Establishment of an Evaluation Method for Gene Silencing by Serial Pulmonary Administration of siRNA and pDNA Powders: Naked siRNA Inhalation Powder Suppresses Luciferase Gene Expression in the Lung

In order to evaluate the in vivo effect of inhaled formulations, it is a gold standard to create a lung metastasis model by intravenously injecting cancer cells into an animal. Because the cancer grows from the blood vessel side, there is a possibility of underestimating the effect of an inhaled formulation administered to the lung epithelium side. In addition, the metastasis model has disadvantages in terms of preparation time and expense. The present study aimed to establish a new method to evaluate the effect of an inhaled small interfering RNA (siRNA) formulation that is more correct, more rapid, and less expensive. We investigated whether siRNA can suppress gene expression of plasmid DNA (pDNA) by serial pulmonary administration of siRNA and pDNA powders prepared by spray-freeze-drying. We revealed that formulations of dry siRNA powder significantly suppressed gene expression of pDNA powder compared with a control group with no siRNA. Naked siRNA inhalation powder with no vector showed the suppression of gene expression equivalent to that of an siRNA-polyethyleneimine complex without damaging tissues. These results show that the present method is suitable for evaluating the gene-silencing effect of inhaled siRNA powders.     

Outcomes for patients with infantile idiopathic scoliosis by casting table type

Purpose:Serial casting is an effective treatment for infantile idiopathic scoliosis. The most common casting table types are Mehta, Risser, and spica tables. We compared major curve correction between patients with infantile idiopathic scoliosis treated using pediatric hip spica tables versus Risser or Mehta tables.Methods:In this multicenter retrospective study, we included 52 children younger than 3 years (mean ± standard deviation age, 1.6 ± 0.68 years) treated with ≥2 consecutive casts for infantile idiopathic scoliosis between September 2011 and July 2018. We compared major curve angle (measured using the Cobb method) before and after treatment and improvement in curve angle between the spica tables group (n = 12) and the Risser or Mehta tables group (n = 40). The primary outcome was the difference in percentage correction of the major curve according to radiographs taken after first casting and at final follow-up.Results:The mean major curve was 47° ± 18° before casting. A median of six casts (range: 2–14) were applied. Mean follow-up after treatment initiation was 22 months (range: 7–86 months). At baseline, the major curve was significantly larger in the spica tables group (58°) than in the Risser or Mehta tables group (43°) (p = 0.01). We found no differences in the percentage curve correction in the spica tables group versus Risser or Mehta tables group after first casting or at final follow-up.Conclusion:Serial casting was associated with substantial major curve correction in patients with infantile idiopathic scoliosis. Curve correction did not differ between patients treated with a spica table versus a Risser or Mehta table.Level of Evidence:Level III, retrospective cohort study     

Multiple Invagination Patterns and Synaptic Efficacy in Primate and Mouse Rod Synaptic Terminals

PurposeOptical retina images are scaled based on eye size, which results in a linear scale ratio of 10:1 for human versus mouse and 7:1 for macaque monkey versus mouse. We examined how this scale difference correlates with the structural configuration of synaptic wiring in the rod spherule (RS) between macaque and mouse retinas compared with human data.MethodsRod bipolar cell (BC) dendrites and horizontal cell (HC) axonal processes, which invaginate the RS to form synaptic ribbon-associated triads, were examined by serial section transmission electron microscopy.ResultsThe number of rod BC invaginating dendrites ranged 1∼4 in the macaque RS but only 1∼2 in the mouse. Approximately 40% of those dendrites bifurcated into two central elements in the macaque, but 3% of those dendrites did in the mouse. Both factors gave rise to 10 invagination patterns of BC and HC neurites in the macaque RS but only two in the mouse. Five morphological parameters: the lengths of arciform densities and ribbons, the area of the BC–RS contact, and the surface areas of BC and HC invaginating neurites, were all independent of the invagination patterns in the macaque RS. However, those parameters were significantly greater in the macaque than in the mouse by ratios of 1.5∼1.8.ConclusionsThe primate RS provides a more expansive BC–RS interface associated with the longer arciform density and more branched invaginating neurites of BCs and HCs than the mouse RS. The resulting greater synaptic contact area may contribute to more efficient signal transfer.     

Clusters of proliferating endothelial cells and smooth muscle cells in rabbit carotid arteries

Schwarz and Benditt found clustering of replicating cells in aortic endothelium in 1976 and discussed how homeostasis of the arterial wall is maintained through this nonrandom distribution of replicating cells. However, it is still unclear how cells of vascular walls turnover. In order to address this issue, we evaluated distribution of the cells in mitotic cycle, labeled by Ki67‐immunostaining, in serial histological sections of twelve carotid arteries of six adult male Japanese rabbits. As a result, a total of 1713 Ki67‐positive endothelial cells (ECs) and 1247 Ki67‐positive smooth muscle cells (SMCs) were identified. The Ki67‐positivity rate in ECs and SMCs were about 0.048% and 0.0027%, respectively. Many of the Ki67‐positive cells clustered in two (EC, 37%; SMC, 33%), three to four (EC, 8%; SMC, 28%), and five to eight cells (EC, 5%; SMC, 10%). Clusters having more than eight cells were not found. Thus, it can be speculated that the cell division of proliferating ECs and SMCs occur four times at most. These novel findings offer great insights for better understanding of the mechanism that underlies cell number regulation of the blood vessel.     

How many sputum smears are necessary for case finding in pulmonary tuberculosis?

We reviewed the laboratory registers of 42 tuberculosis (TB) diagnostic centres in the southern region of Ethiopia to determine the value of submitting serial sputum samples for the diagnosis of pulmonary TB (PTB) and estimate the proportion of suspects that are smear positive. A total of 15,821 TB suspects submitted three smears each (47,463 smears) in 2000 with a median of 228 per centre. The smear positivity rate (two or more positive smears) was 25%, with a range of 16.8-36.4% per zone. This exceeds the international recommendations of examining 10 suspects to identify one case. A total of 4099 (26%) of the suspects had at least one positive smear with 3753 (91.6%) of the first specimens being positive. A further 303 (7.4%) were negative in the first specimen but had a positive second specimen and 42 (1%) suspects had two negative specimens followed by a positive third smear. The value of the third sputum is negligible as 99% of the cases were identified from the first and second specimens. Reducing the number of specimens to two or even one would have multiple advantages in countries where laboratories are usually over-burdened and are not easily accessible to the population. Submission of two specimens on the same day could improve compliance in submitting samples and collecting results as the number of diagnostic visits would be reduced without significant loss of sensitivity.     

Input and output modes modulate phonological and semantic contributions to immediate serial recall: Evidence from a brain-damaged patient

Psycholinguistic models of short-term retention suggest that performance at verbal short-term memory (STM) tasks relies on the activation of phonological, lexical, and semantic representations, the relative impact of each depending on task variables. This was tested in normal individuals and in I.R., a brain-damaged patient with a phonological deficit. In Experiment 1, the effect of phonological and semantic similarity was assessed under different presentation formats (words, pictures) and recall modes (oral, picture pointing, and picture pointing among distractors). In Experiment 2, effects were compared using reproduction and reconstruction responses. When words were used at input, controls showed robust phonological similarity effects irrespective of response mode. In contrast, I.R. showed a reliable semantic effect. However, both studies indicated that when response mode promoted order recuperation (reconstruction and picture pointing modes), I.R. showed a typical phonological similarity effect with no semantic contribution. The data support current psycholinguistic views suggesting that the short-term retention of verbal items depends on the temporary activation of word representations. In healthy controls, presentation mode appears to modulate the role of those representations but in I.R., it was the output condition—particularly whether order was or was not required—that was found to be crucial with respect to the appearance of semantic or phonological effects. This supports the important role that order information plays in short-term memory tasks.     

基于单片机的功能磁共振同步器的设计

目的：设计一种处理功能磁共振成像( fMRI)同步输出信号的同步器,对其解决同步问题的性能进行鉴定。方法fMRI在采集图像数据时,有两种同步信号输出方式：其一,采集一幅完整脑图时,每采集一层图像输出一个同步方波信号;其二,采集完一幅脑图输出一个同步信号。首先设定一幅完整脑图的采集层数,也称同步参数( SP);然后采集第一种同步方波信号,用单片机外部中断方法对第一种同步方波信号的上升沿计数,并保持初始输出为高电平;直到最后一层脑图时,将输出信号置为低电平并延时一段时间。结果同步器成功将第一种同步方波信号处理成第二种同步信号;matlab编程串口程序自动化设定SP,通过串口传输至单片机;结果表明该同步器可自动化设置SP。结论设计的同步器能使某类功能磁共振输出同步信号的同步问题得以解决,设备兼容性得到提升,其自动化设置SP,可减少医护人员的工作量。     

Protein crystal structure obtained at 2.9 ? resolution from injecting bacterial cells into an X-ray free-electron laser beam

It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.The advent of X-ray free-electron lasers (XFELs) has made it possible to obtain atomic resolution macromolecular structures from crystals with sizes approximating only 1/60th of the volume of a single red blood cell. Brief, intense pulses of coherent X-rays, focused on a spot of 3-μm diameter, have produced 1.9-Å-resolution diffraction data from a stream of lysozyme crystals, each crystal no bigger than 3 μm³ (1). A stream of crystals, not just one crystal, is required to collect the many tens of thousands of diffraction patterns that compose a complete data set. No single crystal can contribute more than one diffraction pattern because the XFEL beam is so intense and the crystals so small that the crystals are typically vaporized after a single pulse. Impressively, a photosystem I crystal no bigger than 10 unit cells (300 nm) on an edge produced observable subsidiary diffraction peaks between Bragg reflections, details which would be unobservable from conventionally sized crystals (2). With this new ability to collect diffraction patterns from crystals of unprecedentedly small dimensions, it is conceivable that high-resolution diffraction data could be collected from crystals in vivo. The structure obtained in this manner would be unaltered from that occurring naturally in a living cell, free from distortion that might otherwise potentially arise from nonphysiological conditions imposed by recrystallization. A practical advantage would also be gained by eliminating the need for a protein purification step, whether the in vivo grown crystals were naturally, or heterologously expressed (3).The nascent field of serial femtosecond crystallography (SFX) has published results on nine different macromolecular systems since its inception in 2009 (3, 9). The crystals for this study were not grown in artificial crystallization chambers as has been the protocol of conventional macromolecular crystallography since the 1950s. Instead, crystals were grown in cells. Specifically, they were grown in Sf9 insect cells, heterologously expressing Trypanosoma brucei cathepsin B. These in vivo-grown crystals were used for the XFEL diffraction experiment. To this end, the cells were lysed and the crystals were extracted before injecting them in the XFEL beam for data collection. This last purification step seems to be the only major departure from our goal of obtaining high-resolution structural information from crystal inclusions in vivo, without requiring the crystal to be extracted from the cell that assembled it. Here we attempt to go one step further than previous studies—to record diffraction from crystals within living cells.

Table 1.

SFX publications from XFEL sources to date