Tobacco smoking and somatic mutations in human bronchial epithelium.

Tobacco smoking causes lung cancer1-3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6-10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

Simultaneous Optimization of MP2RAGE T1 -weighted (UNI) and FLuid And White matter Suppression (FLAWS) brain images at 7T using Extended Phase Graph (EPG) Simulations.

PURPOSE: The MP2RAGE sequence is typically optimized for either T1 -weighted uniform image (UNI) or gray matter-dominant fluid and white matter suppression (FLAWS) contrast images. Here, the purpose was to optimize an MP2RAGE protocol at 7 Tesla to provide UNI and FLAWS images simultaneously in a clinically applicable acquisition time at <0.7 mm isotropic resolution. METHODS: Using the extended phase graph formalism, the signal evolution of the MP2RAGE sequence was simulated incorporating T2 relaxation, diffusion, RF spoiling, and B1 + variability. Flip angles and TI were optimized at different TRs (TRMP2RAGE ) to produce an optimal contrast-to-noise ratio for UNI and FLAWS images. Simulation results were validated by comparison to MP2RAGE brain scans of 5 healthy subjects, and a final protocol at TRMP2RAGE  = 4000 ms was applied in 19 subjects aged 8-62 years with and without epilepsy. RESULTS: FLAWS contrast images could be obtained while maintaining >85% of the optimal UNI contrast-to-noise ratio. Using TI1 /TI2 /TRMP2RAGE of 650/2280/4000 ms, 6/8 partial Fourier in the inner phase-encoding direction, and GRAPPA factor = 4 in the other, images with 0.65 mm isotropic resolution were produced in <7.5 min. The contrast-to-noise ratio was around 20% smaller at TRMP2RAGE  = 4000 ms compared to that at TRMP2RAGE  = 5000 ms; however, the 20% shorter duration makes TRMP2RAGE  = 4000 ms a good candidate for clinical applications example, pediatrics. CONCLUSION: FLAWS and UNI images could be obtained in a single scan with 0.65 mm isotropic resolution, providing a set of high-contrast images and full brain coverage in a clinically applicable scan time. Images with excellent anatomical detail were demonstrated over a wide age range using the optimized parameter set.

Host transmission dynamics of first- and third-stage Angiostrongylus cantonensis larvae in Bullastra lessoni.

Given the importance of angiostrongyliasis as an emerging infectious disease of humans, companion animals, and wildlife, the current study focused on the transmission dynamics of first- and third-stage larvae of the parasitic nematode, Angiostrongylus cantonensis. The migration of infective larvae and their subsequent distribution within the Lymnaeidae snail, Bullastra lessoni, were investigated over time using microscopic examination of histological sections and fresh tissue. Snails were divided into four anatomical regions: (i) anterior and (ii) posterior cephalopedal masses, (iii) mantle skirt and (iv) visceral mass. The viability of free-swimming third-stage larvae, after their release from snail tissues, was evaluated in vitro by propidium iodide staining and infectivity by in vivo infection of Wistar rats. Snails were sequentially dissected over time to assess the number and anatomical distribution of larvae within each snail and hence infer their migration pathway. Herein, ongoing larval migratory activity was detected over 28 days post-infection. A comparison of infection rates and the larval distribution within the four designated snail regions demonstrated a significant relationship between anatomical region and density of infective larvae, with larvae mostly distributed in the anterior cephalopedal mass (43.6 ± 10.8%) and the mantle skirt (33.0 ± 8.8%). Propidium iodide staining showed that free-swimming third-stage larvae retained viability for between 4 and 8 weeks when stored under laboratory conditions. In contrast to viability, larval infectivity in rats remained for up to 2 weeks only. Knowledge gained from the current work could provide information on the development of new approaches to controlling the transmission of this parasite.

Adolescent idiopathic scoliosis 3D vertebral morphology, progression and nomenclature: a current concepts review.

PURPOSE: There has been a recent shift toward the analysis of the pathoanatomical variation of the adolescent idiopathic scoliosis (AIS) spine with the three dimensions, and research of level-wise vertebral body morphology in single anatomical planes is now replete within the field. In addition to providing a precise description of the osseous structures that are the focus of instrumented surgical interventions, understanding the anatomical variation between vertebral bodies will elucidate possible pathoaetiological mechanisms of the onset of scoliotic deformity. METHODS: This review aimed to discuss the current landscape of AIS segmental vertebral morphology research and provide a comprehensive report of the typical patterns observed at the individual vertebral level. RESULTS: We have detailed how these vertebrae are typically characterised by lateral wedging to the convexity, have a marked degree of anterior overgrowth, are rotated towards the convexity, have inherent gyratory mechanical torsion created within them and are associated with pedicles on the concave side being narrower, longer and more laterally angled. For the most part, these findings are most pronounced at and around the apex of a scoliotic curve, with these deformations reducing towards junctional vertebrae. We have also summarised a nomenclature defined by the Scoliosis Research Society, highlighting the need for more consistent reporting of these level-wise dimensional anatomical changes. CONCLUSION: Finally, we emphasised how a marked degree of heterogeneity exists between the included investigations, namely in scoliotic curve-type inclusion, imaging modality and timepoint of analysis within scoliosis' longitudinal development, and how improvement in these study design characteristics will enhance ongoing research.

Evaluating and comparing the green wall retrofit suitability across major Australian cities.

Urban densification continues to present a unique set of economic and environmental challenges. A growing shortage of green space and infrastructure is intrinsically linked with urban growth and development. With this comes the loss of ecosystem services such as urban heat island effects, reduction of air quality and biodiversity loss. Vertical greenery systems (VGS) offer an adaptive solution to space-constrained areas that are characteristic of dense urban areas, and can potentially improve the sustainability of cities. However, in order to promote VGS uptake, methods are required to enable systematic appraisal of whether existing walls can be retrofitted with VGS. Further, feasibility studies that quantify the potential for retrofit suitability of VGS across entire urban areas are lacking. This study established an evaluation tool for green wall constructability in urban areas and validated the assessment tool by determining the quantity of walls in five major Australian cities that could potentially have VGS incorporated into the existing infrastructure. Each wall was analysed using an exclusionary set of criteria that evaluated and ranked a wall based on its suitability to VGS implementation. Sydney and Brisbane recorded the greatest proportional length of walls suitable for VGS, with 33.74% and 34.12% respectively. Conversely, Perth's urban centre was the least feasible site in which to incorporate VGS, with over 97% of surveyed walls excluded, mainly due to the prevalence of <1 m high fence lines and glazed shopfronts. This study aimed to evaluate feasibility assessments of green wall retrofitability in highly urbanised areas with the intention of creating an analytical method that is accessible to all. This method, coupled with the promising number of feasible walls found in this study, emphasises the need for more government policy and incentives encouraging green wall uptake and could play a pivotal role in the expansion of green infrastructure and urban forestry.

Lepidosaur ß-keratin chains with four 34-residue repeats: Modelling reveals a potential filament-crosslinking role.

ß-keratin chains contain a characteristic and homologous 34-residue sequence, which is believed to adopt a twisted ß-sheet conformation that assembles in an antiparallel manner with a similar sheet in a second chain to form a ß-sandwich. These sandwiches are, in turn, related to one another by a left-handed four-fold screw axis to generate a helical structure that forms the core of the 3.4 nm diameter filaments observed by electron microscopy and deduced from X-ray fibre diffraction. Recently, it has been shown that one ß-keratin chain, with a molecular weight approximately twice that of the majority of ß-keratin chains, is conserved across the lepidosaurs (lizards, snakes and tuatara). Uniquely, it contains four 34-residue repeats. Although this chain is a minor component the observation that the entire chain shows a high degree of sequence conservation between species suggests an important structural/functional role in vivo. Modelling shows that only six families of structures are physically possible. In three of these the repeats exist within a single filament and might therefore act in a filament nucleation role. In the second three families the repeats exist in two, three or four filaments, implying that their function may be to act as an inter-filament crosslinker, thereby providing lateral reinforcement to the epidermal appendage. The favoured model is one in which the first two repeats form a ß-sandwich in one filament and the second two repeats form a ß-sandwich in a neighbouring filament. Links between alternating up- and down-pointing ß-sheets would provide optimum connectivity.

Prevalence and long-term outcome of sub-clinical primary sclerosing cholangitis in patients with ulcerative colitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is closely associated with inflammatory bowel disease, particularly ulcerative colitis (UC), with an increased risk of biliary and colorectal malignancy. We sought to clarify the prevalence, characteristics and long-term outcome of sub-clinical PSC diagnosed by magnetic resonance cholangiogram (MRC) in patients with UC and normal liver biochemistry, with or without colorectal dysplasia (CRD). METHODS: In this prospective case-control study, 70 patients with UC and normal liver function (51 extensive UC, 19 CRD), 28 healthy volunteers (negative controls) and 28 patients with PSC and cholestasis (positive controls) underwent MRC and blood evaluation. MRC scans were interpreted blindly by two radiologists who graded individually, the scans as definitive for PSC, possible for PSC or normal. Clinical outcome was assessed by blood monitoring, abdominal imaging and endoscopic surveillance. RESULTS: 7/51 (14%) with extensive UC and 4/19 (21%) with CRD had biliary abnormalities on MRC consistent with PSC. 7/11 (64%) with sub-clinical PSC had isolated intrahepatic duct involvement. Sub-clinical PSC was associated with advanced age (P = .04), non-smoking (P = .03), pANCA (P = .04), quiescent colitis (P = .02), absence of azathioprine (P = .04) and high-grade CRD (P = .03). Inter-observer (kappa = 0.88) and intra-observer (kappa = 0.96) agreement for MRC interpretation was high. No negative controls were assessed as definite PSC, 4/28 were considered on blinding as possible PSC. During follow-up of sub-clinical PSC (median 10.1(3.1-11.9) years), four patients developed abnormal liver biochemistry, two had radiological progression of PSC and seven developed malignancy, including two biliary and one colorectal carcinoma. CONCLUSIONS: Prevalence of sub-clinical PSC appears high in patients with extensive UC and normal liver biochemistry, with or without CRD. Disease progression and malignancy were identified on long-term follow-up. MRC should be considered for all patients with extensive UC or CRD to stratify surveillance.

Airborne particulate matter accumulation on common green wall plants.

In order to better design greening systems for effective particulate matter (PM) removal, it is important to understand the impact leaf traits have on PM deposition. There are however, inconsistences amongst the leaf traits that have previously been correlated with PM accumulation. The aim of this paper was to identify vegetation characteristics of green wall plants that were associated with the accumulation of particulate matter. To determine patterns associated with different leaf morphologies, eleven common ornamental plant species were sampled across 15 sites, over a 6 month duration. PM deposition was determined gravimetrically and its associated size fractions determined microscopically. Linear mixed models were used to identify statistical patterns relating to differences in PM deposition across plant species. PM deposition and the relative frequencies of particle size fractions were found to be statistically different among species, sites and months. Green wall plants were shown to be effective at PM accumulation as all of the assessed plant species had equivalent PM removal efficiency, with minimal evidence of influential leaf characteristics that could enhance PM removal.

Molecular structure of sauropsid ß-keratins from tuatara (Sphenodon punctatus).

The birds and reptiles, collectively known as the sauropsids, can be subdivided phylogenetically into the archosaurs (birds, crocodiles), the testudines (turtles), the squamates (lizards, snakes) and the rhynchocephalia (tuatara). The structural framework of the epidermal appendages from the sauropsids, which include feathers, claws and scales, has previously been characterised by electron microscopy, infrared spectroscopy and X-ray diffraction analyses, as well as by studies of the amino acid sequences of the constituent ß-keratin proteins (also referred to as the corneous ß-proteins). An important omission in this work, however, was the lack of sequence and structural data relating to the epidermal appendages of the rhynchocephalia (tuatara), one of the two branches of the lepidosaurs. Considerable effort has gone into sequencing the tuatara genome and while this is not yet complete, there are now sufficient sequence data for conclusions to be drawn on the similarity of the ß-keratins from the tuatara to those of other members of the sauropsids. These results, together with a comparison of the X-ray diffraction pattern of tuatara claw with those from seagull feather and goanna claw, confirm that there is a common structural plan in the ß-keratins of all of the sauropsids, and not just those that comprise the archosaurs (birds and crocodiles), the testudines (turtles) and the squamates (lizards and snakes).

Characterization of underwater scattering layers based on variance components of LiDAR backscattering.

Range-resolved detection of submerged scattering layers was investigated in the Gulf of Mexico based on vertical profiles made with a LiDAR (Light detection and range) system having a green laser (wavelength λ = 532 nm). The backscattering power (Sd) variability was decomposed in principal components (PCs) and related to non-polarized Sd, the Sd ratio between cross- and co-polarized waveforms, the chlorophyll-a fluorescence (Fchl), and the ratio between volume scattering angles of 150° and 100°. The variance of PCs was dominated by non-polarized Sd followed by Fchl. Correlation between PC1 scores and Fchl anomalies suggested that Sd was mainly originated from pigmented particulates.

Association of Genetic Variants in NUDT15 With Thiopurine-Induced Myelosuppression in Patients With Inflammatory Bowel Disease.

Importance: Use of thiopurines may be limited by myelosuppression. TPMT pharmacogenetic testing identifies only 25% of at-risk patients of European ancestry. Among patients of East Asian ancestry, NUDT15 variants are associated with thiopurine-induced myelosuppression (TIM). Objective: To identify genetic variants associated with TIM among patients of European ancestry with inflammatory bowel disease (IBD). Design, Setting, and Participants: Case-control study of 491 patients affected by TIM and 679 thiopurine-tolerant unaffected patients who were recruited from 89 international sites between March 2012 and November 2015. Genome-wide association studies (GWAS) and exome-wide association studies (EWAS) were conducted in patients of European ancestry. The replication cohort comprised 73 patients affected by TIM and 840 thiopurine-tolerant unaffected patients. Exposures: Genetic variants associated with TIM. Main Outcomes and Measures: Thiopurine-induced myelosuppression, defined as a decline in absolute white blood cell count to 2.5 × 109/L or less or a decline in absolute neutrophil cell count to 1.0 × 109/L or less leading to a dose reduction or drug withdrawal. Results: Among 1077 patients (398 affected and 679 unaffected; median age at IBD diagnosis, 31.0 years [interquartile range, 21.2 to 44.1 years]; 540 [50%] women; 602 [56%] diagnosed as having Crohn disease), 919 (311 affected and 608 unaffected) were included in the GWAS analysis and 961 (328 affected and 633 unaffected) in the EWAS analysis. The GWAS analysis confirmed association of TPMT (chromosome 6, rs11969064) with TIM (30.5% [95/311] affected vs 16.4% [100/608] unaffected patients; odds ratio [OR], 2.3 [95% CI, 1.7 to 3.1], P = 5.2 × 10-9). The EWAS analysis demonstrated an association with an in-frame deletion in NUDT15 (chromosome 13, rs746071566) and TIM (5.8% [19/328] affected vs 0.2% [1/633] unaffected patients; OR, 38.2 [95% CI, 5.1 to 286.1], P = 1.3 × 10-8), which was replicated in a different cohort (2.7% [2/73] affected vs 0.2% [2/840] unaffected patients; OR, 11.8 [95% CI, 1.6 to 85.0], P = .03). Carriage of any of 3 coding NUDT15 variants was associated with an increased risk (OR, 27.3 [95% CI, 9.3 to 116.7], P = 1.1 × 10-7) of TIM, independent of TPMT genotype and thiopurine dose. Conclusions and Relevance: Among patients of European ancestry with IBD, variants in NUDT15 were associated with increased risk of TIM. These findings suggest that NUDT15 genotyping may be considered prior to initiation of thiopurine therapy; however, further study including additional validation in independent cohorts is required.

Direct evidence supporting the existence of a helical dislocation in protofilament packing in the intermediate filaments of oxidized trichocyte keratin.

The X-ray diffraction patterns of quill and hair, as well as other trichocyte keratin appendages, contain meridional reflections that can be indexed on an axial repeat of 470 Å. Unusually, however, many of the expected orders are not observed. A possible explanation, proposed by Fraser and MacRae (1983), was that the intermediate filaments (IF) that constitute the fibrillar component of the filament/matrix texture consist of 4-chain protofilaments arranged on a surface lattice subject to a helical dislocation. The radial projection of the resulting 8-protofilament ribbon was defined in terms of a two-dimensional unit cell characterized by vectors (a, b) with axial projections za ∼ 74 Šand zb ∼ 198 Å. This situation resembles that found in microtubules, where helical dislocations in subunit packing are also encountered, leading to a so-called "seam" along their length (Metoz and Wade, 1997). In keratin, however, the protofilaments are helical so the seam is inclined to the axis of the IF. Here we report details of the Patterson function that provides independent evidence for both the helical dislocation and the dimensions of the surface lattice. In addition, the observed meridional X-ray amplitudes have been compared with those predicted by various models of the axial distribution of electron density. A new model, adapted from one previously proposed, fits the data significantly better than has heretofore proved possible. An interpretation of the model in terms of either specific keratin-associated-protein (KAP) binding or the retention of IF symmetry by a portion of the head and/or tail domains is suggested.

Identification of polymorphisms in the bovine collagenous lectins and their association with infectious diseases in cattle.

Infectious diseases are a significant issue in animal production systems, including both the dairy and beef cattle industries. Understanding and defining the genetics of infectious disease susceptibility in cattle is an important step in the mitigation of their impact. Collagenous lectins are soluble pattern recognition receptors that form an important part of the innate immune system, which serves as the first line of host defense against pathogens. Polymorphisms in the collagenous lectin genes have been shown in previous studies to contribute to infectious disease susceptibility, and in cattle, mutations in two collagenous lectin genes (MBL1 and MBL2) are associated with mastitis. To further characterize the contribution of variation in the bovine collagenous lectins to infectious disease susceptibility, we used a pooled NGS approach to identify short nucleotide variants (SNVs) in the collagenous lectins (and regulatory DNA) of cattle with (n = 80) and without (n = 40) infectious disease. Allele frequency analysis identified 74 variants that were significantly (p < 5 × 10-6) associated with infectious disease, the majority of which were clustered in a 29-kb segment upstream of the collectin locus on chromosome 28. In silico analysis of the functional effects of all the variants predicted 11 SNVs with a deleterious effect on protein structure and/or function, 148 SNVs that occurred within potential transcription factor binding sites, and 31 SNVs occurring within potential miRNA binding elements. This study provides a detailed look at the genetic variation of the bovine collagenous lectins and identifies potential genetic markers for infectious disease susceptibility.

Unexpected differential metabolic responses of Campylobacter jejuni to the abundant presence of glutamate and fucose.

INTRODUCTION: Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity. OBJECTIVES: For instance, the C. jejuni 11168 strain can utilize both L-fucose and L-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of L-fucose and L-glutamate in the growth medium. METHODS: To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h. RESULTS: This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon L-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy. CONCLUSIONS: Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon L-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.

Conservation mycology in Australia and the potential role of citizen science.

Fungi are undoubtedly important for ecosystem functioning; however, they have been omitted or given scant attention in most biodiversity policy documents, management plans, and formal conservation schedules throughout the world. This oversight may be due to a general lack of awareness in the scientific community and compounded by a scarcity of mycology-associated curricula at the tertiary level and a lack of mycologists in research institutions. Although molecular techniques advance the systematic cataloging of fungi and facilitate insights into fungal communities, the scarcity of professional mycologists in the environmental sciences hampers conservation efforts. Conversely, citizen science initiatives are making significant contributions to the mycology discipline by increasing awareness and extending the scope of fungal surveys. Future research by professional and amateur mycologists into the distribution of fungi and their function in ecosystems will help identify wider and more effective conservation goals.

Orthopaedic Surgeon Density in South Africa.

BACKGROUND: In the era of global surgery, there are limited data regarding the available surgical workforce in South Africa. METHODS: This aim of this study was to determine the orthopaedic surgeon density in South Africa. This involved a quantitative descriptive analysis of all registered specialist orthopaedic surgeons in South Africa, using data collected from various professional societal national databases. RESULTS: The results showed 1.63 orthopaedic surgeons per 100,000 population. The vast majority were male (95%) with under two-thirds (65%) being under the age of 55 years. The majority of the orthopaedic surgeons were found in Gauteng, followed by the Western Cape and Kwa-Zulu Natal. The majority of specialists reportedly worked either full time or part time in the private sector (95%), and the orthopaedic surgeon density per uninsured population (0.36) was far below that of the private sector (8.3). CONCLUSION: Interprovincial differences as well as intersectoral differences were marked indicating geographic and socio-economic maldistribution of orthopaedic surgeons. This parallels previous studies which looked at other surgical sub-disciplines in South Africa. Addressing this maldistribution requires concerted efforts to expand public sector specialist posts as well as quantifying the burden of orthopaedic disease in both private and public sectors before recommendations can be made regarding workforce allocation in the future. LEVEL OF EVIDENCE: IV.

Structural Transition of Trichocyte Keratin Intermediate Filaments During Development in the Hair Follicle.

The intermediate filaments (IF) in trichocyte (hard α-) keratin are unique amongst the various classes of IF in having not one but two topologically-distinct structures. The first is formed at an early stage of hair development in a reducing environment within the cells in the lower part of the follicle. The second structure occurs at a later stage of hair development in the upper part of the follicle, where there is a transition to an oxidizing environment. Crosslinking studies reveal that molecular slippage occurs within the IF upon oxidation and that this results in many cysteine residues lying in near axial alignment, thereby facilitating disulphide bond formation. The disulphide bonds so formed stabilize the assembly of IF molecules and convert the keratin fibre into a tough, resilient and insoluble structure suitable for its function in vivo as a thermo-regulator and a protector of the animal against its external environment.

Filamentous Structure of Hard ß-Keratins in the Epidermal Appendages of Birds and Reptiles.

The structures of avian and reptilian epidermal appendages, such as feathers, claws and scales, have been modelled using X-ray diffraction and electron microscopy data, combined with sequence analyses. In most cases, a family of closely related molecules makes up the bulk of the appendage, and each of these molecules contains a central ß-rich 34-residue segment, which has been identified as the principal component of the framework of the 3.4 nm diameter filaments. The N- and C-terminal segments form the matrix component of the filament/matrix complex. The 34-residue ß-rich central domains occur in pairs, related by either a parallel dyad or a perpendicular dyad axis, and form a ß-sandwich stabilized by apolar interactions. They are also twisted in a right-handed manner. In feather, the filaments are packed into small sheets and it is possible to determine their likely orientation within the sheets from the low-angle X-ray diffraction data. The physical properties of the various epidermal appendages can be related to the amino acid sequence and composition of defined molecular segments characteristic of the chains concerned.

Trichocyte Keratin-Associated Proteins (KAPs).

The trichocyte (hard α-) keratins are epidermal appendages (hair, wool, hoof, horn, claw, baleen and quill) with a classic filament-matrix composite structure. In human hair, for example, keratin intermediate filaments (IF) of diameter 7.5 nm are embedded in a matrix formed from at least 89 different types of keratin-associated proteins (KAPs). The latter fall into three families, generally defined in terms of their cysteine residue or glycine plus tyrosine residue content. The KAPs, which infiltrate the space between the IF, are recognized as having especially important roles in the organisation of the IF into macrofibrils, in determining some of the most important physical attributes of the fully-keratinised hair fibre, including its hardness, toughness and pliability, and in linking IF to one another, either directly or indirectly, with a resultant increase in durability and resistance to degradation by microorganisms. Sequence data for many KAPs are now available, and repeating motifs of varying extent have been observed in a number of them. Little, however, is known about their three-dimensional structures, though modelling has indicated that some local structural regularity is likely to exist. Current data suggest that the KAPs in vivo may adopt a variety of energetically-similar conformations stabilized predominantly by intramolecular disulfide bonds. The role of KAPs in hair diseases relates more to modulation in gene expression than to point mutations, in contrast to that observed for the IF proteins.

Structural Hierarchy of Trichocyte Keratin Intermediate Filaments.

Although trichocyte keratins (hair, wool, quill, claw) have been studied since the 1930s it is only over the last 30 years or so that major advances have been made in our understanding of the complex structural hierarchy of the filamentous component of this important filament-matrix composite. A variety of techniques, including amino acid sequence analysis, computer modelling, X-ray fibre diffraction and protein crystallography, various forms of electron microscopy, and crosslinking methods have now combined to reveal much of the structural detail. The heterodimeric structure of the keratin molecule is clear, as are the highly-specific modes by which these molecules aggregate to form functionally viable IF. The observation that hair keratin can adopt not one but two structurally-distinct conformations, one formed in the living cells at the base of the hair follicle in a reducing environment and the second in the fully differentiated hair in dead cells in an oxidized state, was unexpected but has major implications for the mechanism of hair growth. Insights have also been made into the mechanism of the uppermost level of hair superstructure, relating to the assembly of the IF in the paracortical and orthocortical macrofibrils.