Optimized Protocol for Imaging Cleared Neural Tissues Using Light Microscopy

Understanding physical and chemical processes at an organismal scale is a fundamental goal in biology. While science is adept at explaining biological phenomena at both molecular and cellular levels, understanding how these processes translate to organismal functions remains a challenging problem. This issue is particularly significant for the nervous system where cell signaling and synaptic activities function in the context of broad neural networks. Recent progress in tissue clearing technologies lessens the barriers that previously prevented the study of large tissue samples while maintaining molecular and cellular resolution. While these new methods open vast opportunities and exciting new questions, the logistics of analyzing cellular processes in intact tissue have to be carefully considered. In this protocol, we outline a procedure to rapidly image intact brain tissue up to thousands of cubic millimeters. This experimental pipeline involves three steps: tissue clearing, tissue imaging, and data analysis. In an attempt to streamline the process for researchers entering this field, we address important considerations for each of these stages and describe an integrated solution to image intact biological tissues. Hopefully, this optimized protocol will lower the barrier of implementing high-resolution tissue imaging and facilitate the investigations of mesoscale questions at molecular and cellular resolution

The effect of elevated carbon dioxide on the sinking and swimming of the shelled pteropod Limacina retroversa

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in ICES Journal of Marine Science 74 (2017): 1893–1905, doi:10.1093/icesjms/fsx008.Shelled pteropods are planktonic molluscs that may be affected by ocean acidification. Limacina retroversa from the Gulf of Maine were used to investigate the impact of elevated carbon dioxide (CO2) on shell condition as well as swimming and sinking behaviours. Limacina retroversa were maintained at either ambient (ca. 400 μatm) or two levels of elevated CO2 (800 and 1200 μatm) for up to four weeks, and then examined for changes in shell transparency, sinking speed, and swimming behaviour assessed through a variety of metrics (e.g., speed, path tortuosity, wing beat frequency). After exposures to elevated CO2 for as little as four days, the pteropod shells were significantly darker and more opaque in the elevated CO2 treatments. Sinking speeds were significantly slower for pteropods exposed to medium and high CO2 in comparison to the ambient treatment. Swimming behaviour showed less clear patterns of response to treatment and duration of exposure, but overall, swimming did not appear to be hindered under elevated CO2. Sinking is used by L. retroversa for predator evasion, and altered speeds and increased visibility could increase the susceptibility of pteropods to predation.Funding for this research was provided by a National Science Foundation grant to Lawson, Maas, and Tarrant (OCE-1316040). Additional support for field sampling was provided by the WHOI Coastal Ocean Institute, Pickman Foundation, and the Tom Haas Fund at the New Hampshire Charitable Foundation

Exposure to CO2 influences metabolism, calcification and gene expression of the thecosome pteropod Limacina retroversa

Author Posting. © The Company of Biologists, 2018. This article is posted here by permission of The Company of Biologists for personal use, not for redistribution. The definitive version was published in Journal of Experimental Biology 221 (2018): jeb164400, doi:10.1242/jeb.164400.Thecosomatous pteropods, a group of aragonite shell-bearing zooplankton, are becoming an important sentinel organism for understanding the influence of ocean acidification on pelagic organisms. These animals show vulnerability to changing carbonate chemistry conditions, are geographically widespread, and are both biogeochemically and trophically important. The objective of this study was to determine how increasing duration and severity of CO2 treatment influence the physiology of the thecosome Limacina retroversa, integrating both gene expression and organism-level (respiration and calcification) metrics. We exposed pteropods to over-saturated, near-saturated or under-saturated conditions and sampled individuals at 1, 3, 7, 14 and 21 days of exposure to test for the effect of duration. We found that calcification was affected by borderline and under-saturated conditions by week two, while respiration appeared to be more strongly influenced by an interaction between severity and duration of exposure, showing complex changes by one week of exposure. The organismal metrics were corroborated by specific gene expression responses, with increased expression of biomineralization-associated genes in the medium and high treatments throughout and complex changes in metabolic genes corresponding to both captivity and CO2 treatment. Genes associated with other physiological processes such as lipid metabolism, neural function and ion pumping had complex responses, influenced by both duration and severity. Beyond these responses, our findings detail the captivity effects for these pelagic organisms, providing information to contextualize the conclusions of previous studies, and emphasizing a need for better culturing protocols.Funding for this research was provided by a National Science Foundation grant to G.L.L., A.E.M. and A.M.T. (OCE-1316040). Additional support for field sampling was provided by theWoods Hole Oceanographic Institution, Coastal Ocean Institute and the Pickman Foundation.2019-02-1

A Phase I Trial of Aminolevulinic Acid-Photodynamic Therapy for Treatment of Oral Leukoplakia

Background Photodynamic therapy with aminolevulinic acid (ALA PDT) for oral leukoplakia has shown promising effects in regression of oral leukoplakia. Although ALA has been extensively studied and is an ideal photosensitizer, the optimal light dose for treatment of oral leukoplakia has not been determined. We conducted a phase I study to determine MTD and DLT of PDT in patients treated with ALA for leukoplakia. Methods Patients with histologically confirmed oral leukoplakia received a single treatment of ALA PDT in cohorts with escalating doses of light (585 nm). Clinical, histologic, and biologic markers were assessed. Results Analysis of 11 participants is reported. No significant toxicity from ALA PDT was observed in patients who received ALA with a light dose of up to 4 J/cm2. One participant experienced transient grade 3 transaminase elevation due to ALA. One participant had a partial clinical response 3 months after treatment. Biologic mucosal risk markers showed no significant associations. Determination of MTD could not be accomplished within a feasible timeframe for completion of the study. Conclusions ALA PDT could be safely administered with a light dose up to 4 J/cm2 and demonstrated activity. Larger studies are needed to fully elucidate the MTD and efficacy of ALA-PDT

Defining the role of extended saphenofemoral junction ligation: A prospective comparative study

AbstractObjective: This study explores the added effect of extended saphenofemoral junction (SFJ) ligation when the greater saphenous vein (GSV) has been eliminated from participating in thigh reflux by means of endovenous obliteration. GSV obliteration, unlike surgical stripping, can be done with or without SFJ ligation to isolate and study SFJ ligation’s specific contribution to treatment results. Methods: Sixty limbs treated with SFJ ligation and 120 limbs treated without high ligation were selected from an ongoing, multicenter, endovenous obliteration trial on the basis of their having primary varicose veins, GSV reflux, and early treatment dates. Results: Five (8%) high-ligation limbs and seven (6%) limbs without high ligation with patent veins at 6 weeks or less were excluded as unsuccessful obliterations. Treatment significantly reduced symptoms and CEAP clinical class in both groups (P =.0001). Recurrent reflux developed in one (2%) of 49 high-ligation limbs and eight (8%) of 97 limbs without high ligation by 6 months (P =.273). New instances of reflux did not appear thereafter in 57 limbs followed to 12 months. Recurrent varicose veins occurred in three high-ligation limbs and four limbs without high ligation by 6 months and in one additional high-ligation limb and two additional limbs without high ligation by 12 months. Actuarial recurrence curves were not statistically different with or without SFJ ligation (P >.156), predicting greater than 90% freedom from recurrent reflux and varicosities at 1 year for both groups. Conclusion: These early results suggest that extended SFJ ligation may add little to effective GSV obliteration, but our findings are not sufficiently robust to warrant abandonment of SFJ ligation as currently practiced in the management of primary varicose veins associated with GSV vein reflux. (J Vasc Surg 2000;32:941-53.

Direct detection of Mycobacterium tuberculosis in respiratory samples from patients in Scandinavia by polymerase chain reaction

ObjectiveTo investigate the use of DNA amplification by the polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis directly in human respiratory specimens.MethodsThe PCR assay employed was the Amplicor M. tuberculosis Test (Roche Diagnostics, Switzerland), which uses the 16S rDNA as the target template. Nine hundred and sixty samples from 741 patients in two clinical microbiology laboratories in Norway and Sweden were processed by routine culture analysis and PCR.ResultsOf the 56 specimens containing cultivatable M. tuberculosis, 49 (87.5%) were detected by PCR. Among the 904 culture-negative specimens, 897 samples were negative also by PCR and seven (0.8%) were positive by PCR. In comparison with culture, the sensitivity, specificity, and positive and negative predictive values of PCR were 91.7%, 99.6%, 94.2% and 99.4% for laboratory 1 and 80.0%, 98.7%, 76.2% and 99.0% for laboratory 2, respectively. For both laboratories combined the values were 87.5%, 99.2%, 87.5% and 99.2%.ConclusionsThese results indicate that multiple (two or three) respiratory samples from each patient should be tested, to allow sufficient accuracy in detecting M. tuberculosis in the specimens. Still, the labor-intensive format of this test necessitates strong clinical indications and patient prioritization to provide a service feasible within the current limits of routine laboratories