Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1) selection of fluorescent dyes having minimal spectral overlap is complicated, and 2) quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH³ resin composites. Specimens were subsequently immersed for 60 sec in either Biotène PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; α=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p<0.05) on both resin composites, whereas LTO did not produce differences (p>0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.     

即时扩展型背阔肌肌皮瓣乳房再造在保留乳头乳晕复合体乳癌术后的运用分析

目的：探讨分析即时扩展型背阔肌肌皮瓣乳房再造在保留乳头乳晕复合体乳癌术后的运用。方法：回顾性分析我院2008年2月-2012年4月收治的乳腺癌术后患者106例,采用乳癌术即时扩展型背阔肌肌皮瓣乳房再造保留乳头乳晕复合体,观察手术效果以及满意度。结果：术后患者乳房美容优良率为88．68％明显大于对照组的47．17％,并且术后6个月治疗组生活质量评价总分明显大于对照组总分术后6个月患者生活质量评价总分明显大于术前评价总分,差异具有条件下意义（P〈0．05）,差异均具有统计学意义（P〈0．05）。结论：即时扩展型背阔肌肌皮瓣乳房再造在保留乳头乳晕复合体乳癌术后患者乳房美容效果较好,提高患者生活质量高,值得在临床上推广,但在手术后需积极处理可能存在的并发症情况。     

全胃切除术后四种消化道重建方式的对照研究

目的:全胃切除术后常常会发生明显的营养不良、各种术后不良症状以及生活质量的下降.本研究通过对比全胃切除术后四种消化道重建方式,探讨全胃切除术后理想的消化道重建术式.方法:选取自2007年11月至2011年3月间我院行全胃切除术治疗胃癌患者196例,分为A组(行功能性空肠间置代胃术)、B组(P型空肠间置代胃术)、C组(P型Roux-en-Y空肠代胃术)、D组(改良Longmire法空肠代胃术)4种消化道重建术.比较4组患者手术时间、出血量、住院时间和死亡率以及术后1年生活质量、营养状况和术后并发症情况.结果:4组患者在平均手术时间、出血量、住院时间和死亡率方面无明显差异(P＞0.05).A组患者倾倒综合征、反流性食管炎、Roux-en-Y滞留综合征发生率分别为2.1％(1/48)、4.2％(2/48)和2.1％(1/48),均显著低于其他3组(均P＜0.05).术后1年生活质量(Visick分级指数)的比较,A组明显优于其他3组(均P＜0.05).术后1年营养预后指数(PNI)分别为55.40± 3.6,52.30± 2.8,41.5±4.3和40.7±5.5,A组和B组明显高于C组和D组(P＜0.05),A组和B组组间数据差异无统计学意义(P＞0.05),C组和D组组间数据差异无统计学意义(P＞0.05).结论:全胃切除术术后消化道重建应保持空肠的连续性并保持摄入食物通过十二指肠.功能性空肠间置代胃术安全可靠,对于减少全胃切除术后并发症的发生率,提高生活质量和营养状况有着重要的意义.     

Explicit tests of palaeodrainage connections of southeastern North America and the historical biogeography of Orangethroat Darters (Percidae: Etheostoma: Ceasia)

The alteration in palaeodrainage river connections has shaped patterns of speciation, genetic diversity and the geographical distribution of the species‐rich freshwater fauna of North America. The integration of ancestral range reconstruction methods and divergence time estimates provides an opportunity to infer palaeodrainage connectivity and test alternative palaeodrainage hypotheses. Members of the Orangethroat Darter clade, Ceasia, are endemic to southeastern North America and occur north and south of the Pleistocene glacial front, a distributional pattern that makes this clade of closely related species an ideal system to investigate the number and location of glacial refugia and compare alternative hypotheses regarding the proposed evolution of the Teays‐Mahomet palaeodrainage. This study utilized time‐calibrated mitochondrial and nuclear gene phylogenies and present‐day geographical distributions to investigate hypothesized Teays‐Mahomet River connections through time using a dispersal–extinction–cladogenesis (DEC) framework. Results of DEC ancestral area reconstructions indicate that the Teays‐Mahomet River was a key dispersal route between disjunct highland regions connecting the Mississippi River tributaries to the Old‐Ohio Drainage minimally at two separate occasions during the Pleistocene. There was a dynamic interplay between palaeodrainage connections through time and postglacial range expansion from three glacial refugia that shaped the current genetic structure and geographical distributions of the species that comprise Ceasia.     

Ancient host shifts followed by host conservatism in a group of ant parasitoids

While ant colonies serve as host to a diverse array of myrmecophiles, few parasitoids are able to exploit this vast resource. A notable exception is the wasp family Eucharitidae, which is the only family of insects known to exclusively parasitize ants. Worldwide, approximately 700 Eucharitidae species attack five subfamilies across the ant phylogeny. Our goal is to uncover the pattern of eucharitid diversification, including timing of key evolutionary events, biogeographic patterns and potential cophylogeny with ant hosts. We present the most comprehensive molecular phylogeny of Eucharitidae to date, including 44 of the 53 genera and fossil-calibrated estimates of divergence dates. Eucharitidae arose approximately 50 Ma after their hosts, during the time when the major ant lineages were already established and diversifying. We incorporate host association data to test for congruence between eucharitid and ant phylogenies and find that their evolutionary histories are more similar than expected at random. After a series of initial host shifts, clades within Eucharitidae maintained their host affinity. Even after multiple dispersal events to the New World and extensive speciation within biogeographic regions, eucharitids remain parasitic on the same ant subfamilies as their Old World relatives, suggesting host conservatism despite access to a diverse novel ant fauna.     

Repeated evolution of uniparental reproduction in Sellaphora (Bacillariophyceae)

Diatoms possess a remarkable life cycle in which cell size decreases slowly during vegetative cell division and then increases rapidly via special expanding cells called ‘auxospores’, which are usually formed as a result of biparental sexual reproduction. However, auxospores are sometimes produced by single unpaired cells, i.e. uniparentally. We examined the nature of uniparental auxosporulation in Sellaphora and used a two-gene dataset to study phylogenetic relationships between uniparental and biparental Sellaphora demes and species; we tested whether uniparental reproduction has evolved once or repeatedly in the genus. In at least two of the uniparental demes auxosporulation occurred through autogamy (i.e. intra-tetrad mating within an undivided cell). Maximum likelihood phylogenies indicated four lineages of uniparental Sellaphora and significance tests of alternative topologies, in which combinations of uniparental Sellaphora were constrained to be monophyletic, coupled with likelihood reconstruction of ancestral character states, led to rejection of the hypothesis that uniparental auxosporulation evolved only once in the genus. Uniparentally reproducing lineages appear to arise not infrequently in diatoms but do not persist. Two small extranuclear bodies, apparently containing DNA and lying outside the chloroplast (one close to each pole of the cell), were revealed by DAPI staining.     

Accuracy of computer-aided geometric 3D reconstruction based on histological serial microgrinding preparation

Purpose: For our research on computer-optimised and automated cochlear implant surgery, we pursue a model-based approach to overcome the limitations of currently available clinical imaging modalities. A serial cross section preparation procedure has been developed and evaluated concerning accuracy to serve for modelling of a digital anatomic atlas to make delicate soft tissue structures available for pre-operative planning.

Methods: A special grinding tool was developed allowing the setting of a specific amount of abrasion as equidistant slice thickness was considered a crucial step. Additionally, each actual abrasion was accurately measured and used during three-dimensional reconstruction of the serial cross-sectional images obtained via digital photo documentation after each microgrinding step. A well-known reference object was prepared using this procedure and evaluated in terms of accuracy.

Results: Reconstruction of the whole sample was achieved with an error less than 0.4%, and the edge lengths in the direction of abrasion could be reconstructed with an average error of 0.6 ± 0.3 mm; both prove the realisation of equidistant abrasion. Using artificial registration fiducials and a custom-made algorithm for image alignment, parallelism and rectangularity could be preserved with average errors less than 0.4° ± 0.3°.

Conclusion: We present a systematic, practicable and reliable method for the geometrically accurate reconstruction of anatomical structures, which is especially suitable for the middle and inner ear anatomy including soft tissue structures. For the first time, the quality of such a reconstruction process has been quantified and successfully proven for its usability.     

Biomechanical Study on the Novel Biomimetic Hemi-Pelvis Prosthesis

A pelvic endoprosthesis is the primary means of pelvic reconstruction after internal hemipelvectomy. In this study, a novel biomimetic hemipelvic prosthesis, including an artificial ilium, an artificial acetabulum, and an artificial pubis, was developed. A Finite Element Method （FEM） was carried out to investigate the biomechanical performance of a pelvis reconstructed with biomimetic hemipelvic prosthesis. Two models, including the reconstructed pelvis and the original pelvis （control model）, were established according to the geometry from CT data of a human male patient with pelvic bone sarcomas. The FE models predict that the biomeehanical function of the pelvic ring can be reestablished using this prosthesis. Results show that the body force loaded on the S 1 vertebra is restored and transferred towards the sacro-iliac joint, and along the ilium onto the bearing surface of the artificial ilium, then to the artificial acetabulum and pubis. Von Mises stresses observed in this reconstructed pelvis model are still within a low and elastic range below the yielding strength of cortical bone and Ti6A14V. The values of deformation and strain of the reconstructed pelvis are close to the data obtained in the original pelvis. With the partial replacement of the pubis, little influence is found towards the pubis symphysis. However, the interface between the prosthesis and pelvic bone may become the critical part of the reconstructed pelvis due to the discontinuity in the material properties, which results in stress shielding and deformation constraining. So a biomimetic flexible connection or inter layer to release the deformation of pelvis is suggested in future designing.     

The Open Microcirculation in Human Spleens: A Three-Dimensional Approach

It has long been debated whether the red pulp of human spleens harbors an open or a closed microcirculation or both. To solve this issue, the authors differentially stained the endothelium in red pulp arterial microvessels and in venous sinuses using brightfield and fluorescence immunohistology with reagents against CD34 and CD141. Three-dimensional models of red pulp arterial microvessels and sinuses were derived from serial double-stained paraffin sections with the help of license-free open-access software. In each model, arterial microvascular ends were traced and verified by reference to the original serial sections. In total, 142 ends were analyzed in the specimens of three individuals. None of these ends was connected to a sinus, suggesting that the human splenic red pulp harbors an entirely open circulatory system. Thus, the spleen is the only human organ where blood passes through spaces not lined by endothelia or other barrier-forming cells.     

Ecology and evolution of the diaspore "burial syndrome"

Hygroscopically active awns or "bristles" have long intrigued scientists. Experimental evidence shows that they are important for diaspore burial in the correct orientation, thereby increasing successful seed germination and seedling survival. Despite these ecological advantages, 38 of the 280 species of grasses in Danthonioideae lack awns. We provide the first study of awns in a phylogenetic context and show that although the awnless state has arisen ca. 25 times independently, the ecological disadvantage of not having an awn also applies in an evolutionary context. Only in Tribolium and Schismus have awnless ancestors diversified to form a clade of primarily awnless descendents. Several of the awnless species in these genera are annual and we find a significant correlation between the evolution of awns and the evolution of life history. A suite of other diaspore traits accompany the awned or awnless states. We interpret the awn as being the visible constituent of a compound "burial syndrome," the two ecological extremes of which may explain the correlation between awns and life history and provide an explanation why awnless species in Tribolium and Schismus persist.