Posterior Vitreous Detachment and the Posterior Hyaloid Membrane

PURPOSE: Despite posterior vitreous detachment being a common ocular event affecting most individuals in an aging population, there is little consensus regarding its precise anatomic definition. We investigated the morphologic appearance and molecular composition of the posterior hyaloid membrane to determine whether the structure clinically observed enveloping the posterior vitreous surface after posterior vitreous detachment is a true basement membrane and to postulate its origin. Understanding the relationship between the vitreous (in both its attached and detached state) and the internal limiting membrane of the retina is essential to understanding the cause of rhegmatogenous retinal detachment and vitreoretinal interface disorders, as well as potential future prophylactic and treatment strategies. DESIGN: Clinicohistologic correlation study. PARTICIPANTS: Thirty-six human donor globes. METHODS: Vitreous bodies identified to have posterior vitreous detachment were examined with phase-contrast microscopy and confocal microscopy after immunohistochemically staining for collagen IV basement membrane markers, in addition to extracellular proteins that characterize the vitreoretinal junction (fibronectin, laminin) and vitreous gel (opticin) markers. The posterior retina similarly was stained to evaluate the internal limiting membrane. Findings were correlated to the clinical appearance of the posterior hyaloid membrane observed during slit-lamp biomicroscopy after posterior vitreous detachment and compared with previously published studies. MAIN OUTCOME MEASURES: Morphologic appearance and molecular composition of the posterior hyaloid membrane. RESULTS: Phase-contrast microscopy consistently identified a creased and distinct glassy membranous sheet enveloping the posterior vitreous surface, correlating closely with the posterior hyaloid membrane observed during slit-lamp biomicroscopy in patients with posterior vitreous detachment. Immunofluorescent confocal micrographs demonstrated the enveloping membranous structure identified on phase-contrast microscopy to show positive stain results for type IV collagen. Immunofluorescence of the residual intact internal limiting membrane on the retinal surface also showed positive stain results for type IV collagen. CONCLUSIONS: The results of this study provide immunohistochemical evidence that the posterior hyaloid membrane is a true basement membrane enveloping the posterior hyaloid surface. Because this membranous structure is observed only after posterior vitreous detachment, the results of this study indicate that it forms part of the internal limiting membrane when the vitreous is in its attached state

Alternative splicing modifies the effect of mutations in COL11A1 and results in recessive type 2 Stickler syndrome with profound hearing loss.

BACKGROUND: Stickler syndromes types 1, 2 and 3 are usually dominant disorders caused by mutations in the genes COL2A1, COL11A1 and COL11A2 that encode the fibrillar collagens types II and XI present in cartilage and vitreous. Rare recessive forms of Stickler syndrome exist that are due to mutations in genes encoding type IX collagen (COL9A1 type 4 Stickler syndrome and COL9A2 type 5 Stickler syndrome). Recently, recessive mutations in the COL11A1 gene have been demonstrated to result in fibrochondrogenesis, a much more severe skeletal dysplasia, which is often lethal. Here we demonstrate that some mutations in COL11A1 are recessive, modified by alternative splicing and result in type 2 Stickler syndrome rather than fibrochondrogenesis. METHODS: Patients referred to the national Stickler syndrome diagnostic service for England, UK were assessed clinically and subsequently sequenced for mutations in COL11A1. Additional in silico and functional studies to assess the effect of sequence variants on pre-mRNA processing and collagen structure were performed. RESULTS: In three different families, heterozygous COL11A1 biallelic null, null/missense or silent/missense mutations, were found. They resulted in a recessive form of type 2 Stickler syndrome characterised by particularly profound hearing loss and are clinically distinct from the recessive types 4 and 5 variants of Stickler syndrome. One mutant allele in each family is capable of synthesising a normal α1(XI) procollagen molecule, via variable pre-mRNA processing. CONCLUSION: This new variant has important implications for molecular diagnosis and counselling families with type 2 Stickler syndrome

Auditory dysfunction in type 2 Stickler Syndrome.

PURPOSE: To present the extent and site of lesion of auditory dysfunction in a large cohort of individuals with type 2 Stickler Syndrome. Type 2 Stickler Syndrome results from a mutation in the gene coding for α-1 type XI pro-collagen, which has been identified in the human vitreous, cartilage and the cochlea of the mouse. The condition is characterised by classic ocular abnormalities, auditory dysfunction, osteoarthropathy and oro-facial dysplasia. METHODS: This is a population study which used a combination of audiometric, tympanometric, and self-report measures on a series of 65 individuals (mean age 29.2 years, range 3-70, female 63.1%) with genetically confirmed type 2 Stickler Syndrome. RESULTS: Hearing impairment was identified in at least one ear for 69% of individuals. Analysis against age-matched normative data showed that reduced hearing sensitivity was present across all test frequencies. Sensorineural hearing loss was most common (77% of ears), with conductive (3%), mixed (7%) and no hearing loss (13%), respectively. The proportion of hypermobile tympanic membranes (24%) was less than previously documented in type 1 Stickler Syndrome. When present, this appears to arise as a direct result of collagen abnormalities in the middle ear. Self-report measures of speech and spatial hearing in sound were comparable to a non-syndromic cohort with similar audiometric thresholds. CONCLUSIONS: Auditory impairment in type 2 Stickler Syndrome is predominantly associated with cochlear hearing loss of varying severities across affected individuals. The impact on hearing thresholds can be seen across the frequency range, suggesting a contribution of defective collagen throughout the cochlea. Self-report questionnaires showed that difficulties understanding speech, and spatial information in sound (such as that used for localisation), were worse than a young, normal-hearing population but comparable to a non-syndromic cohort with similar audiometric thresholds. Therefore, it is likely that hearing loss in type 2 Stickler Syndrome arises in the auditory periphery, without significant central processing deficits

Research enrichment: evaluation of structured research in the curriculum for dental medicine students as part of the vertical and horizontal integration of biomedical training and discovery

<p>Abstract</p> <p>Background</p> <p>Research programs within medical and dental schools are important vehicles for biomedical and clinical discovery, serving as effective teaching and learning tools by providing situations in which predoctoral students develop problem-solving and critical-thinking skills. Although research programs at many medical and dental schools are well-established, they may not be well integrated into the predoctoral curriculum to effectively support the learning objectives for their students.</p> <p>Methods</p> <p>A series of structured seminars, incorporating faculty research, was designed for first-year dental students at the University of Nevada, Las Vegas, School of Dental Medicine to reinforce and support the concepts and skills taught in concurrent courses. A structured research enrichment period was also created to facilitate student engagement in active research using faculty and student curricular release time. Course evaluations and surveys were administered to gauge student perceptions of the curricular integration of research, the impact of these seminars on recruitment to the research program, and overall levels of student satisfaction with research enrichment.</p> <p>Results</p> <p>The analysis of course surveys revealed that students perceived the research-containing seminars effectively illustrated concepts, were logically sequenced, and were well-integrated into their curriculum. In addition, analysis of surveys revealed that the Integration Seminar courses motivated students to engage in research enrichment. Finally, this analysis provided evidence that students were very satisfied with their overall learning experience during research enrichment.</p> <p>Conclusion</p> <p>Curricular integration is one method of improving the teaching and learning of complicated and inter-related concepts, providing an opportunity to incorporate research training and objectives into traditionally separate didactic courses. Despite the benefits of curricular integration, finding the most appropriate points of integration, obtaining release time for curricular development and for research engagement, and funding predoctoral student research remain issues to be addressed in ways that reflect the character of the faculty and the goals of each institution.</p

A broad distribution of the alternative oxidase in microsporidian parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of ironsulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosome

Shell we cook it? An experimental approach to the microarchaeological record of shellfish roasting

In this paper, we investigate the microarchaeological traces and archaeological visibility of shellfish cooking activities through a series of experimental procedures with direct roasting using wood-fueled fires and controlled heating in a muffle furnace. An interdisciplinary geoarchacological approach, combining micromorphology, FTIR (in transmission and ATR collection modes), TGA and XRD, was used to establish a baseline on the mineralogical transformation of heated shells from aragonite to calcite and diagnostic sedimentary traces produced by roasting fire features. Our experimental design focused on three main types of roasting procedures: the construction of shallow depressions with heated rocks (pebble cuvette experiments), placing shellfish on top of hot embers and ashes (fire below experiment), and by kindling short-lived fires on top of shellfish (fire above experiments). Our results suggest that similar shellfish roasting procedures will largely create microstratigraphic signatures of anthropogenically reworked combusted material spatially "disconnected" from the actual combustion locus. The construction of shallow earth ovens might entail an increased archaeological visibility, and some diagnostic signatures of in situ hearths can be obtained by fire below roasting activities. We also show that macroscopic visual modifications and mineralogical characterization of discarded shellfish might be indicative of specific cooking activities versus secondary burning.Max Planck Societyinfo:eu-repo/semantics/publishedVersio

Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3

Background: Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described. Results: Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates. Conclusion: We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution

Laser capture microdissection of gonads from juvenile zebrafish

<p>Abstract</p> <p>Background</p> <p>Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation.</p> <p>Methods</p> <p>The laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.</p> <p>Results</p> <p>The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.</p> <p>Conclusion</p> <p>The study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.</p

Human Milk Protein Production in Xenografts of Genetically Engineered Bovine Mammary Epithelial Stem Cells

BACKGROUND: In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer. METHODS AND FINDINGS: We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo. CONCLUSIONS: These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption

The influence of self-owned home blood pressure monitoring (HBPM) on primary care patients with hypertension: A qualitative study

<p>Abstract</p> <p>Background</p> <p>Home blood pressure monitoring (HBPM) is gaining popularity among hypertensive patients. This study aimed to explore the influence of self-initiated HBPM on primary care patients with hypertension.</p> <p>Methods</p> <p>Six in-depth interviews and two focus group discussions were conducted, taking into consideration the experiences of 24 primary care patients with hypertension. These patients had been using HBPM as part of their hypertension management. The overriding influences were grouped under themes which emerged from analyzing the data using the grounded theory approach.</p> <p>Results</p> <p>There are both positive and negative influences of self-initiated HBPM. Patients used the readings of their HBPM to decide on many aspects of their hypertension management. The HBPM readings both influenced their adherence to diet and exercise and provided certain reassurance when they experienced symptoms. In addition, the act of discussing their HBPM readings with their health care providers resulted in an enhanced doctor-patient therapeutic relationship. Nevertheless, HBPM created confusion at times in some patients, particularly with regard to the target blood pressure level and the need for medication. This led to some patients making their own medical decisions based on their own standards.</p> <p>Conclusions</p> <p>HBPM is becoming an integral part of hypertension management. Primary care patients who self-initiated HBPM reported being more self-efficacious, but lack of participation and guidance from their doctors created confusion, and hindered the true benefit of HBPM.</p