Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events.

Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.

Global patterns of antigen receptor repertoire disruption across adaptive immune compartments in COVID-19.

Whereas pathogen-specific T and B cells are a primary focus of interest during infectious disease, we have used COVID-19 to ask whether their emergence comes at a cost of broader B cell and T cell repertoire disruption. We applied a genomic DNA-based approach to concurrently study the immunoglobulin-heavy (IGH) and T cell receptor (TCR) ß and δ chain loci of 95 individuals. Our approach detected anticipated repertoire focusing for the IGH repertoire, including expansions of clusters of related sequences temporally aligned with SARS-CoV-2-specific seroconversion, and enrichment of some shared SARS-CoV-2-associated sequences. No significant age-related or disease severity-related deficiencies were noted for the IGH repertoire. By contrast, whereas focusing occurred at the TCRß and TCRδ loci, including some TCRß sequence-sharing, disruptive repertoire narrowing was almost entirely limited to many patients aged older than 50 y. By temporarily reducing T cell diversity and by risking expansions of nonbeneficial T cells, these traits may constitute an age-related risk factor for COVID-19, including a vulnerability to new variants for which T cells may provide key protection.

Genetic Architecture of Salt Tolerance in Cowpea (Vigna unguiculata (L.) Walp.) at Seedling Stage Using a Whole Genome Resequencing Approach.

Cowpea (Vigna unguiculata (L.) Walp.) is a diploid legume crop used for human consumption, feed for livestock, and cover crops. Earlier reports have shown that salinity has been a growing threat to cowpea cultivation. The objectives of this study were to conduct a genome-wide association study (GWAS) to identify SNP markers and to investigate candidate genes for salt tolerance in cowpea. A total of 331 cowpea genotypes were evaluated for salt tolerance by supplying a solution of 200 mM NaCl in our previous work. The cowpea panel was genotyped using a whole genome resequencing approach, generating 14,465,516 SNPs. Moreover, 5,884,299 SNPs were used after SNP filtering. GWAS was conducted on a total of 296 cowpea genotypes that have high-quality SNPs. BLINK was used for conducting GWAS. Results showed (1) a strong GWAS peak on an 890-bk region of chromosome 2 for leaf SPAD chlorophyll under salt stress in cowpea and harboring a significant cluster of nicotinamide adenine dinucleotide (NAD) dependent epimerase/dehydratase genes such as Vigun02g128900.1, Vigun02g129000.1, Vigun02g129100.1, Vigun02g129200.1, and Vigun02g129500.1; (2) two GWAS peaks associated with relative tolerance index for chlorophyll were identified on chromosomes 1 and 2. The peak on chromosome 1 was defined by a cluster of 10 significant SNPs mapped on a 5 kb region and was located in the vicinity of Vigun01g086000.1, encoding for a GATA transcription factor. The GWAS peak on chromosome 2 was defined by a cluster of 53 significant SNPs and mapped on a 68 bk region of chromosome 2, and (3) the highest GWAS peak was identified on chromosome 3, and this locus was associated with leaf score injury. This peak was within the structure of a potassium channel gene (Vigun03g144700.1). To the best of our knowledge, this is one the earliest reports on the salt tolerance study of cowpea using whole genome resequencing data.

Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2.

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR-Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR-Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

Immunoglobulin light chain allelic inclusion in systemic lupus erythematosus.

The principles of allelic exclusion state that each B cell expresses a single light and heavy chain pair. Here, we show that B cells with both kappa and lambda light chains (Igκ and Igλ) are enriched in some patients with the systemic autoimmune disease systemic lupus erythematosus (SLE), but not in the systemic autoimmune disease control granulomatosis with polyangiitis. Detection of dual Igκ and Igλ expression by flow cytometry could not be abolished by acid washing or by DNAse treatment to remove any bound polyclonal antibody or complexes, and was retained after two days in culture. Both surface and intracytoplasmic dual light chain expression was evident by flow cytometry and confocal microscopy. We observed reduced frequency of rearrangements of the kappa-deleting element (KDE) in SLE and an inverse correlation between the frequency of KDE rearrangement and the frequency of dual light chain expressing B cells. We propose that dual expression of Igκ and Igλ by a single B cell may occur in some patients with SLE when this may be a consequence of reduced activity of the KDE.

Mitomycin-C in corneal surface excimer laser ablation techniques: a report by the American Academy of Ophthalmology.

OBJECTIVE: To review the published literature assessing the efficacy and safety of mitomycin-C (MMC) as an adjunctive treatment in corneal surface excimer laser ablation procedures. METHODS: Literature searches of the PubMed and Cochrane Library databases were last conducted on August 19, 2014, without language or date limitations. The searches retrieved a total of 239 references. Of these, members of the Ophthalmic Technology Assessment Committee Refractive Management/Intervention Panel selected 26 articles that were considered to be of high or medium clinical relevance, and the panel methodologist rated each article according to the strength of evidence. Ten studies were rated as level I evidence, 5 studies were rated as level II evidence, and the remaining 11 studies were rated as level III evidence. RESULTS: The majority of the articles surveyed in this report support the role of MMC as an adjunctive treatment in surface ablation procedures. When MMC is applied in the appropriate concentration and confined to the central cornea, the incidence of post-surface ablation haze is decreased. Although a minority of studies that evaluated endothelial cell density (ECD) reported an MMC-related decrease in ECD, no clinical adverse outcomes were reported. CONCLUSIONS: Over the past 15 years, the use of MMC during surgery in surface ablation has become widespread. There is good evidence of the effectiveness of MMC when used intraoperatively as prophylaxis against haze in higher myopic ablations. Although there are reports of decreased endothelial counts after the administration of MMC during surgery, the clinical significance of this finding remains uncertain, because no adverse outcomes were reported with as much as 5 years of follow-up. Optimal dosage, effectiveness as prophylaxis in lower myopic and hyperopic ablations, and long-term safety, particularly in eyes with reduced corneal endothelial cell counts from prior intraocular surgery, have yet to be established.

Femtosecond lasers for LASIK flap creation: a report by the American Academy of Ophthalmology.

OBJECTIVE: To review the published literature to assess the safety, efficacy, and predictability of femtosecond lasers for the creation of corneal flaps for LASIK; to assess the reported outcomes of LASIK when femtosecond lasers are used to create corneal flaps; and to compare the differences in outcomes between femtosecond lasers and mechanical microkeratomes. METHODS: Literature searches of the PubMed and Cochrane Library databases were last conducted on October 12, 2011, without language or date limitations. The searches retrieved a total of 636 references. Of these, panel members selected 58 articles that they considered to be of high or medium clinical relevance, and the panel methodologist rated each article according to the strength of evidence. Four studies were rated as level I evidence, 14 studies were rated as level II evidence, and the remaining studies were rated as level III evidence. RESULTS: The majority of published studies evaluated a single laser platform. Flap reproducibility varied by device and the generation of the device. Standard deviations in flap thicknesses ranged from 4 to 18.4 µm. Visual acuities and complications reported with LASIK flaps created using femtosecond lasers are within Food and Drug Administration safety and efficacy limits. Of all complications, diffuse lamellar keratitis is the most common after surgery but is generally mild and self-limited. Corneal sensation was reported to normalize by 1 year after surgery. Unique complications of femtosecond lasers included transient light-sensitivity syndrome, rainbow glare, opaque bubble layer, epithelial breakthrough of gas bubbles, and gas bubbles within the anterior chamber. CONCLUSIONS: Available evidence (levels I and II) indicates that femtosecond lasers are efficacious devices for creating LASIK flaps, with accompanying good visual results. Overall, femtosecond lasers were found to be as good as or better than mechanical microkeratomes for creating LASIK flaps. There are unique complications that can occur with femtosecond lasers, and long-term follow-up is needed to evaluate the technology fully.

Wavefront-guided PRK treatment of myopia using a refractive aberrometer.

PURPOSE: To evaluate the safety, effectiveness, and patient-reported outcomes of wavefront-guided photorefractive keratectomy (PRK) for the correction of myopic refractive errors with and without astigmatism. SETTING: U.S. multicenter study. DESIGN: Prospective, nonrandomized clinical investigation. METHODS: 334 eyes (167 patients) underwent wavefront-guided PRK with the STAR S4 IR Excimer Laser System. Patients had preoperative myopic refractive errors with sphere up to -8.00 diopters (D) and cylinder up to -4.00 D with a maximum spherical equivalent (SE) of -10.00 D. All eyes were targeted for emmetropia, and treatment plans were derived from the iDESIGN system wavefront measurements. RESULTS: At 6 months, the point of refractive stability, 99.4% of eyes achieved 20/20 or better uncorrected distance visual acuity (UDVA), 92% of eyes achieved 20/16 or better UDVA, 85.5% of eyes achieved manifest refraction SE (MRSE) within 0.50 D of target, mean SE was -0.06, and less than 1% of eyes lost more than 2 lines of corrected distance visual acuity. Glare and halos occurred with similar or lower frequencies at 6 months vs preoperative. Results from the National Eye Institute Refractive Error Quality of life questionnaire showed statistically significant improvements at 6 months vs preoperative across most measures of vision-related functioning and well-being. Approximately 98% of patients reported high satisfaction with their overall vision at 6 months. CONCLUSIONS: Wavefront-guided PRK with the iDESIGN aberrometer was safe, effective, and predictable for the correction of low to moderate myopia with and without astigmatism and led to high patient satisfaction.

Quality of Vision Following LASIK and PRK-MMC for Treatment of Myopia.

INTRODUCTION: Femtosecond-assisted thin flap, laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy with mitomycin-C (PRK-MMC) are the two most common refractive surgical procedures used to enhance visual capability in the U.S military. The purposes of the study were to examine effects on quality of vision following LASIK and PRK-MMC using a novel computer-based quick contrast sensitivity function (qCSF) test. MATERIALS AND METHODS: This prospective clinical study included 58 active duty U.S. military service members who elected LASIK (n = 29) or PRK-MMC (n = 29) refractive surgery for myopia (nearsightedness) treatment. Monocular photopic and mesopic quality of vision of the right eyes in spectacle correction preoperatively and unaided right eyes at four postoperative follow-up visits (1 week, 2 weeks, 1 month, and 3 months) were examined using the qCSF device. Two qCSF parameters, area under a log CSF (AULCSF) between 1.5 and 18 cycles per degree, and CSF cutoff acuity (CSF Acuity), were collected using a 50-trial setting at a 4-m testing distance. General linear model (GLM) Repeated-measures Analysis of Covariance was used to examine effects on quality of vision following LASIK and PRK-MMC. Post hoc testing with Bonferroni correction was used for pairwise comparisons, and preoperative cylinder refraction was used as a covariate. Two-tailed independent t-test was used to compare preoperative and postoperative parameters between LASIK and PRK-MMC. Pearson's correlation, Bland-Altman plots, and multiple linear regression were used to examine the relationship among the qCSF and other vision tests. RESULTS: Quality of vision, AULCSF, and CSF Acuity returned to the preoperative baseline at postoperative 2 weeks under mesopic condition and at postoperative 1 month under photopic condition after PRK-MMC. In comparison, photopic and mesopic quality of vision were not significantly different from the baseline at any of the four postoperative visits following LASIK. Changes of CSF Acuity from the baseline after LASIK were significantly better under photopic than mesopic condition by 0.067 ± 0.014 logarithm of the minimum angle of resolution (logMAR); P < .001). Quality of vision was not significantly different between the LASIK and PRK-MMC groups at postoperative 1 and 3 months. When predicting photopic AULCSF (overall model fit R2 = 0.47), 5% contrast acuity (beta = -0.43), visual acuity in 100% contrast (beta = -0.18), and residual refraction in spherical equivalent (beta = 0.20) were significant predictors (P ≤ .001), while high-order aberrations (beta = -0.07, P = .22) were not significant predictors. Visual acuity (beta = -0.12, P = .07) and high-order aberrations (beta = -0.04, P = .58) were not significant predictors of mesopic AULCSF. Bland-Altman plots show that photopic CSF Acuity and visual acuity had a mean difference of 0.19 ± 0.01 logMAR with limits of agreement (LOAs) at -0.01 and 0.39 logMAR. Photopic CSF Acuity and 5% contrast acuity had a mean difference of -0.06 ± 0.01 logMAR with LOAs at -0.33 and 0.21 logMAR. CONCLUSION: Quality of vision recovers at postoperative 1 week after LASIK and at postoperative 1 month after PRK-MMC. The standard black-on-white high-contrast, chart-based visual acuity test is weak in predicting quality of vision. The qCSF detects mild-to-moderate visual changes and is suitable for quality of vision assessment following refractive eye surgery.

Diagnosis and management of postrefractive surgery ectasia.

Postrefractive surgery ectasia is a serious, sight-threatening complication seen after the following procedures: laser in situ keratomileusis, photorefractive keratectomy, small-incision lenticule extraction, radial keratotomy, and/or arcuate keratotomy. Specific risk factors may include age, corneal thickness, degree of refractive error, corneal topographic changes including irregular astigmatism, percent tissue ablation, and residual stromal bed. Biomarkers may be a new option to help indicate who is at greatest risk for ectasia. Visual aids including spectacles or contacts lenses are often required to achieve optimal vision. Collagen crosslinking is the only treatment believed to stop progression of ectasia and prevent keratoplasty. Other surgical options may include topography-guided phototherapeutic keratectomy and intrastromal corneal ring segments. Ultimately, an "ounce of prevention is a pound of cure," so careful preoperative screening and ultimately offering the safest and most effective treatments for patients is arguably the most important job of the refractive surgeon.

Raman spectroscopy-based diagnostics of water deficit and salinity stresses in two accessions of peanut.

Water deficit and salinity are two major abiotic stresses that have tremendous effect on crop yield worldwide. Timely identification of these stresses can help limit associated yield loss. Confirmatory detection and identification of water deficit stress can also enable proper irrigation management. Traditionally, unmanned aerial vehicle (UAV)-based imaging and satellite-based imaging, together with visual field observation, are used for diagnostics of such stresses. However, these approaches can only detect salinity and water deficit stress at the symptomatic stage. Raman spectroscopy (RS) is a noninvasive and nondestructive technique that can identify and detect plant biotic and abiotic stress. In this study, we investigated accuracy of Raman-based diagnostics of water deficit and salinity stresses on two greenhouse-grown peanut accessions: tolerant and susceptible to water deficit. Plants were grown for 76 days prior to application of the water deficit and salinity stresses. Water deficit treatments received no irrigation for 5 days, and salinity treatments received 1.0 L of 240-mM salt water per day for the duration of 5-day sampling. Every day after the stress was imposed, plant leaves were collected and immediately analyzed by a hand-held Raman spectrometer. RS and chemometrics could identify control and stressed (either water deficit or salinity) susceptible plants with 95% and 80% accuracy just 1 day after treatment. Water deficit and salinity stressed plants could be differentiated from each other with 87% and 86% accuracy, respectively. In the tolerant accessions at the same timepoint, the identification accuracies were 66%, 65%, 67%, and 69% for control, combined stresses, water deficit, and salinity stresses, respectively. The high selectivity and specificity for presymptomatic identification of abiotic stresses in the susceptible line provide evidence for the potential of Raman-based surveillance in commercial-scale agriculture and digital farming.

A Proof-of-Principle Study of Non-invasive Identification of Peanut Genotypes and Nematode Resistance Using Raman Spectroscopy.

Identification of peanut cultivars for distinct phenotypic or genotypic traits whether using visual characterization or laboratory analysis requires substantial expertise, time, and resources. A less subjective and more precise method is needed for identification of peanut germplasm throughout the value chain. In this proof-of-principle study, the accuracy of Raman spectroscopy (RS), a non-invasive, non-destructive technique, in peanut phenotyping and identification is explored. We show that RS can be used for highly accurate peanut phenotyping via surface scans of peanut leaves and the resulting chemometric analysis: On average 94% accuracy in identification of peanut cultivars and breeding lines was achieved. Our results also suggest that RS can be used for highly accurate determination of nematode resistance and susceptibility of those breeding lines and cultivars. Specifically, nematode-resistant peanut cultivars can be identified with 92% accuracy, whereas susceptible breeding lines were identified with 81% accuracy. Finally, RS revealed substantial differences in biochemical composition between resistant and susceptible peanut cultivars. We found that resistant cultivars exhibit substantially higher carotenoid content compared to the susceptible breeding lines. The results of this study show that RS can be used for quick, accurate, and non-invasive identification of genotype, nematode resistance, and nutrient content. Armed with this knowledge, the peanut industry can utilize Raman spectroscopy for expedited breeding to increase yields, nutrition, and maintaining purity levels of cultivars following release.

Colour vision classification - comparing CAD and CIE 143:2001 International recommendations for colour vision requirements in transport.

PURPOSE: To evaluate the colour vision severity classification standard 'CIE 143:2001 International recommendations for colour vision requirements in transport' (CIE 143:2001), which has become out of date because of the lack of commercial availability of required colour vision tests. METHODS: One-hundred-five subjects had colour vision tested and colour vision severity classified according to a modified CIE 143:2001 algorithm that included pseudoisochromatic plates (Ishihara's test and Hardy Rand Rittler (HRR) 4th edition), Optec 900 lantern and Farnsworth D-15. Subject's results and colour vision severity classification were compared to performance and colour vision severity classification on the computerized 'Colour Assessment and Diagnosis' (CAD) test. RESULTS: According to CIE 143:2001, using Ishihara's test, Optec lantern and Farnsworth D 15, 11 subjects (10%) were category I (normal), 16 (15%) were category II (mild), 48 (46%) were category III (poor), and 30 (29%) were category IV (severe). Classified by CAD score, 10 (10%) were category I, 11 (10%) were category II, 41 (39%) were category III, and 43 (41%) were category IV. The correlation between the two estimates of the severity of colour vision loss (i.e. CIE 143:2001 and CAD) was high, with a Kendall's Tau test of 0.81 (τ = 0.81 p < 0.001). A suggested CIE 143:2001 classification including new CAD score limits improves the classification correlation to 0.90 (τ = 0.90 p < 0.001) for all diagnoses. CONCLUSION: The colour vision severity classification standard 'CIE 143:2001 International recommendations for colour vision requirements in transport', has not implemented new diagnostic tools with better accuracy. We propose three possible revisions to the CIE 143:2001 algorithm, based on the availability of CAD: (1) Replacing the current CIE 143:2001 algorithm using new CAD threshold limits, (2) Use of CAD as a secondary test to Ishihara's test and HRR or (3) Revising the current CIE 143:2001 algorithm using Ishihara's test, HRR, Optec 900 and FD15.

LASIK flap stability after severe ocular injury.

PURPOSE: To report two cases of LASIK flap stability after severe ocular trauma.Observations: Two patients suffered open globe injuries several years after undergoing uneventful LASIK with femtosecond laser corneal flap. Both underwent primary ruptured globe repair, during which no LASIK flap dislocation was identified. Histopathologic examination of one of the cornea specimens confirmed an intact LASIK flap. CONCLUSION AND IMPORTANCE: In these cases, the femtosecond LASIK flap remained in place despite significant injury to the cornea. The presence of a femtosecond LASIK flap did not complicate surgical management of the injury, and did not contribute to the patient's loss of BCVA.

Raman Spectroscopy Enables Non-Invasive Identification of Peanut Genotypes and Value-Added Traits.

Identification of specific genotypes can be accomplished by visual recognition of their distinct phenotypical appearance, as well as DNA analysis. Visual identification (ID) of species is subjective and usually requires substantial taxonomic expertise. Genotyping and sequencing are destructive, time- and labor-consuming. In this study, we investigate the potential use of Raman spectroscopy (RS) as a label-free, non-invasive and non-destructive analytical technique for the fast and accurate identification of peanut genotypes. We show that chemometric analysis of peanut leaflet spectra provides accurate identification of different varieties. This same analysis can be used for prediction of nematode resistance and oleic-linoleic oil (O/L) ratio. Raman-based analysis of seeds provides accurate genotype identification in 95% of samples. Additionally, we present data on the identification of carbohydrates, proteins, fiber and other nutrients obtained from spectroscopic signatures of peanut seeds. These results demonstrate that RS allows for fast, accurate and non-invasive screening and selection of plants which can be used for precision breeding.

A dynamic COVID-19 immune signature includes associations with poor prognosis.

Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.

Author Correction: A dynamic COVID-19 immune signature includes associations with poor prognosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.