An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal [mu]-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. [mu]-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

Exploring retinal and functional markers of diabetic neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most debilitating complications of diabetes. DPN is a major cause of foot ulceration and lower limb amputation. Early diagnosis and management is a key factor in reducing morbidity and mortality. Current techniques for clinical assessment of DPN are relatively insensitive for detecting early disease or involve invasive procedures such as skin biopsies. There is a need for less painful, non-invasive and safe evaluation methods. Eye care professionals already play an important role in the management of diabetic retinopathy; however recent studies have indicated that the eye may also be an important site for the diagnosis and monitoring of neuropathy. Corneal nerve morphology has been shown to be a promising marker of diabetic neuropathy occurring elsewhere in the body, and emerging evidence tentatively suggests that retinal anatomical markers and a range of functional visual indicators could similarly provide useful information regarding neural damage in diabetes – although this line of research is, as yet, less well established. This review outlines the growing body of evidence supporting a potential diagnostic role for retinal structure and visual functional markers in the diagnosis and monitoring of peripheral neuropathy in diabetes.

Mild systemic hypoxia and photopic visual field sensitivity

Purpose: Flickering stimuli increase the metabolic demand of the retina,making it a sensitive perimetric stimulus to the early onset of retinal disease. We determine whether flickering stimuli are a sensitive indicator of vision deficits resulting from to acute, mild systemic hypoxia when compared to standard static perimetry. Methods: Static and flicker visual perimetry were performed in 14 healthy young participants while breathing 12% oxygen (hypoxia) under photopic illumination. The hypoxia visual field data were compared with the field data measured during normoxia. Absolute sensitivities (in dB) were analysed in seven concentric rings at 1°, 3°, 6°, 10°, 15°, 22° and 30° eccentricities as well as mean defect (MD) and pattern defect (PD) were calculated. Preliminary data are reported for mesopic light levels. Results: Under photopic illumination, flicker and static visual field sensitivities at all eccentricities were not significantly different between hypoxia and normoxia conditions. The mean defect and pattern defect were not significantly different for either test between the two oxygenation conditions. Conclusion: Although flicker stimulation increases cellular metabolism, flicker photopic visual field impairment is not detected during mild hypoxia. These findings contrast with electrophysiological flicker tests in young participants that show impairment at photopic illumination during the same levels of mild hypoxia. Potential mechanisms contributing to the difference between the visual fields and electrophysiological flicker tests including variability in perimetric data, neuronal adaptation and vascular autoregulation, are considered. The data have implications for the use of visual perimetry in the detection of ischaemic/hypoxic retinal disorders under photopic and mesopic light levels.

Functional status, postural stability and falls among older adults with glaucoma

Visual impairment is an important contributing factor in falls among older adults, which is one of the leading causes of injury and injury-related death in this population. Visual impairment is also associated with greater disability among older adults, including poorer health-related quality of life, increased frailty and reduced postural stability. The majority of this evidence, however, is based on measures of central visual function, rather than peripheral visual function. As such, there is comparatively limited research on the associations between peripheral visual function, disability and falls, and even fewer studies involving older adults with specific diseases which affect peripheral visual function, the most common of which is glaucoma. Glaucoma is one of the leading causes of irreversible vision loss among older adults, affecting around 3 per cent of adults aged over 60 years. The condition is characterised by retinal nerve fibre loss, primarily affecting peripheral visual function. Importantly, the number of older adults with glaucomatous visual impairment is projected to increase as the ageing population grows. The first component of the thesis examined the cross-sectional association between glaucomatous visual impairment and health-related quality of life (Study 1a), functional status (Study 1b) and postural stability (Study 1c) among older adults. A cohort of 74 community-dwelling adults with glaucoma (mean age 74.2 ± 5.9 years) was recruited and completed a baseline assessment. A number of visual function measures was assessed, including central visual function (visual acuity and contrast sensitivity), motion sensitivity, retinal nerve fibre analysis and monocular and binocular visual field measures (monocular 24-2 and binocular integrated visual fields (IVF): IVF-60 and IVF-120). The analyses focused on the associations between the outcomes measures and severity and location of visual field loss, as this is the primary visual function affected by glaucoma. In Study 1a, we examined the association between visual field loss and health-related quality of life, measured by the Short Form 36-item Health Survey (SF-36). Greater binocular visual field loss, on both IVF measures, was associated with lower SF-36 physical component scores, adjusted for age and gender (Pearson's r =|0.32| to |0.36|, p<0.001). Furthermore, inferior visual field loss was more strongly associated with the SF-36 physical component than superior field loss. No association was found between visual field loss and SF-36 mental component scores. The association between visual field loss and functional status was examined in Study 1b. Functional status outcomes measures included a physical activity questionnaire (Physical Activity Scale for the Elderly, PASE), performance tests (six-minute walk test, timed up and go test and lower leg strength) and an overall functional status score. Significant, but weak, correlations were found between binocular visual field loss and PASE and overall functional status scores, adjusted for age and gender (Pearson's r =|0.24| to |0.33|, p<0.05). Greater inferior visual field loss, independent of superior visual field loss, was significantly associated with poorer physical performance results and lower overall functional status scores. In Study 1c, we examined the association between visual field loss and postural stability, using a swaymeter device which recorded body movement during four conditions: eyes open and closed, on a firm and foam surface. Greater binocular visual field loss was associated with increased postural sway, both on firm and foam surfaces, independent of age and gender (Pearson’s r =|0.44| to |0.46|, p <0.001). Furthermore, inferior visual field was a stronger contributor to postural stability, more so than the superior visual field, particularly on the foam condition with the eyes open. Greater visual field loss was associated with a reduction in the visual contribution to postural sway, which underlies the observed association with postural sway. The second component of the thesis examined the association between severity and location of visual field loss and falls during a 12-month longitudinal follow-up. The number of falls was assessed prospectively using monthly fall calendars. Of the 71 participants who successfully completed the follow up (mean age 73.9 ± 5.7 years), 44% reported one or more falls, and around 20% reported two or more falls. After adjusting for age and gender, every 10 points missed on the IVF-120 increased the rate of falls by 25% (rate ratio 1.25, 95% confidence interval 1.08 - 1.44) or every 5dB reduction in IVF-60 increased the rate of falls by 47% (rate ratio 1.47, 95% confidence interval 1.16 - 1.87). Inferior visual field loss was a significant predictor of falls, more so than superior field loss, highlighting the importance of the inferior visual field area in safe and efficient navigation. Further analyses indicated that postural stability, more so than functional status, may be a potential mediating factor in the relationship between visual field loss and falls. Future research is required to confirm this causal pathway. In addition, the use of topical beta-blocker medications was not associated with an increased rate of falls in this cohort, compared with the use of other topical anti-glaucoma medications. In summary, greater binocular visual field loss among older adults with glaucoma was associated with poorer health-related quality of life in the physical domain, reduced functional status, greater postural instability and higher rates of falling. When the location of visual field loss was examined, inferior visual field loss was consistently more strongly associated with these outcomes than superior visual field loss. Insights gained from this research improve our understanding of the association between glaucomatous visual field loss and disability, and its link with falls among older adults. The clinical implications of this research include the need to include visual field screening in falls risk assessments among older adults and to raise awareness of these findings to eye care practitioners and adults with glaucoma. The findings also assist in developing further research to examine strategies to reduce disability and prevent falls among older adults with glaucoma to promote healthy ageing and independence for these individuals.

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families : functional analysis in heterozygous individuals

A protein-truncating variant of CHEK2, 1100delC, is associated with a moderate increase in breast cancer risk. We have determined the prevalence of this allele in index cases from 300 Australian multiple-case breast cancer families, 95% of which had been found to be negative for mutations in BRCA1 and BRCA2. Only two (0.6%) index cases heterozygous for the CHEK2 mutation were identified. All available relatives in these two families were genotyped, but there was no evidence of co-segregation between the CHEK2 variant and breast cancer. Lymphoblastoid cell lines established from a heterozygous carrier contained approximately 20% of the CHEK2 1100delC mRNA relative to wild-type CHEK2 transcript. However, no truncated CHK2 protein was detectable. Analyses of expression and phosphorylation of wild-type CHK2 suggest that the variant is likely to act by haploinsufficiency. Analysis of CDC25A degradation, a downstream target of CHK2, suggests that some compensation occurs to allow normal degradation of CDC25A. Such compensation of the 1100delC defect in CHEK2 might explain the rather low breast cancer risk associated with the CHEK2 variant, compared to that associated with truncating mutations in BRCA1 or BRCA2.

Multiple human single-stranded DNA binding proteins function in genome maintenance : structural, biochemical and functional analysis

DNA exists predominantly in a duplex form that is preserved via specific base pairing. This base pairing affords a considerable degree of protection against chemical or physical damage and preserves coding potential. However, there are many situations, e.g. during DNA damage and programmed cellular processes such as DNA replication and transcription, in which the DNA duplex is separated into two singlestranded DNA (ssDNA) strands. This ssDNA is vulnerable to attack by nucleases, binding by inappropriate proteins and chemical attack. It is very important to control the generation of ssDNA and protect it when it forms, and for this reason all cellular organisms and many viruses encode a ssDNA binding protein (SSB). All known SSBs use an oligosaccharide/oligonucleotide binding (OB)-fold domain for DNA binding. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating strand-exchange proteins and helicases, and mediation of protein–protein interactions. Recently two additional human SSBs have been identified that are more closely related to bacterial and archaeal SSBs. Prior to this it was believed that replication protein A, RPA, was the only human equivalent of bacterial SSB. RPA is thought to be required for most aspects of DNA metabolism including DNA replication, recombination and repair. This review will discuss in further detail the biological pathways in which human SSBs function.

Physical and functional interaction of the archaeal single-stranded DNA-binding protein SSB with RNA polymerase

Archaeal transcription utilizes a complex multisubunit RNA polymerase and the basal transcription factors TBP and TF(II)B, closely resembling its eukaryal counterpart. We have uncovered a tight physical and functional interaction between RNA polymerase and the single-stranded DNA-binding protein SSB in Sulfolobus solfataricus. SSB stimulates transcription from promoters in vitro under TBP-limiting conditions and supports transcription in the absence of TBP. SSB also rescues transcription from repression by reconstituted chromatin. We demonstrate the potential for promoter melting by SSB, suggesting a plausible basis for the stimulation of transcription. This stimulation requires both the single-stranded DNA-binding domain and the acidic C-terminal tail of the SSB. The tail forms a stable interaction with RNA polymerase. These data reveal an unexpected role for single-stranded DNA-binding proteins in transcription in archaea.

Inferior visual field reductions are associated with poorer functional status among older adults with glaucoma

Purpose: To examine the relationship between visual impairment and functional status in a community-dwelling sample of older adults with glaucoma. Methods: This study included 74 community-dwelling older adults with open-angle glaucoma (aged 74 ± 6 years). Assessment of central vision included high-contrast visual acuity and Pelli-Robson contrast sensitivity. Binocular integrated visual fields were derived from merged monocular Humphrey Field Analyser visual field plots. Functional status outcome measures included physical performance tests (6-min walk test, timed up and go test and lower limb strength), a physical activity questionnaire (Physical Activity Scale for the Elderly) and an overall functional status score. Correlation and linear regression analyses, adjusting for age and gender, examined the association between visual impairment and functional status outcomes. Results: Greater levels of visual impairment were significantly associated with lower levels of functional status among community-dwelling older adults with glaucoma, independent of age and gender. Specifically, lower levels of visual function were associated with slower timed up and go performance, weaker lower limb strength, lower self-reported physical activity, and lower overall functional status scores. Of the components of vision examined, the inferior visual field and contrast factors were the strongest predictors of these functional outcomes, whereas the superior visual field factor was not related to functional status. Conclusions: Greater visual impairment, particularly in the inferior visual field and loss of contrast sensitivity, was associated with poorer functional status among older adults with glaucoma. The findings of this study highlight the potential links between visual impairment and the onset of functional decline. Interventions which promote physical activity among older adults with glaucoma may assist in preventing functional decline, frailty and falls, and improve overall health and well-being.

Functional role of cell surface CUB domain-containing protein 1 in tumour cell dissemination

The function of CUB domain-containing protein 1 (CDCP1), a recently described transmembrane protein expressed on the surface of hematopoietic stem cells and normal and malignant cells of different tissue origin, is not well defined. The contribution of CDCP1 to tumor metastasis was analyzed by using HeLa carcinoma cells overexpressing CDCP1 (HeLa-CDCP1) and a high-disseminating variant of prostate carcinoma PC-3 naturally expressing high levels of CDCP1 (PC3-hi/diss). CDCP1 expression rendered HeLa cells more aggressive in experimental metastasis in immunodeficient mice. Metastatic colonization by HeLa-CDCP1 was effectively inhibited with subtractive immunization-generated, CDCP1-specific monoclonal antibody (mAb) 41-2, suggesting that CDCP1 facilitates relatively late stages of the metastatic cascade. In the chick embryo model, time- and dose-dependent inhibition of HeLa-CDCP1 colonization by mAb 41-2 was analyzed quantitatively to determine when and where CDCP1 functions during metastasis. Quantitative PCR and immunohistochemical analyses indicated that CDCP1 facilitated tumor cell survival soon after vascular arrest. Live cell imaging showed that the function-blocking mechanism of mAb 41-2 involved enhancement of tumor cell apoptosis, confirmed by attenuation of mAb 41-2–mediated effects with the caspase inhibitor z-VAD-fmk. Under proapoptotic conditions in vitro, CDCP1 expression conferred HeLa-CDCP1 cells with resistance to doxorubicin-induced apoptosis, whereas ligation of CDCP1 with mAb 41-2 caused additional enhancement of the apoptotic response. The functional role of naturally expressed CDCP1 was shown by mAb 41-2–mediated inhibition of both experimental and spontaneous metastasis of PC3-hi/diss. These findings confirm that CDCP1 functions as an antiapoptotic molecule and indicate that during metastasis CDCP1 facilitates tumor cell survival likely during or soon after extravasation.