Optimization of Whole-Body Zebrafish Sectioning Methods for Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) methods and protocols have become widely adapted to a variety of tissues and species. However, the MSI literature contains minimal information on whole-body cryosection preparation for the zebrafish (ZF; Danio rerio), a model organism routinely used in developmental, toxicity, and carcinogenicity studies. The optimal medium for embedding and cryosectioning a whole organism or soft-tissue specimen for histological examination is a synthetic polymer mixture that is incompatible with MSI as a result of ion suppression. We describe the optimal methods and results for embedding and cryosectioning whole-body ZF for MALDI-MSI. We evaluated 13 distinct embedding media formulations and found a supportive hydrogel with the consistency of cartilage to be the optimal embedding medium. The hydrogel medium does not interfere with MSI data collection, aids in tissue stability, is readily available for purchase, and is easy to prepare and handle during cryosectioning. Additionally, we decreased the matrix cluster interference commonly caused by α-cyano-4-hydroxycinnamic acid by adding ammonium phosphate to the solvent spray solution. The optimized methods developed in our laboratory produced high-quality cryosections, as well as high-quality mass spectral images of sectioned ZF.     

Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (∼2 °C) and the gut contents somewhat lower levels (∼0.6 °C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14 kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.     

An image-based method to measure joint deformity in inflammatory arthritis: development and pilot study

Quantifying joint deformity in people with rheumatoid (RA) and psoriatic arthritis (PsA) remains challenging. Here, we demonstrate a new method to measure bone erosions and abnormal periosteal growths, based on the difference between a predicted healthy and actual diseased joint surface. We optimized the method by creating and measuring artificial bone erosions and growths. Then we measured 46 healthy and diseased patient surfaces. We found average sensitivity errors of ≤0.27?mm when measuring artificial erosions and growths. Patients had significantly more bone erosion than healthy subjects. Surface based outcomes are a novel way to interpret and quantify bone changes in PsA and RA.     

BioID Performed on Golgi Enriched Fractions Identify C10orf76 as a GBF1 Binding Protein Essential for Golgi Maintenance and Secretion

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Highlights

• •BioID with Golgi fractions identified C10orf76 as proximal to GBF1.
• •Tagged C10orf76 overlaps with Golgi markers.
• •C10orf76 binds GBF1 and exchanges rapidly between free and bound forms.
• •C10orf76 is essential for maintenance of the Golgi and for secretion.

     

Detection and destruction of HER2-positive cancer cells by Ultra Quenchbody-siRNA complex

Ultra Quenchbody (UQ-body) is a biosensor that utilizes the quenching behavior of the fluorescent dye linked to the antibody V region. When the corresponding antigen is bound to the UQ-body, the fluorescence is restored and allows the detection of target molecules easily and sensitively. In this paper, we constructed UQ-bodies to sensitively detect the human epidermal growth factor receptor 2 (HER2) cancer marker in solution or on cancer cells, which was further used to kill the cancer cells. A synthetic Fab fragment of anti-HER2 antibody Fab37 with many Trp residues at hypervariable region was prepared and labeled with fluorescent dyes to obtain the UQ-bodies. The UQ-body could detect HER2 in solution at concentrations as low as 20 pM with an EC₅₀ of 0.3 nM with a fourfold response. Fluorescence imaging of HER2-positive cells was successfully performed without any washing steps. To deliver small interfering RNA (siRNA) to cancer cells, a modified UQ-body with C-terminal 9R sequence was also prepared. HER2-positive cancer cells were effectively killed by polo-like kinase 1 siRNA intracellularly delivered by the UQ-body-9R. The novel approach employing siRNA-empowered UQ-body could detect and image the HER2 antigen easily and sensitively, and effectively kill the HER2-positive cancer cells.     

In vivo dynamics and kinetics of pKi-67: transition from a mobile to an immobile form at the onset of anaphase

A cell proliferation marker protein, pKi-67, distributes to the chromosome periphery during mitosis and nucleolar heterochromatin in the interphase. We report here on the structural domains of pKi-67 that are required for its correct distribution. While both the LR domain and the conserved domain were involved in localization to the nucleolar heterochromatin, both the LR domain and the Ki-67 repeat domain were required for its distribution to the mitotic chromosome periphery. Using in vivo time-lapse microscopy, GFP-pKi-67 was dynamically tracked from the mitotic chromosome periphery to reforming nucleoli via prenucleolar bodies (PNBs). The signals in PNBs then moved towards and fused into the reforming nucleoli with a thin string-like fluorescence during early G1 phase. An analysis of the in vivo kinetics of pKi-67 using photobleaching indicated that the association of pKi-67 with chromatin was progressively altered from "loose" to "tight" after the onset of anaphase. These findings indicate that pKi-67 dynamically alters the nature of the interaction with chromatin structure during the cell cycle, which is closely related to the reformation process of the interphase nucleolar chromatin.     

在320排螺旋CT双下肢动脉成像中的护理配合效应

目的：探讨320排螺旋CT血管成像（CTA）在行双下肢动脉血管成像过程中,护理配合对的图像质量的影响。方法：将138例患者分为护理组（82例）及对照组（56例）,分别进行320排螺旋CT双下肢血管造影检查,经高压团注造影剂欧乃派克,进行三维重组,获取容积再观（VR）、曲面重组（CPR）和最大密度投影（MIP）图像。对照组的患者只进行口头的训练,没有其他的护理干预措施;实验组进行一系列的护理干预以提高患者的配合,减低在成像过程中的非生理性运动。用工作站进行图像后处理,显示双下肢动脉图像,对其图像质量和影响因素进行分析,并在检查过程中的护理干预加以总结。结果：对照组有2例患者穿刺部位不合理而影响图像质量,对双下肢的病变显示具有一定的影响,3例患者注射压力过高引起外渗,流速在2.5-3.5 ml/s,影响了图像的清晰度,其余均获得满意效果。结论：良好的护理配合有利于320排CT双下肢成像的顺利进行,精心的护理操作是取得检查成功的动脉成像保证。     

Dementia from the ABCD1 mutation c.1415-1416delAG in a female carrier

Objectives

Progressive dementia is a rare phenotypic feature of female X-ALD carriers. Even rarer is the additional presence of further risk factors for dementia, such as diabetes, hypothyroidism, and hepatopathy. We report a unique female X-ALD carrier presenting with severe, progressive dementia, paraspasticity, sphincteric dysfunction, and multisystem disease.

Case report

A 79 years-old female with a history of strumectomy, diabetes, hepatopathy, hypothyroidism, arterial hypertension, hiatal hernia, left retinal ablation, ovariectomy, hysterectomy, osteoporosis, bilateral hip endoprosthesis, and neurogenic bladder dysfunction developed slowly progressive cognitive decline since age of 77 years. She had been identified as a female carrier of X-ALD in 12/2010 upon a family screening. At age of 79 years she presented with severe dementia, anxiety, unsteadiness, helplessness, hypertelorism, exaggerated patella tendon reflexes, reduced Achilles tendon reflexes, club feet, contractures of the ankles, the knees, and the hips, and the inability to stay or walk. Cerebral CT showed diffuse atrophy, demyelination periventricularly, small lacunas in the basal ganglia, and small calcifications of the basal ganglia and the temporal lobe on the right side. Differential diagnoses of dementia were considered but were all excluded upon the clinical presentation, blood chemical investigations, imaging studies, and the pattern of neuropsychological deficits.

Conclusions

With progression of the disease manifesting X-ALD carriers may develop progressive severe dementia, severe paraspasticity, and sphincteric dysfunction. Female carriership of X-ALD can be a differential diagnosis of dementia.     

A Transient Rise in Free Mg2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation

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Mid-infrared spectroscopy and short wave infrared hyperspectral imaging—A novel approach in the qualitative assessment of Harpagophytum procumbens and H. zeyheri (Devil's Claw)

Harpagophytum procumbens (Burch.) DC. ex Meisn. subsp. procumbens (Pedaliaceae) is an important African medicinal plant growing in the Kalahari region of southern Africa. This species, together with its close taxonomic ally Harpagophytum zeyheri are collectively referred to as Devil's Claw and are used interchangeably for the treatment of inflammation-related disorders. Although the two taxa are botanically and chemically similar, H. zeyheri contains lower levels of harpagoside and these two species have not been proven to exhibit equipotent pharmacological activity. Due to these taxonomic similarities, effective quality control methods are required to distinguish between the two species. Differentiation between the two species was achieved using single point mid-infrared spectroscopy in combination with chemometric data analysis. The orthogonal projections to latent structures discriminant analysis (OPLS-DA) model had good predictive ability, as illustrated by the model statistics: R²X (cum predictive + orthogonal) = 0.86 and Q² (cum) = 0.63. Short wave infrared (SWIR) hyperspectral imaging could distinguish between the two species with acceptable model statistics: R²X and R²Y of 0.99 and 0.78, respectively. This study demonstrated that both MIR single point spectroscopy and SWIR hyperspectral imaging coupled with chemometric modelling are reliable and rapid methods to determine the authenticity of Harpagophytum spp.