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Christopher Okunseri,1 Elaye Okunseri,1 Melissa Christine Fischer,1 Saba Noori Sadeghi,1 Qun Xiang,2 Aniko Szabo21Department of Clinical Services, School of Dentistry, Marquette University, Milwaukee, WI, USA; 2Division of Biostatistics, Institute of Health and Society, Medical College of Wisconsin, Milwaukee, WI, USAObjective: To determine whether the rates of nontraumatic dental condition (NTDC)-related emergency department (ED) visits are higher during the typical working hours of dental offices and lower during night hours, as well as the associated factors.Methods: We analyzed data from the National Hospital Ambulatory Medical Care Survey for 1997 through 2007 using multivariate binary and polytomous logistic regression adjusted for survey design to determine the effect of predictors on specified outcome variables.Results: Overall, 4,726 observations representing 16.4 million NTDC-related ED visits were identified. Significant differences in rates of NTDC-related ED visits were observed with 40%–50% higher rates during non-working hours and 20% higher rates on weekends than the overall average rate of 170 visits per hour. Compared with 19–33 year olds, subjects

Closely related species often use the same genes to adapt to similar environments. However, we know little about why such genes possess increased adaptive potential and whether this is conserved across deeper evolutionary lineages. Adaptation to climate presents a natural laboratory to test these ideas, as even distantly related species must contend with similar stresses. Here, we re-analyse genomic data from thousands of individuals from 25 plant species as diverged as lodgepole pine and Arabidopsis (~300 Myr). We test for genetic repeatability based on within-species associations between allele frequencies in genes and variation in 21 climate variables. Our results demonstrate significant statistical evidence for genetic repeatability across deep time that is not expected under randomness, identifying a suite of 108 gene families (orthogroups) and gene functions that repeatedly drive local adaptation to climate. This set includes many orthogroups with well-known functions in abiotic stress response. Using gene co-expression networks to quantify pleiotropy, we find that orthogroups with stronger evidence for repeatability exhibit greater network centrality and broader expression across tissues (higher pleiotropy), contrary to the 'cost of complexity' theory. These gene families may be important in helping wild and crop species cope with future climate change, representing important candidates for future study., (© 2024. The Author(s).)

Background: There is limited literature regarding equine immune mediated keratitis (IMMK) in Europe. North America-based publications describe minimal blepharospasm, rare corneal ulceration and no uveitis; clinical impression suggests these are seen in Europe., Objectives: Assess the prevalence of blepharospasm, corneal ulceration and uveitis and their impact on outcome in horses diagnosed with IMMK in Europe (UK and Finland)., Study Design: Retrospective case series., Methods: Clinical records of 94 horses with IMMK were evaluated. The UK and Finland populations were comparable; therefore, descriptive statistics were performed on combined data on subtypes of IMMK and clinical features. Odds ratios (OR) and confidence intervals (CI) were calculated for impact of blepharospasm, ulceration or presence of uveitis on the outcome of enucleation and treatment duration., Results: IMMK subtype was classified as 10/94 (10.6%) epithelial, 50/94 (53.2%) anterior stromal, 14/94 (14.9%) mid-stromal, 4/94 (4.25%) endothelial and 16/94 (17.0%) unrecorded. After excluding three horses with incidental corneal ulceration, blepharospasm was documented in 34/91 (37.4%), corneal ulceration in 26/91 (28.6%), and signs of uveitis in 23/91 (25.3%) horses. Increased odds of enucleation were significantly associated with the presence of blepharospasm (OR 5.5, 95% CI 1.6-19.4, p = 0.008) and signs of uveitis (OR 8.9, 95% CI 2.6-30.8, p < 0.001), but not corneal ulceration. The presence of blepharospasm, corneal ulceration or uveitis did not significantly alter the odds of ongoing medication., Main Limitations: Data were collected over a wide timeframe; the diagnosis was mainly made without histopathology; a broad definition of uveitis was used and there was a bias towards complicated cases being retained for follow-up., Conclusions: The clinical features of IMMK were similar between two European countries but differed to USA descriptions. Blepharospasm, corneal ulceration and signs of uveitis can occur with IMMK; presence of blepharospasm and uveitis increase the odds of enucleation., (© 2024 The Author(s). Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

In optical experiments, shutters are devices that open or close a path of light. They are often used to limit the duration of light exposure onto a target or onto a detector to reduce possible light-induced damage. Many commercial shutters are available for different applications - some provide very fast opening and closing times, some can handle large optical powers, and others allow for fail-safe operation. Many of these devices are costly and offer limited control options. Here we provide an open-source design for a low-cost, general purpose shutter system based on ubiquitous actuators (servo motors or solenoids) that are connected to an Arduino-based controller. Several shutters can be controlled by one controller, further reducing system cost. The state of the shutters can be controlled via a display built into the controller, by serial commands via USB, or by electrical control lines. The use of a microcontroller makes the shutter controller adaptable - only control options that are used need to be included, and the design accommodates a selection of display and actuator options. We provide designs for all required components, including 3D print files for the actuator holders and cases, the Arduino code, libraries for serial communication (C and python), and example graphical user interfaces for testing., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Martin Fischer reports financial support was provided by National Science Foundation. Martin Fischer reports financial support was provided by The Chan Zuckerberg Initiative. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The millions of specimens stored in entomological collections provide a unique opportunity to study historical insect diversity. Current technologies allow to sequence entire genomes of historical specimens and estimate past genetic diversity of present-day endangered species, advancing our understanding of anthropogenic impact on genetic diversity and enabling the implementation of conservation strategies. A limiting challenge is the extraction of historical DNA (hDNA) of adequate quality for sequencing platforms. We tested four hDNA extraction protocols on five body parts of pinned false heath fritillary butterflies, Melitaea diamina, aiming to minimise specimen damage, preserve their scientific value to the collections, and maximise DNA quality and yield for whole-genome re-sequencing. We developed a very effective approach that successfully recovers hDNA appropriate for short-read sequencing from a single leg of pinned specimens using silica-based DNA extraction columns and an extraction buffer that includes SDS, Tris, Proteinase K, EDTA, NaCl, PTB, and DTT. We observed substantial variation in the ratio of nuclear to mitochondrial DNA in extractions from different tissues, indicating that optimal tissue choice depends on project aims and anticipated downstream analyses. We found that sufficient DNA for whole genome re-sequencing can reliably be extracted from a single leg, opening the possibility to monitor changes in genetic diversity maintaining the scientific value of specimens while supporting current and future conservation strategies., (© 2024. The Author(s).)

Background: Using mouse genetic studies and systematic assessments of brain neuroanatomical phenotypes, we set out to identify which of the 30 genes causes brain defects at the autism-associated 16p11.2 locus., Results: We show that multiple genes mapping to this region interact to regulate brain anatomy, with female mice exhibiting far fewer brain neuroanatomical phenotypes. In male mice, among the 13 genes associated with neuroanatomical defects (Mvp, Ppp4c, Zg16, Taok2, Slx1b, Maz, Fam57b, Bola2, Tbx6, Qprt, Spn, Hirip3, and Doc2a), Mvp is the top driver implicated in phenotypes pertaining to brain, cortex, hippocampus, ventricles, and corpus callosum sizes. The major vault protein (MVP), the main component of the vault organelle, is a conserved protein found in eukaryotic cells, yet its function is not understood. Here, we find MVP expression highly specific to the limbic system and show that Mvp regulates neuronal morphology, postnatally and specifically in males. We also recapitulate a previously reported genetic interaction and show that Mvp +/- ;Mapk3 +/- mice exhibit behavioral deficits, notably decreased anxiety-like traits detected in the elevated plus maze and open field paradigms., Conclusions: Our study highlights multiple gene drivers in neuroanatomical phenotypes, interacting with each other through complex relationships. It also provides the first evidence for the involvement of the major vault protein in the regulation of brain size and neuroanatomy, specifically in male mice., (© 2023. The Author(s).)

Objective: To investigate the prevalence and surgical outcome of lens capsule disruption (LCD) in dogs undergoing cataract removal., Animals Studied: Medical records of 924 eyes undergoing phacoemulsification were analyzed retrospectively., Procedures: Routine cataract surgeries with or without LCD were included. Any LCD other than routine anterior capsulorhexis was defined as LCD and classified according to location and etiology. Odds ratios (OR) were calculated for maintaining vision, implantation of an artificial intraocular lens (IOL), and enucleation., Results: In total, 520 eyes were included. A LCD occurred in 145 eyes (27.8%; 145/520) and affected the posterior (85.5%; 124/145), anterior (6.2%; 9/145), and equatorial lens capsule (4.8%; 7/145) and at multiple locations (3.4%; 5/145). The etiology of the LCD was spontaneous preoperative in 41 eyes (28.3%; 41/145), accidental intraoperative in 57 eyes (39.3%; 57/145), and planned in 47 eyes (32.4%; 47/145). Disruption did not increase the odds of enucleation (OR = 1.48, 95% confidence interval [CI] 0.56-3.67; p = .36). The presence of LCD significantly increased the risk of losing vision 1 year post-operatively (OR = 8.17, 95% CI 1.41-84.93; p = .007) associated with retinal detachment. However, this was not present at 2 years follow-up or in PCCC cases at any time point. An IOL was implanted in 108 eyes (108/145; 75.2%) with LCD and in 45/47 (95.7%) eyes with a PCCC., Conclusion: Increased surgeon awareness of possible intraoperative, accidental LCDs is important, as LCDs were relatively common and associated with increased odds for vision loss after 1 year in the present study. A prospective study investigating the causes of intraoperative, accidental LCD is warranted., (© 2023 The Authors. Veterinary Ophthalmology published by Wiley Periodicals LLC on behalf of American College of Veterinary Ophthalmologists.)

Pump-probe microscopy of melanin in tumors has been proposed to improve diagnosis of malignant melanoma, based on the hypothesis that aggressive cancers disaggregate melanin structure. However, measured signals of melanin are complex superpositions of multiple nonlinear processes, which makes interpretation challenging. Polarization control during measurement and data fitting are used to decompose signals of melanin into their underlying molecular mechanisms. We then identify the molecular mechanisms that are most susceptible to melanin disaggregation and derive false-coloring schemes to highlight these processes in biological tissue. We demonstrate that false-colored images of a small set of melanoma tumors correlate with clinical concern. More generally, our systematic approach of decomposing pump-probe signals can be applied to a multitude of different samples.

A nonsense allele at rs1343879 in human MAGEE2 on chromosome X has previously been reported as a strong candidate for positive selection in East Asia. This premature stop codon causing ∼80% protein truncation is characterized by a striking geographical pattern of high population differentiation: common in Asia and the Americas (up to 84% in the 1000 Genomes Project East Asians) but rare elsewhere. Here, we generated a Magee2 mouse knockout mimicking the human loss-of-function mutation to study its functional consequences. The Magee2 null mice did not exhibit gross abnormalities apart from enlarged brain structures (13% increased total brain area, P = 0.0022) in hemizygous males. The area of the granular retrosplenial cortex responsible for memory, navigation, and spatial information processing was the most severely affected, exhibiting an enlargement of 34% (P = 3.4×10-6). The brain size in homozygous females showed the opposite trend of reduced brain size, although this did not reach statistical significance. With these insights, we performed human association analyses between brain size measurements and rs1343879 genotypes in 141 Chinese volunteers with brain MRI scans, replicating the sexual dimorphism seen in the knockout mouse model. The derived stop gain allele was significantly associated with a larger volume of gray matter in males (P = 0.00094), and smaller volumes of gray (P = 0.00021) and white (P = 0.0015) matter in females. It is unclear whether or not the observed neuroanatomical phenotypes affect behavior or cognition, but it might have been the driving force underlying the positive selection in humans., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Traumatic brain injury (TBI) is a common neurological disorder whose outcomes vary widely depending on a variety of environmental factors, including diet. Using a Drosophila melanogaster TBI model that reproduces key aspects of TBI in humans, we previously found that the diet consumed immediately following a primary brain injury has a substantial effect on the incidence of mortality within 24 h (early mortality). Flies that receive equivalent primary injuries have a higher incidence of early mortality when fed high-carbohydrate diets versus water. Here, we report that flies fed high-fat ketogenic diet (KD) following TBI exhibited early mortality that was equivalent to that of flies fed water and that flies protected from early mortality by KD continued to show survival benefits weeks later. KD also has beneficial effects in mammalian TBI models, indicating that the mechanism of action of KD is evolutionarily conserved. To probe the mechanism, we examined the effect of KD in flies mutant for Eip75B, an ortholog of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) that contributes to the mechanism of action of KD and has neuroprotective effects in mammalian TBI models. We found that the incidence of early mortality of Eip75B mutant flies was higher when they were fed KD than when they were fed water following TBI. These data indicate that Eip75B/PPARγ is necessary for the beneficial effects of KD following TBI. In summary, this work provides the first evidence that KD activates PPARγ to reduce deleterious outcomes of TBI and it demonstrates the utility of the fly TBI model for dissecting molecular pathways that contribute to heterogeneity in TBI outcomes., Competing Interests: The authors have declared that no competing interests exist.

Traumatic brain injury (TBI) frequently leads to non-neurological consequences such as intestinal permeability. The beta-blocker drug labetalol, which inhibits binding of catecholamine neurotransmitters to adrenergic receptors, reduces intestinal permeability in a rat TBI model. Using a Drosophila melanogaster TBI model, we previously found a strong positive correlation between intestinal permeability and mortality within 24 hours of TBI in a standard laboratory line ( w 1118 ) and across genetically diverse inbred lines from the Drosophila Genetic Reference Panel (DGRP). Here, we report that feeding injured w 1118 flies the beta-blockers labetalol and metoprolol reduced intestinal permeability and mortality. Additionally, metoprolol reduced intestinal permeability when 18 DGRP fly lines were analyzed in aggregate, but neither beta-blocker affected mortality. These data indicate that the mechanism underlying disruption of the intestinal barrier by adrenergic signaling following TBI is conserved between humans and flies and that mortality following TBI in flies is not strictly dependent on disruption of the intestinal barrier. Thus, the fly TBI model is useful for shedding light on the mechanism and consequences of adrenergic signaling between the brain and intestine following TBI in humans., (Copyright: © 2021 by the authors.)

Quaternary chalcogenide materials have long been a source of semiconductors for optoelectronic applications. Recent studies on I 2 -II-IV-X 4 (I = Ag, Cu, Li; II = Ba, Sr, Eu, Pb; IV = Si, Ge, Sn; X = S, Se) materials have shown particular versatility and promise among these compounds. These semiconductors take advantage of a diverse bonding scheme and chemical differences among cations to target a degree of antisite defect resistance. Within this set of compounds, the materials containing both Ag and Sr have not been experimentally studied and leave a gap in the full understanding of the family. Here, we have synthesized powders and single crystals of two Ag- and Sr-containing compounds, Ag 2 SrSiS 4 and Ag 2 SrGeS 4 , each found to form in the tetragonal I 4̅2 m structure of Ag 2 BaGeS 4 . During the synthesis targeting the title compounds, two additional materials, Ag 2 Sr 3 Si 2 S 8 and Ag 2 Sr 3 Ge 2 S 8 , have also been identified. These cubic compounds represent impurity phases during the synthesis of Ag 2 SrSiS 4 and Ag 2 SrGeS 4 . We show through hybrid density functional theory calculations that Ag 2 SrSiS 4 and Ag 2 SrGeS 4 have highly dispersive band-edge states and indirect band gaps, experimentally measured as 2.08(1) and 1.73(2) eV, respectively. Second-harmonic generation measurements on Ag 2 SrSiS 4 and Ag 2 SrGeS 4 powders show frequency-doubling capabilities in the near-infrared range.

Pump-probe microscopy is an emerging nonlinear imaging technique based on high repetition rate lasers and fast intensity modulation. Here, we present new methods for pump-probe microscopy that keep the beam intensity constant and instead modulate the inter-pulse time delay or the relative polarization. These techniques can improve image quality for samples that have poor heat dissipation or long-lived radiative states and can selectively address nonlinear interactions in the sample. We experimentally demonstrate this approach and point out the advantages over conventional intensity modulation.

Objectives: To describe the use of corneal autografts for repair of deep corneal defects in dogs., Materials and Methods: Retrospective analysis of clinical records of dogs that received autologous corneal grafts., Results: Fifteen dogs (16 eyes) of different breed, age and gender were included. Brachycephalic breeds were overrepresented (10/15 dogs). Defects were unilateral in 14 dogs and bilateral in one dog, extended to at least 80% of the stromal thickness in all eyes, with descemetoceles in four eyes and corneal perforations in five eyes. Most ulcers (13/16 eyes) were located centrally. Corneal autografts were harvested from healthy peripheral cornea of the ipsilateral eye. The thickness of the autograft was limited to a set depth of 0.3 mm. The autograft was sutured into a previously debrided ulcer bed with a continuous or simple interrupted suture pattern using absorbable or non-absorbable suture material. Additional interventions included a partial temporary tarsorrhaphy and bandage contact lenses. Postoperatively patients received topical antibiotics and systemic anti-inflammatory drugs, and 12/15 dogs also received systemic antibiotics. Mean follow-up time was 54 days (2 to 462). In all eyes the donor site healed uneventfully with mild, persistent corneal fibrosis. Postoperative complications included autograft keratomalacia, graft dehiscence and corneal pigmentation. No patient required additional surgery. Good structural and functional outcome was accomplished in 14 of 16 eyes., Clinical Significance: Autologous corneal grafts provide tectonic support and result in good corneal transparency in selected cases of dogs with deep to perforated corneal ulcerations., (© 2020 British Small Animal Veterinary Association.)

It has long been discussed to what extent related species develop similar genetic mechanisms to adapt to similar environments. Most studies documenting such convergence have either used different lineages within species or surveyed only a limited portion of the genome. Here, we investigated whether similar or different sets of orthologous genes were involved in genetic adaptation of natural populations of three related plant species to similar environmental gradients in the Alps. We used whole-genome pooled population sequencing to study genome-wide SNP variation in 18 natural populations of three Brassicaceae (Arabis alpina, Arabidopsis halleri, and Cardamine resedifolia) from the Swiss Alps. We first de novo assembled draft reference genomes for all three species. We then ran population and landscape genomic analyses with ~3 million SNPs per species to look for shared genomic signatures of selection and adaptation in response to similar environmental gradients acting on these species. Genes with a signature of convergent adaptation were found at significantly higher numbers than expected by chance. The most closely related species pair showed the highest relative over-representation of shared adaptation signatures. Moreover, the identified genes of convergent adaptation were enriched for nonsynonymous mutations, suggesting functional relevance of these genes, even though many of the identified candidate genes have hitherto unknown or poorly described functions based on comparison with Arabidopsis thaliana. We conclude that adaptation to heterogeneous Alpine environments in related species is partly driven by convergent evolution, but that most of the genomic signatures of adaptation remain species-specific., (© 2020 John Wiley & Sons Ltd.)

Mandates for mask use in public during the recent coronavirus disease 2019 (COVID-19) pandemic, worsened by global shortage of commercial supplies, have led to widespread use of homemade masks and mask alternatives. It is assumed that wearing such masks reduces the likelihood for an infected person to spread the disease, but many of these mask designs have not been tested in practice. We have demonstrated a simple optical measurement method to evaluate the efficacy of masks to reduce the transmission of respiratory droplets during regular speech. In proof-of-principle studies, we compared a variety of commonly available mask types and observed that some mask types approach the performance of standard surgical masks, while some mask alternatives, such as neck gaiters or bandanas, offer very little protection. Our measurement setup is inexpensive and can be built and operated by nonexperts, allowing for rapid evaluation of mask performance during speech, sneezing, or coughing., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

It is generally accepted that the spatial distribution of neutral genetic diversity within a species' native range mostly depends on effective population size, demographic history, and geographic position. However, it is unclear how genetic diversity at adaptive loci correlates with geographic peripherality or with habitat suitability within the ecological niche. Using exome-wide genomic data and distribution maps of the Alpine range, we first tested whether geographic peripherality correlates with four measures of population genetic diversity at > 17,000 SNP loci in 24 Alpine populations (480 individuals) of Swiss stone pine (Pinus cembra) from Switzerland. To distinguish between neutral and adaptive SNP sets, we used four approaches (two gene diversity estimates, F ST outlier test, and environmental association analysis) that search for signatures of selection. Second, we established ecological niche models for P. cembra in the study range and investigated how habitat suitability correlates with genetic diversity at neutral and adaptive loci. All estimates of neutral genetic diversity decreased with geographic peripherality, but were uncorrelated with habitat suitability. However, heterozygosity (H e ) at adaptive loci based on Tajima's D declined significantly with increasingly suitable conditions. No other diversity estimates at adaptive loci were correlated with habitat suitability. Our findings suggest that populations at the edge of a species' geographic distribution harbour limited neutral genetic diversity due to demographic properties. Moreover, we argue that populations from suitable habitats went through strong selection processes, are thus well adapted to local conditions, and therefore exhibit reduced genetic diversity at adaptive loci compared to populations at niche margins., (© 2020 John Wiley & Sons Ltd.)

We present a new imaging method for pump-probe microscopy that explores non-collinear excitation. This method (crossed-beam pump-probe microscopy, or CBPM) can significantly improve the axial resolution when imaging through low-NA lenses, providing an alternative way for depth-resolved, large field-of-view imaging. We performed a proof-of-concept demonstration, characterized CBPM's resolution using different imaging lenses, and measured an enhanced axial resolution for certain types of low-NA lenses.

The spatial heterogeneity of carrier dynamics in mixed halide perovskite CH 3 NH 3 PbI 3-x Cl x thin films with a range of different chloride additions is mapped using femtosecond transient absorption microscopy (TAM). The comparison of TAM images of fibrous and granular polycrystalline CH 3 NH 3 PbI 3-x Cl x films indicates that the impact of chloride addition on the local heterogeneity of carrier dynamics is highly dependent on the film preparation method and the resulting morphology. In addition to signals of pristine CH 3 NH 3 PbI 3 , CH 3 NH 3 PbI 3-x Cl x films with a fibrous structure show long-lived excited state absorption (ESA) signals in localized, microscopic regions. The ESA signal exhibits transient absorption with a rise time of about 5 ps after the excitation pulse, indicating that these distinct micrograins have preferential carrier trapping properties. The chemical composition of these micrograins does not differ detectably from their surroundings. In contrast, in CH 3 NH 3 PbI 3-x Cl x films with a granular structure, Cl addition does not seem to affect the charge carrier dynamics. These results provide insight into the localized effects of halide mixing and on the resulting photophysical properties of mixed halide perovskite materials on the micrometer length scale.

Here, we demonstrate the use of pump-probe microscopy for high-resolution studies of vermilion degradation. Vermilion (mostly α-HgS), an important red pigment used in historical paintings, blackens over time, and metallic Hg and β-HgS have been implicated as possible degradation products. Conventional analysis techniques have trouble differentiating α- and β-HgS with sufficiently high spatial resolution. However, pump-probe microscopy can differentiate metallic mercury, α- and β-HgS, and map each distribution on the microscopic scale. We studied artificial degradation of α-HgS; femtosecond-pulsed laser irradiation induces an irreversible phase shift of α- to β-HgS, in which the initial presence of β-HgS grains can increase the rate of conversion in their vicinity. Continuous ultraviolet exposure instead generates both liquid Hg and β-HgS, with a conversion rate that increases with elevated temperatures. Last, we reveal the presence of β-HgS as a natural degradation product in discolored vermilion layers in a 14th century Italian painting.

More people die from melanoma after a stage I diagnosis than after a stage IV diagnosis, because the tools available to clinicians do not readily identify which early-stage cancers will be aggressive. Near-infrared pump-probe microscopy detects fundamental differences in melanin structure between benign human moles and melanoma and also correlates with metastatic potential. However, the biological mechanisms of these changes have been difficult to quantify, as many different mechanisms can contribute to the pump-probe signal. We use model systems (sepia, squid, and synthetic eumelanin), cellular uptake studies, and a range of pump and probe wavelengths to demonstrate that the clinically observed effects come from alterations of the aggregated mode from "thick oligomer stacks" to "thin oligomer stacks" (due to changes in monomer composition) and (predominantly) deaggregation of the assembled melanin structure. This provides the opportunity to use pump-probe microscopy for the detection and study of melanin-associated diseases.

Objectives: To describe MRI features of canine retrobulbar inflammation, their association with clinical findings and outcome and to assess the value of MRI in detecting orbital foreign bodies., Materials and Methods: Clinical records of dogs with confirmed (23 of 31) and suspected (eight of 31) retrobulbar inflammation that underwent low field MRI studies were analysed retrospectively., Results: Of the 31 dogs included in the study there was abscessation in 19. Extraocular myositis (27 of 31) was concurrent with strabismus in three cases. Of 25 patients with exophthalmos, 14 had concurrent optic nerve swelling and, of these, five had permanent loss of vision. There was no vision loss in patients without nerve swelling. One case of suspected retinal detachment on MRI was confirmed clinically. Extensive abnormal contrast enhancement in the temporalis, masseter and pterygoideus muscles was associated with facial (n=3) and trigeminal nerve deficits (n=1). Three patients with inflammation extending into the nasal cavity and frontal sinus (one of 31) or meningeal contrast enhancement (two of 31), showed optic and oculomotor nerve deficits. On MRI a foreign body was not visible in 20 of 31 case or "appeared likely" in 11 of 31 dogs. A foreign body was found at surgery in one case., Clinical Significance: MRI outlines the extent of retrobulbar inflammation. Clinical findings were associated with imaging findings. MRI overestimated the presence of foreign bodies., (© 2018 British Small Animal Veterinary Association.)

Variation in local herbivore pressure along elevation gradients is predicted to drive variation in plant defense traits. Yet, the extent of intraspecific variation in defense investment along elevation gradients, and its effects on both herbivore preference and performance, remain relatively unexplored. Using populations of Arabidopsis halleri (Brassicaceae) occurring at different elevations in the Alps, we tested for associations between elevation, herbivore damage in the field, and constitutive chemical defense traits (glucosinolates) assayed under common-garden conditions. Additionally, we examined the feeding preferences and performance of a specialist herbivore, the butterfly Pieris brassicae , on plants from different elevations in the Alps. Although we found no effect of elevation on the overall levels of constitutive glucosinolates in leaves, relative amounts of indole glucosinolates increased significantly with elevation and were negatively correlated with herbivore damage in the field. In oviposition preference assays, P. brassicae females laid fewer eggs on plants from high-elevation populations, although larval performance was similar on populations from different elevations. Taken together, these results support the prediction that species distributed along elevation gradients exhibit genetic variation in chemical defenses, which can have consequences for interactions with herbivores in the field.

Pulmonary edema is a common ailment of heart failure patients and has remained an unmet medical need due to dose-limiting side effects associated with current treatments. Preclinical studies in rodents have suggested that inhibition of transient receptor potential vanilloid-4 (TRPV4) cation channels may offer an alternative-and potentially superior-therapy. Efforts directed toward small-molecule antagonists of the TRPV4 receptor have led to the discovery of a novel sulfone pyrrolidine sulfonamide chemotype exemplified by lead compound 6. Design elements toward the optimization of TRPV4 activity, selectivity, and pharmacokinetic properties are described. Activity of leading exemplars 19 and 27 in an in vivo model suggestive of therapeutic potential is highlighted herein.

In this work, we investigate the relationship between the complex hierarchical assembly structure of eumelanin, its characteristic broad absorption band, and the highly unusual nonlinear dynamics revealed by pump-probe or transient absorption microscopy. Melanin-like nanoparticles (MelNPs), generated by spontaneous oxidation of dopamine, were created with uniform but adjustable size distributions, and kinetically controlled oxidation was probed with a wide range of characterization methods. This lets us explore the broad absorption bands of eumelanin models at different assembly levels, such as small subunit fractions (single monomeric and oligomeric units and small oligomer stacks), stacked oligomer fractions (protomolecules), and large-scale aggregates of protomolecules (parental particles). Both the absorption and pump-probe dynamics are very sensitive to these structural differences or to the size of intact particles (a surprising result for an organic polymer). We show that the geometric packing order of protomolecules in long-range aggregation is key secondary interactions to extend the absorption band of eumelanin to the low energy spectrum and produce drastic changes in the transient absorption spectrum.

Edaphic conditions are important determinants of plant fitness. While much has been learnt in recent years about plant adaptation to heavy metal contaminated soils, the genomic basis underlying adaptation to calcareous and siliceous substrates remains largely unknown. We performed a reciprocal germination experiment and whole-genome resequencing in natural calcareous and siliceous populations of diploid Arabidopsis lyrata to test for edaphic adaptation and detect signatures of selection at loci associated with soil-mediated divergence. In parallel, genome scans on respective diploid ecotypes from the Arabidopsis arenosa species complex were undertaken, to search for shared patterns of adaptive genetic divergence. Soil ecotypes of A. lyrata display significant genotype-by-treatment responses for seed germination. Sequence (SNPs) and copy-number variants (CNVs) point towards loci involved in ion transport as the main targets of adaptive genetic divergence. Two genes exhibiting high differentiation among soil types in A. lyrata further share trans-specific single nucleotide polymorphisms with A. arenosa. This work applies experimental and genomic approaches to study edaphic adaptation in A. lyrata and suggests that physiological response to elemental toxicity and deficiency underlies the evolution of calcareous and siliceous ecotypes. The discovery of shared adaptive variation between sister species indicates that ancient polymorphisms contribute to adaptive evolution., (© 2018 John Wiley & Sons Ltd.)

Objective: To describe concurrent ophthalmic diseases in dogs with retrobulbar cellulitis and abscessation., Materials and Methods: Retrospective analysis of clinical records of dogs with retrobulbar inflammation., Results: Forty-one dogs were diagnosed with retrobulbar inflammation; of these, 23 presented with abscessation and two with zygomatic sialoadenitis. Diagnosis was based on orbital ultrasound, MRI, CT and cytological and microbiological examination of fluid or tissue samples. Management involved evacuation of fluid contents using ultrasound-guided fine-needle aspiration via the pterygopalatine fossa or orbitotomy. Patients received systemic antibiotics (except for one with sialoadenitis), glucocorticoids, non-steroidal anti-inflammatory drugs, opioids and fluid therapy. At initial presentation one or more ophthalmic complications were reported in 19 dogs (46%) including internal ophthalmoplegia (n=5), blindness due to optic nerve damage (n=5), facial nerve paralysis (n=3), prolapse of the third eyelid gland (n=3), corneal ulceration (n=8), anterior uveitis (n=4), chorioretinitis (n=3), retinal detachment (n=2) and increased intraocular pressure (n=7). Information on ophthalmic complications after cessation of active inflammation was available for 33 patients. One or more concurrent disease was found in 10 cases (30%): in addition to persistent neurological deficits and third eyelid gland prolapse reported at initial presentation, visual deficits after retinal re-attachment (n=2), loss of corneal sensation (n=1), corneal oedema (n=1), corneal fibrosis (n=4), corneal lipidosis (n=1) and strabismus after suspected fibrosis (n=2) were diagnosed., Clinical Significance: Ophthalmic complications are common in patients with retrobulbar inflammation indicating that these patients should undergo ophthalmic assessment and follow-up., (© 2018 British Small Animal Veterinary Association.)

A 10-year-old Rottweiler presented with right-sided moderately painful exophthalmia, blindness, absence of dazzle and pupillary light reflexes, a swollen optic nerve head and ventrolateral indentation of the globe. On magnetic resonance imaging, a 3 x 2 x 2 cm mass with a fluid filled center and contrast-enhancing periphery was noted posteriolateral of the globe. Orbital ultrasound was used for a guided fine needle aspirate of the mass. Cytology revealed moderate numbers of polygonal cells with lightly basophilic cytoplasm. Several cells showed nuclear pseudoinclusions. Histopathology following exenteration of the orbit revealed an infiltrative, extradural neoplasm surrounding the optic nerve. Cells were arranged in packets. Neoplastic cells were immunopositive for neuron specific enolase, synaptophysin and chromogranin A and immunonegative for cytokeratin. Findings were consistent with an extra-adrenal paraganglioma (neuroendocrine tumour). Although complete excision could not be confirmed on histopathology, the owners reported no apparent tumour recurrence 25 months after surgery. In conclusion a paraganglioma should be considered as a differential diagnosis of an orbital mass., Competing Interests: The authors declare no conflict of interest with respect to the publication of this manuscript., (Georg Thieme Verlag KG Stuttgart · New York.)

Analysis of red organic pigments in artworks (and in forensics applications) is challenging, because conventional nondestructive mapping techniques provide little contrast, and most chemical analyses with high specificity require sample removal. Here we demonstrate a new optical approach, pump-probe microscopy, for the analysis of red organic pigments. We investigate Carmine naccarat, Lac dye, purpurin, alizarin, madder lake, and eosin Y and show that their intrinsic photophysical properties produce distinctive pump-probe spectra. We utilize this contrast for high-resolution, three-dimensional imaging without the need for physical sample removal. Lastly, we highlight the potential of pump-probe microscopy as an analytical tool for forensics of other types of organic colorants by investigating a series of automotive paints.

When plants adapt to local environments, strong signatures of selection are expected in the genome, particularly in high-stress environments such as trace metal element enriched (metalliferous) soils. Using Arabidopsis halleri, a model species for metal homeostasis and adaptation to extreme environments, we identifid genes, gene variants, and pathways that are associated with soil properties and may thus contribute to adaptation to high concentrations of trace metal elements. We analysed whole-genome Pool-seq data from two metallicolous (from metalliferous soils) and two non-metallicolous populations (in total 119 individuals) and associated allele frequencies of the identified single-nucleotide polymorphisms (SNPs) with soil variables measured on site. Additionally, we accounted for polygenic adaptation by searching for gene pathways showing enrichment of signatures of selection. Out of >2.5 million SNPs, we identified 57 SNPs in 19 genes that were significantly associated with soil variables and are members of three enriched pathways. At least three of these candidate genes and pathways are involved in transmembrane transport and/or associated with responses to various stresses such as oxidative stress. We conclude that both allocation and detoxification processes play a crucial role in A. halleri for coping with these unfavourable conditions.

In this article, we present a standardized protocol for fast and robust neuroanatomical phenotyping of the adult mouse brain, which complements a previously published article (doi: 10.1002/cpmo.12) in Current Protocols in Mouse Biology. It is aimed at providing an experimental pipeline within an academic research setting from experimental work to data analysis. Our analysis focuses on one single parasagittal plane, covering the majority of brain regions involved in higher order cognitions such as the cortex, hippocampus, and cerebellum, for a total of 166 parameters of area, length, and cell-level measurements in contrast to 78 parameters in our previously published coronal screen. Benefits of using parasagittal analysis for large-scale neuroanatomic screens are discussed. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

Objectives: To evaluate progression and complications of various cataract aetiologies and stages of development., Materials and Methods: In all, 447 eyes of 250 dogs with cataracts were monitored between 2012 and 2015. Breed, gender, age, vision, types of cataract, stage of cataract development (incipient, immature, mature and hypermature), complications of cataracts, ocular ultrasonography and electroretinography findings were recorded. Re-examinations were performed after a minimum of 1, 6 and 12 months. A relative rate of progression within 1 month was determined and progression was categorised as stationary, slow, moderate or rapid., Results: Overall, 44∙7% cataracts progressed and 55∙3% remained stationary. Incipient, immature, mature and hypermature cataracts progressed in 34∙1, 72∙7, 66∙7 and 44∙6% of cases, respectively. Hereditary, congenital, diabetic, contusive trauma-related, perforating trauma-related, radiation-related, senile cataracts and cataracts secondary to concurrent ocular diseases progressed in 47∙1, 66∙7, 66∙7, 50, 44∙4, 0, 29∙9 and 48∙9% of cases, respectively. Rapid progression was identified in 13∙3% of diabetic and 4∙3% of hereditary cataracts. Complications were diagnosed in 43∙5% and occurred at any stage but more frequently the further the cataract had developed. Lens-induced uveitis was the most common complication. Diabetic, traumatic, secondary and hereditary cataracts were associated with more complications than senile cataracts., Clinical Significance: Stage of development and cataract aetiology are associated with different rates of progression and complications., (© 2018 British Small Animal Veterinary Association.)

Papillary thyroid cancer (PTC) is the most prevalent endocrine neoplasia. The increased incidence of PTC in patients with thyroiditis and the frequent immune infiltrate found in PTC suggest that inflammation might be a risk factor for PTC development. The CXCR3-ligand system is involved in thyroid inflammation and CXCR3 has been found upregulated in many tumors, suggesting its pro-tumorigenic role under the inflammatory microenvironment. CXCR3 ligands (CXCL4, CXCL9, CXCL10 and CXCL11) trigger antagonistic responses partly due to the presence of two splice variants, CXCR3A and CXCR3B. Whereas CXCR3A promotes cell proliferation, CXCR3B induces apoptosis. However, the relation between CXCR3 variant expression with chronic inflammation and PTC development remains unknown. Here, we characterized the expression pattern of CXCR3 variants and their ligands in benign tumors and PTC. We found that CXCR3A and CXCL10 mRNA levels were increased in non-metastatic PTC when compared to non-neoplastic tissue. This increment was also observed in a PTC epithelial cell line (TPC-1). Although elevated protein levels of both isoforms were detected in benign and malignant tumors, the CXCR3A expression remained greater than CXCR3B and promoted proliferation in Nthy-ori-3-1 cells. In non-metastatic PTC, inflammation was conditioning for the CXCR3 ligands increased availability. Consistently, CXCL10 was strongly induced by interferon gamma in normal and tumor thyrocytes. Our results suggest that persistent inflammation upregulates CXCL10 expression favoring tumor development via enhanced CXCR3A-CXCL10 signaling. These findings may help to further understand the contribution of inflammation as a risk factor in PTC development and set the basis for potential therapeutic studies., Competing Interests: CONFLICTS OF INTEREST The authors don't have any duality of interests to declare.

Microscopic variations in melanin composition can be mapped through linear and nonlinear optical responses. Though instrumentation to measure linear attenuation is simple and inexpensive, the nonlinear response provides more degrees of freedom with which to spectroscopically resolve pigments. The objective of this study is to assess differences in imaging melanin contrast by comparing hyperspectral (linear) versus pump-probe (nonlinear) microscopy of unstained histology sections of pigmented lesions. The images and analysis we have presented here show that pump-probe uncovers a greater variation in pigment composition, compared with hyperspectral microscopy, and that the two methods yield complimentary biochemical information.

Objectives: Melanomas of the female genital tract present a unique clinical challenge. Not only are these lesions in an anatomically sensitive area, but also they tend to be multifocal and have high recurrence rates. Furthermore, several benign melanocytic proliferations resemble early-stage melanoma clinically and/or histopathologically. Thus, there is a significant need for additional tools that can help correctly diagnose and stage these lesions. Here, we quantitatively and nondestructively analyze the chemical composition of melanin in excised pigmented lesions of the female genital tract using pump-probe microscopy, a high-resolution optical imaging technique that is sensitive to many biochemical properties of melanin., Materials and Methods: Thirty-one thin (~5 μm) tissue sections previously excised from female genital tract melanocytic lesions were imaged with pump-probe microscopy and analyzed., Results: We find significant quantitative differences in melanin type and structure between melanoma and nonmalignant melanocytic proliferations. Our analysis also suggests a link between the molecular signatures of melanins and lesion-specific genetic mutations. Finally, significant differences are found between metastatic and nonmetastatic melanomas. The limitations of this work include the fact that molecular information is restricted to melanin pigment and the sample size is relatively small., Conclusions: Pump-probe microscopy provides unique information regarding the biochemical composition of genital tract melanocytic lesions, which can be used to improve the diagnosis and staging of vulvar melanomas.

A simplistic view of the adaptive process pictures a hillside along which a population can climb: when ecological 'demands' change, evolution 'supplies' the variation needed for the population to climb to a new peak. Evolutionary ecologists point out that this simplistic view can be incomplete because the fitness landscape changes dynamically as the population evolves. Geneticists meanwhile have identified complexities relating to the nature of genetic variation and its architecture, and the importance of epigenetic variation is under debate. In this review, we highlight how complexity in both ecological 'demands' and the evolutionary 'supply' influences organisms' ability to climb fitness landscapes that themselves change dynamically as evolution proceeds, and encourage new synthetic effort across research disciplines towards ecologically realistic studies of adaptation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Numerous landscape genomic studies have identified single-nucleotide polymorphisms (SNPs) and genes potentially involved in local adaptation. Rarely, it has been explicitly evaluated whether these environmental associations also hold true beyond the populations studied. We tested whether putatively adaptive SNPs in Arabidopsis halleri (Brassicaceae), characterized in a previous study investigating local adaptation to a highly heterogeneous environment, show the same environmental associations in an independent, geographically enlarged set of 18 populations. We analysed new SNP data of 444 plants with the same methodology (partial Mantel tests, PMTs) as in the original study and additionally with a latent factor mixed model (LFMM) approach. Of the 74 candidate SNPs, 41% (PMTs) and 51% (LFMM) were associated with environmental factors in the independent data set. However, only 5% (PMTs) and 15% (LFMM) of the associations showed the same environment-allele relationships as in the original study. In total, we found 11 genes (31%) containing the same association in the original and independent data set. These can be considered prime candidate genes for environmental adaptation at a broader geographical scale. Our results suggest that selection pressures in highly heterogeneous alpine environments vary locally and signatures of selection are likely to be population-specific. Thus, genotype-by-environment interactions underlying adaptation are more heterogeneous and complex than is often assumed, which might represent a problem when testing for adaptation at specific loci.

We integrate spectroscopic optical coherence tomography (SOCT) with stimulated Raman scattering (SRS) to enable simultaneously multiplexed spatial and spectral imaging with sensitivity to many endogenous biochemical species that play an important role in biology and medicine. The combined approach, termed SRS-SOCT, overcomes the limitations of each individual method. Ultimately, SRS-SOCT has the potential to achieve fast, volumetric, and highly sensitive label-free molecular imaging. We demonstrate the approach by imaging excised human adipose tissue and detecting the lipids' Raman signatures in the high-wavenumber region.

Background: Microsatellite markers are widely used for estimating genetic diversity within and differentiation among populations. However, it has rarely been tested whether such estimates are useful proxies for genome-wide patterns of variation and differentiation. Here, we compared microsatellite variation with genome-wide single nucleotide polymorphisms (SNPs) to assess and quantify potential marker-specific biases and derive recommendations for future studies. Overall, we genotyped 180 Arabidopsis halleri individuals from nine populations using 20 microsatellite markers. Twelve of these markers were originally developed for Arabidopsis thaliana (cross-species markers) and eight for A. halleri (species-specific markers). We further characterized 2 million SNPs across the genome with a pooled whole-genome re-sequencing approach (Pool-Seq)., Results: Our analyses revealed that estimates of genetic diversity and differentiation derived from cross-species and species-specific microsatellites differed substantially and that expected microsatellite heterozygosity (SSR-H e ) was not significantly correlated with genome-wide SNP diversity estimates (SNP-H e and θ Watterson ) in A. halleri. Instead, microsatellite allelic richness (A r ) was a better proxy for genome-wide SNP diversity. Estimates of genetic differentiation among populations (F ST ) based on both marker types were correlated, but microsatellite-based estimates were significantly larger than those from SNPs. Possible causes include the limited number of microsatellite markers used, marker ascertainment bias, as well as the high variance in microsatellite-derived estimates. In contrast, genome-wide SNP data provided unbiased estimates of genetic diversity independent of whether genome- or only exome-wide SNPs were used. Further, we inferred that a few thousand random SNPs are sufficient to reliably estimate genome-wide diversity and to distinguish among populations differing in genetic variation., Conclusions: We recommend that future analyses of genetic diversity within and differentiation among populations use randomly selected high-throughput sequencing-based SNP data to draw conclusions on genome-wide diversity patterns. In species comparable to A. halleri, a few thousand SNPs are sufficient to achieve this goal.

We hypothesized that GnRH would increase pregnancy risk (PR) in a split-time AI program for cows in which estrus was not detected. A total of 1,236 suckled beef cows at 12 locations in 3 states (Colorado, Kansas, and North Dakota) were enrolled. Before applying the fixed-time AI program, BCS was assessed. Cows were treated on d -7 with a progesterone insert concurrent with 100 μg GnRH and on d 0 with 25 mg PGF plus removal of the insert. Estrus-detection patches were affixed to cows at insert removal. Estrus was defined to have occurred when an estrus-detection patch was >50% colored (activated). Cows in estrus by 65 h ( = 758; 61.3% of all cows) were randomly allocated to 2 treatments: 1) 100 μg GnRH and early + GnRH (E+G; = 373) or 2) AI only at 65 h (early - no GnRH [E-G]; = 385). The remaining cows were randomly allocated to 2 treatments: 1) 5(L+G; = 252) or 2) AI only at 84 h (late no GnRH [L-G]; = 226). Pregnancy was determined 35 d after AI via transrectal ultrasound. Pregnancy risk did not differ ( = 0.68) between E+G and E-G cows (61.9 vs. 60.4%, respectively). Conversely, for cows inseminated at 84 h, PR was greater ( = 0.01) in cows that received GnRH (L+G) compared with their herd mates not receiving GnRH (L- G; 41.7 vs. 30.8%, respectively). Of those cows not detected in estrus by 65 h, 42.1% were detected by 84 h, for a total expression of estrus by all cows of 77.6%. Administration of GnRH increased ( < 0.01) PR in cows not detected in estrus by 84 h (+GnRH = 33.4% [ = 146] vs. no GnRH = 15.0% [ = 128]) but had no effect in cows expressing estrus by 84 h (+GnRH = 65.3% [ = 103] vs. no GnRH = 61.7% [ = 97]). Neither estrus expression by 65 or 84 h nor PR was influenced by BCS, parity, or days postpartum at AI. Cows had greater PR when they had been detected in estrus before AI, and PR was improved by administration of GnRH at 65 h after insert removal in cows that were not detected in estrus and inseminated at 84 h.

A multilocation study examined pregnancy risk (PR) after delaying AI in suckled beef cows from 60 to 75 h when estrus had not been detected by 60 h in response to a 7-d CO-Synch + progesterone insert (CIDR) timed AI (TAI) program (d -7: CIDR insert concurrent with an injection of GnRH; d 0: PGF injection and removal of CIDR insert; and GnRH injection at TAI [60 or 75 h after CIDR removal]). A total of 1,611 suckled beef cows at 15 locations in 9 states (CO, IL, KS, MN, MS, MT, ND, SD, and VA) were enrolled. Before applying the fixed-time AI program, BCS was assessed, and blood samples were collected. Estrus was defined to have occurred when an estrus detection patch was >50% colored (activated). Pregnancy was determined 35 d after AI via transrectal ultrasound. Cows ( = 746) detected in estrus by 60 h (46.3%) after CIDR removal were inseminated and treated with GnRH at AI (Control). Remaining nonestrous cows were allocated within location to 3 treatments on the basis of parity and days postpartum: 1) GnRH injection and AI at 60 h (early-early = EE; = 292), 2) GnRH injection at 60 h and AI at 75 h (early-delayed = ED; = 282), or 3) GnRH injection and AI at 75 h (delayed-delayed = DD; = 291). Control cows had a greater ( < 0.01) PR (64.2%) than other treatments (EE = 41.7%, ED = 52.8%, DD = 50.0%). Use of estrus detection patches to delay AI in cows not in estrus by 60 h after CIDR insert removal (ED and DD treatments) increased ( < 0.05) PR to TAI when compared with cows in the EE treatment. More ( < 0.001) cows that showed estrus by 60 h conceived to AI at 60 h than those not showing estrus (64.2% vs. 48.1%). Approximately half (49.2%) of the cows not in estrus by 60 h had activated patches by 75 h, resulting in a greater ( < 0.05) PR than their nonestrous herd mates in the EE (46.1% vs. 34.5%), ED (64.2% vs. 39.2%), and DD (64.8% vs. 31.5%) treatments, respectively. Overall, cows showing estrus by 75 h (72.7%) had greater ( < 0.001) PR to AI (61.3% vs. 37.9%) than cows not showing estrus. Use of estrus detection patches to allow for a delayed AI in cows not in estrus by 60 h after removal of the CIDR insert improved PR to TAI by optimizing the timing of the AI in those cows.

Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.

The effects of nose flap devices in calves before dam separation on cow BCS, pre- and postseparation calf performance, and humoral immune response were compared with traditional weaning. Primiparous and multiparous Angus and Hereford cows ( = 113) and their Angus, Hereford, and Angus × Hereford calves (179.4 ± 3.92 kg and 161 ± 22.7 d of age) were used. Cow-calf pairs were allocated to 1 of 2 treatments in a completely randomized design: 1) nose flap for 21 d before separation from the dam (NF) or 2) no nose flap for 21 d before separation from the dam (CON). Calves were separated from dams on d 0, and calves were placed in group feed-yard pens for 28 d. A subset ( = 75) of weaned calves were placed into 1 of 8 pens to evaluate DMI. Cow BCS was measured on d -21 and 56, and calves were given modified live vaccinations (d -21 and 1), challenged with ovalbumin (OVA; d 1), and weighed (d -21, 1, 7, 14, 21, and 28). In addition, blood samples were collected (d -21, 1, 14, and 28) to measure primary humoral immune response. Control calves tended to have greater BW on d 14 ( = 0.09) and 21 ( = 0.07) than NF calves, and CON calves had greater ( < 0.05) ADG from d -21 to 1 vs. NF calves. Treatments did not differ ( ≥ 0.27) for postweaning DMI, G:F, or morbidity. Serum neutralization tests for bovine viral diarrhea virus type 1 (BVDV-1) and bovine herpesvirus type 1 (BHV-1) were used to measure humoral response to a viral vaccination. Serum antibody titers to BVDV-1 for CON calves tended ( = 0.08) to be greater on d 1 and were greater ( < 0.05) by d 28 vs. NF calves. By d 28, a greater percentage ( < 0.05) of CON calves seroconverted for BVDV-1 than NF calves (82.1 vs. 66.7%, respectively). Serum antibody titers for BHV-1 were greater ( < 0.05) on d 1 and 28 for CON vs. NF calves. Humoral immune response to OVA during the 28-d postseparation period from the dam was evaluated in a subset ( = 57) of calves. There was no difference ( = 0.92) in OVA-specific IgG between treatments on d 14 or 28 ( = 0.76); however, OVA-specific IgM was greater ( < 0.05) in CON vs. NF calves on d 28. Results indicate that nose flap devices did not influence feed intake, feed efficiency, or morbidity during the initial postseparation period from the dam. However, preweaning ADG, serum BVDV-1 and BHV-1 titers, and humoral immune response to OVA were decreased in calves that received the nose flap treatment.

Stimulated Raman scattering (SRS) enables fast, high resolution imaging of chemical constituents important to biological structures and functional processes, both in a label-free manner and using exogenous biomarkers. While this technology has shown remarkable potential, it is currently limited to point scanning and can only probe a few Raman bands at a time (most often, only one). In this work we take a fundamentally different approach to detecting the small nonlinear signals based on dispersion effects that accompany the loss/gain processes in SRS. In this proof of concept, we demonstrate that the dispersive measurements are more robust to noise compared to amplitude-based measurements, which then permit spectral or spatial multiplexing (potentially both, simultaneously). Finally, we illustrate how this method may enable different strategies for biochemical imaging using phase microscopy and optical coherence tomography.

Metastatic melanoma is associated with a poor prognosis, but no method reliably predicts which melanomas of a given stage will ultimately metastasize and which will not. While sentinel lymph node biopsy (SLNB) has emerged as the most powerful predictor of metastatic disease, the majority of people dying from metastatic melanoma still have a negative SLNB. Here we analyze pump-probe microscopy images of thin biopsy slides of primary melanomas to assess their metastatic potential. Pump-probe microscopy reveals detailed chemical information of melanin with subcellular spatial resolution. Quantification of the molecular signatures without reference standards is achieved using a geometrical representation of principal component analysis. Melanin structure is analyzed in unison with the chemical information by applying principles of mathematical morphology. Results show that melanin in metastatic primary lesions has lower chemical diversity than non-metastatic primary lesions, and contains two distinct phenotypes that are indicative of aggressive disease. Further, the mathematical morphology analysis reveals melanin in metastatic primary lesions has a distinct "dusty" quality. Finally, a statistical analysis shows that the combination of the chemical information with spatial structures predicts metastatic potential with much better sensitivity than SLNB and high specificity, suggesting pump-probe microscopy can be an important tool to help predict the metastatic potential of melanomas.

The lack of DNA sequence information for most non-model organisms impairs the design of primers that are universally applicable for the study of molecular polymorphisms in nuclear markers. Next-generation sequencing (NGS) techniques nowadays provide a powerful approach to overcome this limitation. We present a flexible and inexpensive method to identify large numbers of nuclear primer pairs that amplify in most Brassicaceae species. We first obtained and mapped NGS transcriptome sequencing reads from two of the distantly related Brassicaceae species, Cardamine hirsuta and Arabis alpina, onto the Arabidopsis thaliana reference genome, and then identified short conserved sequence motifs among the three species bioinformatically. From these, primer pairs to amplify coding regions (nuclear protein coding loci, NPCL) and exon-primed intron-crossing sequences (EPIC) were developed. We identified 2,334 universally applicable primer pairs, targeting 1,164 genes, which provide a large pool of markers as readily usable genomic resource that will help addressing novel questions in the Brassicaceae family. Testing a subset of the newly designed nuclear primer pairs revealed that a great majority yielded a single amplicon in all of the 30 investigated Brassicaceae taxa. Sequence analysis and phylogenetic reconstruction with a subset of these markers on different levels of phylogenetic divergence in the mustard family were compared with previous studies. The results corroborate the usefulness of the newly developed primer pairs, e.g., for phylogenetic analyses or population genetic studies. Thus, our method provides a cost-effective approach for designing nuclear loci across a broad range of taxa and is compatible with current NGS technologies.

The lock-in amplifier is a critical component in many different types of experiments, because of its ability to reduce spurious or environmental noise components by restricting detection to a single frequency and phase. One example application is pump-probe microscopy, a multiphoton technique that leverages excited-state dynamics for imaging contrast. With this application in mind, we present here the design and implementation of a high-speed lock-in amplifier on the field-programmable gate array (FPGA) coprocessor of a data acquisition board. The most important advantage is the inherent ability to filter signals based on more complex modulation patterns. As an example, we use the flexibility of the FPGA approach to enable a novel pump-probe detection scheme based on spread-spectrum communications techniques.