Revisiting Rat Spermatogenesis with MALDI Imaging at 20-μm Resolution

Matrix-assisted laser desorption/ionization (MALDI) molecular imaging technology attracts increasing attention in the field of biomarker discovery. The unambiguous correlation between histopathology and MALDI images is a key feature for success. MALDI imaging mass spectrometry (MS) at high definition thus calls for technological developments that were established by a number of small steps. These included tissue and matrix preparation steps, dedicated lasers for MALDI imaging, an increase of the robustness against cell debris and matrix sublimation, software for precision matching of molecular and microscopic images, and the analysis of MALDI imaging data using multivariate statistical methods. The goal of these developments is to approach single cell resolution with imaging MS. Currently, a performance level of 20-μm image resolution was achieved with an unmodified and commercially available instrument for proteins detected in the 2–16-kDa range. The rat testis was used as a relevant model for validating and optimizing our technological developments. Indeed, testicular anatomy is among the most complex found in mammalian bodies. In the present study, we were able to visualize, at 20-μm image resolution level, different stages of germ cell development in testicular seminiferous tubules; to provide a molecular correlate for its well established stage-specific classification; and to identify proteins of interest using a top-down approach and superimpose molecular and immunohistochemistry images.MALDI imaging mass spectrometry (IMS)¹ is increasingly recognized as a powerful approach for the discovery of tissue biomarkers candidates and tissue leakage products in clinical proteomics (1–6). The technology holds an enormous potential for differentiation of disease states, classification of tumors, and early diagnosis or prognosis but also for elucidating pathogenesis pathways and as a follow-up for pharmacological treatments (1, 7–9).IMS offers some unique advantages that can support or complement most conventional techniques. It allows multiplex spatial localization of biomolecules on tissue sections without the need for time-consuming processing steps, specific probes, or labeling of analytes that can alter their native localization or function (10). Interestingly, in the field of biomarker detection, it is now recognized that in many cases a single protein cannot serve as a reliable disease-specific marker, whereas a panel of biomarkers candidates can potentially provide a clear diagnosis (8, 11). Although well established and extremely powerful, conventional differential proteomics approaches based on the analysis of tissue homogenates represent an oversimplification of tissue morphology in a “black versus white” manner (even if combined with tissue microdissection). In particular, cancer tissue consists of several different cell types at various developmental stages (12). An efficient, sensitive, and selective analysis of tumor tissue, therefore, ultimately requires the ability to perform molecular histological analyses on the level of individual cells.Many technical challenges remain to be faced in IMS among which the improvement of lateral resolution, which is currently in the range of 100–200 μm corresponding, at the best, to a few cells. Lateral resolution is mainly limited by the matrix application step and the laser beam diameter and shape, whereas reduction of the irradiated sample area naturally reduces the ion yield. Consequently, a compromise between high lateral resolution and spectral quality has generally to be found (13). However, the adequate evaluation of histological images after conventional hematoxylin and eosin (H&E) or immunohistological stainings routinely requires higher resolution down to the level of individual cellular nuclei. To beneficially integrate MALDI images into the histo(patho)logical evaluation, there are two key requirements. 1) MALDI images should have utmost resolution to correlate them with the tissue morphology at least on the level of single cells (i.e. 10 μm). 2) Overlapping of the transmission microscopic and MALDI images is mandatory to correlate molecular marker distributions with tissue morphology at typical microscopic image resolution (i.e. ∼100 nm). One of the driving forces behind improvement of lateral resolution is that the detection of biomarker candidates can thus be confined to very fine structures of the tissue and even single cells. Improvements in resolution, which is indeed a methodological challenge, must be demonstrated using a tissue sample that on the one hand has clear morphological features of appropriate size and that is well described by classical histology.In the present study, we combined readily available technologies to achieve 20-μm lateral image resolution and to match this information to high quality optical microscopy images as they are used in the histological practice. Because of its highly complex anatomical features, we used the mammalian testis as a model to establish the complete work flow from the acquisition of MALDI images with lateral resolution near the single cell level, the proper match with histological information derived from standard optical microscopy, to the identification of proteins of interest using a top-down approach.     

Chimeric evolution of the 2-μm genome in Saccharomyces cerevisiae

We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 m) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-m variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-m plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection. Correspondence to: G.H. Rank     

Polymorphism of 2-μm plasmids in industrial strains of Saccharomyces cerevisiae

Restriction fragment length polymorphism (RFLP) analyses of industrial Saccharomyces yeast DNA have identified eight 2-m plasmidsvariants that fall into two distinct types. Type-I plasmids are of unique form, whereas type-II plasmids exist in seven distinct RFLP forms. Only two different 2-m variants were observed in 35 bakers' strains analysed. One variant was the unique type-I whereas the second variant represents an ancestral form of the type-II plasmid. Sixteen of nineteen wine yeasts carried a distinctive type-II plasmid with a homeologous STB repeat whereas ale and lager yeasts had a wide range of type-II variants. Relative to nuclear and mtDNA, 2-m polymorphism is less diverse and not diagnostic for a specific strain. This 2-m DNA polymorphism is a convenient and useful addendum to nuclear and mtDNA RFLP analyses but cannot serve as the sole marker for strain identification. A tentative phylogeny of industrial S. cerevisiae yeasts is suggested with origins in bakers' yeast carrying the ancestral type-II form. Correspondence to: G. H. Rank     

Enantiomeric separation of triazole fungicides with 3-μm and 5-μml particle chiral columns by reverse-phase high-performance liquid chromatography

This study used chiral columns packed with 3‐μm and 5‐μm particles to comparatively separate enantiomers of 9 triazole fungicides, and Lux Cellulose‐1 columns with chiral stationary phase of cellulose‐tris‐(3,5‐dimethylphenylcarbamate) were used on reverse‐phase high‐performance liquid chromatography with flow rates of 0.3 and 1.0 mL min⁻¹ for 3‐μm and 5‐μm columns, respectively. The (+)‐enantiomers of hexaconazole ( 1 ) , tetraconazole ( 4 ) , myclobutanil ( 7 ) , fenbuconazole ( 8 ) and the (−)‐enantiomers of flutriafol ( 2 ) diniconazole ( 3 ) , epoxiconazole ( 5 ) , penconazole ( 6 ) , triadimefon ( 9 ) were firstly eluted from both columns, the elution orders identified with an optical rotation detector didn't change with variety of column particles and mobile phases (acetronitrile/water and methanol/water). The plots of natural logarithms of the selectivity factors (ln α) for all fungicides except penconazole ( 6 ) versus the inverse of temperature (1/T) were linear in range of 5–40°C. The thermodynamic parameters (ΔH°, ΔS°, ΔΔH° and ΔΔS°) were calculated using Van't Hoff equations to understand the thermosynamic driving forces for enantioseparation. This work will be very helpful to obtain good enantiomeric separation and establish more efficient analytical method for triazole fungicides. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

脉冲1.338μm激光角膜损伤效应研究

目的:实验研究脉冲1.338μm激光的角膜损伤效应,确定其损伤阈值,并与10.6μm激光角膜损伤特点进行比较。方法:采用输出波长1.338μm、脉冲宽度5 ms的Nd:YAG激光为照射光源,角膜光斑直径1.7 mm,以不同剂量的激光照射新西兰白兔角膜,于照后1 h观察角膜损伤情况,统计损伤发生率,采用加权概率单位法计算损伤发生率为50%时所对应的激光剂量,即损伤阈值ED50。在1.5倍阈值剂量下比较该激光与10.6μm激光角膜损伤的特点。实验中注意观察晶状体和眼底是否有损伤。结果:脉冲1.338μm激光角膜损伤阈值为27.0 J/cm2(95%置信区间25.8～28.1 J/cm2)。阈值水平下,角膜损伤斑肉眼观察呈淡淡的灰白色,裂隙灯下可见一与角膜同厚的灰白色反光带,晶状体或视网膜正常;1.5倍阈值剂量下,角膜损伤斑为清晰的瓷白色,裂隙灯下可见一与角膜同厚的白色反光带,同时观察到晶状体前表面白色反光点,但视网膜无变化。与之相比,10.6μm激光角膜损伤在裂隙灯下仅观察到一很窄的亮白反光条,位于角膜表层。结论:脉冲1.338μm激光在光斑直径1.7 mm,脉冲宽度5 ms条件下的角膜损伤阈值为27.0 J/cm2。角膜损伤特点是,损伤斑呈灰白色或瓷白色,累及角膜全层,而10.6μm激光损伤仅累及角膜浅层。     

Evidence forCis- andTrans-acting element coevolution of the 2-μm circle genome inSaccharomyces cerevisiae

Summary We compared the DNA sequence of the yeas 2-μm plasmidcis-actingSTB andtrans-actingREP1 partition loci of laboratory haploid and industrial amphiploid strains. Several industrial strains had a uniqueSTB sequence (type 1) sharing only 70% homology with laboratorySTB (type 2). Type 1 plasmids had a REP1 protein with 6–10% amino acid substitutions when compared to REP1 of type 2 plasmids. All 2-μm variants that shared a similarSTB consensus sequence exhibited a high degree ofREP1 nucleotide and amino acid sequence conservation. These observations suggest molecular coevolution oftrans-acting elements with cognate target DNA structure. Based on DNA sequencing and Southern hybridization analyses, we classified 2-μm variants into two main evolutionary lineages that differ atSTB as well asREP1 loci. The role of molecular coevolution in yeast intra- and interspecies plasmid evolution was discussed.     

Inheritance of the 2μm DNA Plasmid from Saccharomyces

A variety of Saccharomyces strains were examined for the presence of 2micro DNA and, if present, for the pattern of fragments produced by its digestion with site-specific (restriction) endonucleases. Two strains were found that did not contain detectable levels of 2micro DNA, and two strains contained 2micro DNA molecules having only one EcoRI restriction endonuclease recognition site rather than the usual two.-A haploid containing 2micro DNA with one EcoRI restriction site was mated with a haploid containing 2micro DNA with two EcoRI restriction sites and the resulting diploid maintained both types during vegetative growth. Sporulation of the diploid produced four spores, and the clones from these spores contained both types.-A haploid lacking 2micro DNA was mated with a haploid containing 2micro DNA and the resulting diploid contained 2micro DNA. The four clones derived from the haploid spores after sporulation of this diploid all contained 2micro DNA. A rho(-) strain without 2micro DNA was mated to a rho(+) strain with 2micro DNA, and heteroplasmons were selected that had received the nucleus from the strain without 2micro DNA and the mitochondria from the strain with 2micro DNA. Twelve of twenty-four such clones contained 2micro DNA.-I conclude that: (1) the different types of 2micro DNA identified in these strains do not restrict one another, (2) the different types are inherited extrachromosomally, (3) lack of 2micro DNA in two strains is not due to the absence of genes needed for maintenance and (4) the approximately 100 copies of 2micro DNA contained within a single cell are probably clustered within one or a few cytoplasmic organelles.     

3.74μm远红外激光致小鼠角膜损伤修复观察

为观察3.74μm远红外激光致角膜损伤的特点和损伤修复过程,利用该激光在光斑直径为2 mm、照射时间为0.8 s、辐照量为23.2 J/cm²的条件下照射C57BL/6J小鼠角膜,采用大体观察、裂隙灯显微镜、光学相干断层扫描(OCT)以及组织病理方法,在角膜损伤后3 h、6 h、12 h、1 d、3 d、7 d、14 d和21 d进行观察。大体观察角膜损伤即刻可见灰白色损伤斑,表面凹凸不平,角膜混浊随时间逐渐加重,1 d达到顶峰,3～7 d混浊减轻,14～21 d再次加重。裂隙灯下角膜损伤累及全层,角膜厚度随时间先增大后逐渐恢复。OCT观察角膜损伤后明显外凸,反射光带全层性增强,3 h角膜显著增厚,12 h达到最厚,后逐渐恢复至正常。经组织切片观察：上皮层损伤后3 h核固缩深染,6～12 h核染色变淡消失,1 d时1～2层新生上皮完全覆盖损伤区,3～7 d上皮细胞增至3～4层,14～21 d恢复正常;基质层损伤后3～6 h核染色质大量脱失,12 h出现浸润细胞,后浸润细胞增多,由基质深层向浅层迁移,14～21 d浸润细胞减少,纤维排列仍不规则;内皮层损伤后3 h细...     

一种可用于500μm脑片的高分辨率细胞内标记技术

目的：建立简便高效的、与脑片盲法膜片钳记录相结合的生物胞素细胞内标记染色方法。方法：制备大鼠听皮层脑片（500 μm）,采用盲法脑片膜片钳全细胞记录,泳入生物胞素（0.2％）对记录细胞进行标记,经组织化学显色和甘油封片后,沿显微镜Z轴,每隔30 μm拍摄一帧显微数码图像,利用Adobe Photoshop软件对神经元进行三维重建。结果：标记的神经元层次清楚,可在光镜下分辨出胞体、轴树突分支、棘突等细微结构,而且非特异性背景染色浅;不需要进行厚脑片的二次切片即可对神经元进行三维重建。结论：本方法简便易行,结果可靠,分辨率高,而且对设备要求不高。     

1．06μm激光致皮肤痛觉效应的研究

目的：研究1．06μm激光所致人手背皮肤的痛觉效应。方法：以输出波长为1．06μm的脉冲Nd：YAG激光照射人手背皮肤,记录每次刺激激光的能量以及受试者的反应。采用加权概率单位算法计算诱发痛觉概率为50％时对应的激光剂量ED50,即为痛觉阈值。改变光斑大小和脉冲宽度,测定三种不同刺激条件下的痛觉阈值,并探索温度对激光所致痛觉效应的影响。结果：当皮肤温度约为30℃,分别使用光斑直径1．20mm、脉冲宽度85μs,光斑直径1．20mm、脉冲宽度20ns和光斑直径2．56mm、脉冲宽度20ns的激光刺激时,痛觉阈值分别为394mJ／mm^2、36．4mJ／mm^2和8．92mJ／mm^2。在第一种刺激条件下,当皮肤温度为25℃时,剂量为383mJ／mm^2的激光诱发痛觉的概率为16．7％;当皮肤温度为39℃时,剂量为361mJ／mm^2的激光诱发痛觉的概率为56．7％。结论：1．06“m激光所致痛觉的阈值随脉冲宽度的减小、光斑面积的增大和皮肤表面温度的增加而减小。     

Erbium(III) ion-doped borate-based glasses for 1.53 μm broad band applications

Novel erbium(III) ion-doped borate-based glasses (Er³⁺:BCNF) by conventional melt-quenching technique were designed and synthesized. The glasses were characterized for their structural, vibrational and spectroscopic properties. The nephelauxetic ratio, bonding parameters, and Judd–Ofelt (JO) intensity parameters (Ω_λ λ = 2, 4 and 6) were determined by using absorption spectrum of 1 mol% Er₂O₃ doped glass. These JO parameters were utilized to derive radiative properties for various excited states of erbium(III) ions. Emission cross-section for ⁴I_13/2 → ⁴I_15/2 transition of erbium(III) ions was computed through McCumber theory. The decay curves for (²H_11/2, ⁴S_3/2) and ⁴I_13/2 levels were recorded and analysed. All the results of Er³⁺:BCNF glasses revealed that the studied glasses are efficient and thermally stable and could be suitable for display devices, optical amplification and green laser applications.     

The seasonal abundance and vertical distribution of the <3-μm phytoplakton in the north basin of Lake Biwa

The seasonal changes in the size-fractionated chlorophylla concentrations (<3 μm, 3 to 25 μm, and >25 μm) were investigated at a pelagic site of the north basin of Lake Biwa during June to December 1985. Autofluorescing plankton cells in the <3-μm fractions were also examined using the fluorescein isothiocyanate staining epifluorescence microscopic technique. The <3-μm phytoplankton (usually dominated by chroococcoid cyanobacteria except for a few cases dominated by small eukaryotes) showed a clearly different pattern of seasonal change compared with the larger fractions. That is, from August to early September, chlorophylla of the larger fractions declined considerably, while the <3-μm chlorophylla did not decrease significantly. Moreover, cyanobacterial cell density in the <3-μm fraction showed a maximum value (2–3.5×10⁵ cells·ml⁻¹) during this period. The relative contribution of the <3-μm chlorophylla to the total chlorophylla increased from <5% to 45% during the course of this change. No clear vertical trend in the distribution and composition of the <3-μm phytoplankton was found, except that relatively large cyanobacteria (>4 μm³) appeared at a depth of 15m but not at 0,5 and 10 m from late July to August. These large cells were also found in November and December. The drastic seasonal change of phytoplankton size structure occurring in this basin was discussed in relation to grazing, nutrient depletion and sinking. Contribution from Otsu Hydrobiological Station, Kyoto Univeristy (No. 308, foreign language series).     

Average hydroxyapatite concentration is uniform in the extracollagenous ultrastructure of mineralized tissues: evidence at the 1–10-μm scale

At the ultrastructural observation scale of fully mineralized tissues (l=1-10 mum), transmission electron micrographs (TEM) reveal that hydroxyapatite (HA) is situated both within the fibrils and extrafibrillarly, and that the majority of HA lies outside the fibrils. The extrafibrillar amount of HA varies from tissue to tissue. By means of mathematical modeling, we here provide strong indications that there exists a physical quantity that is the same inside and outside the fibrils, for all different fully mineralized tissues. This quantity is the average mineral concentration in the non-collagenous space. This space is the sum of the extrafibrillar volume and of the volume of the fibrils that is not occupied by collagen molecules. Two independent sets of experimental observations covering a large range of tissue mass densities establish the relevance of our proposition: (i) mass density measurements and diffraction spacing measurements, re-analyzed through a dimensionally consistent packing model; (ii) optical density measurements of TEMs. The aforementioned average uniform HA-concentration in the extracollagenous space of the ultrastructure may emphasize the putative role played by a number of non-collagenous organic molecules in providing the chemical boundary conditions for mineralization of HA in the extracollagenous space. The probable existence of an average uniform extracollagenous HA concentration has far-reaching consequences for the mechanical behavior of mineralized tissues.     

Simultaneous determination of twelve benzodiazepines in human serum using a new reversed-phase chromatographic column on a 2-μm porous microspherical silica gel

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of twelve frequently used benzodiazepines (BZPs) (bromazepam, clonazepam, chlordiazepoxide, estazolam, etizolam, flutazoram, haloxazolam, lorazepam, nitrazepam, oxazolam, triazolam and diazepam, internal standard) by using commerically available 2 or 5 μm particle size reversed-phase columns and a microflow cell-equipped ultraviolet detector. The separation was achieved using a C₁₈ reversed-phase column (condition 1: 100×4.6 mm I.D., particle size 2 μm, TSK gel Super-ODS; condition 2: 100×4.6 mm I.D., particle size 5 μm, Hypersil ODS-C₁₈). The mobile phase was composed of methanol-5 mM NaH₂PO₄ (pH 6) (45:55, v/v), and the flow-rate was 0.65 ml/min (conditions 1 and 2). The absorbance of the eluent was monitored at 254 nm. Retention times under condition 1 were shorter than those of condition 2. When the twelve benzodiazepines were determined, sensitivity and limits of quantification were about four to ten times better under condition 1 than under condition 2. The rate of recovery and linearity in condition 1 were approximately the same as those in condition 2. These results show that a new ODS filler with a particle size of 2 μm was more sensitive, provided better separation and was more rapid than that with conventional ODS filler.     

Diagnosing Adrenal Insufficiency: Which Test is Best—The 1-μG or the 250-μG Cosyntropin Stimulation Test?

ObjectiveTo review the available literature on the low-dose cosyntropin stimulation test (CST) for the diagnosis of primary and secondary adrenal insufficiency in both nonstressed and acutely ill patients.MethodsWe performed a MEDLINE search of all English-language literature, published between 1965 and 2007, in which the 1-μg and the 250-μg CSTs were compared in patients with primary and secondary adrenal insufficiency.ResultsThe majority of published evidence suggests that the 1-μg CST is more sensitive than the 250-μg CST for the diagnosis of secondary adrenal insufficiency in nonstressed patients. In patients with primary adrenal insufficiency, the low-dose CST is unlikely to add any diagnostic sensitivity to the high-dose CST. In critically ill patients, the 1-μg test is also likely to be more sensitive than the 250-μg test when an appropriate cutoff value is used (25 μg/dL).ConclusionThe 1-μg CST with a cortisol level determined at 30 minutes after stimulation, with use of a cutoff level of 18 to 20 μg/dL in nonstressed patients and less than 25 μg/dL or an increment of less than 9 μg/dL from baseline in critically ill patients, is the best test that is currently available for establishing the diagnosis of secondary adrenal insufficiency. (Endocr Pract. 2008;14: 233-238)     

羟甲芬太尼──高选择性μ阿片受体激动剂及与μ-阿片受体相互作用的研究

吗啡的强效镇痛功能及广泛的生理、药理作用是几百年来药学、药理学研究的重要对象,但其作用机理未能得到阐明。70年代初发现脑内存在阿片受体以来,随之在脑内找到内源性阿片肽──甲硫氨酸脑啡肽和亮氨酸脑啡肽;接着又发现了β内啡肽、强啡肽等多种内啡肽（endophines）,从而在学术界里掀起一股研究神经肽的热潮,对吗啡药理机理研究又进入一个新的阶段,对阿片肽的生理功能成为研究的热点。利用各种阿片受体配体特性功能表达的差异,证明脑内存在多种阿片受体类型。公认的主要有μ、三种亚型。要深入研究阿片受体亚型的结构功能,必须…