A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the ...most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.
Persistently high, worldwide mortality from cancer highlights the unresolved challenges of disease surveillance and detection that impact survival. Development of a non-invasive, blood-based ...biomarker would transform survival from cancer. We demonstrate the functionality of ultra-high content analyses of a newly identified population of tumor cells that are hybrids between neoplastic and immune cells in patient matched tumor and peripheral blood specimens. Using oligonucleotide conjugated antibodies (Ab-oligo) permitting cyclic immunofluorescence (cyCIF), we present analyses of phenotypes among tumor and peripheral blood hybrid cells. Interestingly, the majority of circulating hybrid cell (CHC) subpopulations were not identified in tumor-associated hybrids. These results highlight the efficacy of ultra-high content phenotypic analyses using Ab-oligo based cyCIF applied to both tumor and peripheral blood specimens. The combination of a multiplex phenotypic profiling platform that is gentle enough to analyze blood to detect and evaluate disseminated tumor cells represents a novel approach to exploring novel tumor biology and potential utility for developing the population as a blood-based biomarker in cancer.
One of the bottlenecks for commercial implementation of integrated multi‐trophic aquaculture (IMTA) is the difficulty in quantifying its environmental performance. We reviewed a large body of ...literature to determine the variability in nutrient dynamics within different IMTA systems (open sea‐cages, land‐based flow‐through and recirculating aquaculture systems), with the aim to provide a generic framework to quantify nutrient retention efficiencies in integrated aquaculture systems. Based on the eco‐physiological requirements of the cultured species, as well as the response of “extractive” species to waste from “fed” species, the maximum retention efficiency was defined for a conceptual four‐species marine IMTA system (fish–seaweed–bivalve–deposit feeder). This demonstrated that 79%–94% of nitrogen, phosphorus and carbon supplied with fish feed could theoretically be retained. In practice, however, various biological and environmental factors may limit retention efficiencies and thereby influence the bioremediation of IMTA systems. These biological (waste production, stoichiometry in nutrient requirements) and environmental (temporal and spatial connectivity) factors were therefore evaluated against the theoretical reference frame and showed that efficiencies of 45%–75% for closed systems and 40%–50% for open systems are more realistic. This study is thereby the first to provide quantitative estimates for nutrient retention across IMTA systems, demonstrating that a substantial fraction of nutrients released from fish culture units can be retained by extractive species and subsequently harvested. Furthermore, by adapting this framework to the design and the condition prevailing for a specific IMTA system, it becomes a generic tool to analyse the system's bioremediation potential and explore options for further improvement.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in a zoological institution were initially reported in March 2020. Since then, at least 94 peer-reviewed cases have been ...reported in zoos worldwide. Among the affected animals, nonhuman primates, carnivores, and artiodactyls appear to be most susceptible to infection, with the Felidae family accounting for the largest number of reported cases. Clinical symptoms tend to be mild across taxa; although, certain species exhibit increased susceptibility to disease. A variety of diagnostic tools are available, allowing for initial diagnostics and for the monitoring of infectious risk. Whilst supportive therapy proves sufficient in most cases, monoclonal antibody therapy has emerged as a promising additional treatment option. Effective transmission of SARS-CoV-2 in some species raises concerns over potential spillover and the formation of reservoirs. The occurrence of SARS-CoV-2 in a variety of animal species may contribute to the emergence of variants of concern due to altered viral evolutionary constraints. Consequently, this review emphasizes the need for effective biosecurity measures and surveillance strategies to prevent and control SARS-CoV-2 infections in zoological institutions.
This review summarizes the current understanding of how brevetoxins, produced by
during harmful algal blooms, impact sea turtle health. Sea turtles may be exposed to brevetoxins through ingestion, ...inhalation, maternal transfer, and potentially absorption through the skin. Brevetoxins bind to voltage-gated sodium channels in the central nervous system, disrupting cellular function and inducing neurological symptoms in affected sea turtles. Moreover, the current evidence suggests a broader and longer-term impact on sea turtle health beyond what is seen during stranding events. Diagnosis relies on the detection of brevetoxins in tissues and plasma from stranded turtles. The current treatment of choice, intravenous lipid emulsion therapy, may rapidly reduce symptoms and brevetoxin concentrations, improving survival rates. Monitoring, prevention, and control strategies for harmful algal blooms are discussed. However, as the frequency and severity of blooms are expected to increase due to climate change and increased environmental pollution, continued research is needed to better understand the sublethal effects of brevetoxins on sea turtles and the impact on hatchlings, as well as the pharmacokinetic mechanisms underlying brevetoxicosis. Moreover, research into the optimization of treatments may help to protect endangered sea turtle populations in the face of this growing threat.
Marmosets are routinely used in biomedical research, therefore there is an increasing need for updated reference intervals calculated using a large sample size, correct statistics, and considering ...different variables. Hematological and biochemical values from 472 healthy common marmosets sedated with alphaxalone were collected over a ten-year period (2013-2023). The variables assumed to have influenced the blood-based parameters were compared, i.e., sex, age, housing condition, pregnancy, and contraceptive use. Reference intervals were calculated based on observed percentiles without parametric assumptions, and with parametric assumptions following Box-Cox transformation. Juvenile marmosets showed increased ALP, phosphate, WBC, lymphocyte count, and basophil count and decreased levels of GGT and Fe compared to adults. Marmosets housed strictly indoors showed increased ALT and GGT levels and decreased levels of total bilirubin and neutrophil count compared to marmosets housed with outdoor access. Pregnant marmosets showed increased ALP, total bilirubin, neutrophil count, monocyte count, and basophil count, and decreased levels of AST, ALT, cholesterol, Fe, and lymphocyte count compared to non-pregnant marmosets. Etonogestrel contracepted marmosets showed decreased P-LCR compared to females who were not contracepted. Updated reference intervals will aid researchers and veterinarians in identifying physiological and pathological changes, as well as improve the reproducibility of research in this species.
Capitella sp. and Ophryotrocha craigsmithi received a diet of salmon feces to evaluate their potential to convert fish waste into valuable marine products, e.g. ingredients for fish feed formulation. ...Production rate and body composition (focusing on fatty acid FA profiles) were determined for polychaetes fed fresh, acid-preserved or oven-dried salmon feces to evaluate their application in (de)coupled integrated multi-trophic aquaculture (IMTA) systems. Coupled production refers to direct integration of fish and polychaetes within the same (eco)system, while in decoupled production, units can be spatially or functionally separated. For decoupled production, preservation of fish waste is recommended. Although diets contained relatively low polyunsaturated FA (PUFA) levels (5–9% of total FAs), both species were rich in PUFAs (>30% of total FAs) and contained the essential FAs for fish. Feeding Capitella sp. the acid-preserved diet enriched its FA profile. Accumulation of PUFAs, de novo synthesis and/or transfer via bacterial biomass could have played a role in the upregulation of PUFA content. Amino acid profiles indicated that these polychaetes contained the amino acids essential for fish. Highest growth for both species was observed when fed fresh feces, whereas preserved diets resulted in negative growth rates for O. craigsmithi, suggesting an important role of microbes in polychaete diets. Our results indicate that both species are potential valuable marine products. Given growth rates with different diets, O. craigsmithi seems more suitable for integration in coupled systems, while Capitella sp. is interesting for both coupled and decoupled integrated systems.
The growth, tissue content and nutrient removal rates of
Ulva
spp., when exposed to moderate to high nitrogen (0.5–5 mmol L
−1
) and phosphorus (0.01–0.9 mmol L
−1
) concentrations, were examined to ...get a better understanding of recirculating IMTA (Integrated Multi-Trophic Aquaculture) systems with fish and seaweed. It was hypothesized that fish waste effluents might lead to unfavorable nutrient stoichiometry and/or toxic conditions, which might harm seaweeds and, specifically for the present study, reduce
Ulva
spp. performance. Results demonstrate that: (I) the unfavorable N:P stoichiometry (N:P ≠ Atkinson atomic ratio of 30:1) did not restrict
Ulva
spp. growth nor tissue content; this indicates that supply of both nutrients exceeded the minimum requirements; (II) a high orthophosphate concentration (0.9 mmol L
−1
) was toxic to
Ulva
spp.
,
whereas (III) a high nitrate concentration (5 mmol L
−1
) did not inhibit phosphorus uptake; (IV)
Ulva
’s growth was not enhanced when nitrate was exchanged for similarly high ammonium concentrations. However, tissue nitrogen content was 1.4 times higher when exposed to ammonium than nitrate, suggesting that the former N-form was stored faster in the seaweed’s tissue. Therefore, other factors must have limited growth with the high ammonium concentrations. This study also highlights the importance of relatively long acclimatization periods (one week) when maintenance uptake (V
m
) is evaluated, as surge uptake (V
s
) may result in considerably different and more variable rates. Results of this study contribute to a better understanding of the application of
Ulva
spp. as extractive component in closed IMTA systems, thus advancing sustainable and circular production techniques.
Applications of seaweeds require the supply of uniform biomass, yet performance of
Ulva
is generally characterised by a wide variation across study sites and seasons, as well as among species or ...strains. This study aimed to determine the variation in growth, biochemical composition and nutrient uptake of five
Ulva
strains collected in the Eastern Scheldt estuary (the Netherlands) and cultivated under standardised outdoor conditions from July to November. Surprisingly, only two strains performed well (maximum 291 ± 95 and 570 ± 122 kg FW ha
−1
day
−1
for VEE and YER strain), one strain showed slow growth (maximum 155 ± 91 kg FW ha
−1
day
−1
for SCH strain), and the last two strains (JAC and KOM) did not grow at all. Chemical composition was only determined for the well-performing strains. For most of the parameters, strong seasonality was observed; growth, crude fat (0.4–1.3%), ash (16–25%) and starch (3–11%) decreased, whereas fibres (37–45%) and proteins (7–22% crude; 6–18% true) increased. To evaluate the bio-remediation potential, nutrient uptake of YER, VEE and SCH strains was determined from in- and outflowing water (453–2027 µmol DIN; 4–101 µmol PO
4
kg FW h
−1
). Highest rates were observed for the SCH strain which was not in accordance with growth nor composition, suggesting that nutrients were not (all) directed towards vegetative growth. This study demonstrates a large variation in performance between different
Ulva
strains collected in relatively close proximity and thereby highlights the importance to test a variety of local seaweed strains prior to commercial farming.
Significance: Advanced genetic characterization has informed cancer heterogeneity and the challenge it poses to effective therapy; however, current methods lack spatial context, which is vital to ...successful cancer therapy. Conventional immunolabeling, commonplace in the clinic, can provide spatial context to protein expression. However, these techniques are spectrally limited, resulting in inadequate capacity to resolve the heterogenous cell subpopulations within a tumor.
Aim: We developed and optimized oligonucleotide conjugated antibodies (Ab-oligo) to facilitate cyclic immunofluorescence (cyCIF), resulting in high-dimensional immunostaining.
Approach: We employed a site-specific conjugation strategy to label antibodies with unique oligonucleotide sequences, which were hybridized in situ with their complementary oligonucleotide sequence tagged with a conventional fluorophore. Antibody concentration, imaging strand concentration, and configuration as well as signal removal strategies were optimized to generate maximal staining intensity using our Ab-oligo cyCIF strategy.
Results: We successfully generated 14 Ab-oligo conjugates and validated their antigen specificity, which was maintained in single color staining studies. With the validated antibodies, we generated up to 14-color imaging data sets of human breast cancer tissues.
Conclusions: Herein, we demonstrated the utility of Ab-oligo cyCIF as a platform for highly multiplexed imaging, its utility to measure tumor heterogeneity, and its potential for future use in clinical histopathology.
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