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1. Enumeration of marine microbial organisms by flow cytometry using near‐ UV excitation of Hoechst 34580‐stained DNA

   https://doi.org/10.1002/lom3.10454  

   Selph, Karen E. (August 2021, Limnology and Oceanography: Methods)

   Abstract A flow cytometry method for enumerating marine heterotrophic bacteria and phytoplankton in a living or preserved sample using a low power solid state near‐ultraviolet laser is described. The method uses Hoechst 34580 to stain DNA in microbial cells in seawater samples. This stain is optimally excited at 375 nm unlike the similar Hoechst 33342, which requires ~ 350 nm excitation only available on more expensive lasers. Phytoplankton abundances from the Hoechst 34580 method are comparable to those of unstained samples and when analyzed by the Hoechst 33342 staining method. With this new method, nonpigmented marine bacteria and phytoplankton abundances are obtained simultaneously in a single sample as the Hoechst emission wavelength (~ 450 nm) is well separated from the emission wavelengths of chlorophyll and phycoerythrin fluorescence. Bacteria abundances are similar between this new method and those obtained with established Hoechst 33342 and SybrGreen I methods. Precision estimates (coefficient of variation) on populations with abundances near ~ 10⁵cells mL⁻¹are 1–3%, increasing to 3–9% at lower cell concentrations of 10³cells mL⁻¹. The Hoechst 34580 method is simple, requiring no heating or pretreatment with RNAse, can be used on unpreserved and formaldehyde‐preserved cells, and is amenable to at‐sea use with portable, compact, low power‐requiring flow cytometers. 

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2. Towards an effective method for melting ice cores for microbiological analysis

   Michaud, Alexander B; Santibanez, Pamela A; Winski, Dominic A.; Chellman, Nathan; McConnell, Joseph R (May 2023, 2nd Annual US Ice Core Open Science Workshop)

   Ice cores contain stratigraphic records of microbial cells, buried through thousands of years of snow accumulation and spanning significant climatic periods. It is well established that microorganisms are transported to and preserved within the West Antarctic Ice Sheet. From the total assemblage of microorganisms that land on the ice sheet, we do not know how or if microorganisms survive burial and persist long-term in glacial ice equally. We cannot accurately interpret microbial cell stratigraphic records or utilize these cellular records as proxies until we understand post-depositional processes and the genomic adaptations of microbial cells in glacial ice. Here, we quantify cell concentrations in meltwater from four flow paths of a continuous flow analysis melter system in order to evaluate the efficacy of these flow paths for the successful collection of intact cells archived in ice cores. Using this information, we melted eight sections from the WAIS Divide ice core and quantified the cell concentrations, assayed the viability of the microbial cells, and sorted individual cells for genome sequencing. We will present preliminary data from the flow path cell recovery experiment, and genomic and viability results from the WAIS Divide ice core, with the hope to stimulate further discussion around single cell genomes and how they can be leveraged to complement paleoclimate information from ice cores. 

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3. On Single-Cell Enzyme Assays in Marine Microbial Ecology and Biogeochemistry

   https://doi.org/10.3389/fmars.2022.846656  

   Traving, Sachia J.; Balmonte, John Paul; Seale, Dan; Arnosti, Carol; Glud, Ronnie N.; Hallam, Steven J.; Middelboe, Mathias (February 2022, Frontiers in Marine Science)

   Extracellular enzyme activity is a well-established parameter for evaluating microbial biogeochemical roles in marine ecosystems. The presence and activity of extracellular enzymes in seawater provide insights into the quality and quantity of organic matter being processed by the present microorganisms. A key challenge in our understanding of these processes is to decode the extracellular enzyme repertoire and activities of natural communities at the single-cell level. Current measurements are carried out on bulk or size-fractionated samples capturing activities of mixed populations. This approach – even with size-fractionation – cannot be used to trace enzymes back to their producers, nor distinguish the active microbial members, leading to a disconnect between measured activities and the producer cells. By targeting extracellular enzymes and resolving their activities at the single-cell level, we can investigate underlying phenotypic heterogeneity among clonal or closely related organisms, characterize enzyme kinetics under varying environmental conditions, and resolve spatio-temporal distribution of individual enzyme producers within natural communities. In this perspective piece, we discuss state-of-the-art technologies in the fields of microfluidic droplets and functional screening of prokaryotic cells for measuring enzyme activity in marine seawater samples, one cell at a time. We further elaborate on how this single-cell approach can be used to address research questions that cannot be answered with current methods, as pertinent to the enzymatic degradation of organic matter by marine microorganisms. 

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4. The Medium is the Message: How Secure Messaging Apps Leak Sensitive Data to Push Notification Services

   https://doi.org/10.56553/popets-2024-0151  

   Samarin, Nikita; Sanchez, Alex; Chung, Trinity; Juleemun, Akshay_Dan Bhavish; Gilsenan, Conor; Merrill, Nick; Reardon, Joel; Egelman, Serge (October 2024, Proceedings on Privacy Enhancing Technologies)

   Like most modern software, secure messaging apps rely on third-party components to implement important app functionality. Although this practice reduces engineering costs, it also introduces the risk of inadvertent privacy breaches due to misconfiguration errors or incomplete documentation. Our research investigated secure messaging apps' usage of Google's Firebase Cloud Messaging (FCM) service to send push notifications to Android devices. We analyzed 21 popular secure messaging apps from the Google Play Store to determine what personal information these apps leak in the payload of push notifications sent via FCM. Of these apps, 11 leaked metadata, including user identifiers (10 apps), sender or recipient names (7 apps), and phone numbers (2 apps), while 4 apps leaked the actual message content. Furthermore, none of the data we observed being leaked to FCM was specifically disclosed in those apps' privacy disclosures. We also found several apps employing strategies to mitigate this privacy leakage to FCM, with varying levels of success. Of the strategies we identified, none appeared to be common, shared, or well-supported. We argue that this is fundamentally an economics problem: incentives need to be correctly aligned to motivate platforms and SDK providers to make their systems secure and private by default. 

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5. Selective Uptake of Pelagic Microbial Community Members by Caribbean Reef Corals

   https://doi.org/10.1128/AEM.03175-20  

   Hoadley, Kenneth D.; Hamilton, Maria; Poirier, Camille L.; Choi, Chang Jae; Yung, Cheuk-Man; Worden, Alexandra Z. (April 2021, Applied and Environmental Microbiology)

   Johnson, Karyn N. (Ed.)

   ABSTRACT Coral reefs are possible sinks for microbes; however, the removal mechanisms at play are not well understood. Here, we characterize pelagic microbial groups at the CARMABI reef (Curaçao) and examine microbial consumption by three coral species: Madracis mirabilis , Porites astreoides , and Stephanocoenia intersepta . Flow cytometry analyses of water samples collected from a depth of 10 m identified 6 microbial groups: Prochlorococcus , three groups of Synechococcus , photosynthetic eukaryotes, and heterotrophic bacteria. Minimum growth rates (μ) for Prochlorococcus , all Synechococcus groups, and photosynthetic eukaryotes were 0.55, 0.29, and 0.45 μ day −1 , respectively, and suggest relatively high rates of productivity despite low nutrient conditions on the reef. During a series of 5-h incubations with reef corals performed just after sunset or prior to sunrise, reductions in the abundance of photosynthetic picoeukaryotes, Prochlorococcus and Synechococcus cells, were observed. Of the three Synechococcus groups, one decreased significantly during incubations with each coral and the other two only with M. mirabilis. Removal of carbon from the water column is based on coral consumption rates of phytoplankton and averaged between 138 ng h −1 and 387 ng h −1 , depending on the coral species. A lack of coral-dependent reduction in heterotrophic bacteria, differences in Synechococcus reductions, and diurnal variation in reductions of Synechococcus and Prochlorococcus , coinciding with peak cell division, point to selective feeding by corals. Our study indicates that bentho-pelagic coupling via selective grazing of microbial groups influences carbon flow and supports heterogeneity of microbial communities overlying coral reefs. IMPORTANCE We identify interactions between coral grazing behavior and the growth rates and cell abundances of pelagic microbial groups found surrounding a Caribbean reef. During incubation experiments with three reef corals, reductions in microbial cell abundance differed according to coral species and suggest specific coral or microbial mechanisms are at play. Peaks in removal rates of Prochlorococcus and Synechococcus cyanobacteria appear highest during postsunset incubations and coincide with microbial cell division. Grazing rates and effort vary across coral species and picoplankton groups, possibly influencing overall microbial composition and abundance over coral reefs. For reef corals, use of such a numerically abundant source of nutrition may be advantageous, especially under environmentally stressful conditions when symbioses with dinoflagellate algae break down. 

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