Minimally destructive hDNA extraction method for retrospective genetics of pinned historical Lepidoptera specimens.

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.

Contrast mechanisms in pump-probe microscopy of melanin.

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.

Structural, Optical, and Electronic Properties of Two Quaternary Chalcogenide Semiconductors: Ag2SrSiS4 and Ag2SrGeS4.

Quaternary chalcogenide materials have long been a source of semiconductors for optoelectronic applications. Recent studies on I2-II-IV-X4 (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, Ag2SrSiS4 and Ag2SrGeS4, each found to form in the tetragonal I4̅2m structure of Ag2BaGeS4. During the synthesis targeting the title compounds, two additional materials, Ag2Sr3Si2S8 and Ag2Sr3Ge2S8, have also been identified. These cubic compounds represent impurity phases during the synthesis of Ag2SrSiS4 and Ag2SrGeS4. We show through hybrid density functional theory calculations that Ag2SrSiS4 and Ag2SrGeS4 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 Ag2SrSiS4 and Ag2SrGeS4 powders show frequency-doubling capabilities in the near-infrared range.

Beyond intensity modulation: new approaches to pump-probe microscopy.

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.

Low-cost measurement of face mask efficacy for filtering expelled droplets during speech.

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.

Genomic signatures of convergent adaptation to Alpine environments in three Brassicaceae species.

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.

Crossed-beam pump-probe microscopy.

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.

Disentangling the effects of geographic peripherality and habitat suitability on neutral and adaptive genetic variation in Swiss stone pine.

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, FST 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 (He ) 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.

Visualizing the impact of chloride addition on the microscopic carrier dynamics of MAPbI3 thin films using femtosecond transient absorption microscopy.

The spatial heterogeneity of carrier dynamics in mixed halide perovskite CH3NH3PbI3-xClx 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 CH3NH3PbI3-xClx 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 CH3NH3PbI3, CH3NH3PbI3-xClx 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 CH3NH3PbI3-xClx 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.

Visualization of vermilion degradation using pump-probe microscopy.

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 ß-HgS have been implicated as possible degradation products. Conventional analysis techniques have trouble differentiating α- and ß-HgS with sufficiently high spatial resolution. However, pump-probe microscopy can differentiate metallic mercury, α- and ß-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 ß-HgS, in which the initial presence of ß-HgS grains can increase the rate of conversion in their vicinity. Continuous ultraviolet exposure instead generates both liquid Hg and ß-HgS, with a conversion rate that increases with elevated temperatures. Last, we reveal the presence of ß-HgS as a natural degradation product in discolored vermilion layers in a 14th century Italian painting.

Unraveling the molecular nature of melanin changes in metastatic cancer.

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.

Divergence in Glucosinolate Profiles between High- and Low-Elevation Populations of Arabidopsis halleri Correspond to Variation in Field Herbivory and Herbivore Behavioral Preferences.

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.

Understanding the Role of Aggregation in the Broad Absorption Bands of Eumelanin.

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.

The genomic basis of adaptation to calcareous and siliceous soils in Arabidopsis lyrata.

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.

Transmembrane transport and stress response genes play an important role in adaptation of Arabidopsis halleri to metalliferous soils.

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.

Spectroscopic Differentiation and Microscopic Imaging of Red Organic Pigments Using Optical Pump-Probe Contrast.

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.

Differential expression profile of CXCR3 splicing variants is associated with thyroid neoplasia. Potential role in papillary thyroid carcinoma oncogenesis?

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.

Comparison of pump-probe and hyperspectral imaging in unstained histology sections of pigmented lesions.

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.

Label-Free Imaging of Female Genital Tract Melanocytic Lesions With Pump-Probe Microscopy: A Promising Diagnostic Tool.

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.

Can Evolution Supply What Ecology Demands?

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.