A clinical and molecular characterisation of CRB1-associated maculopathy

To date, over 150 disease-associated variants in CRB1 have been described, resulting in a range of retinal disease phenotypes including Leber congenital amaurosis and retinitis pigmentosa. Despite this, no genotype–phenotype correlations are currently recognised. We performed a retrospective review of electronic patient records to identify patients with macular dystrophy due to bi-allelic variants in CRB1. In total, seven unrelated individuals were identified. The median age at presentation was 21 years, with a median acuity of 0.55 decimalised Snellen units (IQR = 0.43). The follow-up period ranged from 0 to 19 years (median = 2.0 years), with a median final decimalised Snellen acuity of 0.65 (IQR = 0.70). Fundoscopy revealed only a subtly altered foveal reflex, which evolved into a bull’s-eye pattern of outer retinal atrophy. Optical coherence tomography identified structural changes—intraretinal cysts in the early stages of disease, and later outer retinal atrophy. Genetic testing revealed that one rare allele (c.498_506del, p.(Ile167_Gly169del)) was present in all patients, with one patient being homozygous for the variant and six being heterozygous. In trans with this, one variant recurred twice (p.(Cys896Ter)), while the four remaining alleles were each observed once (p.(Pro1381Thr), p.(Ser478ProfsTer24), p.(Cys195Phe) and p.(Arg764Cys)). These findings show that the rare CRB1 variant, c.498_506del, is strongly associated with localised retinal dysfunction. The clinical findings are much milder than those observed with bi-allelic, loss-of-function variants in CRB1, suggesting this in-frame deletion acts as a hypomorphic allele. This is the most prevalent disease-causing CRB1 variant identified in the non-Asian population to date

Expression and Function of Ccbe1 in the Chick Early Cardiogenic Regions Are Required for Correct Heart Development

During the course of a differential screen to identify transcripts specific for chick heart/hemangioblast precursor cells, we have identified Ccbe1 (Collagen and calcium-binding EGF-like domain 1). While the importance of Ccbe1 for the development of the lymphatic system is now well demonstrated, its role in cardiac formation remained unknown. Here we show by whole-mount in situ hybridization analysis that cCcbe1 mRNA is initially detected in early cardiac progenitors of the two bilateral cardiogenic fields (HH4), and at later stages on the second heart field (HH9-18). Furthermore, cCcbe1 is expressed in multipotent and highly proliferative cardiac progenitors. We characterized the role of cCcbe1 during early cardiogenesis by performing functional studies. Upon morpholino-induced cCcbe1 knockdown, the chick embryos displayed heart malformations, which include aberrant fusion of the heart fields, leading to incomplete terminal differentiation of the cardiomyocytes. cCcbe1 overexpression also resulted in severe heart defects, including cardia bifida. Altogether, our data demonstrate that although cardiac progenitors cells are specified in cCcbe1 morphants, the migration and proliferation of cardiac precursors cells are impaired, suggesting that cCcbe1 is a key gene during early heart development.FCT [SFRH/BD/65628/2009, SFRH/BPD/86497/2012, SFRH/BPD/41081/2007]; F.C.T.B.I. fellowship [PTDC/SAU-BID/114902/ 2009]; FCT; Institute for Biotechnology Bioengineering (Centro Biomedicina Molecular e Celular (IBB/CBME), Laboratorio Associado (LA) in the frame of Project [PestOE/EQB/LA0023/2013]info:eu-repo/semantics/publishedVersio

Three-dimensional holographic optical manipulation through a high-numerical-aperture soft-glass multimode fibre

Holographic optical tweezers (HOT) hold great promise for many applications in biophotonics, allowing the creation and measurement of minuscule forces on biomolecules, molecular motors and cells. Geometries used in HOT currently rely on bulk optics, and their exploitation in vivo is compromised by the optically turbid nature of tissues. We present an alternative HOT approach in which multiple three-dimensional (3D) traps are introduced through a high-numerical-aperture multimode optical fibre, thus enabling an equally versatile means of manipulation through channels having cross-section comparable to the size of a single cell. Our work demonstrates real-time manipulation of 3D arrangements of micro-objects, as well as manipulation inside otherwise inaccessible cavities. We show that the traps can be formed over fibre lengths exceeding 100 mm and positioned with nanometric resolution. The results provide the basis for holographic manipulation and other high-numerical-aperture techniques, including advanced microscopy, through single-core-fibre endoscopes deep inside living tissues and other complex environments

Population‐based cohort study of outcomes following cholecystectomy for benign gallbladder diseases

Background The aim was to describe the management of benign gallbladder disease and identify characteristics associated with all‐cause 30‐day readmissions and complications in a prospective population‐based cohort. Methods Data were collected on consecutive patients undergoing cholecystectomy in acute UK and Irish hospitals between 1 March and 1 May 2014. Potential explanatory variables influencing all‐cause 30‐day readmissions and complications were analysed by means of multilevel, multivariable logistic regression modelling using a two‐level hierarchical structure with patients (level 1) nested within hospitals (level 2). Results Data were collected on 8909 patients undergoing cholecystectomy from 167 hospitals. Some 1451 cholecystectomies (16·3 per cent) were performed as an emergency, 4165 (46·8 per cent) as elective operations, and 3293 patients (37·0 per cent) had had at least one previous emergency admission, but had surgery on a delayed basis. The readmission and complication rates at 30 days were 7·1 per cent (633 of 8909) and 10·8 per cent (962 of 8909) respectively. Both readmissions and complications were independently associated with increasing ASA fitness grade, duration of surgery, and increasing numbers of emergency admissions with gallbladder disease before cholecystectomy. No identifiable hospital characteristics were linked to readmissions and complications. Conclusion Readmissions and complications following cholecystectomy are common and associated with patient and disease characteristics

Influenza vaccination for immunocompromised patients: systematic review and meta-analysis from a public health policy perspective.

Immunocompromised patients are vulnerable to severe or complicated influenza infection. Vaccination is widely recommended for this group. This systematic review and meta-analysis assesses influenza vaccination for immunocompromised patients in terms of preventing influenza-like illness and laboratory confirmed influenza, serological response and adverse events

Tracing ancestry with methylation patterns: most crypts appear distantly related in normal adult human colon

BACKGROUND: The ability to discern ancestral relationships between individual human colon crypts is limited. Widely separated crypts likely trace their common ancestors to a time around birth, but closely spaced adult crypts may share more recent common ancestors if they frequently divide by fission to form clonal patches. Alternatively, adult crypts may be long-lived structures that infrequently divide or die. METHODS: Methylation patterns (the 5' to 3' order of methylation) at CpG sites that exhibit random changes with aging were measured from isolated crypts by bisulfite genomic sequencing. This epigenetic drift may be used to infer ancestry because recently related crypts should have similar methylation patterns. RESULTS: Methylation patterns were different between widely separated ("unrelated") crypts greater than 15 cm apart. Evidence for a more recent relationship between directly adjacent or branched crypts could not be found because their methylation pattern distances were not significantly different than widely separated crypt pairs. Methylation patterns are essentially equally different between two adult human crypts regardless of their relative locations. CONCLUSIONS: Methylation patterns appear to record somatic cell trees. Starting from a single cell at conception, sequences replicate and may drift apart. Most adult human colon crypts appear to be long-lived structures that become mosaic with respect to methylation during aging

Age-related human small intestine methylation: evidence for stem cell niches

BACKGROUND: The small intestine is constructed of many crypts and villi, and mouse studies suggest that each crypt contains multiple stem cells. Very little is known about human small intestines because mouse fate mapping strategies are impractical in humans. However, it is theoretically possible that stem cell histories are inherently written within their genomes. Genomes appear to record histories (as exemplified by use of molecular clocks), and therefore it may be possible to reconstruct somatic cell dynamics from somatic cell errors. Recent human colon studies suggest that random somatic epigenetic errors record stem cell histories (ancestry and total numbers of divisions). Potentially age-related methylation also occurs in human small intestines, which would allow characterization of their stem cells and comparisons with the colon. METHODS: Methylation patterns in individual crypts from 13 small intestines (17 to 78 years old) were measured by bisulfite sequencing. The methylation patterns were analyzed by a quantitative model to distinguish between immortal or niche stem cell lineages. RESULTS: Age-related methylation was observed in the human small intestines. Crypt methylation patterns were more consistent with stem cell niches than immortal stem cell lineages. Human large and small intestine crypt niches appeared to have similar stem cell dynamics, but relatively less methylation accumulated with age in the small intestines. There were no apparent stem cell differences between the duodenum and ileum, and stem cell survival did not appear to decline with aging. CONCLUSION: Crypt niches containing multiple stem cells appear to maintain human small intestines. Crypt niches appear similar in the colon and small intestine, and the small intestinal stem cell mitotic rate is the same as or perhaps slower than that of the colon. Although further studies are needed, age-related methylation appears to record somatic cell histories, and a somatic epigenetic molecular clock strategy may potentially be applied to other human tissues to reconstruct otherwise occult stem cell histories

Sleep-Disordered Breathing in Alcoholics

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66229/1/j.1530-0277.1999.tb04034.x.pd

The selective Cox-2 inhibitor Celecoxib suppresses angiogenesis and growth of secondary bone tumors: An intravital microscopy study in mice

BACKGROUND: The inhibition of angiogenesis is a promising strategy for the treatment of malignant primary and secondary tumors in addition to established therapies such as surgery, chemotherapy, and radiation. There is strong experimental evidence in primary tumors that Cyclooxygenase-2 (Cox-2) inhibition is a potent mechanism to reduce angiogenesis. For bone metastases which occur in up to 85% of the most frequent malignant primary tumors, the effects of Cox-2 inhibition on angiogenesis and tumor growth remain still unclear. Therefore, the aim of this study was to investigate the effects of Celecoxib, a selective Cox-2 inhibitor, on angiogenesis, microcirculation and growth of secondary bone tumors. METHODS: In 10 male severe combined immunodeficient (SCID) mice, pieces of A549 lung carcinomas were implanted into a newly developed cranial window preparation where the calvaria serves as the site for orthotopic implantation of the tumors. From day 8 after tumor implantation, five animals (Celecoxib) were treated daily with Celecoxib (30 mg/kg body weight, s.c.), and five animals (Control) with the equivalent amount of the CMC-based vehicle. Angiogenesis, microcirculation, and growth of A549 tumors were analyzed by means of intravital microscopy. Apoptosis was quantified using the TUNEL assay. RESULTS: Treatment with Celecoxib reduced both microvessel density and tumor growth. TUNEL reaction showed an increase in apoptotic cell death of tumor cells after treatment with Celecoxib as compared to Controls. CONCLUSION: Celecoxib is a potent inhibitor of tumor growth of secondary bone tumors in vivo which can be explained by its anti-angiogenic and pro-apoptotic effects. The results indicate that a combination of established therapy regimes with Cox-2 inhibition represents a possible application for the treatment of bone metastases

Reduced Bone Mass and Muscle Strength in Male 5α-Reductase Type 1 Inactivated Mice

Androgens are important regulators of bone mass but the relative importance of testosterone (T) versus dihydrotestosterone (DHT) for the activation of the androgen receptor (AR) in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2), encoded by separate genes (Srd5a1 and Srd5a2). 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1−/− mice. Four-month-old male Srd5a1−/− mice had reduced trabecular bone mineral density (−36%, p<0.05) and cortical bone mineral content (−15%, p<0.05) but unchanged serum androgen levels compared with wild type (WT) mice. The cortical bone dimensions were reduced in the male Srd5a1−/− mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05) in orchidectomized WT mice but not in orchidectomized Srd5a1−/− mice. Male Srd5a1−/− mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05). Female Srd5a1−/− mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1−/− mice, is an indirect effect mediated by elevated circulating androgen levels