Yersinia pestis, the causative agent of plague, binds host cells to deliver cytotoxic Yop proteins into the cytoplasm that prevent phagocytosis and generation of proinflammatory cytokines. Ail is an eight-stranded β-barrel outer membrane protein with four extracellular loops that mediates cell binding and resistance to human serum. Following the deletion of each of the four extracellular loops that potentially interact with host cells, the Ail-Δloop 2 and Ail-Δloop 3 mutant proteins had no cell-binding activity while Ail-Δloop 4 maintained cell binding (the Ail-Δloop 1 protein was unstable). Using the codon mutagenesis scheme SWIM (selection without isolation of mutants), we identified individual residues in loops 1, 2, and 3 that contribute to host cell binding. While several residues contributed to the binding of host cells and purified fibronectin and laminin, as well as Yop delivery, three mutations, F80A (loop 2), S128A (loop 3), and F130A (loop 3), produced particularly severe defects in cell binding. Combining these mutations led to an even greater reduction in cell binding and severely impaired Yop delivery with only a slight defect in serum resistance. These findings demonstrate that Y. pestis Ail uses multiple extracellular loops to interact with substrates important for adhesion via polyvalent hydrophobic interactions.

Laboratory cadaver dissection is essential for three-dimensional understanding of anatomical structures and variability, but there are many challenges to teaching gross anatomy in medical and dental schools, including a lack of available space and qualified anatomy faculty. The aim of this study was to determine the efficacy of high-definition audiovisual educational technology in the gross anatomy laboratory in improving dental students' learning outcomes and satisfaction. Exam scores were compared for two classes of first-year students at one U.S. dental school: 2012-13 (no audiovisual technology) and 2013-14 (audiovisual technology), and section exams were used to compare differences between semesters. Additionally, an online survey was used to assess the satisfaction of students who used the technology. All 284 first-year students in the two years (2012-13 N=144; 2013-14 N=140) participated in the exams. Of the 140 students in the 2013-14 class, 63 completed the survey (45% response rate). The results showed that those students who used the technology had higher scores on the laboratory exams than those who did not use it, and students in the winter semester scored higher (90.17±0.56) than in the fall semester (82.10±0.68). More than 87% of those surveyed strongly agreed or agreed that the audiovisual devices represented anatomical structures clearly in the gross anatomy laboratory. These students reported an improved experience in learning and understanding anatomical structures, found the laboratory to be less overwhelming, and said they were better able to follow dissection instructions and understand details of anatomical structures with the new technology. Based on these results, the study concluded that the ability to provide the students a clear view of anatomical structures and high-quality imaging had improved their learning experience.

The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca2+ release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appeared by 12-24 h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24-48 h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRDdog and JCTdog, demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQD-sRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed (delTRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRDdog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRDdog and del TRD were therefore able to elucidate areas of ER-SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER-SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic.

Calsequestrin-2 (CSQ2) is a resident glycoprotein of junctional sarcoplasmic reticulum that functions in the regulation of SR Ca(2+) release. CSQ2 is biosynthesized in rough ER around cardiomyocyte nuclei and then traffics transversely across SR subcompartments. During biosynthesis, CSQ2 undergoes N-linked glycosylation and phosphorylation by protein kinase CK2. In mammalian heart, CSQ2 molecules subsequently undergo extensive mannose trimming by ER mannosidase(s), a posttranslational process that often regulates protein breakdown. We analyzed the intact purified CSQ2 from mongrel canine heart tissue by electrospray mass spectrometry. The average molecular mass of CSQ2 in normal mongrel dogs was 46,306 ± 41 Da, corresponding to glycan trimming of 3-5 mannoses, depending upon the phosphate content. We tested whether CSQ2 glycan structures would be altered in heart tissue from mongrel dogs induced into heart failure (HF) by two very different experimental treatments, rapid ventricular pacing or repeated coronary microembolizations. Similarly dramatic changes in mannose trimming were found in both types of induced HF, despite the different cardiomyopathies producing the failure. Unique to all samples analyzed from HF dog hearts, 20-40 % of all CSQ2 contained glycans that had minimal mannose trimming (Man9,8). Analyses of tissue samples showed decreases in CSQ2 protein levels per unit levels of mRNA for tachypaced heart tissue, also indicative of altered turnover. Quantitative immunofluorescence microscopy of frozen tissue sections suggested that no changes in CSQ2 levels occurred across the width of the cell. We conclude that altered processing of CSQ2 may be an adaptive response to the myocardium under stresses that are capable of inducing heart failure.

No abstract