Antler mineral composition of Iberian red deer <Emphasis Type="Italic">Cervus elaphus hispanicus</Emphasis> is related to mineral profile of diet

Previous studies have suggested that antlers are costly bone structures whose mineral composition may change depending on physiological and other factors. This study examined whether nutrition variation associated with deer management influences antler mineral composition and structural characteristics of whole antler. Mineral distribution and bone structure were examined in antlers from two groups of adult Iberian red deer Cervus elaphus hispanicus Hilzheimer, 1909. They were kept under different feeding regimes at an experimental deer farm and a game estate in southeastern Spain. Protein and mineral contents differed between the diet of captive deer and that of deer in the wild. Significant differences were found for Na, Mg, K and protein. Antler composition seems to reflect the diet, as antlers of deer differed in protein, Na, Mg and K, but not in total mineral content, Ca, Fe or Zn. Thus, management conditions related to nutrition are reflected on antler composition.     

Acrosomal exocytosis of mouse sperm progresses in a consistent direction in response to zona pellucida

Sperm acrosomal exocytosis is essential for successful fertilization, and the zona pellucida (ZP) has been classically considered as the primary initiator in vivo. At present, following what is referred to as primary binding of the sperm to the ZP, the acrosome reaction paradigm posits that the outer acrosomal membrane and plasma membrane fuse at random points, releasing the contents of the acrosome. It is then assumed that the inner acrosomal membrane mediates secondary binding of the sperm to the ZP. In the present work we used a live fluorescence imaging system and mouse sperm containing enhanced green fluorescent protein (EGFP) in their acrosomes. We compared the processes of acrosomal exocytosis stimulated by the calcium ionophore ionomycin or by solubilized ZP. As monitored by the loss of EGFP from the sperm, acrosomal exocytosis driven by these two agents occurred differently. When ionomycin was used, exocytosis started randomly (no preference for the anterior, middle or posterior acrosomal regions). In contrast, following treatment with solubilized ZP, the loss of acrosomal components always started at the posterior zone of the acrosome and progressed in an anterograde direction. The exocytosis was slower when stimulated with ZP and on the order of 10 sec, which is in accordance with other reports. These results demonstrate that ZP stimulates acrosomal exocytosis in an orderly manner and suggest that a receptor‐mediated event controls this process of membrane fusion and release of acrosomal components. These findings are incorporated into a model. J. Cell. Physiol. 220: 611–620, 2009. © 2009 Wiley‐Liss, Inc.     

Crystal Structure of LipL32, the Most Abundant Surface Protein of Pathogenic Leptospira spp.

Spirochetes of the genus Leptospira cause leptospirosis in humans and animals worldwide. Proteins exposed on the bacterial cell surface are implicated in the pathogenesis of leptospirosis. However, the biological role of the majority of these proteins is unknown; this is principally due to the lack of genetic systems for investigating Leptospira and the absence of any structural information on leptospiral antigens. To address this, we have determined the 2.0-Å-resolution structure of the lipoprotein LipL32, the most abundant outer-membrane and surface protein present exclusively in pathogenic Leptospira species. The extracellular domain of LipL32 revealed a compact, globular, “jelly-roll” fold from which projected an unusual extended β-hairpin that served as a principal mediator of the observed crystallographic dimer. Two acid-rich patches were also identified as potential binding sites for positively charged ligands, such as laminin, to which LipL32 has a propensity to bind. Although LipL32 shared no significant sequence identity to any known protein, it possessed structural homology to the adhesins that bind components of the extracellular matrix, suggesting that LipL32 functions in an analogous manner. Moreover, the structure provides a framework for understanding the immunological role of this major surface lipoprotein.     

Genetically Encoded Sensors to Elucidate Spatial Distribution of Cellular Zinc

Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn²⁺ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn²⁺, these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn²⁺ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn²⁺ is taken up into mitochondria following glutamate/Zn²⁺ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn²⁺ signals under different cellular conditions.Although mammalian cells are known to concentrate transition metals, it is now well established that under resting conditions, “free” (e.g. unbound) metals are maintained at extremely low levels. Estimates of the total Zn²⁺ concentration in mammalian cells typically range from 100 to 500 μm (1); yet free Zn²⁺ concentrations are tightly buffered by proteins such as metallothionein to maintain cytosolic Zn²⁺ concentrations in the picomolar to nanomolar range (2–5). However, there is emerging evidence that this static picture is dramatically altered by different cellular conditions, such as redox perturbations caused by oxidative stress (6, 7) and cellular signals such as nitric oxide (8). Consequently, there is a pool of labile Zn²⁺ that, if mobilized by cellular signals, would result in the generation of transient Zn²⁺ signals. Recent studies suggest that these Zn²⁺ signals influence critical biological processes, such as mitochondrial function (7, 9, 10). Elucidation of the sources and dynamics of these Zn²⁺ signals would greatly advance our understanding of the interplay between metal regulation and cellular function.There has been a huge effort in the past few years to develop sensitive and selective fluorescent probes to monitor Zn²⁺ in biological systems. The majority of this work has focused on the generation of small molecule fluorescent indicators (reviewed by Que et al. (11)). Yet there are also examples of sensors based partially on Zn²⁺-binding proteins, such as carbonic anhydrase (12) and metallothionein (13), and peptide scaffolds (14). Although many of these sensors have begun to provide insight into Zn²⁺ concentrations within cells, one limitation is that it is challenging to explicitly target them to subdomains within the cell. Localized probes are necessary to generate a complete picture of cellular Zn²⁺ homeostasis in mammalian cells. For this reason, sensors that are genetically encoded (i.e. generated by translation of a nucleic acid sequence) are attractive platforms for engineering metal-specific sensors. Encoded sensors provide additional benefits such as retention of the sensor over days to weeks permitting long term imaging and the ability to systematically vary the sensor concentration to evaluate the extent to which the sensor perturbs resting Zn²⁺ concentrations.Here we present genetically encoded sensors designed with a “Zn²⁺-sensing domain” sandwiched between two fluorescent proteins. The fluorescent proteins are chosen so that they are capable of undergoing fluorescence resonance energy transfer (FRET).² Because the mechanism of FRET involves dipole-dipole coupling, it is exquisitely dependent on the distance and orientation of the fluorophores with respect to one another. Therefore, if the binding of Zn²⁺ induces a conformational change in the sensor, it will alter the energy transfer between the two fluorescent proteins. The advantage of using FRET as the optical readout is that the donor emission will decrease and the acceptor emission will increase upon Zn²⁺ binding. Hence, by taking the ratio of the acceptor to the donor emission, we can create a ratiometric sensor. These sensors are targeted to the cytosol, mitochondria, and plasma membrane by attachment of signal sequences and fusion to other proteins. These sensors reveal differences in the spatial distribution of Zn²⁺ and highlight the power and utility of localized probes.     

Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.In Gram-negative bacteria, the type 2 secretion system (T2SS)² is used for the secretion of several important proteins across the outer membrane (1). The T2SS is also called the terminal branch of the general secretory pathway (Gsp) (2) and, in Vibrio species, the extracellular protein secretion (Eps) apparatus (3). This sophisticated multiprotein machinery spans both the inner and the outer membrane of Gram-negative bacteria and contains 11–15 different proteins. The T2SS consists of three major subassemblies (4–9): (i) the outer membrane complex comprising mainly the crucial multisubunit secretin GspD; (ii) the pseudopilus, which consists of one major and several minor pseudopilins; and (iii) an inner membrane platform, containing the cytoplasmic secretion ATPase GspE and the membrane proteins GspL, GspM, GspC, and GspF.The pseudopilus is a key element of the T2SS that forms a helical fiber spanning the periplasm. The fiber is assembled from multiple subunits of the major pseudopilin GspG (4, 5, 10–14). The pseudopilus is thought to form a plug of the secretin pore in the outer membrane and/or to function as a piston during protein secretion. In recent years, studies of the T2SS pseudopilins led to structure determinations of all individual pseudopilins (13, 15–17). The recent structure of the helical ternary complex of GspK-GspI-GspJ suggested that these three minor pseudopilins form the tip of the pseudopilus (17). A crystal structure of GspG from Klebsiella oxytoca was in a previous study combined with electron microscopy data to arrive at a helical arrangement, with no evidence for special features, such as disulfide bridges, other covalent links, or metal-binding sites, for stabilizing this major pseudopilin or the pseudopilus (13).The pseudopilins of the T2SS share a common fold with the type 4 pilins (15–21). Pilins are proteins incorporated into pili, long appendages on the surface of bacteria forming thin, strong fibers with multiple functions (19, 21). Type 4 pilins and pseudopilins contain a prepilin leader sequence that is cleaved off by a prepilin peptidase, yielding mature protein (10, 11, 22). A distinct feature of the type 4 pilins is the occurrence of a disulfide bridge connecting β4 to a Cys in the so-called “D-region” near the C terminus (21). In a recent study (23) on the thin fibers of Gram-positive bacteria, isopeptide units appeared to be essential for providing these filaments sufficient cohesion and stability. A key question was therefore whether the major pseudopilin GspG also requires a special feature to obtain sufficient stability to perform its function.     

Rodent phylogeny revised: analysis of six nuclear genes from all major rodent clades

Background

Bacterial biofilm is ubiquitous in nature. However, it is not clear how this crowded habitat would impact the evolution of bacteriophage (phage) life history traits. In this study, we constructed isogenic λ phage strains that only differed in their adsorption rates, because of the presence/absence of extra side tail fibers or improved tail fiber J, and maker states. The high cell density and viscosity of the biofilm environment was approximated by the standard double-layer agar plate. The phage infection cycle in the biofilm environment was decomposed into three stages: settlement on to the biofilm surface, production of phage progeny inside the biofilm, and emigration of phage progeny out of the current focus of infection.

Results

We found that in all cases high adsorption rate is beneficial for phage settlement, but detrimental to phage production (in terms of plaque size and productivity) and emigration out of the current plaque. Overall, the advantage of high adsorption accrued during settlement is more than offset by the disadvantages experienced during the production and emigration stages. The advantage of low adsorption rate was further demonstrated by the rapid emergence of low-adsorption mutant from a high-adsorption phage strain with the side tail fibers. DNA sequencing showed that 19 out of the 21 independent mutant clones have mutations in the stf gene, with the majority of them being single-nucleotide insertion/deletion mutations occurring in regions with homonucleotide runs.

Conclusion

We conclude that high mutation rate of the stf gene would ensure the existence of side tail fiber polymorphism, thus contributing to rapid adaptation of the phage population between diametrically different habitats of benthic biofilm and planktonic liquid culture. Such adaptability would also help to explain the maintenance of the stf gene in phage λ's genome.     

Impact of an educational video-based strategy on the behavior process associated with colorectal cancer screening: A randomized controlled study

Background: Low public awareness is an important barrier for colorectal cancer screening participation. Aim: To evaluate the impact of educational intervention on the health behavior process, patient knowledge and compliance with colorectal cancer screening in the average-risk population. Methods: 158 subjects (aged 50–79 years) were randomly assigned either to watch a non-medical video or a colorectal cancer educational video. Before and after watching the experimental or control videotape, participants completed a five-item questionnaire that assessed their knowledge about risk factors for colorectal cancer, age of risk, warning symptoms, 5-year prognosis, and incidence. Subjective risk perception for developing colorectal cancer, barriers or benefits of screening, and intention to be screened were also investigated. Finally, subjects received a faecal occult blood test kit and were requested to use and return it within 2 weeks. Results: Participants in the video-based intervention group showed significant improvement in knowledge of colorectal cancer scores (P < 0.001) and decreased barrier scores. The intervention group returned significantly more faecal occult blood tests than controls (69.6% vs. 54.4%, P = 0.035). The intervention had a positive effect on modifying attitudes and intention to take part in screening. Additionally, the intervention was a predictor of compliance (OR 2.0; 95% CI = 1.02–3.84, P = 0.044). Conclusion: Video-based intervention significantly reduced barriers to screening and improved participant awareness and compliance with colorectal cancer screening with faecal occult blood test.     

Comparison of Prognostic Gene Profiles Using qRT-PCR in Paraffin Samples: A Retrospective Study in Patients with Early Breast Cancer

Introduction

Gene profiling may improve prognostic accuracy in patients with early breast cancer, but this technology is not widely available. We used commercial assays for qRT-PCR to assess the performance of the gene profiles included in the 70-Gene Signature, the Recurrence Score and the Two-Gene Ratio.

Methods

153 patients with early breast cancer and a minimum follow-up of 5 years were included. All tumours were positive for hormonal receptors and 38% had positive lymph nodes; 64% of patients received adjuvant chemotherapy. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) specimens using a specific kit. qRT-PCR amplifications were performed with TaqMan Gene Expression Assays products. We applied the three gene-expression-based models to our patient cohort to compare the predictions derived from these gene sets.

Results

After a median follow-up of 91 months, 22% of patients relapsed. The distant metastasis-free survival (DMFS) at 5 years was calculated for each profile. For the 70-Gene Signature, DMFS was 95% -good prognosis- versus 66% -poor prognosis. In the case of the Recurrence Score, DMFS was 98%, 81% and 69% for low, intermediate and high-risk groups, respectively. Finally, for the Two-Gene Ratio, DMFS was 86% versus 70%. The 70-Gene Signature and the Recurrence Score were highly informative in identifying patients with distant metastasis, even in multivariate analysis.

Conclusion

Commercially available assays for qRT-PCR can be used to assess the prognostic utility of previously published gene expression profiles in FFPE material from patients with early breast cancer. Our results, with the use of a different platform and with different material, confirm the robustness of the 70-Gene Signature and represent an independent test for the Recurrence Score, using different primer/probe sets.     

Population-Based Surveillance for Invasive Pneumococcal Disease in Homeless Adults in Toronto

Background

Identification of high-risk populations for serious infection due to S. pneumoniae will permit appropriately targeted prevention programs.

Methods

We conducted prospective, population-based surveillance for invasive pneumococcal disease and laboratory confirmed pneumococcal pneumonia in homeless adults in Toronto, a Canadian city with a total population of 2.5 M, from January 1, 2002 to December 31, 2006.

Results

We identified 69 cases of invasive pneumococcal disease and 27 cases of laboratory confirmed pneumococcal pneumonia in an estimated population of 5050 homeless adults. The incidence of invasive pneumococcal disease in homeless adults was 273 infections per 100,000 persons per year, compared to 9 per 100,000 persons per year in the general adult population. Homeless persons with invasive pneumococcal disease were younger than other adults (median age 46 years vs 67 years, P<.001), and more likely than other adults to be smokers (95% vs. 31%, P<.001), to abuse alcohol (62% vs 15%, P<.001), and to use intravenous drugs (42% vs 4%, P<.001). Relative to age matched controls, they were more likely to have underlying lung disease (12/69, 17% vs 17/272, 6%, P = .006), but not more likely to be HIV infected (17/69, 25% vs 58/282, 21%, P = .73). The proportion of patients with recurrent disease was five fold higher for homeless than other adults (7/58, 12% vs. 24/943, 2.5%, P<.001). In homeless adults, 28 (32%) of pneumococcal isolates were of serotypes included in the 7-valent conjugate vaccine, 42 (48%) of serotypes included in the 13-valent conjugate vaccine, and 72 (83%) of serotypes included in the 23-valent polysaccharide vaccine. Although no outbreaks of disease were identified in shelters, there was evidence of clustering of serotypes suggestive of transmission of pathogenic strains within the homeless population.

Conclusions

Homeless persons are at high risk of serious pneumococcal infection. Vaccination, physical structure changes or other program to reduce transmission in shelters, harm reduction programs to reduce rates of smoking, alcohol abuse and infection with bloodborne pathogens, and improved treatment programs for HIV infection may all be effective in reducing the risk.