Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres.

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five- to sixfold ploidy increase approximately 60 million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1-2 Myr ago, conferred about 30-36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum A(t)D(t) (in which 't' indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.

Visible-light optical coherence tomography platform for the characterization of the skin barrier.

We demonstrate a free-space, trolley-mountable Fourier domain visible-light optical coherence tomography (OCT) system for studying the stratum corneum in non-palmar human skin. An axial resolution of 1 µm in tissue and at least -75 dB sensitivity have been achieved. High-quality B-scans, containing 1600 A-scans, are acquired at a rate of 39 Hz. Images from the dorsal hand, ventral wrist and ventral forearm areas are obtained, with a clearly resolved stratum corneum layer (typically 5-15 µm thick) presenting as a hypoechogenic dark layer below the bright entrance signal, similar to that found in palmar skin with traditional OCT systems. We find that the appearance of the stratum corneum layer strongly depends on its water content, becoming brighter after occlusive hydration.

Potential application of PS-OCT in the safety assessment of non-steroidal topical creams for atopic dermatitis treatment.

Crisaborole 2% ointment is a non-steroidal treatment for mild-moderate atopic dermatitis (AD) and may produce fewer adverse effects than topical corticosteroids (TCS). We used PS-OCT to quantify dermal collagen at baseline and after 29 days of treatment with crisaborole and betamethasone valerate (BMV), in 32 subjects. PS-OCT detected a mean increase 1 × 10-6, 95% CI (6.3, 1.37) × 10-6 in dermal birefringence following TCS use (p < 0.0001, ad-hoc, not powered), whereas a change of -4 × 10-6, 95% CI (-32, 24) × 10-6 was detected for crisaborole (p = 0.77, ad-hoc, not powered). These results could suggest a differential effect on dermal collagen between the two compounds. PS-OCT may thus find an important role in safety assessment of novel AD treatment' and larger trials are warranted.

Development and mapping of SNP assays in allotetraploid cotton.

A narrow germplasm base and a complex allotetraploid genome have made the discovery of single nucleotide polymorphism (SNP) markers difficult in cotton (Gossypium hirsutum). To generate sequence for SNP discovery, we conducted a genome reduction experiment (EcoRI, BafI double digest, followed by adapter ligation, biotin-streptavidin purification, and agarose gel separation) on two accessions of G. hirsutum and two accessions of G. barbadense. From the genome reduction experiment, a total of 2.04 million genomic sequence reads were assembled into contigs with an N(50) of 508 bp and analyzed for SNPs. A previously generated assembly of expressed sequence tags (ESTs) provided an additional source for SNP discovery. Using highly conservative parameters (minimum coverage of 8× at each SNP and 20% minor allele frequency), a total of 11,834 and 1,679 non-genic SNPs were identified between accessions of G. hirsutum and G. barbadense in genome reduction assemblies, respectively. An additional 4,327 genic SNPs were also identified between accessions of G. hirsutum in the EST assembly. KBioscience KASPar assays were designed for a portion of the intra-specific G. hirsutum SNPs. From 704 non-genic and 348 genic markers developed, a total of 367 (267 non-genic, 100 genic) mapped in a segregating F(2) population (Acala Maxxa × TX2094) using the Fluidigm EP1 system. A G. hirsutum genetic linkage map of 1,688 cM was constructed based entirely on these new SNP markers. Of the genic-based SNPs, we were able to identify within which genome ('A' or 'D') each SNP resided using diploid species sequence data. Genetic maps generated by these newly identified markers are being used to locate quantitative, economically important regions within the cotton genome.

Imaging striae distensae: a comparison between PS-OCT and digital dermoscopy.

Stretch marks or striae distensae (SD) cause emotional distress and negatively affect the psychological well-being of patients. We investigate and compare two methods for quantifying the severity of SD: visual scoring of images captured using a clinical visible-light dermatological camera (C-Cube, Pixience Inc) and measuring the local birefringence of skin using polarization-sensitive optical coherence tomography (PS-OCT). Data on skin visually affected by SD and visually normal skin were collected from 19 human volunteers. Our results show a weak correlation between visual scores of the C-Cube images and the birefringence values obtained from the PS-OCT system. SD datasets have a significantly larger birefringence values compared to visually normal datasets.

Genetic Analysis of the Transition from Wild to Domesticated Cotton (Gossypium hirsutum L.).

The evolution and domestication of cotton is of great interest from both economic and evolutionary standpoints. Although many genetic and genomic resources have been generated for cotton, the genetic underpinnings of the transition from wild to domesticated cotton remain poorly known. Here we generated an intraspecific QTL mapping population specifically targeting domesticated cotton phenotypes. We used 466 F2 individuals derived from an intraspecific cross between the wild Gossypium hirsutum var. yucatanense (TX2094) and the elite cultivar G. hirsutum cv. Acala Maxxa, in two environments, to identify 120 QTL associated with phenotypic changes under domestication. While the number of QTL recovered in each subpopulation was similar, only 22 QTL were considered coincident (i.e., shared) between the two locations, eight of which shared peak markers. Although approximately half of QTL were located in the A-subgenome, many key fiber QTL were detected in the D-subgenome, which was derived from a species with unspinnable fiber. We found that many QTL are environment-specific, with few shared between the two environments, indicating that QTL associated with G. hirsutum domestication are genomically clustered but environmentally labile. Possible candidate genes were recovered and are discussed in the context of the phenotype. We conclude that the evolutionary forces that shape intraspecific divergence and domestication in cotton are complex, and that phenotypic transformations likely involved multiple interacting and environmentally responsive factors.

Attenuation of stripe artifacts in optical coherence tomography images through wavelet-FFT filtering.

The use of polarization-maintaining (PM) fibers for polarization-sensitive optical coherence tomography (PS-OCT) can result in numerous image artifacts which degrade the reliability of birefringence measurements. Similar artifacts can also arise in conventional OCT, due to stray reflections from optical surfaces, a problem which is increasing in tandem with the steady rise in source coherence lengths. Here, a recently presented wavelet-FFT filter[Opt. Express17(10), 8567 (2009).] is combined with surface flattening displacement fields in order to suppress ghost artifacts following either a duplicate or inverse profile to that of the sample surface. In addition, horizontal coherence stripes originating from Fresnel reflections of optical components are suppressed in order to facilitate accurate surface detection. The result is an improved visualization of the phase-retardance profile within tissue, which may improve the reliability of curve-fitting methods for localized birefringence estimation. While the results are presented with a focus towards PS-OCT, the filtering method can also be applied to the removal of stray reflection artifacts in conventional OCT images.

Quantitative Assessment of the Anatomical Footprint of the C1 Pedicle Relative to the Lateral Mass: A Guide for C1 Lateral Mass Fixation.

STUDY DESIGN: Anatomic study. OBJECTIVES: To determine the relationship of the anatomical footprint of the C1 pedicle relative to the lateral mass (LM). METHODS: Anatomic measurements were made on fresh frozen human cadaveric C1 specimens: pedicle width/height, LM width/height (minimum/maximum), LM depth, distance between LM's medial aspect and pedicle's medial border, distance between LM's lateral aspect to pedicle's lateral border, distance between pedicle's inferior aspect and LM's inferior border, distance between arch's midline and pedicle's medial border. The percentage of LM medial to the pedicle and the distance from the center of the LM to the pedicle's medial wall were calculated. RESULTS: A total of 42 LM were analyzed. The C1 pedicle's lateral aspect was nearly confluent with the LM's lateral border. Average pedicle width was 9.0 ± 1.1 mm, and average pedicle height was 5.0 ± 1.1 mm. Average LM width and depth were 17.0 ± 1.6 and 17.2 ± 1.6 mm, respectively. There was 6.9 ± 1.5 mm of bone medial to the medial C1 pedicle, which constituted 41% ± 9% of the LM's width. The distance from C1 arch's midline to the medial pedicle was 13.5 ± 2.0 mm. The LM's center was 1.6 ± 1 mm lateral to the medial pedicle wall. There was on average 3.5 ± 0.6 mm of the LM inferior to the pedicle inferior border. CONCLUSIONS: The center of the lateral mass is 1.6 ± 1 mm lateral to the medial wall of the C1 pedicle and approximately 15 mm from the midline. There is 6.9 ± 1.5 mm of bone medial to the medial C1 pedicle. Thus, the medial aspect of C1 pedicle may be used as an anatomic reference for locating the center of the C1 LM for screw fixation.

Sub-clinical assessment of atopic dermatitis severity using angiographic optical coherence tomography.

Measurement of sub-clinical atopic dermatitis (AD) is important for determining how long therapies should be continued after clinical clearance of visible AD lesions. An important biomarker of sub-clinical AD is epidermal hypertrophy, the structural measures of which often make optical coherence tomography (OCT) challenging due to the lack of a clearly delineated dermal-epidermal junction in AD patients. Alternatively, angiographic OCT measurements of vascular depth and morphology may represent a robust biomarker for quantifying the severity of clinical and sub-clinical AD. To investigate this, angiographic data sets were acquired from 32 patients with a range of AD severities. Deeper vascular layers within skin were found to correlate with increasing clinical severity. Furthermore, for AD patients exhibiting no clinical symptoms, the superficial plexus depth was found to be significantly deeper than healthy patients at both the elbow (p = 0.04) and knee (p<0.001), suggesting that sub-clinical changes in severity can be detected. Furthermore, the morphology of vessels appeared altered in patients with severe AD, with significantly different vessel diameter, length, density and fractal dimension. These metrics provide valuable insight into the sub-clinical severity of the condition, allowing the effects of treatments to be monitored past the point of clinical remission.

Racial/ethnic and age disparities in HIV prevalence and disease progression among men who have sex with men in the United States.

OBJECTIVES: We examined HIV diagnosis rates and disease progression among men who have sex with men (MSM) according to race/ethnicity and age. METHODS: Using data obtained from the national HIV/AIDS surveillance system, we examined trends in HIV diagnosis rates for 2001 through 2004 using Poisson regression. We used a standardized Kaplan-Meier method to determine differences in time of progression from HIV to AIDS and AIDS survival. RESULTS: HIV diagnosis rates were higher for Black and Hispanic than for White MSM, but trends within age groups from 2001 to 2004 did not differ by race/ethnicity. Diagnosis rates increased among MSM aged 13 to 19 years (14% per year), 20 to 24 years (13%), 25 to 29 years, and 40 to 54 years (3%-6%; P< or = .01 for each). The percentage of MSM who did not have AIDS 3 years after HIV diagnosis was lower among Black (66.8%; 95% confidence interval [CI]=66.1, 67.4) and Hispanic (68.1%; 95% CI=67.5, 68.8) than among White MSM (74.7%; 95% CI=74.2, 75.1). Three-year survival after AIDS diagnosis was lower for Black than for White or Hispanic MSM. CONCLUSIONS: HIV prevention efforts should target young and middle-aged MSM and must offer early diagnosis and treatment for all MSM.

Vascular patterning of subcutaneous mouse fibrosarcomas expressing individual VEGF isoforms can be differentiated using angiographic optical coherence tomography.

Subcutaneously implanted experimental tumors in mice are commonly used in cancer research. Despite their superficial location, they remain a challenge to image non-invasively at sufficient spatial resolution for microvascular studies. Here we evaluate the capabilities of optical coherence tomography (OCT) angiography for imaging such tumors directly through the murine skin in-vivo. Data sets were collected from mouse tumors derived from fibrosarcoma cells genetically engineered to express only single splice variant isoforms of vascular endothelial growth factor A (VEGF); either VEGF120 or VEGF188 (fs120 and fs188 tumors respectively). Measured vessel diameter was found to be significantly (p<0.001) higher for fs120 tumors (60.7 ± 4.9µm) compared to fs188 tumors (45.0 ± 4.0µm). The fs120 tumors also displayed significantly higher vessel tortuosity, fractal dimension and density. The ability to differentiate between tumor types with OCT suggests that the visible abnormal vasculature is representative of the tumor microcirculation, providing a robust, non-invasive method for observing the longitudinal dynamics of the subcutaneous tumor microcirculation.

A new mode of contrast in biological second harmonic generation microscopy.

Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can produce large changes in the wavelength-dependency of epi-generated SHG. However, in the case of forward SHG, although the same changes impact upon absolute intensity at a given wavelength, they have very little effect on wavelength-dependency beyond the expected monotonic fall. We also propose that this difference between forward and epi-generated SHG provides an explanation for many of the wavelength-dependency discrepancies in the published literature.

In vivo measurement of skin surface strain and sub-surface layer deformation induced by natural tissue stretching.

Stratum corneum and epidermal layers change in terms of thickness and roughness with gender, age and anatomical site. Knowledge of the mechanical and tribological properties of skin associated with these structural changes are needed to aid in the design of exoskeletons, prostheses, orthotics, body mounted sensors used for kinematics measurements and in optimum use of wearable on-body devices. In this case study, optical coherence tomography (OCT) and digital image correlation (DIC) were combined to determine skin surface strain and sub-surface deformation behaviour of the volar forearm due to natural tissue stretching. The thickness of the epidermis together with geometry changes of the dermal-epidermal junction boundary were calculated during change in the arm angle, from flexion (90°) to full extension (180°). This posture change caused an increase in skin surface Lagrange strain, typically by 25% which induced considerable morphological changes in the upper skin layers evidenced by reduction of epidermal layer thickness (20%), flattening of the dermal-epidermal junction undulation (45-50% reduction of flatness being expressed as Ra and Rz roughness profile height change) and reduction of skin surface roughness Ra and Rz (40-50%). The newly developed method, DIC combined with OCT imaging, is a powerful, fast and non-invasive methodology to study structural skin changes in real time and the tissue response provoked by mechanical loading or stretching.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

Syphilis increases HIV viral load and decreases CD4 cell counts in HIV-infected patients with new syphilis infections.

BACKGROUND: Syphilitic ulcers are known to facilitate the transmission of HIV infection, but the effect of syphilis infection on HIV viral loads and CD4 cell counts is poorly understood. METHODS: We abstracted medical records for HIV-infected male syphilis patients seen at three clinics in San Francisco and Los Angeles from January 2001 to April 2003. We compared plasma HIV-RNA levels and CD4 cell counts during syphilis infection with those before syphilis infection and after syphilis treatment, using the Wilcoxon signed rank test. RESULTS: Fifty-two HIV-infected men with primary or secondary syphilis had HIV viral load and CD4 cell count data available for analysis; 30 (58%) were receiving antiretroviral therapy. Viral loads were higher during syphilis compared with pre-syphilis levels by a mean of 0.22 RNA log10 copies/ml (P = 0.02) and were lower by a mean of -0.10 RNA log10 copies/ml (P = 0.52) after syphilis treatment. CD4 cell counts were lower during syphilis infection than before by a mean of -62 cells/mm3 (P = 0.04), and were higher by a mean of 33 cells/mm3 (P = 0.23) after syphilis treatment. Increases in the HIV viral load and reductions in the CD4 cell count were most substantial in men with secondary syphilis and those not receiving antiretroviral therapy. CONCLUSION: Syphilis infection was associated with significant increases in the HIV viral load and significant decreases in the CD4 cell count. The findings underscore the importance of preventing and promptly treating syphilis in HIV-infected individuals.

Evaluation of a sensitive/less sensitive testing algorithm using the bioMérieux Vironostika-LS assay for detecting recent HIV-1 subtype B' or E infection in Thailand.

The performance of the bioMérieux Vironostika-LS EIA (less sensitive enzyme immunoassay) was assessed to detect recent seroconversion among injecting drug users (IDUs) in Bangkok, Thailand who were infected with either HIV-1 subtypes B' or E (also known as circulating recombinant form CRF01_AE). To evaluate the Vironostika-LS EIA in non-B subtypes, we collected longitudinal specimens (n = 796) from 115 IDUs (subtype B' infection, n = 24; subtype E infection, n = 91). After testing HIV-positive specimens with the Vironostika-LS EIA, standardized optical densities (SODs) were calculated using median values to determine the window period, which is the time from seroconversion on a standard EIA to seroconversion on the Vironostika-LS EIA for a given SOD, for either subtype. For an SOD cutoff of 1.0, Vironostika-LS EIA results showed a mean window period of 239 days (95% confidence interval [95% CI], 208-287 days) for subtype B' and 356 days (95% CI, 318-402 days) for subtype E in Thailand. This outcome demonstrates that the Vironostika-LS EIA has significantly different performance characteristics in detecting recent seroconversion between different HIV-1 subtypes. Accurate identification of recent infection and estimation of incidence for HIV-1 strains other than North American subtype B, using the Vironostika-LS EIA, requires knowledge of specimen subtype and use of appropriate cutoffs and mean window periods.

Quantitative detection of increasing HIV type 1 antibodies after seroconversion: a simple assay for detecting recent HIV infection and estimating incidence.

We have devised a simple enzyme immunoassay (EIA) that detects increasing levels of anti-HIV IgG after seroconversion and can be used for detecting recent HIV-1 infection. Use of a branched peptide that included gp41 immunodominant sequences from HIV-1 subtypes B, E, and D allowed similar detection of HIV-specific antibodies among various subtypes. Because of the competitive nature of the capture EIA, a gradual increase in the proportion of HIV-1-specific IgG in total IgG was observed for 2 years after seroconversion. This was in contrast to results obtained with the conventional EIA using the same antigen in solid phase, which plateaus soon after seroconversion. The assay was used to test 622 longitudinal specimens from 139 incident infections in the United States (subtype B) and in Thailand (subtypes B and E). The assay was also performed with an additional 8 M urea incubation step to assess the contribution of high-avidity antibodies. Normalized optical density (OD-n) was calculated (ODspecimen/ODcalibrator), using a calibrator specimen. An incremental analysis indicated that a cutoff of 1.0 OD-n and a seroconversion period of 160 days offered the best combination of sensitivity and specificity for classifying incident or long-term infections. The urea step increased the seroconversion period to 180 days with similar sensitivity and specificity. Separate analysis of B and E subtype specimens yielded the same optimal OD-n threshold and similar seroconversion periods. The assay was further validated in African specimens (subtypes A, C, and D) where the observed incidence was within 10% of the expected incidence. This assay should be useful for detecting recent HIV-1 infection and for estimating incidence among diverse HIV-1 subtypes worldwide.

HIV type 1 incidence estimates by detection of recent infection from a cross-sectional sampling of injection drug users in Bangkok: use of the IgG capture BED enzyme immunoassay.

Development of serologic tests to detect recent HIV-1 infection has generated worldwide interest in applying this approach to estimate incidence. We previously devised an IgG-capture BED-EIA (or BED-CEIA) that detects increasing levels of anti-HIV IgG following seroconversion to identify recent infection and to estimate incidence among persons infected with diverse HIV-1 subtypes worldwide. Injection drug users (IDUs; n = 1969) were screened in 1996 for participation in a prospective cohort study. Serum specimens from 594 IDUs were HIV-1 seropositive (30.2%) and were tested with the BED-CEIA. The proportion of recent infections and estimated incidence by different epidemiological risk factors were compared with incidence data measured from the prospective cohort. Of 594 HIV-1-seropositive specimens, 113 (19%) were identified as recent infections. Overall, the estimated annual incidence among persons screened was 17.3%/year (95% CI, 12.8-24.2%/year) compared with 9.0%/year (95% CI, 6.7-11.9%/year) measured from the prospective cohort during the same time period. Estimated incidence was higher among younger aged and unemployed IDUs as well as among those who injected more frequently, confirming previously reported risk factors from this prospective cohort. As persons screened from a cross-sectional sampling probably have higher risk for HIV than selected uninfected individuals who choose to participate and receive risk reduction counseling in a longitudinal cohort study, use of this or other serologic testing strategies to identify populations with high incidence (such as for HIV vaccine trials) may overestimate incidence measured from prospective cohorts.

Targeted amplicon sequencing (TAS): a scalable next-gen approach to multilocus, multitaxa phylogenetics.

Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach.