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Coral reefs are invaluable ecosystems that are under threat from various anthropogenic stressors. There has been a recent increase in the diagnostic tools utilized to understand how these threats impact coral reef health. Unfortunately, the application of diagnostic tools like transmission electron microscopy (TEM) is not as standardized or developed in coral research as in other research fields. Utilizing TEM in conjunction with other diagnostic methods can aid in understanding the impact of these stressors on the cellular level because TEM offers valuable insight into the structures and microsymbionts associated with coral tissue that cannot be obtained with a conventional light microscope. Additionally, a significant amount of coral tissue ultrastructure has not yet been extensively described, causing a considerable gap in our understanding of cellular structures that could relate to the immune response, cellular function, or symbioses. Moreover, additional standardization is needed for TEM in coral research to increase comparability and reproducibility of findings across studies. Here, we present standardized TEM sample fixation, embedding, and sectioning techniques for coral studies that ensure consistent ultrastructural preservation and minimize artifacts, enhancing the reliability and accuracy of TEM observations. We also demonstrate that these TEM protocols allow for the observation and quantification of bacterial and viral-like particles within the coral tissue as well as the endosymbiotic microalgae, potentially providing insight into their interactions within coral cells and how they relate to overall coral health and resilience. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Primary fixation Basic Protocol 2: Decalcification Basic Protocol 3: Sample dissection, secondary fixation, dehydration, and embedding Basic Protocol 4: Sectioning and grid staining Basic Protocol 5: Imaging., (© 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

Background: Klebsiella pneumoniae carbapenemase-producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection., Methods: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model., Results: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice., Conclusions: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rationale: Complement is crucial for host defense but may also drive dysregulated inflammation. There is limited understanding of alternative complement function, which can amplify all complement activity, during critical illness. Objectives: We examined the function and key components of the alternative complement pathway in a series of critically ill patients and in a mouse pneumonia model. Methods: Total classical (CH50) and alternative complement (AH50) function were quantified in serum from 321 prospectively enrolled critically ill patients and compared with clinical outcomes. Alternative pathway (AP) regulatory factors were quantified by ELISA ( n  = 181) and examined via transcriptomics data from external cohorts. Wild-type, Cfb -/- , and C3 -/- mice were infected intratracheally with Klebsiella pneumoniae (KP) and assessed for extrapulmonary dissemination. Measurements and Main Results: AH50 greater than or equal to median, but not CH50 greater than or equal to median, was associated with decreased 30-day mortality (adjusted odds ratio [OR], 0.53 [95% confidence interval (CI), 0.31-0.91]), independent of chronic liver disease. One-year survival was improved in patients with AH50 greater than or equal to median (adjusted hazard ratio = 0.59 [95% CI, 0.41-0.87]). Patients with elevated AH50 had increased levels of AP factors B, H, and properdin, and fewer showed a "hyperinflammatory" subphenotype (OR, 0.30 [95% CI, 0.18-0.49]). Increased expression of proximal AP genes was associated with improved survival in two external cohorts. AH50 greater than or equal to median was associated with fewer bloodstream infections (OR, 0.67 [95% CI, 0.45-0.98). Conversely, depletion of AP factors, or AH50 less than median, impaired in vitro serum control of KP that was restored by adding healthy serum. Cfb -/- mice demonstrated increased extrapulmonary dissemination and serum inflammatory markers after intratracheal KP infection compared with wild type. Conclusions: Elevated AP function is associated with improved survival during critical illness, possibly because of enhanced immune capacity.

Characterizing the thermal niche of harmful algae is crucial for understanding and projecting the effects of future climate change on harmful algal blooms. The effects of 6 different temperatures (18-32 °C) on the growth, photophysiology, and toxicity were examined in the dinoflagellate Karlodinium veneficum, and the raphidophytes, Heterosigma akashiwo and Chattonella subsalsa from the Delaware Inland Bays (DIB). K. veneficum and H. akashiwo had skewed unimodal growth patterns, with temperature optima (Topt) at 28.6 and 27.3 °C respectively and an upper thermal niche limit of 32 °C. In contrast, C. subsalsa growth increased linearly with temperature, suggesting Topt and upper thermal boundaries >32 °C. K. veneficum photosystem II (PSII) photochemical efficiency remained stable across all temperatures, while H. akashiwo PSII efficiency declined at higher temperature and C. subsalsa was susceptible to low temperature (~18 °C) photoinactivation. Cell toxicity thermal response was species-specific such that K. veneficum toxicity increased with temperature above Topt. Raphidophyte toxicity peaked at 25-28 °C and was in close agreement with Topt for growth in H. akashiwo but below C. subsalsa maximal growth. The mode of toxicity was markedly different between the dinoflagellate and the raphidophytes such that K. veneficum had greater hemolytic activity while the raphidophytes had pronounced fish gill cell toxicity. These results and patterns of natural abundance for these algae in the DIB suggest that continued ocean warming may contribute to C. subsalsa bloom formation while possibly promoting highly toxic blooms of K. veneficum., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Kidneys are highly vascularized and contain many distinct vascular beds. However, the origins of renal endothelial cells and roles of the developing endothelia in the formation of the kidney are unclear. We have shown that the Foxd1-positive renal stroma gives rise to endothelial marker-expressing progenitors that are incorporated within a subset of peritubular capillaries; however, the significance of these cells is unclear. The purpose of this study was to determine whether deletion of Flk1 in the Foxd1 stroma was important for renal development. To that end, we conditionally deleted Flk1 (critical for endothelial cell development) in the renal stroma by breeding-floxed Flk1 mice ( Flk1 fl/fl ) with Foxd1cre mice to generate Foxd1cre; Flk1 fl/fl ( Flk1 ST-/- ) mice. We then performed FACsorting, histological, morphometric, and metabolic analyses of Flk1 ST-/- vs. control mice. We confirmed decreased expression of endothelial markers in the renal stroma of Flk1 ST-/- kidneys via flow sorting and immunostaining, and upon interrogation of embryonic and postnatal Flk1 ST-/- mice, we found they had dilated peritubular capillaries. Three-dimensional reconstructions showed reduced ureteric branching and fewer nephrons in developing Flk1 ST-/- kidneys vs., Controls: Juvenile Flk1 ST-/- kidneys displayed renal papillary hypoplasia and a paucity of collecting ducts. Twenty-four-hour urine collections revealed that postnatal Flk1 ST-/- mice had urinary-concentrating defects. Thus, while lineage-tracing revealed that the renal cortical stroma gave rise to a small subset of endothelial progenitors, these Flk1-expressing stromal cells are critical for patterning the peritubular capillaries. Also, loss of Flk1 in the renal stroma leads to nonautonomous-patterning defects in ureteric lineages., (Copyright © 2017 the American Physiological Society.)