Validation of the Knowledge of Genome Sequencing (KOGS) scale in cancer patients.

INTRODUCTION: The Knowledge of Genome Sequencing (KOGS) questionnaire was recently developed to measure knowledge of genomic sequencing (GS), with preliminary psychometric data supporting its reliability and validity. The aim of this study was to test the reliability and validity of the KOGS in a larger sample, and to confirm its utility in a cancer setting. METHODS: The Genetic Cancer Risk in the Young (RisC) study recruits participants with a personal history of cancer, to investigate heritable cancer causes and future cancer risk using germline GS. Participants (n = 261) in a psychosocial substudy of RisC completed a questionnaire after consent to RisC but before GS, including the KOGS, the Intolerance of Uncertainty Scale, the Chew health literacy scale and items assessing demographic and disease variables. Confirmatory factor analysis (CFA), Cronbach alpha and correlational analyses were undertaken. RESULTS: The CFA testing a single-factor model yielded a good model fit, χ2/df = 2.43, comparative fit index (CFI) = 0.97, root mean square error of approximation (RMSEA) = 0.07 and weighted mean root square (WRMR) = 1.03. Factor loadings of all items were above 0.60 and ranged between.66 and.93. The single factor score demonstrated excellent internal consistency (α = 0.82). KOGS scores were significantly associated with health literacy (r = 0.23, p < .001), having a university education [t(258) = -4.53, p < .001] and having a medical or science background [t(259) = -3.52, p < .001] but not with speaking a language other than English at home, time since diagnosis, previous genetic counselling/testing or intolerance of uncertainty. DISCUSSION: This study confirmed a single-factor structure for the KOGS, and its reliability and validity in a cancer population. Associations with measures of health literacy and education were significant and positive as expected, supporting the KOG's construct validity. Previous genetic counselling may not be sufficient to provide specific knowledge of GS.

Sociodemographic, psychosocial and clinical factors associated with uptake of genetic counselling for hereditary cancer: a systematic review.

Evidence suggests that a significant proportion of individuals referred to cancer genetic counselling (GC) do not attend, and thus may not be engaged in adequate cancer risk management. We aimed to review the literature to better understand barriers to accessing GC and how they may be overcome. We conducted a systematic literature search for articles examining factors influencing cancer GC uptake as well as motivators and barriers to GC attendance. Factors were categorised as sociodemographic, psychosocial or clinical. The literature search identified 1413 citations, 35 of which met the inclusion criteria. GC uptake ranged from 19% to 88%. With the exceptions of education level, socioeconomic status, cancer-specific distress, personal cancer diagnosis and actual and perceived risk of cancer, support was lacking for most sociodemographic, clinical and psychosocial factors as predictors of GC uptake. Cost and logistical barriers, emotional concerns, family concerns and low perceived personal relevance were reported as important considerations for those declining GC. We conclude that there is poor understanding of GC and a lack of decision support among those referred to GC. Research into ways of providing education and support to referred individuals will be important as the scope and availability of GC and genetic testing broaden.

Glycosaminoglycan synthesis and structure as targets for the prevention of calcific aortic valve disease.

Calcific aortic valve disease is frequently driven by ageing and the obesity-associated metabolic syndrome, and the increasing impact of these factors indicates that valve disease will become a cardiovascular disease of considerable significance. This disease is now thought to be an active cell-based disease process, which may therefore be amenable to therapeutic intervention. Some similarities are apparent with atherosclerosis. The accumulation of lipid, possibly by retention by proteoglycans and the attraction of inflammatory cells by hyaluronan, may be common to the early stages of both pathologies. The synthesis and structure of glycosaminoglycans, proteoglycans, and hyaluronan are exquisitely regulated, and the signalling pathways controlling these processes may provide tissue-specific opportunities for concomitant prevention of atherosclerosis and calcific aortic valve disease.

Fenofibrate modifies human vascular smooth muscle proteoglycans and reduces lipoprotein binding.

AIMS/HYPOTHESIS: Vascular disease in type 2 diabetes is associated with an up-regulation of atherogenic growth factors, which stimulate matrix synthesis including proteoglycans. We have examined the direct actions of fenofibrate on human vascular smooth muscle cells (VSMCs) and have specifically investigated proteoglycan synthesis and binding to LDL. METHODS: Proteoglycans synthesised by human VSMCs treated with fenofibrate (30 micromol/l) were assessed for binding to human LDL using a gel mobility shift assay, metabolically labelled with [(35)S]-sulphate and quantitated by cetylpyridinium chloride. They were then assessed for electrophoretic mobility by SDS-PAGE, for size by gel filtration, for sulphation pattern by fluorophore-assisted carbohydrate electrophoresis, and for glycosaminoglycan (GAG) composition by enzyme digestion. RESULTS: Proteoglycans synthesised in the presence of fenofibrate showed an increase in the half-maximum saturation concentration of LDL from 36.8+/-12.4 microg/ml to 77.7+/-17 microg/ml under basal conditions, from 24.9+/-4.6 microg/ml to 39.1+/-6.1 microg/ml in the presence of TGF-beta1, and from 9.5+/-4.4 microg/ml to 31.1+/-3.4 microg/ml in the presence of platelet-derived growth factor/insulin. Fenofibrate treatment in the presence of TGF-beta1 inhibited the incorporation of [(35)S]-sulphate into secreted and cell-associated proteoglycans synthesised by human VSMCs by 59.2% (p<0.01) and 39.8% (p<0.01) respectively. The changes in sulphate incorporation following treatment with fenofibrate were associated with a concentration-related increase in the electrophoretic mobility due to a reduction in GAG length. There was no change in the sulphation pattern; however, there was an alteration in the disaccharide composition of the GAGs. CONCLUSIONS/INTERPRETATION: Fenofibrate modifies the structure of vascular proteoglycans by reducing the length of the GAG chains and GAG composition, resulting in reduced binding to human LDL, a mechanism which may lead to a reduction of atherosclerosis and cardiovascular disease in people with diabetes treated with fenofibrate.

Regulation of glycosaminoglycan structure and atherogenesis.

Cardiovascular disease is the major cause of premature death in modern society, and its impact is increasing due to rising rates of obesity and type 2 diabetes. Clinical studies based on targeting metabolic abnormalities and biomarkers demonstrate significant benefits, but always an element of disease remains which is resistant to treatment. Recent evidence has strongly implicated an early interaction of atherogenic lipoproteins with vascular matrix proteoglycans as the initiating step in atherogenesis. Expert commentary has pointed to the need for vascular directed therapies to provide reductions in the residual disease component. We propose that the regulation of synthesis and thus structure of glycosaminoglycans on proteoglycans provides a potential pathway to this reduction. We review existing evidence that the vascular synthesis of glycosaminoglycan chains can be regulated in a manner which reduces lipoprotein binding and the potential application of this strategy to attenuation of the current cardiovascular disease pandemic.

Structural changes at cut ends of earthworm giant axons in the interval between dye barrier formation and neuritic outgrowth.

We describe structural changes at the cut ends of invertebrate myelinated earthworm giant axons beginning with the formation of a dye barrier (15 minutes posttransection or postcalcium addition) and ending with the formation of a neuritic outgrowth (2-10 days posttransection). The morphology of the cut end, and the location and morphological configuration of the dye barrier, were assessed by time-lapse confocal, fluorescence microscopy and by electron microscopy. During the interval from 15 to 35 minutes postcalcium addition, the dye barrier continuously migrated away from a cut axonal end; the dye barrier then remained stable for up to 5 hours. The size, packing density, and arrangement of membranous structures were correlated with changes in the dye barrier from 15 to 35 minutes postcalcium addition. During this interval, uptake of an externally placed hydrophilic dye by these membranous structures was also variable. After 35 minutes postcalcium addition, the membranous structures remained stable until they completely disappeared between 1 and 2 days posttransection. The disappearance of membranous structures always preceded neuritic outgrowth, which only arose from cut axonal ends. These results demonstrate that the dye barrier and associated membranous structures, which form after transection of earthworm giant axons, are very dynamic in the short term (35 minutes) with respect to their location and morphological configuration and suggest that axolemmal repair must be completed before neuritic outgrowth can occur.

Calcium entry initiates processes that restore a barrier to dye entry in severed earthworm giant axons.

After severance, axons can restore structural barriers that are necessary for recovery of their electrical function. In earthworm myelinated axons, such a barrier to dye entry is mediated by many vesicles and myelin-derived membranous structures. From time-lapse confocal fluorescence and DIC images, we now report that Ca2+ entry and not axonal injury per se initiates the processes that form a dye barrier, as well as the subsequent structural changes in this barrier and associated membranous structures. The time required to restore a dye barrier after transection also depends only on the time of Ca2+ entry.

Rapid induction of functional and morphological continuity between severed ends of mammalian or earthworm myelinated axons.

The inability to rapidly restore the loss of function that results from severance (cutting or crushing) of PNS and CNS axons is a severe clinical problem. As a novel strategy to help alleviate this problem, we have developed in vitro procedures using Ca2+-free solutions of polyethylene glycol (PEG solutions), which within minutes induce functional and morphological continuity (PEG-induced fusion) between the cut or crushed ends of myelinated sciatic or spinal axons in rats. Using a PEG-based hydrogel that binds to connective tissue to provide mechanical strength at the lesion site and is nontoxic to nerve tissues in earthworms and mammals, we have also developed in vivo procedures that permanently maintain earthworm myelinated medial giant axons whose functional and morphological integrity has been restored by PEG-induced fusion after axonal severance. In all these in vitro or in vivo procedures, the success of PEG-induced fusion of sciatic or spinal axons and myelinated medial giant axons is measured by the restored conduction of action potentials through the lesion site, the presence of intact axonal profiles in electron micrographs taken at the lesion site, and/or the intra-axonal diffusion of fluorescent dyes across the lesion site. These and other data suggest that the application of polymeric fusiogens (such as our PEG solutions), possibly combined with a tissue adherent (such as our PEG hydrogels), could lead to in vivo treatments that rapidly and permanently repair cut or crushed axons in the PNS and CNS of adult mammals, including humans.

Endocytotic formation of vesicles and other membranous structures induced by Ca2+ and axolemmal injury.

Vesicles and/or other membranous structures that form after axolemmal damage have recently been shown to repair (seal) the axolemma of various nerve axons. To determine the origin of such membranous structures, (1) we internally dialyzed isolated intact squid giant axons (GAs) and showed that elevation of intracellular Ca2+ >100 microM produced membranous structures similar to those in axons transected in Ca2+-containing physiological saline; (2) we exposed GA axoplasm to Ca2+-containing salines and observed that membranous structures did not form after removing the axolemma and glial sheath but did form in severed GAs after >99% of their axoplasm was removed by internal perfusion; (3) we examined transected GAs and crayfish medial giant axons (MGAs) with time-lapse confocal fluorescence microscopy and showed that many injury-induced vesicles formed by endocytosis of the axolemma; (4) we examined the cut ends of GAs and MGAs with electron microscopy and showed that most membranous structures were single-walled at short (5-15 min) post-transection times, whereas more were double- and multi-walled and of probable glial origin after longer (30-150 min) post-transection times; and (5) we examined differential interference contrast and confocal images and showed that large and small lesions evoked similar injury responses in which barriers to dye diffusion formed amid an accumulation of vesicles and other membranous structures. These and other data suggest that Ca2+ inflow at large or small axolemmal lesions induces various membranous structures (including endocytotic vesicles) of glial or axonal origin to form, accumulate, and interact with each other, preformed vesicles, and/or the axolemma to repair the axolemmal damage.

Heat-shock proteins in axoplasm: high constitutive levels and transfer of inducible isoforms from glia.

To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1-3% of the total protein in the axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock.

Delaminating myelin membranes help seal the cut ends of severed earthworm giant axons.

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5-60 min post-transection in physiological salines and Ca2+-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20-100 microm from the cut end at 5-60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15-30 min post-transection. At 30-60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca2+-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca2+-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma.

Calpain activity promotes the sealing of severed giant axons.

A barrier (seal) must form at the cut ends of a severed axon if a neuron is to survive and eventually regenerate. Following severance of crayfish medial giant axons in physiological saline, vesicles accumulate at the cut end and form a barrier (seal) to ion and dye diffusion. In contrast, squid giant axons do not seal, even though injury-induced vesicles form after axonal transection and accumulate at cut axonal ends. Neither axon seals in Ca2+-free salines. The addition of calpain to the bath saline induces the sealing of squid giant axons, whereas the addition of inhibitors of calpain activity inhibits the sealing of crayfish medial giant axons. These complementary effects involving calpain in two different axons suggest that endogenous calpain activity promotes plasmalemmal repair by vesicles or other membranes which form a plug or a continuous membrane barrier to seal cut axonal ends.

Repair of plasmalemmal lesions by vesicles.

Crayfish medial giant axons (MGAs) transected in physiological saline form vesicles which interact with each other, pre-existing vesicles, and/or with the plasmalemma to form an electrical and a physical barrier that seals a cut axonal end within 60 min. The formation of this barrier (seal) was assessed by measuring the decay of injury current at the cut end; its location at the cut end was determined by the exclusion of fluorescent hydrophilic dye at the cut end. When a membrane-incorporating styryl dye was placed in the bath prior to axonal transection and a hydrophilic dye was placed in the bath just after axonal transection, many vesicles near the barrier at the cut axonal end had their limiting membrane labeled with the styryl dye and their contents labeled with the hydrophilic dye, indicating that these vesicles originated from the axolemma by endocytosis. This barrier does not form in Ca2+-free salines. Similar collections of vesicles have been observed at regions of plasmalemmal damage in many cell types. From these and other data, we propose that plasmalemmal lesions in most eukaryotic cells (including axons) are repaired by vesicles, at least some of which arise by endocytosis induced by Ca2+ inflow resulting from the plasmalemmal damage. We describe several models by which vesicles could interact with each other and/or with intact or damaged regions of the plasmalemma to repair small (1-30 microm) plasmalemmal holes or a complete transection of the plasmalemma.

Cooling of peripheral myelinated axons retards Wallerian degeneration.

The histological and ultrastructural status of intact and severed axons was examined in the ventral tail nerve of rats whose tails were maintained at 32, 23, and 13 degrees C. Compared to contralateral intact nerves, distal (anucleate) portions of severed myelinated axons morphologically and ultrastructurally degenerated within 3 days at 32 degrees C and within 6 days at 23 degrees C. In contrast, anucleate myelinated axons in ventral tail nerves maintained at 13 degrees C did not degenerate for at least 10 days. These and other data suggest that rapid Wallerian degeneration of anucleate myelinated axons is not an inevitable result of axonal severance in mammals.

Extent and mechanism of sealing in transected giant axons of squid and earthworms.

Transected axons are often assumed to seal at their cut ends by the formation of continuous membrane barriers that allow for the restoration of function in the axonal stumps. We have used several electrophysiological measures (membrane potential, input resistance, injury current density) and several morphological measures (phase-contrast, video-enhanced differential interference contrast, light, and electron microscopies) of living and fixed material to assess the extent and mechanism of sealing within hours after transecting giant axons of squid (Loligo pealei and Sepioteuthis lessoniana) and earthworms (Lumbricus terrestris). Our electrophysiological data suggest that the proximal and distal ends of transected squid giant axons do not completely seal within 2.5 hr in physiological saline. In contrast, the same set of measures suggest that proximal and distal ends of transected earthworm giant axons seal within 1 hr in physiological saline. Our morphological data show that the cut ends of both squid and earthworm axons constrict, but that a 20-70-microns-diameter opening always remains at the cut end that is filled with vesicles. Axonal transection induces the formation of vesicles that are observed in the axoplasm within minutes in standard salines and that rapidly migrate to the cut ends. These injury-induced vesicles are loosely packed near the cut ends of squid giant axons, which do not functionally seal within 2.5 hr of transection. In contrast, vesicles formed a tightly packed plug at the cut ends of earthworm medial giant axons, which do functionally seal within 1 hr of transection in physiological saline. Since we detect no single continuous membrane that spans the cut end, sealing does not appear to occur by the fusion of constricted axolemmal membrane or the formation of a membranous partition at the cut end. Rather, our data are consistent with the hypothesis that a tightly packed vesicular plug is responsible for sealing of earthworm giant axons.

Long-term survival of severed crayfish giant axons is not associated with an incorporation of glial nuclei into axoplasm.

Glial nuclei have been reported to be incorporated into the axoplasm of surviving distal stumps (anucleate axons) weeks to months after lesioning abdominal motor axons in rock lobsters. We have not observed this phenomenon in crayfish medial giant axons (MGAs) which also survive for weeks to months after lesioning. Glial nuclei were not observed within MGAs perfused with a physiological intracellular saline. However, incorporation of glial nuclei was observed after MGAs were perfused with intracellular salines containing Fast green. From these and previously published data, we confirm that glial incorporation into axoplasm can occur, but we suggest that is is not a common mechanism used by crustaceans to provide for long-term survival of anucleate axons.

Rapid artificial restoration of electrical continuity across a crush lesion of a giant axon.

Action potentials never conducted through a crush lesion to the medial giant axon in the earthworm (Lumbricus terrestris) if the axon was exposed to normal or hypotonic salines that did not contain polyethylene glycol. However, action potentials, as well as electrotonic potentials, often conducted through a crush lesion exposed for 1 min to polyethylene glycol in hypotonic saline.

Reconnection of severed nerve axons with polyethylene glycol.

Severed medial giant axons in crayfish can be rejoined in vitro with polyethylene glycol (PEG) to produce axoplasmic continuity and through transmission of action potentials. Severed axon-like processes of a mammalian neuroblastoma/glioma cell line seem to be rejoined to the cell body using PEG in tissue culture. Our data suggest that PEG might be used to rejoin severed axons in vivo in various organisms.

Ultrastructural changes at gap junctions between lesioned crayfish axons.

In crayfish, the severed distal segment of single lateral giant axon (SLGA) often survives for at least 10 months after lesioning if this segme;t retains a septal region of apposition with an adjacent, intact SLGA. In control (unsevered) SLGAs, this septal usually contains gap junctions and 50-60 nm vesicles near the axolemma of both SLGAs. From 1-14 days after lesioning, the distal segment of a severed SLGA undergoes obvious ultrastructural changes in mitochondria and neurotubular organization compared to control SLGAs or to adjacent, intact SLGAs in the same animal. Gap junctions are very difficult to locate in severed SLGAs within 24 h after lesioning. From two weeks to ten months after lesioning, the surviving stumps of severed SLGAs often appear remarkably normal except that structures normally associated with the presence of gap junctions remain very difficult to find. These and other data suggest that SLGA distal segments receive trophic support from adjacent, intact SLGAs. The mechanism of this support probably could not be via diffusion across gap junctions between intact and severed SLGAs since gap junctions largely disappear after lesioning. However, trophic maintenance could occur via the exocytotic - pinocytotic action of 50-60 nm vesicles which are always present on both sides of the septum between an intact SLGA and a severed SLGA distal segment.