Yes / Photobiomodulation-based (LLLT) therapies show tantalizing promise for treatment of skin diseases. Confidence in this approach is blighted however by lamentable inconsistency in published experimental designs, and so complicates interpretation. Here we interrogate the appropriateness of a range of previously-reported treatment parameters, including light wavelength, irradiance and radiant exposure, as well as cell culture conditions (e.g., serum concentration, cell confluency, medium refreshment, direct/indirect treatment, oxygen concentration, etc.), in primary cultures of normal human dermal fibroblasts exposed to visible and near infra-red (NIR) light. Apart from irradiance, all study parameters impacted significantly on fibroblast metabolic activity. Moreover, when cells were grown at atmospheric O2 levels (i.e. 20%) short wavelength light inhibited cell metabolism, while negligible effects were seen with long visible and NIR wavelength. By contrast, NIR stimulated cells when exposed to dermal tissue oxygen levels (approx. 2%). The impact of culture conditions was further seen when inhibitory effects of short wavelength light were reduced with increasing serum concentration and cell confluency. We conclude that a significant source of problematic interpretations in photobiomodulation reports derives from poor optimization of study design. Further development of this field using in vitro/ex vivo models should embrace significant standardization of study design, ideally within a design-of-experiment setting.

<p>Environmental DNA (eDNA) surveys utilize DNA shed from animals in order to detect their presence. Since it was developed, this technique has been applied to numerous species across several taxa. In some cases, it has been shown to be superior to traditional survey methods at detecting rare or cryptic species. It allows for the detection of animals in low numbers and does not require direct capture of an animal. This allows eDNA to be more effective at detecting rare or cryptic species that require high survey effort to find. This often reduces survey costs as many eDNA samples can be collected quickly with little equipment required.</p> <p>The Kirtland’s Snake (<i>Clonophis kirtlandii</i>) is a small Natricine snake endemic to the Midwest. It is a species of conservation concern since it is threatened throughout its range. Due to its cryptic and fossorial lifestyle, it is also a notoriously difficult snake to survey. This has resulted in a poor understanding of Kirtland’s Snake life history and population status. Applying eDNA surveys to this species may increase detection probability, offering a more efficient way to survey for them. </p> <p>In this study, a quantitative PCR (qPCR) assay was designed and tested alongside traditional coverboard surveys. The assay had a limit of detection of 166 copies of Kirtland’s Snake DNA. In crayfish burrow sediment, eDNA was found to be detectable up to 10 days and may persist for up to 25 days. However, only one detection occurred out of 380 field samples. Coverboard surveys revealed temporal and spatial variation in Kirtland’s Snake abundance. More snakes were captured in the spring, during the first field season, and at the south coverboard transects. Kirtland’s Snake abundance was also found to be related to the presence of grass and herbaceous vegetation as well as close proximity to shrubs. Comparing survey methods, coverboards resulted in far better snake detection, suggesting that eDNA does not offer an advantage over traditional survey methods for this species. </p>

<p>Loop-mediated Isothermal Amplification (LAMP) is a promising technology to address diagnostic and surveillance testing during public health crises, such as the current COVID-19 pandemic; however, primer design and assay optimization remain a barrier to enabling rapid deployment of assays based on LAMP. Herein, we introduce a design and screening process that allows for strategic determination of optimally performing primer sets and standardized assay conditions which enable execution of LAMP at point-of-care (POC) settings using complex human samples such as saliva. A total of 20 primer sets targeting the N, E, RdRP, and orf1ab genes of the SARS-CoV-2 were designed, screened, and selected based on performance metrics such as reaction time, sensitivity, and specificity. Of these 20 primer sets, only two primer sets (orf1ab.2 and orf1ab.4) proved to be viable for use in the final assay. Colorimetric RT-LAMP of the selected primer set, orf1ab.2 was shown to produce a distinct color change in contrived samples containing heat-inactivated SARS-CoV-2 in 5% saliva. The limit of detection of our assay using primer set orf1ab.2 was determined to be 1000 copies/µL of saliva collected. Furthermore, methods are introduced which allow for the high-throughput design of LAMP primers using standard software tools and the <i>in-silico</i> performance of LAMP primer sets. </p>

<div>Sea turtles are integral components of many marine ecosystems. Green turtles (Chelonia mydas) are generally herbivorous, feed primarily on seagrasses, and are endangered in the Caribbean. The species utilizes extensive marine habitats for foraging and migratory routes, and because of its broad distribution, it is difficult to conduct population assessments. Here, I assessed commonly used techniques for monitoring green turtles in the wild. Specifically: (1) biopsy sampling for molecular assays and (2) unoccupied aerial vehicles (UAVs) deployment and boat-based surveys for population monitoring.</div><div><br></div><div>Skin biopsies are collected from sea turtles for a variety of molecular analyses; however, very little information exists on the natural healing rates at the site of the biopsy in the wild. In Chapter 2, I monitored the healing rates of 17 juvenile green turtles in Eleuthera, The Bahamas, for up to 488 d after taking a 6mm biopsy tissue sample. Complete tissue repair and maturation was observed after a year and a half, and there was no evidence of infection at any point during the healing process. While scarring persisted for several months, biopsy sampling had minimal long-term impact.</div><div><br></div><div>UAVs are increasingly being used to monitor marine megafauna. In Chapter 3, I evaluated the efficacy of using UAVs to detect sea turtles when compared to boat-based surveys. During UAV surveys, the UAV was flown along preprogrammed routes in four creek systems. A boat survey was conducted simultaneously on the same path. I used regression analyses for each survey type to assess the effects of environmental variables on turtle detection rates My results indicate that there were no statistically significant difference between the numbers of turtle detected via boat or UAV surveys; however, there were clear differences in the time and potential cost associated with either method.</div>

O trabalho teve por objetivo diagnosticar a infecção experimental e natural por Toxoplasma gondii em pombos (Columba livia), por técnicas sorológicas, biológicas e moleculares. Pombos foram infectados com oocistos esporulados de T. gondii e acompanhados por 60 dias com coleta de soro semanal para o acompanhamento da curva de anticorpos séricos e eutanásia quinzenal para avaliar a presença do parasito em diferentes tecidos. Observou-se concordância em todas as técnicas utilizadas, indicando serem eficazes no diagnóstico da infecção nessa espécie. Pombos de vida livre foram capturados nos municípios de São Paulo, Sorocaba e Ibiúna e anticorpos anti-T. gondii não foram observados. Nestas aves o bioensaio em camundongos foi realizado, independente da ausência de anticorpos e em nenhuma foi possível o isolamento do parasito. / The study aimed to diagnose the experimental and natural infection by Toxoplasma gondii in pigeons (Columba livia), by serological, biological and molecular techniques. Pigeons were infected with sporulated oocysts of T. gondii and monitored for 60 days with weekly serum collection for monitoring the curve of serum antibodies and euthanasia two weeks to assess the presence of parasites in different tissues. Agreement was observed in all the techniques used, indicating to be effective in the diagnosis of infection in this species. Free-living pigeons were captured in the municipalities of São Paulo, Sorocaba and Ibiúna and anti-T. gondii antibodies were not observed. In birds the bioassay was conducted in mice, regardless of the absence of antibodies and none was possible to isolate the parasite.

No description available.

O trabalho teve por objetivo diagnosticar a infecção experimental e natural por Toxoplasma gondii em pombos (Columba livia), por técnicas sorológicas, biológicas e moleculares. Pombos foram infectados com oocistos esporulados de T. gondii e acompanhados por 60 dias com coleta de soro semanal para o acompanhamento da curva de anticorpos séricos e eutanásia quinzenal para avaliar a presença do parasito em diferentes tecidos. Observou-se concordância em todas as técnicas utilizadas, indicando serem eficazes no diagnóstico da infecção nessa espécie. Pombos de vida livre foram capturados nos municípios de São Paulo, Sorocaba e Ibiúna e anticorpos anti-T. gondii não foram observados. Nestas aves o bioensaio em camundongos foi realizado, independente da ausência de anticorpos e em nenhuma foi possível o isolamento do parasito. / The study aimed to diagnose the experimental and natural infection by Toxoplasma gondii in pigeons (Columba livia), by serological, biological and molecular techniques. Pigeons were infected with sporulated oocysts of T. gondii and monitored for 60 days with weekly serum collection for monitoring the curve of serum antibodies and euthanasia two weeks to assess the presence of parasites in different tissues. Agreement was observed in all the techniques used, indicating to be effective in the diagnosis of infection in this species. Free-living pigeons were captured in the municipalities of São Paulo, Sorocaba and Ibiúna and anti-T. gondii antibodies were not observed. In birds the bioassay was conducted in mice, regardless of the absence of antibodies and none was possible to isolate the parasite.

<p>Metastasis of primary mammary tumors to vital secondary organs is the primary cause of breast cancer-associated death, with no effective treatment. Metastasis is a highly selective process that requires cancer cells to overcome multiple barriers to escape the primary tumor, survive in circulation, and eventually colonize distant secondary organs. One of the important aspects of metastatic cancers is the ability to undergo epithelial-mesenchymal transition (EMT) and the reverse process mesenchymal-epithelial transition (MET) process. Constant interconversion of tumor cells between these phenotypes creates epithelial-mesenchymal heterogeneity (EMH) and interaction between these tumor cell types and the stromal cell compartment is clearly important to metastasis. In healthy tissues, stromal cells maintain the composition and structure of the tissue through the production of extracellular matrix (ECM) proteins and paracrine signaling with epithelial cells. However, little is known about how EMH promotes changes in the ECM to promote breast cancer progression and metastasis. Cancer cells also secret exosomes, nano-size extracellular vesicles, to establish intercellular communication with distant organs in order to induce metastasis. These exosomes contain a plethora of different proteins including extracellular matrix proteins and matrix crosslinking enzymes. Fibronectin, an important ECM protein, plays an active role in tumor progression and is often crosslinked by tissue transglutaminase 2 (TGM2) to promote fibrosis in cancer. Both FN and TGM2 exist in exosomes and are expressed by heterogenous breast tumors. Although FN and TGM2 have been reported to play essential roles in cancer, their involvement in metastasis remains unclear. This work utilizes a variety of approaches to investigate the role of tumor heterogeneity and ECM proteins in promoting breast cancer metastasis. In this dissertation, we establish that mesenchymal cells expressing intracellular FN are held in a stable non-metastatic mesenchymal phenotype and produce cellular fibrils containing functionalized FN capable of supporting the growth of metastatic competent epithelial cells. We introduce a novel 3D culture system consisting of a tessellated scaffold which is capable of recapitulating cellular and matrix phenotypes <i>in vivo. </i>Further, we also demonstrate breast tumor cells secrete exosomes containing TGM2 crosslinked FN fibrils to promote premetastatic niche formation and induction of metastasis.<i> </i>Using genetic approaches, we establish TGM2 is essential and sufficient to drive metastasis. Finally, we demonstrate pharmacological inhibition of TGM2 offers a potential therapeutic strategy to treat metastatic breast cancer. Altogether, our research provides insights into the mechanism through which TGM2 promotes metastatic breast cancer. This work will help in developing new drugs to target TGM2 aimed at reducing breast cancer metastasis.<br></p>

<p>Neutrophils are the first cells recruited to an immune stimulus stemming from infection or sterile injuries via a mixture of chemoattractant cues. In addition to eliminating pathogens, neutrophils coordinate the overall inflammation by activating and producing inflammatory signals in the tissue while modulating the activation of other immune cells which in some cases leads to adverse tissue damage. Over amplified or chronic neutrophil recruitment directly leads to autoimmune diseases including rheumatic arthritis, diabetes, neurodegenerative diseases, and cancer. Dampening neutrophil recruitment is a strategy to intervene in neutrophil-orchestrated chronic inflammation. Despite intensive research over the past several decades, clinical studies targeting neutrophil migration have been largely unsuccessful, possibly due to the prominent redundancy of adhesion receptors and chemokines. Additional challenges lie in the balance of dampening detrimental inflammation while preserving immunity. Neutrophils are terminally differentiated cells that are hard to study in cell culture. Mouse models are often used to study hematopoiesis, migration, and chemotaxis of neutrophils but is very labor intensive. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified eight miRNAs as potent suppressors of neutrophil migration. We have generated transgenic zebrafish lines that overexpresses these candidate miRNAs where we recapitulated the mitigation in neutrophil motility and chemotaxis to tissue injury or infection. Among those we further characterized two miRNAs which have not been reported to regulate neutrophil migration, namely miR-722 and miR-199.</p> <p> </p> <p>MiR-722 downregulates the transcript level of <i>rac2</i> through binding to the <i>rac2</i> 3'UTR. Furthermore, miR-722-overexpressing larvae display improved outcomes in both sterile and bacterial systemic models, which correlates with a robust upregulation of the anti-inflammatory cytokines in the whole larvae and isolated neutrophils. miR-722 protects zebrafish from lethal lipopolysaccharide challenge. In addition, overexpression of mir-722 reduced chemotaxis of human neutrophil like cells, indicating that miR-722 is a potential agent to reduce inflammation in humans. </p> <p>MiR-199<i>,</i> decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (<i>cdk2</i>), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. </p> <p> </p> <p>In summary, our results reveal previously unknown functions of these miRNAs, and provide potential avenues to modulate neutrophil migration as well as lead to discoveries of novel factors which can regulate this process. We have also discovered a non-classical role of CDK2 in regulating neutrophil migration which provides directions for alleviating systemic inflammation and a better understanding of neutrophil biology. </p>