysis.     

Gait Analysis in a Mecp2 Knockout Mouse Model of Rett Syndrome Reveals Early-Onset and Progressive Motor Deficits

Rett syndrome (RTT) is a genetic disorder characterized by a range of features including cognitive impairment, gait abnormalities and a reduction in purposeful hand skills. Mice harbouring knockout mutations in the Mecp2 gene display many RTT-like characteristics and are central to efforts to find novel therapies for the disorder. As hand stereotypies and gait abnormalities constitute major diagnostic criteria in RTT, it is clear that motor and gait-related phenotypes will be of importance in assessing preclinical therapeutic outcomes. We therefore aimed to assess gait properties over the prodromal phase in a functional knockout mouse model of RTT. In male Mecp2 knockout mice, we observed alterations in stride, coordination and balance parameters at 4 weeks of age, before the onset of other overt phenotypic changes as revealed by observational scoring. These data suggest that gait measures may be used as a robust and early marker of MeCP2-dysfunction in future preclinical therapeutic studies.     

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.     

Detection of Lung Tumors in Mice Using a 1-Tesla Compact Magnetic Resonance Imaging System

Due to their small size, lung tumors in rodents are typically investigated using high-field magnetic resonance (MR) systems (4.7 T or higher) to achieve higher signal-to-noise ratios, although low-field MR systems are less sensitive to susceptibility artifacts caused by air in the lung. We investigated the feasibility of detecting lung tumors in living, freely breathing mice with a 1-T compact permanent magnet MR system. In total, 4 mice were used, and MR images of mouse lungs were acquired using a T1-weighted three-dimensional fast low-angle shot sequence without cardiac or respiratory gating. The delineation and size of lung tumors were assessed and compared with histopathological findings. Submillimeter lesions were demonstrated as hyperintense, relative to the surrounding lung parenchyma, and were delineated clearly. Among the 13 lesions validated in histopathological sections, 11 were detected in MR images; the MR detection rate was thus 84.6%. A strong correlation was obtained in size measurements between MR images and histological sections. Thus, a dedicated low-field MR system can be used to detect lung tumors in living mice noninvasively without gating.     

基于双荧光系统的HepG2肝细胞癌原位移植裸鼠模型建立及活体荧光成像动态定量分析

目的:建立一种可以实时、定量、动态监测的肝细胞癌原位移植模型,并利用活体荧光成像系统对裸鼠体内原位肝细胞癌生长进行分析。方法:利用慢病毒包装系统包装pCDH-GFP-Luc质粒,将绿色荧光蛋白(GFP)和萤光素酶(Luc)基因通过病毒感染的方式整合到HepG2肝癌细胞染色体中,利用流式细胞术分选GFP+细胞,扩增培养后,将该细胞注射到裸鼠皮下进行成瘤,成瘤后分离肿瘤组织接种裸鼠肝脏,将造模成功的裸鼠分为对照组和治疗组,分别灌胃给与0.5%羧甲基纤维素钠(CMC-Na)和50 mg/kg索拉非尼,2/d,连续28 d,每7 d利用活体荧光成像系统观察肝癌细胞在对照组和治疗组裸鼠肝脏内的生长情况。实验结束后,分离裸鼠肝脏肿瘤,拍照称重。结果:建立了稳定表达双荧光的人肝癌细胞系HepG2-GFP-Luc,体外发光强度与表达萤光素酶的细胞数量呈正相关(R2=0.9945);建立了肝细胞癌原位移植活体荧光成像模型,对照组和治疗组肝脏内肿瘤细胞荧光强度随时间的延长逐渐增加,治疗组荧光强度明显低于对照组。定量分析结果显示,在第24、31和38 d,治疗组荧光总光子数值显著低于对照组;治疗组平均瘤重显著低于对照组。结论:建立了一种肝细胞癌原位移植荧光成像模型,可通过活体成像系统对肿瘤大小进行动态定量分析,为抗肝癌药物的药效学评价提供了实时定量分析动物模型。     

Quantitative Mass Spectrometry Analysis Reveals that Deletion of the TRPV1 Receptor in Mice Alters Substance P and Neurokinin A Expression in the Central Nervous System

Vanilloid receptors have a central role in the processing of nociceptive stimuli. TRPV1 null mice showed significant decrease in response to heat noxious stimuli. However, thermal sensitivity is still present suggesting that the TRPV1 is not an exclusive transducer of thermal stimuli. Additionally, tachykinin peptides play a central role in pain processing and expression levels may also contribute in modifying the pain threshold. The LC?CMS/MS analysis revealed that SP and NKA were significantly down-regulated in TRPV1^?/? in spinal cord and brain tissues. In spinal cord, SP concentrations were 23.4?% lower (p?<?0.0049) and NKA concentrations were 22.0?% lower (p?<?0.0022) in TRPV1 null mice. Additionally, brain SP concentrations were 26.9?% lower (p?<?0.0260) and brain NKA concentrations were 31.9?% lower (p?<?0.0063) in TRPV1 null mice. These results clearly demonstrate that TPRV1 null mice exhibit lower SP and NKA concentrations in the central nervous system. The deficit of thermal responses may also be related to the down-regulations of SP and NKA.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

A New Method of Defense Response Analysis Using a Transient Expression System in Rice Protoplasts

We established a new plant defense response assay using a transient expression system in rice protoplasts. The assay system sensitively detected defense induction by flagellin, which had previously been assigned to a specific elicitor. Our assay system provides a rapid and efficient way to dissect rice defense mechanisms.     

An in Vitro Analysis System Using a Fluorescence Protein Reporter for Evaluating Anti-Inflammatory Effects in Macrophages

Monitoring of inflammation in adipose tissues, which causes insulin resistance, is valuable in evaluating insulin resistance. We developed an in vitro analysis system using a fluorescence protein (FP) as a reporter gene driven by pro-inflammatory cytokine promoters such as monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNFα). In the reporter-transfected RAW264 cells, the protein expression levels of green fluorescence protein (GFP) were increased by inflammatory stimulations such as lipopolysaccharide (LPS), conditioned medium prepared using hypertrophied 3T3-L1 adipocytes, and a co-culture system. The changes in fluorescence intensity were equivalent to those of the mRNA and protein expression levels for each cytokine. Moreover, the effects of 15-deoxy-12,14Δ-prostaglandine J₂, a natural anti-inflammatory compound, were detectable in this system. These data indicate that the FP system developed here is an analysis system of low cost with simple procedures for evaluating inflammation, suggesting usability in the large-scale screening of anti-inflammatory compounds.     

Single Cell FRET Analysis for the Identification of Optimal FRET-Pairs in Bacillus subtilis Using a Prototype MEM-FLIM System

Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET). Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM). For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV)-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.     

Quantitative Image Analysis and Modeling Indicate the Agrobacterium tumefaciens Type IV Secretion System Is Organized in a Periodic Pattern of Foci

The Gram negative plant pathogen Agrobacterium tumefaciens is uniquely capable of genetically transforming eukaryotic host cells during the infection process. DNA and protein substrates are transferred into plant cells via a type IV secretion system (T4SS), which forms large cell-envelope spanning complexes at multiple sites around the bacterial circumference. To gain a detailed understanding of T4SS positioning, the spatial distribution of fluorescently labeled T4SS components was quantitatively assessed to distinguish between random and structured localization processes. Through deconvolution microscopy followed by Fourier analysis and modeling, T4SS foci were found to localize in a non-random periodic pattern. These results indicate that T4SS complexes are dependent on an underlying scaffold or assembly process to obtain an organized distribution suitable for effective delivery of substrates into host cells.     

Using Network Analysis to Explore if Professional Opinions on Japanese Encephalitis Risk Factors in Nepal Reflect a Socio-ecological System Perspective

Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia and a significant public health problem in Nepal. Its epidemiology is influenced by factors affecting its amplifying hosts (pigs), vectors (mosquitoes), and dead-end hosts (including people). While most control efforts target reduced susceptibility to infection either by vaccination of people or pigs or by reduced exposure to mosquitoes; the economic reality of Nepal makes it challenging to implement standard JE control measures. An ecohealth approach has been nominated as a way to assist in finding and prioritizing locally relevant strategies for JE control that may be viable, feasible, and acceptable. We sought to understand if Nepalese experts responsible for JE management conceived of its epidemiology in terms of a socio-ecological system to determine if they would consider ecohealth approaches. Network analysis suggested that they did not conceive JE risk as a product of a socio-ecological system. Traditional proximal risk factors of pigs, mosquitoes, and vaccination predominated experts’ conception of JE risk. People seeking to encourage an ecohealth approach or social change models to JE management in Nepal may benefit from adopting social marketing concepts to encourage and empower local experts to examine JE from a socio-ecological perspective.     

Isolation of a Tobacco cDNA Encoding Sar1 GTPase and Analysis of Its Dominant Mutations in Vesicular Traffic Using a Yeast Complementation System

The cDNA clone of NtSARl, a gene encoding the small GTPase Sar1pwhich is essential for vesicle formation from the endoplasmicreticulum (ER) membrane in yeast, has been isolated from Nicotianatabacum BY-2 cells. NtSAR1 as well as AtSAR1 cDNA isolated fromArabidopsis thaliana [d'Enfert et al. (1992) EMBO J. 11: 4205]could complement the lethality of the disruption of SARI inyeast cells in a temperature-sensitive fashion. They also suppressedyeast sec12 and sec16 temperature-sensitive mutations as yeastSARI does. Using this complementation system, we analyzed thephenotypes of several mutations in plant SAR1 cDNAs in yeastcells. The expression of NtSAR1 H74L and AtSAR1 N129I showeddominant negative effect in growth over the wild-type SARI,which was accompanied by the arrest of ER-to-Golgi transport.Such dominant mutations will be useful to analyze the role ofmembrane trafficking in plant cells, if their expression canbe regulated conditionally. (Received October 29, 1997; Accepted March 17, 1998)     

Statistical and Economical Efficiency in Assessment of Liver Regeneration Using Defined Sample Size and Selection in Combination With a Fully Automated Image Analysis System

Quantification of liver regeneration is frequently based on determining the 5-bromo-2-deoxyuridine labeling index (BrdU-LI). The quantitative result is influenced by preanalytical, analytical, and postanalytical variables such as the region of interest (ROI). We aimed to present our newly developed and validated automatic computer-based image analysis system (AnalySIS-Macro), and to standardize the selection and sample size of ROIs. Images from BrdU-labeled and immunohistochemically stained liver sections were analyzed conventionally and with the newly developed AnalySIS-Macro and used for validation of the system. Automatic quantification correlated well with the manual counting result (r=0.9976). Validation of our AnalySIS-Macro revealed its high sensitivity (>90%) and specificity. The BrdU-LI ranged from 11% to 57% within the same liver (32.96 ± 11.94%), reflecting the highly variable spatial distribution of hepatocyte proliferation. At least 2000 hepatocytes (10 images at 200× magnification) per lobe were required as sample size for achieving a representative BrdU-LI. Furthermore, the number of pericentral areas should be equal to that of periportal areas. The combination of our AnalySIS-Macro with rules for the selection and size of ROIs represents an accurate, sensitive, specific, and efficient diagnostic tool for the determination of the BrdU-LI and the spatial distribution of proliferating hepatocytes. (J Histochem Cytochem 57:1075–1085, 2009)