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18 pages, 669 KiB  
Article
Enzymatic Hydrolysis as an Effective Method for Obtaining Wheat Gluten Hydrolysates Combining Beneficial Functional Properties with Health-Promoting Potential
by Magdalena Mika and Agnieszka Wikiera
Molecules 2024, 29(18), 4407; https://doi.org/10.3390/molecules29184407 - 16 Sep 2024
Abstract
The byproduct from wheat starch production contains approximately 70% gluten (WG) and is an inexpensive but demanding protein raw material for the food industry. This study attempted to determine the optimal hydrolysis conditions for such raw material to obtain peptides combining beneficial functional [...] Read more.
The byproduct from wheat starch production contains approximately 70% gluten (WG) and is an inexpensive but demanding protein raw material for the food industry. This study attempted to determine the optimal hydrolysis conditions for such raw material to obtain peptides combining beneficial functional characteristics with health-promoting activity. The proteases Bromelain, Alcalase, Flavourzyme, and a protease from A. saitoi were used for hydrolysis. It was shown that the tested proteases differ both in terms of the effective hydrolysis conditions of gluten and the profile of the released hydrolysates. Bromelain was particularly effective in converting gluten into peptides, combining beneficial health and functional properties. It achieved maximum activity (189 U/g) against WG at pH 6 and 60 °C, and the best-balanced peptides in terms of desired properties were released at a dose of 2.5 U/g. These peptides were free from most allergenic epitopes, effectively inhibited ACE, and, at 0.34 g, were equivalent to the approved dose of BHT. Their emulsifying activity was higher than that of gluten, and the foaming formation and stabilization potential exceeded that of ovalbumin by 10% and 19%, respectively. It seems that Bromelain-released WG hydrolysates are a promising candidate for a safe fat stabilizer and egg white substitute. Full article
(This article belongs to the Special Issue Bioactive Compounds in Food and Their Applications)
19 pages, 5207 KiB  
Article
Continuous Video Monitoring of Zoo Cheetahs (Acinonyx jubatus jubatus) Reveals Differential Engagement Patterns for Environmental Enrichment Items Based on Sensory Category
by Haley N. Beer, Dustin T. Yates, Trenton C. Shrader and Ty B. Schmidt
J. Zool. Bot. Gard. 2024, 5(3), 520-538; https://doi.org/10.3390/jzbg5030035 (registering DOI) - 16 Sep 2024
Abstract
Strategically enriching the environment of zoo felids to encourage innate behaviors is essential to good welfare. To investigate how engagement differs among sensory-based enrichment, six items representing tactile (firehose log, Jolly Eggs), visual (mirror, movie), and olfactory (cinnamon, cologne) sensory categories were offered [...] Read more.
Strategically enriching the environment of zoo felids to encourage innate behaviors is essential to good welfare. To investigate how engagement differs among sensory-based enrichment, six items representing tactile (firehose log, Jolly Eggs), visual (mirror, movie), and olfactory (cinnamon, cologne) sensory categories were offered to three adult cheetah siblings. Each item was offered four times: twice during daytime hours and twice overnight (≥5 h/occasion). Static video monitoring was used to investigate engagement frequency and duration for each item and to observe grooming behaviors. Cheetahs engaged with visual enrichment more frequently (p < 0.05) and for greater (p < 0.05) periods than other items. Allogrooming occurred most frequently (p < 0.05) when the movie was provisioned at night. Tactile enrichment was only modestly engaged, with fewer but longer engagements (p < 0.05) at nighttime than at daytime. Autogrooming frequency did not vary among enrichment items but was greater (p < 0.05) at night than day. These findings showed that zoo cheetah responses to sensory enrichment items differed based on type and time of day. Visual stimuli were favored, whereas olfactory stimuli were generally ignored. This illustrates the benefit of diverse sensory-specific enrichment strategies. Such dynamic strategies can help overcome the challenge of individual preferences to encourage engagement reflective of the animal’s natural behaviors. Full article
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<p>Environmental enrichment items offered to group-housed zoo cheetahs. Items were chosen from three sensory category: (<b>a</b>) tactile stimuli: firehose log (top) and Jolly Eggs (bottom); (<b>b</b>) visual stimuli: mirror (top) and movie (bottom); and (<b>c</b>) olfactory stimuli: cinnamon (top) and cologne (bottom).</p>
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<p>Enrichment engagement and grooming were documented in group-housed zoo cheetahs provided different enrichment items. The top row shows examples of (<b>A</b>) autogrooming (self-grooming) and (<b>B</b>) allogrooming (grooming of another cheetah). The 2nd row shows examples of engagement with tactile enrichment items: (<b>C</b>) firehose log and (<b>D</b>) Jolly Eggs. The 3rd row shows examples of engagement with visual enrichment items: (<b>E</b>) mirror and (<b>F</b>) movie on a tablet. The bottom row shows examples of engagement with olfactory enrichment items: (<b>G</b>) cinnamon and (<b>H</b>) cologne.</p>
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<p>Proportional time that cheetahs spent engaging environmental enrichment items. Data are expressed as the fraction of total time the item was offered. These data were found to be non-normal by Shapiro–Wilk test (W, 0.466; <span class="html-italic">p</span> &lt; 0.01). Therefore, statistical analyses represented on this graph were performed on data ranks. <sup>a, b</sup> Means with different superscripts differ (<span class="html-italic">p</span> &lt; 0.05). Rank sums are presented in <a href="#app1-jzbg-05-00035" class="html-app">Supplementary Figure S2</a>. Note: Y-axes vary for data visibility.</p>
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<p>Proportion of time that cheetahs spent grooming. Data are expressed as the fraction of total time the item was offered. These data were found to be normal by Shapiro–Wilk test (W = 0.956; <span class="html-italic">p</span> = 0.35). <sup>a, b</sup> Means with different superscripts differ (<span class="html-italic">p</span> &lt; 0.05) within each behavior. Note: Y-axes vary for data visibility.</p>
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<p>Mean duration of engagements with different sensory enrichment items during daytime (top) and nighttime (bottom) periods. These data were found to be non-normal by Shapiro–Wilk test (W, 0.533; <span class="html-italic">p</span> &lt; 0.01). Therefore, statistical analyses represented on this graph were performed on data ranks. <sup>a, b, c, d</sup> Means with different superscripts differ (<span class="html-italic">p</span> &lt; 0.05). Rank sums are presented in <a href="#app1-jzbg-05-00035" class="html-app">Supplementary Figure S3</a>. Note: Y-axes vary for data visibility.</p>
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<p>Mean duration of time spent engaging in autogrooming with different sensory enrichment items during daytime and nighttime applications. These data were found to be non-normal by Shapiro–Wilk test (W, 0.598; <span class="html-italic">p</span> &lt; 0.01). Therefore, statistical analyses represented on this graph were performed on data ranks. <sup>a, b</sup> Means with different superscripts tend to differ (<span class="html-italic">p</span> &lt; 0.10). Rank sums are presented in <a href="#app1-jzbg-05-00035" class="html-app">Supplementary Figure S3</a>. Note: Y-axes vary for data visibility.</p>
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<p>Mean duration of allogrooming during the day and at night. These data were found to be non-normal by Shapiro–Wilk test (W, 0.651; <span class="html-italic">p</span> &lt; 0.01). Therefore, statistical analyses represented on this graph were performed on data ranks. <sup>a, b, c, d</sup> Means with different superscripts differ (<span class="html-italic">p</span> &lt; 0.05). Rank sums are presented in <a href="#app1-jzbg-05-00035" class="html-app">Supplementary Figure S3</a>. Note: Y-axes vary for data visibility.</p>
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<p>Evolution of behavioral responses to a mirror used as environmental enrichment in group-housed zoo cheetahs: (<b>top</b>) initial cautious observation marked by guarded movements and hesitant visitation; (<b>bottom</b>) transition to curiosity with investigative behaviors, such as attempts to peek behind the mirror in search of the source of the reflected images.</p>
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18 pages, 280 KiB  
Article
Complementary Feeding in the Clinical Practice: An Exploratory Survey among Italian Primary Care Pediatricians
by Maria Elena Capra, Nicola Mattia Decarolis, Brigida Stanyevic, Antonella Giudice, Delia Monopoli, Cosimo Neglia, Greta Ramundo, Susanna Esposito and Giacomo Biasucci
Nutrients 2024, 16(18), 3127; https://doi.org/10.3390/nu16183127 - 16 Sep 2024
Abstract
Introduction: Complementary feeding (CF) is the process of introducing solid or liquid foods (complementary foods, CFs) other than human breast milk (HBM) or infant formula into infants’ diet when HBM or infant formula is no longer sufficient to meet infants’ nutritional needs. Primary [...] Read more.
Introduction: Complementary feeding (CF) is the process of introducing solid or liquid foods (complementary foods, CFs) other than human breast milk (HBM) or infant formula into infants’ diet when HBM or infant formula is no longer sufficient to meet infants’ nutritional needs. Primary care pediatricians (PCPs) are paramount in guiding and educating infants’ families during CF. Materials and Methods: Our exploratory survey aimed to investigate PCPs’ current clinical approach to managing CF. From 1 March 2024 to 30 April 2024, a digital questionnaire composed of 32 multiple-choice questions investigating PCPs’ attitudes toward CF in healthy, full-term infants was proposed to 1620 PCPs contacted through scientific societies. Results: The questionnaire was completed voluntarily; 707 PCPs (79.5% female, 66.1% aged over 50 years) fully responded to the survey in the proposed timeframe (participation rate 43.6%). Among the responders, 47.5% recommended traditional CF; 42.1% declared to know the baby-led weaning (BLW) approach and on-demand CF (ODCF), but only 32.8% and 12.5% of them recommended these types of CF, respectively. The vast majority (95%) of participants recommended that CF start between 4 to 6 completed months of age. CF routinely based on vegetarian or plant-based diets was supported by 45/707 (6.1%), only if planned by a specialist by 253/707 (35.8%), and only upon request by caregivers by 257/707 (36.3%). Egg and fish introduction was mostly advised in the first year of life, although in case of a positive family history of food allergy, 13.3% of participants recommended the introduction of egg and fish after 12 months. Conclusions: In conclusion, PCPs did not display a homogenous approach to CF; further studies and educational programs are needed to achieve more flexibility and knowledge on this important nutritional issue. Full article
(This article belongs to the Section Pediatric Nutrition)
18 pages, 2443 KiB  
Article
Biological Properties of the Mucus and Eggs of Helix aspersa Müller as a Potential Cosmetic and Pharmaceutical Raw Material: A Preliminary Study
by Anna Herman, Patrycja Wińska, Małgorzata Białek and Andrzej P. Herman
Int. J. Mol. Sci. 2024, 25(18), 9958; https://doi.org/10.3390/ijms25189958 (registering DOI) - 15 Sep 2024
Viewed by 253
Abstract
In recent years, snail mucus (SM) has become popular as an active ingredient in cosmetic and pharmaceutical products. In turn, snail eggs (SEs) also seem to be a promising active compound, but the biological activities of SEs are significantly less known. Therefore, our [...] Read more.
In recent years, snail mucus (SM) has become popular as an active ingredient in cosmetic and pharmaceutical products. In turn, snail eggs (SEs) also seem to be a promising active compound, but the biological activities of SEs are significantly less known. Therefore, our preliminary study aimed to compare the biological activities of the SEs and SM of Helix aspersa Müller. The metabolomic analysis (LC–MS technique), determination of the antimicrobial activity (agar diffusion test, broth microdilution methods), antioxidant activity (ABTS assay), cytotoxicity assay (MTT), and proapoptotic properties (flow cytometry) of the SEs and SM were evaluated. It was found that the SEs and SM contain 8005 and 7837 compounds, respectively. The SEs showed antibacterial activity against S. aureus (MIC 12.5 mg/mL) and P. aeruginosa (MIC 3.12 mg/mL). The EC50 estimation of the antioxidant activity is 89.64 mg/mL and above 100 mg/mL for the SEs and SM, respectively. The SEs also inhibited the cell proliferation of cancer cell lines (HCT-116, MCF-7, HT-29) more strongly compared to the SM. The highest proportion of apoptotic cells in HCT-116 was observed. The reach composition of the compounds in the SEs and SM may be crucial for the creation of new cosmetic and pharmaceutical raw materials with different biological activities. However, further extended studies on the biological activities of H. aspersa-delivered materials are still necessary. Full article
(This article belongs to the Special Issue New Insights in Natural Bioactive Compounds 3.0)
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<p>Radical scavenging activity of SM and SEs in ABTS methods.</p>
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<p>Viability of MCF-7, HT-29, HCT-116, and Vero cell lines after the treatment with SE (<b>A</b>) or SM (<b>B</b>). After 72 h of incubation, MTT test was performed. The data for the treated cells were analyzed by Dunnett’s multiple comparison test as follows: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 relative to control served as 100%; ns—not significant.</p>
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<p>Induction of apoptosis in HCT-116 cells. The data were determined by Accuri C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA) after 72 h of treatment with SE. Cells were stained with annexin V-FITC and PI (propidium iodide). (<b>A</b>) Mean and standard deviation (SD) of necrosis, as well as viable, early, and late apoptosis, as a percentage from three independent experiments each. (<b>B</b>) Representative cytograms for control HTC-116 cells (CTRL) and after treatment with SE. The data for early and late apoptotic cells were analyzed by Dunnett’s multiple comparison test as follows: *** <span class="html-italic">p</span> &lt; 0.001 relative to control; ns—not significant.</p>
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<p>Induction of apoptosis in MCF-7 cells. The data were determined by Accuri C6 Plus flow cytometer after 72 h of treatment with SE. Cells were stained with annexin V-FITC and PI (propidium iodide). (<b>A</b>) Mean and standard deviation (SD) of necrosis, as well as viable, early, and late apoptosis, as a percentage from three independent experiments each. (<b>B</b>) Representative cytograms for control MCF-7 cells (CTRL) and after treatment with SE. The data for early and late apoptotic cells were analyzed by Dunnett’s multiple comparison test as follows: * <span class="html-italic">p</span> &lt; 0.05, and *** <span class="html-italic">p</span> &lt; 0.001 relative to control.</p>
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15 pages, 2482 KiB  
Article
High-Yield Expressed Human Ferritin Heavy-Chain Nanoparticles in K. marxianus for Functional Food Development
by Xinyi Lu, Liping Liu, Haibo Zhang, Haifang Lu, Tian Tian, Bing Du, Pan Li, Yao Yu, Jungang Zhou and Hong Lu
Foods 2024, 13(18), 2919; https://doi.org/10.3390/foods13182919 - 15 Sep 2024
Viewed by 258
Abstract
The use of Generally Recognized as Safe (GRAS)-grade microbial cell factories to produce recombinant protein-based nutritional products is a promising trend in developing food and health supplements. In this study, GRAS-grade Kluyveromyces marxianus was employed to express recombinant human heavy-chain ferritin (rhFTH), achieving [...] Read more.
The use of Generally Recognized as Safe (GRAS)-grade microbial cell factories to produce recombinant protein-based nutritional products is a promising trend in developing food and health supplements. In this study, GRAS-grade Kluyveromyces marxianus was employed to express recombinant human heavy-chain ferritin (rhFTH), achieving a yield of 11 g/L in a 5 L fermenter, marking the highest yield reported for ferritin nanoparticle proteins to our knowledge. The rhFTH formed 12 nm spherical nanocages capable of ferroxidase activity, which involves converting Fe2+ to Fe3+ for storage. The rhFTH-containing yeast cell lysates promoted cytokine secretion (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1β (IL-1β)) and enhanced locomotion, pharyngeal pumping frequency, egg-laying capacity, and lifespan under heat and oxidative stress in the RAW264.7 mouse cell line and the C. elegans model, respectively, whereas yeast cell lysate alone had no such effects. These findings suggest that rhFTH boosts immunity, holding promise for developing ferritin-based food and nutritional products and suggesting its adjuvant potential for clinical applications of ferritin-based nanomedicine. The high-yield production of ferritin nanoparticles in K. marxianus offers a valuable source of ferritin for the development of ferritin-based products. Full article
(This article belongs to the Section Nutraceuticals, Functional Foods, and Novel Foods)
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<p>Expression of rhFTH in <span class="html-italic">K. marxianus</span>. (<b>a</b>) SDS-PAGE and Western blot analyses of rhFTH expression in <span class="html-italic">K. marxianus</span>. The KM and KM-rhFTH strains are <span class="html-italic">K. marxianus</span> transformed with pUKDN125 and pUKDN125-rhFTH, respectively. The red arrow highlights the bands of rhFTH. Rabbit anti-ferritin monoclonal antibody and secondary antibody goat anti-rabbit IgG were used for Western blot. M: PageRuler prestained protein ladder; T: total cell lysate; S: supernatant of cell lysate; P: precipitate of cell lysate. (<b>b</b>) An orthogonal design with three factors at three levels, including (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> (5, 12, 18 g/L), glucose (10, 40, 60 g/L), and MgSO<sub>4</sub>·7H<sub>2</sub>O (0.5, 7, 10.5 g/L), was used to test the effects on soluble expression of rhFTH in SM medium in shake flasks at 220 rpm at 30 °C for 72 h. The soluble expression of rhFTH under different conditions was separately compared with that in SM medium. R: Range values of orthogonal design experiments. (<b>c</b>) Comparison of the soluble expressions of rhFTH in SM medium and the optimized SMO medium. (<b>d</b>–<b>f</b>) Effects of different concentrations of Triton X-100, NP-40, and Tween 20 in cell lysis solution on the recovery of soluble rhFTH. Statistical differences were analyzed using <span class="html-italic">t</span>-tests. * <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001; <span class="html-italic">p</span> &gt; 0.05 (not significant, ns).</p>
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<p>Fermentation of KM-rhFTH strain and production of rhFTH in 5 L fermenters. (<b>a</b>–<b>c</b>) The fermentation was carried out in three 5 L fermenters fed with 1000 g (Tank F1), 1300 g (Tank F2), and 1500 g (Tank F3) of glucose. Cells were collected every 12 h, lysed after a 5-fold dilution, then subjected to SDS-PAGE analyses for the expression of soluble rhFTH. (<b>d</b>) The growth curves of KM-rhFTH strain in the three fermenters fed with different amounts of glucose. (<b>e</b>) Cell dry weights at 60 h and soluble yields of rhFTH at 72 h in the three fermenters. (<b>f</b>) Observation of rhFTH in the supernatant of the cell lysate collected from Tank F2 at 72 h by transmission electron microscopy. Bar, 100 nm.</p>
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<p>Purification and characterization of rhFTH produced by <span class="html-italic">K. marxianus</span>. (<b>a</b>) SDS-PAGE analysis of rhFTH purified by heat treatment coupled with DEAE chromatography. Lane M: PageRuler prestained protein ladder; Lane 1: Cell lysate supernatant of the KM-rhFTH; Lane 2: Heat-treated supernatant at 75 °C for 10 min; Lane 3: Elution fraction from DEAE column. (<b>b</b>) Native-PAGE. Lane 1: Cell lysate supernatant of the KM-rhFTH strain; Lane 2: Commercial recombinant ferritin expressed in <span class="html-italic">E. coli</span>; Lane 3: Purified rhFTH expressed in <span class="html-italic">K. marxianus</span>; Lane 4: Cell lysate supernatant of the <span class="html-italic">K. marxianus</span> host strain. (<b>c</b>) TEM analysis of purified rhFTH. Scale bar: 50 nm. (<b>d</b>) Iron uptake of the purified rhFTH. The experiments were carried out in solutions containing a fixed concentration of 0.3 mg/mL rhFTH and 0.25, 0.5, and 1 mM FeSO<sub>4</sub>, respectively. The reaction was performed at room temperature for a total of 30 min, and absorbance values at A<sub>310</sub> nm were obtained every 2 s. The initial absorbance was subtracted to obtain ΔA<sub>310</sub> nm. (<b>e</b>) Analysis of iron content in rhFTH by ICP-MS. (<b>f</b>) Analysis of iron release from rhFTH by the ferrozine method. Incubation with FeSO<sub>4</sub> was conducted at a concentration of 1 mM.</p>
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<p>Effects of rhFTH-containing yeast cell lysates on the proliferation and cytokine secretion of RAW264.7 macrophage cell line after 24 h incubation with different treatments. Blank: culture medium; Control: host <span class="html-italic">K. marxianus</span> cell lysate without human ferritin; rhFTH: rhFTH contained in cell lysate; LPS: Lipopolysaccharide at 1 µg/L as the positive control. The biomass of the control host strain was adjusted to an OD<sub>600</sub> equivalent to that of cell lysates containing 250, 500, 750, and 1000 µg/mL rhFTH. (<b>a</b>) Proliferation of the RAW264.7 cell line. (<b>b</b>) Secretion of IL-6 by the RAW264.7 cell line. (<b>c</b>) Secretion of TNF-α by the RAW264.7 cell line. (<b>d</b>) Secretion of IL-1β by the RAW264.7 cell line. Six replicates were set up for each group. Statistical analysis was performed using <span class="html-italic">t</span>-tests to determine significant differences, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p>
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<p>Analysis of biological function of rhFTH-containing yeast cell lysates on the <span class="html-italic">C. elegans</span> model. Blank: culture medium; Control 1: host <span class="html-italic">K. marxianus</span> cell lysate without human ferritin, and the biomass of the control host strain was adjusted to an OD<sub>600</sub> equivalent to that of cell lysates containing 0.5 mg/mL rhFTH; Control 2: host <span class="html-italic">K. marxianus</span> cell lysate without human ferritin, and the biomass of the control host strain was adjusted to an OD<sub>600</sub> equivalent to that of cell lysates containing 2.0 mg/mL rhFTH; rhFTH 1: 0.5 mg/mL rhFTH contained in cell lysate; rhFTH 2: 2 mg/mL rhFTH contained in cell lysate. (<b>a</b>) Proportion of well-motile nematode within 30 s at day 0, 5, 10, and 15. (<b>b</b>) Pharyngeal pumping frequency of nematodes within 1 min. (<b>c</b>) Total spawning number of nematodes. (<b>d</b>) Survival curves of nematodes under normal culture conditions (NGM, 20 °C). (<b>e</b>) Survival curves of nematodes under heat stress at 37 °C. (<b>f</b>) Survival curves of nematodes under H<sub>2</sub>O<sub>2</sub>-induced oxidative stress. (<b>g</b>) T-SOD activity. (<b>h</b>) CAT activity. (<b>i</b>) Quantization of ROS levels. (<b>j</b>) Analysis of the lipofuscin map. Statistical differences were analyzed using <span class="html-italic">t</span>-tests. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001.</p>
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16 pages, 1137 KiB  
Article
Smells Like Anthelmintic Resistance—Gastrointestinal Prevalence, Burden and Diversity in Dogs from Portugal
by Cláudia Luís Batista, Ricardo Cabeças, Cláudio Araújo-Paredes, Maria Aires Pereira and Teresa Letra Mateus
Pathogens 2024, 13(9), 799; https://doi.org/10.3390/pathogens13090799 - 14 Sep 2024
Viewed by 286
Abstract
Anthelmintic resistance has been documented worldwide, but few cases have been reported in dogs. Shelter dogs are a great opportunity to study intestinal helminths and assess their potential resistance to anthelmintic compounds. With these two goals in mind, 361 faecal samples were collected [...] Read more.
Anthelmintic resistance has been documented worldwide, but few cases have been reported in dogs. Shelter dogs are a great opportunity to study intestinal helminths and assess their potential resistance to anthelmintic compounds. With these two goals in mind, 361 faecal samples were collected from dogs in 18 municipalities in Portugal, from 19 shelters and 11 private tutors. These samples were analysed using the Mini-FLOTAC before and 13 days after deworming. The percentage of faecal egg count reduction was calculated. Parasitic forms were identified in 22.4% of the samples collected: Trichuris vulpis (10.5%), Toxocara canis (8.0%), members of the family Ancylostomatidae (6.4%), Toxascaris leonina (0.8%), Dipylidium caninum (0.6%) and members of the family Taeniidae (0.3%). The first three nematode parasites showed the highest intensity of infection (2900, 1325, and 1265 eggs per gram, respectively). In the second faecal collection, parasites were present in 20.8% of the samples. The anthelmintics febendazole + pyrantel embonate + praziquantel and praziquantel + fenbendazole were ineffective for T. vulpis and members of the family Ancylostomatidae in 16.2% and 6.9% of the samples, respectively. The parasites identified are all potentially zoonotic. This study is the first to suggest a potential resistance of T. vulpis to anthelmintics. Full article
(This article belongs to the Section Vaccines and Therapeutic Developments)
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<p>Geographical distribution and diversity of intestinal helminths identified and possible anthelmintic resistance in dogs’ helminths in different municipalities of the north and centre of Portugal.</p>
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20 pages, 9201 KiB  
Article
Epidermal Growth Factor Receptor Emerges as a Viable Target for Reducing Tumorigenicity of MDCK Cells
by Di Yang, Yuejiao Liao, Lingwei Huang, Jiachen Shi, Jiamin Wang, Zilin Qiao, Zhongren Ma and Sijiu Yu
Genes 2024, 15(9), 1208; https://doi.org/10.3390/genes15091208 - 14 Sep 2024
Viewed by 201
Abstract
The MDCK cell line is perceived as better than the embryos of hen eggs for the production of influenza vaccines, but the tumorigenicity of these cells is concerning. Epidermal growth factor receptor (EGFR) is likely to be a crucial target that contributes to [...] Read more.
The MDCK cell line is perceived as better than the embryos of hen eggs for the production of influenza vaccines, but the tumorigenicity of these cells is concerning. Epidermal growth factor receptor (EGFR) is likely to be a crucial target that contributes to the tumorigenicity of MDCK cells. In this study, EGFR-knockdown and EGFR-overexpression cell lines were established. EGFR’s influence on cell growth, migration, clonogenic ability, and flu virus susceptibility was evaluated in vitro, and its role in cell tumorigenicity was examined in nude mice. GST pull-down coupled with mass spectrometry (MS) and bioinformatics analysis identified EGFR-interacting proteins. The expression levels of these proteins, as well as those of PI3K–AKT- and MAPK–ERK-signaling-pathway-related molecules, were confirmed at both gene and protein levels. The result indicates that EGFR overexpression can enhance cell proliferation, migration, and clonal formation; EGFR knockdown could effectively curtail tumorigenesis and amplify the titers of influenza viruses in MDCK cells. An analysis of the underlying mechanism identified a total of 21 interacting proteins implicated in tumor formation, and among these, AKT1, CDK4, GNB2, and MAPK8 were confirmed at both gene and protein levels. EGFR can activate key factors of the PI3K–AKT signaling pathway, AKT and PI3K, and promote their phosphorylation levels. Consequently, we concluded that EGFR interacts with GNB2, facilitating transmembrane signal transduction, activating the PI3K–AKT signaling cascade, controlling cell cycle alterations, stimulating cell proliferation, and promoting tumorigenesis. Full article
(This article belongs to the Section Animal Genetics and Genomics)
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<p>Tumorigenic gene expression in MDCK cell lines from various origins. **, ***, and **** were considered to indicate a highly significant difference (<span class="html-italic">p</span> &lt; 0.01); and ns was considered to indicate no significant difference (<span class="html-italic">p</span> ≥ 0.05).</p>
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<p>(<b>A</b>) Clusters of monoclonal cells at day 1, 4, and 8 after transfection with lv-EGFR lentivirus. (<b>B</b>) Clusters of monoclonal cells at day 1, 4, and 8 after transfection with sh-EGFR lentivirus. (<b>C</b>) Target gene expression in clone cells with EGFR overexpression. (<b>D</b>) Target gene expression in clone cells with EGFR knockdown. (<b>E</b>) Western blotting was used to detect the expression bands and relative expression levels of EGFR protein in each lv-clone cell. (<b>F</b>) Western blotting was used to detect the expression bands and relative expression levels of EGFR protein in each sh-clone cell. **** was considered to indicate a highly significant difference (<span class="html-italic">p</span> &lt; 0.01).</p>
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<p>EGFR can stimulate the proliferation, migration, and clonal establishment of MDCK cells. (<b>A</b>) The effect of EGFR on cell proliferation was detected by CCK8 assay. (<b>B</b>) The morphology of lv-EGFR cells changed after 10 generations. (<b>C</b>) Results from the migration assay of each cell group at 0 and 12 h. (<b>D</b>) The RMR(%) of each cell at 12 h. (<b>E</b>) Graph of the dishes stained for colony formation of cells cultured for 6 days. (<b>F</b>) Number of clones formed from each cell group after 6 days of culture. * was considered to indicate a significant difference (<span class="html-italic">p</span> &lt; 0.05); **, ***, and **** were considered to indicate a highly significant difference (<span class="html-italic">p</span> &lt; 0.01); and ns was considered to indicate no significant difference (<span class="html-italic">p</span> ≥ 0.05).</p>
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<p>EGFR can enhance the tumorigenicity potential of MDCK cells in vivo. (<b>A</b>) Examination of the influence of EGFR on the tumorigenicity of MDCK cells based on xenografts in nude mice. (<b>B</b>) Correlation analysis of the mean tumor volumes among experimental groups in concurrent murine tumorigenesis trials. (<b>C</b>) Analysis of the trends in the changes in the mean mouse body weight across experimental groups within these mouse trials.</p>
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<p>Observations from the GST pull-down assay and subsequent mass spectrometric data evaluation. (<b>A</b>) The concentration of GST antibodies in the protein sample was determined by Western blotting prior to conducting the GST pull-down test. M: marker; EGFR–GST: EGFR–GST fusion protein synthesized via an inclusion body, a protein band was detected at approximately 66 kDa; GST: GST protein, a protein band was detected at ~25 kDa. (<b>B</b>) The expression of GST antibody in protein was detected by Western blotting after the pull-down test. M: marker; pull-down-CG: protein bands evident within the control group’s eluate, which displayed the previously identified ~25-kDa band; pull-down-EG: specific protein bands observed in the test group’s eluate, which corresponded to the preestablished ~66-kDa EGFR–GST protein; input: total protein content of the MDCK cell line employed in the study. (<b>C</b>) The protein expression in each group was detected by silver staining after the pull-down test. As noted earlier, this group mirrors those depicted in panel B. (<b>D</b>) Number of peptides of different lengths detected by MS. The horizontal coordinate is the number of amino acids that compose the peptide segment, namely, the length of the peptide segment, and the vertical coordinate is the number of peptide segments. (<b>E</b>) Number of proteins with different peptide numbers detected by MS. The horizontal coordinate is the number of peptides that make up the protein, and the vertical coordinate is the number of proteins. (<b>F</b>) Venn diagram visualizing the interacting protein network derived by MS analysis of the test versus control groups.</p>
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<p>Comprehensive Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of interacting proteins. (<b>A</b>) The details related to the top five terms in the ‘Biological Process’, ‘Cellular Component’, and ‘Molecular Function’ categories found for the interacting proteins in the GO analysis are presented. (<b>B</b>) The biological pathways in which the interacting genes (proteins) are mapped onto the top 15 positions within a specifically defined pathway.</p>
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<p>Validation of genes and proteins interacting with EGFR. (<b>A</b>) The relative gene expression levels of 21 interacting proteins in EGFR knockdown cells and its control cells. (<b>B</b>) The relative gene expression levels of 21 interacting proteins in EGFR overexpression cells and its control cells. (<b>C</b>) Protein levels were detected by screening interacting proteins in different cell groups. (<b>D</b>) Histogram of quantized analysis of interacting proteins in different cell groups. * was considered to indicate a significant difference (<span class="html-italic">p</span> &lt; 0.05); **, ***, and **** were considered to indicate a highly significant difference (<span class="html-italic">p</span> &lt; 0.01); and ns was considered to indicate no significant difference (<span class="html-italic">p</span> ≥ 0.05).</p>
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<p>Protein expression and phosphorylation degree of key factors in MAPK–ERK and PI3K–AKT signaling pathway. (<b>A</b>) Western blot analysis of JUK1/2/3, ERK1/2, and their phosphorylation levels in different cell groups. (<b>B</b>) Histogram of quantized analysis of each strip in the MAPK–ERK signal path. (<b>C</b>) Western blot analysis of AKT, PI3K, and their phosphorylation levels in different cell groups. (<b>D</b>) Histogram of quantized analysis of each strip in the PI3K–AKT signal path. * was considered to indicate a significant difference (<span class="html-italic">p</span> &lt; 0.05); **** was considered to indicate a highly significant difference (<span class="html-italic">p</span> &lt; 0.01); and ns was considered to indicate no significant difference (<span class="html-italic">p</span> ≥ 0.05).</p>
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14 pages, 261 KiB  
Article
Risk Factors for Salmonella Infection in Children under Five Years: A Hospital-Based Study in Kilimanjaro Region, Tanzania
by Ephrasia A. Hugho, Blandina T. Mmbaga, Abdul-Hamid S. Lukambagire, Grace D. Kinabo, Kate M. Thomas, Happiness H. Kumburu and Tine Hald
Pathogens 2024, 13(9), 798; https://doi.org/10.3390/pathogens13090798 - 14 Sep 2024
Viewed by 275
Abstract
Salmonella is among the causative agents for diarrhea worldwide, but its risk factors in Tanzanian children are poorly understood. A hospital-based cross-sectional study was conducted in Moshi, Kilimanjaro region, from July 2020 to November 2022 among children under five admitted with diarrhea. A [...] Read more.
Salmonella is among the causative agents for diarrhea worldwide, but its risk factors in Tanzanian children are poorly understood. A hospital-based cross-sectional study was conducted in Moshi, Kilimanjaro region, from July 2020 to November 2022 among children under five admitted with diarrhea. A questionnaire was administered to all parents/caretakers of the enrolled children. Logistic regression was utilized to analyze the risk factors, with significance at p < 0.05. A total of 306 children were enrolled in the study. The median age was 13.8 months (IQR 8.4–21.8). The majority (58.5%) were males, and 59.5% were from rural areas. Salmonella was identified in eight (2.6%) stool samples, with a higher prevalence in urban than rural areas (4.8% vs. 1.1%; p-value = 0.044). The significant risk factors associated with Salmonella infection among the children included consuming raw milk (adjusted OR = 30.19; 95% CI: 3.94–231.46), using infant formula (adjusted OR = 15.78; 95% CI: 2.98–83.56), undisclosed household income (adjusted OR = 9.98; 95% CI: 2.46–40.12), purchasing eggs direct from the farms (adjusted OR = 7.58; 95%CI: 1.31–43.96), and contact with chickens (adjusted OR = 6.49; 95%CI: 1.25–33.59). These findings highlight the need for targeted interventions to improve food safety, hygiene practices, and socioeconomic conditions. Full article
26 pages, 1728 KiB  
Article
Eucarid and Peracarid Fauna of the Valencia Seamount, a Deep-Isolated Seamount of the Western Mediterranean: Colonisation Capacity and Historical Changes
by Joan E. Cartes
Diversity 2024, 16(9), 582; https://doi.org/10.3390/d16090582 - 13 Sep 2024
Viewed by 180
Abstract
Seamounts can have a strong influence on the distribution and diversity of species, creating an oasis effect that may favour diversification. In order to assess how and to what extent supra- and epibenthic crustaceans can colonise these environments, the eucarid and peracarid fauna [...] Read more.
Seamounts can have a strong influence on the distribution and diversity of species, creating an oasis effect that may favour diversification. In order to assess how and to what extent supra- and epibenthic crustaceans can colonise these environments, the eucarid and peracarid fauna collected from the summit of the Valencia Seamount (VS), a small deep seamount (summit depth: 1056 m), rising from a depth of ca. 1850 m, in the oligotrophic Balearic Basin, was analysed. Based on a first sampling (beam trawls, plankton nets and stomach contents), and a faunal reconstruction from a sediment core (MC2, at 1151 m), the supra(epi)benthic crustaceans at the VS summit (to 1300 m) were composed of nine Eucarida and 25 Peracarida. Polycheles typhlops, Munida tenuimana, and Aristeus antennatus were the dominant species among eucarids. Among Peracarida the most abundant species were the Mysida Boreomysis arctica, the Amphipoda Rhachtropis caeca, and the Isopoda Munnopsurus atlanticus. Among Decapoda, a species with a wide amplitude in their depth distribution and small eggs (i.e., with planktotrophic larvae), showed a higher colonisation capacity. In the absence of larvae, the colonisation of peracarids depends on the amplitude of their depth distribution and only those species that reach the highest depths in the entire Balearic Basin, at least 1600–1800 m, were able to colonise the summit of VS. The natatory capacity of the species also has some influence and whole groups with low natatory capacity, such as the Desmosomatidae, were completely absent on the VS summit; however, they are distributed throughout the Balearic Basin to depths (up to about 1500 m) exceeding the depth of the seamount summit. Therefore, colonisation by peracarids must not have occurred by swimming through the entire water column, but by swimming along or just above the bottom. Remains of some suprabenthic species (mainly the isopod M. atlanticus) in MC2 and another core collected in NW Mallorca (MC3, 1114 m), i.e., out of the VS, showed how isopod diversity and size distribution changed historically. Also, after the 1960s, a decrease in primary production due to a decrease in rainfall and river runoff associated with river damming could have reduced the abundance of M. atlanticus. These types of historical studies can be useful in interpreting long-term changes in deep-sea communities and optimising the management of these vulnerable areas. Full article
13 pages, 386 KiB  
Article
Nutrients, Diet Quality, and Dietary Patterns in Patients with Inflammatory Bowel Disease: A Comparative Analysis
by Tingting Yin, Wenjing Tu, Yiting Li, Lina Huang, Yamei Bai and Guihua Xu
Nutrients 2024, 16(18), 3093; https://doi.org/10.3390/nu16183093 - 13 Sep 2024
Viewed by 310
Abstract
(1) Background: Diet plays an important role in the development of inflammatory bowel disease (IBD). There are a number of methods available to assess the diets of patients with IBD, including nutrients, dietary patterns, and various appraisal tools of diet quality. However, research [...] Read more.
(1) Background: Diet plays an important role in the development of inflammatory bowel disease (IBD). There are a number of methods available to assess the diets of patients with IBD, including nutrients, dietary patterns, and various appraisal tools of diet quality. However, research on diet quality and dietary patterns in IBD populations is limited, and comparative evaluations of dietary intake in patients with IBD have not been performed. (2) Objectives: The aim of this study was to assess nutrients, the dietary patterns, and diet quality of patients with IBD and to investigate the relationship between dietary patterns, diet quality, and the adequacy of nutrient intake. (3) Methods: Three-day food records of 268 patients with ulcerative colitis (UC) and 126 patients with Crohn’s disease (CD) were collected to estimate nutrients and food groups, while dietary quality was assessed using the Dietary Inflammation Index (DII) and Mediterranean Diet Score (MDS). Dietary patterns were derived using principal component analysis (PCA). Participants’ nutrient intake, diet quality, and dietary patterns were compared. We used binary logistic regression to assess the relationship between dietary patterns (independent variable) and nutritional adequacy (dependent variable). (4) Results: In our sample, patients had inadequate energy, protein, and dietary fiber intake compared with Reference Nutrient Intake (RNI). Regarding micronutrients, intakes of potassium, zinc, selenium, vitamin A, vitamin C, vitamin E, sodium, calcium, iron, niacin, thiamin, and riboflavin were inadequate. Regarding food groups, the highest intakes were fruits, legumes, dairy products, and nuts. PCA revealed four dietary patterns, namely DP1, DP2, DP3, and DP4. Among UC patients, 96, 55, 69, and 48 patients adhered to DP1, DP2, DP3, and DP4 dietary patterns, respectively. Among CD patients, 41, 31, 34, and 20 patients complied with the dietary patterns of DP1, DP2, DP3, and DP4, respectively. There was no significant difference in dietary patterns between UC and CD patients. Compared with DP4 (high intake of mixed legumes and low intake of tubers), DP1 (high intake of cereals, tubers, vegetables and eggs) was more likely to ensure adequate intake of energy (OR, 2.96; 95% CI, 1.55, 5.62), protein (OR, 2.05; 95% CI, 1.06, 3.96), carbohydrates (OR, 3.55; 95% CI, 1.51, 6.59), thiamine (OR, 2.59; 95% CI, 1.36,4.93), niacin (OR, 2.75; 95% CI, 1.39, 5.42), phosphorus (OR, 2.04; 95% CI, 1.08, 3.85), zinc (OR, 2.43; 95% CI, 1.28, 4.63), and manganese (OR, 3.10; 95% CI, 1.60, 5.90), and DP2 (high intake of fruits, poultry, aquatic products, and nuts) was more likely to meet niacin requirements than DP4 (OR, 2.65; 95% CI, 1.28, 5.48). (5) Conclusion: This study clarifies our understanding of dietary intake, diet quality, and dietary patterns in adult patients with IBD. Future attention is needed to improve diet quality, emphasizing the importance of assessing and understanding patient dietary habits and increasing understanding of the factors that influence dietary intake in IBD in order to achieve optimal outcomes for patients with IBD. Full article
(This article belongs to the Topic Ways to Achieve Healthy and Sustainable Diets)
20 pages, 27362 KiB  
Article
SMARTerra, a High-Resolution Decision Support System for Monitoring Plant Pests and Diseases
by Michele Fiori, Giuliano Fois, Marco Secondo Gerardi, Fabio Maggio, Carlo Milesi and Andrea Pinna
Appl. Sci. 2024, 14(18), 8275; https://doi.org/10.3390/app14188275 - 13 Sep 2024
Viewed by 284
Abstract
The prediction and monitoring of plant diseases and pests are key activities in agriculture. These activities enable growers to take preventive measures to reduce the spread of diseases and harmful insects. Consequently, they reduce crop loss, make pesticide and resource use more efficient, [...] Read more.
The prediction and monitoring of plant diseases and pests are key activities in agriculture. These activities enable growers to take preventive measures to reduce the spread of diseases and harmful insects. Consequently, they reduce crop loss, make pesticide and resource use more efficient, and preserve plant health, contributing to environmental sustainability. We illustrate the SMARTerra decision support system, which processes daily measured and predicted weather data, spatially interpolating them at high resolution across the entire Sardinia region. From these data, SMARTerra generates risk predictions for plant pests and diseases. Currently, models for predicting the risk of rice blast disease and the hatching of locust eggs are implemented in the infrastructure. The web interface of the SMARTerra platform allows users to visualize detailed risk maps and promptly take preventive measures. A simple notification system is also implemented to directly alert emergency responders. Model outputs by the SMARTerra infrastructure are comparable with results from in-field observations produced by the LAORE Regional Agency. The infrastructure provides a database for storing the time series and risk maps generated, which can be used by agencies and researchers to conduct further analysis. Full article
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Figure 1

Figure 1
<p>A schematic description of the back-end and front-end of the SMARTerra decision support system.</p>
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<p>The digital elevation model (DEM) of Sardinia, with the island’s main rice-growing areas (<b>left</b>). The DEM was obtained by processing <math display="inline"><semantics> <mrow> <mn>10</mn> <mo> </mo> <mi mathvariant="normal">m</mi> </mrow> </semantics></math> resolution rasters downloaded from the geoportal of the region of Sardinia [<a href="#B14-applsci-14-08275" class="html-bibr">14</a>]. On the (<b>right</b>), the locations of the meteorological stations of the Regional Meteorological Network (RUR) are shown in yellow, and the nodes of the grid points for which the BOLAM model provides weather forecasts are shown in white.</p>
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<p>A schematic description of the InfluxDB time-series-oriented database storing the weather measurements from the RUR network and BOLAM forecasts. After preprocessing, the data are put into the database and organized into measurements and fields. Then, the data become available for the interpolation techniques by query via API.</p>
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<p>Temperature distribution for a selected geographic window at a specific day and time. Enabling the “Station” flag displays all stations (shown as blue dots) within the window. Detailed variations of the selected variable are shown for each station (dark popup), along with the relative trend of the value from the nearest BOLAM grid point (blue popup). The white dots represent the location of the 4 nodes of the BOLAM model closest to the selected station.</p>
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<p>Detail of the rice blast (“brusone”) risk map on a given day, highlighting the paddy field areas. The dark popup allows users to view specific information about the selected parcel, including the name of the farm, technical features like area, the rice cultivar, and other relevant details.</p>
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<p>Daily maximum temperature (<b>left</b>) and daily rainfall accumulation (<b>right</b>) spatially interpolated from measured data with the KED technique.</p>
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<p>Thumbnails from the PDF report. From (<b>top left</b>) to (<b>bottom right</b>), the hourly mean temperature, mean relative humidity and rainfall from the measured and forecast data, the daily maximum and mean temperature and rainfall from the measured data, the risk indices and alert levels for the rice blast disease, and the threshold dates and accumulation totals for the locust egg hatching prediction.</p>
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<p>Risk indices (<b>top</b>, <b>bottom left</b>) and alert levels (<b>bottom right</b>) maps for the rice blast disease obtained from interpolated measured and forecast weather data. See <a href="#sec2dot2dot5-applsci-14-08275" class="html-sec">Section 2.2.5</a> for details.</p>
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<p>The cumulative sum of degree days (<b>left</b>), starting for each pixel of the map once the rain accumulation threshold <math display="inline"><semantics> <mrow> <msub> <mi>R</mi> <mi>th</mi> </msub> <mo>=</mo> <mn>100</mn> <mo> </mo> <mrow> <mi mathvariant="normal">m</mi> <mi mathvariant="normal">m</mi> </mrow> </mrow> </semantics></math> has been reached, and the predicted dates for locust egg hatching (<b>right</b>). The white areas indicate regions where the summation of <math display="inline"><semantics> <mrow> <msub> <mi>D</mi> <mi>th</mi> </msub> <mo>=</mo> <mn>200</mn> <mo> </mo> <mrow> <mo>°</mo> <mi mathvariant="normal">C</mi> </mrow> </mrow> </semantics></math> degree days has not yet occurred. Compare with the former map, where the darkest red areas correspond to regions where the summation of degree days is below the threshold <math display="inline"><semantics> <msub> <mi>D</mi> <mi>th</mi> </msub> </semantics></math>. See <a href="#sec2dot2dot6-applsci-14-08275" class="html-sec">Section 2.2.6</a> for details.</p>
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<p>Comparison of observed and predicted alert levels for rice blast disease in the Oristano rice districts. Observed (<b>top left</b>) and predicted (<b>top right</b>) alert levels for 17 July 2023. Observed (<b>bottom left</b>) and predicted (<b>bottom right</b>) alert levels for 18 July 2023.</p>
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<p>Spatial distribution of <math display="inline"><semantics> <msub> <mo>Δ</mo> <mi>d</mi> </msub> </semantics></math> for egg hatching predicted by the default (literature) model (<b>left</b>) and a slightly modified model (<b>right</b>), both with a superimposed OpenStreetMap layer (<b>top</b>), a map of the dates for <math display="inline"><semantics> <msub> <mi>day</mi> <mn>2</mn> </msub> </semantics></math> when the rain threshold <math display="inline"><semantics> <msub> <mi>R</mi> <mi>th</mi> </msub> </semantics></math> is reached and the accumulation of degree days starts (<b>middle</b>), and a map representing <math display="inline"><semantics> <msub> <mi>day</mi> <mn>3</mn> </msub> </semantics></math>, the predicted dates of the hatching of locust eggs (<b>bottom</b>). The region of interest is partitioned into hexagons, each of which is assigned a color according to the average of the <math display="inline"><semantics> <msub> <mo>Δ</mo> <mi>d</mi> </msub> </semantics></math> values of the records within the area of the hexagon itself.</p>
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22 pages, 49469 KiB  
Article
First Evidence of Reproductive Strategies in Cephalopods Preserved in Phosphate and Siderite Nodules from the Devonian of Uruguay
by Graciela Piñeiro, Magela Rodao and Pablo Núñez Demarco
Foss. Stud. 2024, 2(3), 223-244; https://doi.org/10.3390/fossils2030011 - 13 Sep 2024
Viewed by 593
Abstract
Uruguayan ammonoids are preserved in phosphate and siderite nodules found at the basalmost tillite-like conglomerates of the San Gregorio Formation. This lithostratigraphic unit was deposited under glacial conditions and its age (as well as that of the nodules) has been highly debated because [...] Read more.
Uruguayan ammonoids are preserved in phosphate and siderite nodules found at the basalmost tillite-like conglomerates of the San Gregorio Formation. This lithostratigraphic unit was deposited under glacial conditions and its age (as well as that of the nodules) has been highly debated because glaciations were intermittent in Gondwana during the Late Paleozoic. Reef-builder organisms (e.g., Rugosa and Tabulata), goniatite and orthoceratid cephalopods, brachiopods, sponges, actinopterygians and other indeterminate gnatostomes, as well as fragmentary stems and roots of cf. Lycopsida are the most frequent fossils in the nodules. According to new biostratigraphic and paleoclimatic evidence, these taxa are representative of a reefal environment of a preliminary Devonian age including species that are common in the underlying Early Devonian (Emsian) Durazno Group. Among the ammonoid remains, more than 40 clusters of hatchling goniatites were found in the nodules. Each cluster contains a variable number of shells similar in shape to some of the adults also preserved within the nodules, representing a single species preserved at the same developmental stage (3 mm on size average). The strongly packed shells are enveloped by a substance with a different chemical composition and microstructure with respect to that of the nodule matrix, possibly indicating the presence of a gelatinous-like substance reminiscent of that secreted by the females of some extant cuttlefish and octopuses at the time of the egg spawn. Differing from previously described ammonoid accumulations, our clusters are unique in containing individuals of just a single species preserved in the same ontogenetic stage. That allows us to suggest that they represent a mode of reproduction in which hatchlings were morphologically similar to their parents and occupied the same habitat. Our results are thus one of the oldest known records of reproductive strategies in Paleozoic ammonoids and the phosphate and siderite nodules from the San Gregorio Formation are here classified as a new Konservat-Lagerstätte, which is the oldest known for South America. Full article
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Figure 1
<p>Map showing the distribution of the studied areas of the San Gregorio Formation of Uruguay in the vicinity of the San Gregorio de Polanco city along both the banks of the Negro River (Tacuarembó and Durazno Departments, red circle), and close to the Melo city in the Cerro Largo Department (black symbols) [<a href="#B1-fossstud-02-00011" class="html-bibr">1</a>,<a href="#B2-fossstud-02-00011" class="html-bibr">2</a>,<a href="#B6-fossstud-02-00011" class="html-bibr">6</a>]. The fossiliferous phosphate and siderite nodules described herein are limited to the former area.</p>
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<p>Basic stratigraphy of the nodule bearing San Gregorio Formation of Uruguay and the underlying Early Devonian Durazno Group. (<b>A</b>). Schematic stratigraphic section of one of the outcrops where we found the specimens described herein. The arrow points to the level where striate bedrock surfaces from glacier displacement were found. (<b>B</b>). Photograph of the basal level of tillites that contain the reworked fossiliferous nodules (head arrow). (<b>C</b>). Detail of the bedrock surface scratched by rocks embedded in glacial ice during glacier displacement. It is worth noting that these striae (yellow arrow) are stratigraphically above the tillites that are bringing the nodules, evidencing the periglacial and postglacial origin of these deposits. Scale: Engineer Sebastian Marmol.</p>
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<p>Photographs of early juvenile ammonoid clusters from the Devonian of Uruguay. (<b>A</b>). Specimen JMMO-IMT 0215 represents the largest cluster found (approximately 55 mm in length) and it shows the closely packed ammonoids shells embedded in a possible gelatinous substance that protected and feed the individuals before hatching [<a href="#B39-fossstud-02-00011" class="html-bibr">39</a>]. The ammonitellae average diameter at this stage is near 0.4 to 0.5 mm, and the shell has reached the three additional whorls that would characterize the hatchling phase outside the gelatinous envelope. Scale bar: 10 mm. (<b>B</b>,<b>C</b>). JMMO-IMT 0311. Part and counterpart of a cluster containing well-preserved, early juvenile tiny ammonoids, which allowed us to studied them by SEM and took good microphotographs of the initial chamber, as well as study the morphology that characterize the ammonitella stage for this new species of Uruguayan ammonoids (see <a href="#fossstud-02-00011-f007" class="html-fig">Figure 7</a> and <a href="#fossstud-02-00011-f008" class="html-fig">Figure 8</a>). Scale bars: 5 mm.</p>
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<p>JMMO-IMT 0230. Photograph of early juvenile ammonoid clusters from the Devonian of Uruguay. The image shows some shells inside a phosphate nodule of the San Gregorio Formation (Central Uruguay). Following [<a href="#B40-fossstud-02-00011" class="html-bibr">40</a>], a possible gelatinous substance enclosing the shells seems to have been broken, maybe representing the moment in which the individuals abandon it and escape to the water-column, or the image might show the thin gelatinous envelop unexpectedly broken by a strong environmental event or by a predator attack. Scale bar: 5 mm.</p>
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<p>Size variation in the shells (<b>a</b>) and ammonitella stage (<b>b</b>) recording at the ammonoid clusters found in phosphate and siderite nodules of the San Gregorio Formation of Uruguay. Measurements of the shells were taken from the best preserved specimens in dorsal view from eight clusters (MMO 0156, 171, 215, 225, 230, 304, 305, 311), trying to cover all the represented sizes, ranging from 3 to 5 mm in average. Measurements of diameters of the ammonitella stage were mostly allowed from the best preserved specimens studied by SEM and were taken following [<a href="#B36-fossstud-02-00011" class="html-bibr">36</a>,<a href="#B38-fossstud-02-00011" class="html-bibr">38</a>].</p>
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<p>Comparative chemical composition and microstructure of the substance that encloses the ammonoid early juvenile specimens herein studied with respect of that of the nodule matrix containing them. (<b>A</b>). SEM-EDS analysis of an area of the matrix in specimen JMMO-IMT 0215. The main elements are calcium and phosphorus, with a variable content of iron, which turns the nodules as phosphate or siderite according to the amount of Fe that they contain. (<b>B</b>). Typical composition found in the possible gelatinous substance that encloses the clusters. (<b>C1</b>,<b>C2</b>) are SEM-EDS analyses that show the high content of carbon that is found in the mentioned substance, denoting the high preservation of soft tissues.</p>
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<p>Microanatomy of the ammonitella stage of tiny ammonoid shells inside the clusters from the Devonian of Uruguay. (<b>A</b>). JMMO-IMT 0305. SEM images of one of the best-preserved shells in lateral view. The initial chamber is poorly delimited but the caecum is well identified. (<b>B</b>). Close up of other shell preserved in the same cluster (JMMO-IMT 0305) to show the internal morphology of the initial chamber well delimited by a septum that separates it from the beginning of the first whorl. (<b>C</b>,<b>D</b>). Other specimens from cluster JMMO-IMT 0304 also show the semicircular shape of the initial chamber and the caecum and prosiphon shapes. Abbreviations: c, caecum; ic, initial chamber; prs, proseptum; psi, prosiphon.</p>
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<p>(<b>A</b>). Dorsal view of the initial chamber and the first whorl showing the position and morphology of the proseptum and the second septum (small black arrow). (<b>B</b>). Close up of the proseptum and second septum shown in (<b>A</b>). (<b>C</b>). Ammonitella stage morphology of a specimen found in the cluster JMMO-IMT 0305, where can be seen that the ornamentation of the shell shows a gradual change at the level of what we interpreted is the proseptum 1. (<b>D</b>). Interpretive drawing of (<b>C</b>) at the same scale. Abbreviations: c, caecum, ic, initial chamber, prs, proseptum, psi, prosiphon.</p>
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<p>SEM images showing the morphology and configuration of the siphuncle and the siphuncular tube in the ammonitella ontogenetic stage of the best-preserved specimens in clusters from the Devonian of Uruguay. All specimens shown are part of the cluster JMMO-IMT 0311. (<b>A</b>). Almost complete specimen showing the siphuncle across the last whorl passing through the chambers close to the outer wall of the shell at the middle region of the chamber. Interestingly, it can be seen that the septum is deformed/opened both centrally and dorsally. (<b>B</b>–<b>D</b>). Images taken from different shells confirming the common configuration of the siphuncle by passing through the median opening formed by the septa, particularly clear in (<b>D</b>).</p>
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<p>Ornamentation of the external surface of the ammonitella and early juvenile stages in individual shells from clusters of the Devonian of Uruguay. (<b>A</b>). JMMO-IMT 0304. Photograph under Camera Lucida of part of the gelatinous substance covering the shells. An almost complete and well preserved specimen displays the lirae-like ornamentation across the shell. Note that it is very fine-ribbed at the first whorl, increasing the space between ribs in direction to the aperture. Scale bar: 30 mm. (<b>B</b>). JMMO-IMT 0304. SEM image where both the mold and the outer ornamentation of the ammonitella shell can be appreciated. (<b>C</b>). JMMO-IMT 0311. SEM photograph of one of the cluster components showing the faint lirae-like ornamentation pattern characterizing the first whorls of the phragmocone, which is different from the well marked ribs observed in the outermost whorls.</p>
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<p>Morphology of the suture in ammonoids preserved in siderite and phosphate nodules of the San Gregorio Formation, Devonian of Uruguay. (<b>A</b>): JMMO-IMT 0215, small ammonoid, possible early juvenile individual. (<b>B</b>). JMMO-IMT 0016, young adult individual. (<b>C</b>,<b>D</b>). JMMO-IMT 0092 and 0091, adult individuals. The pattern of the sutures is clearly related to the ontogenetic stage of the analyzed specimens. It seems to be not constant because the walls of the septa form three-dimensional folds that are more or less accentuated or possess more or less irregularities, depending on the height at which the specimen is sectioned. The sutures at the central chambers are not always visible, i.e., they are not always exposed if the cut does not pass exactly to the center of these sections. (<b>E</b>–<b>H</b>). Interpretive drawings of (<b>A</b>–<b>D</b>). (<b>I</b>). JMMO-IMT 0305. Interpretive drawing of the shell of one of the individuals that are components of the cluster showing the first four phragmocone cameral septa and the sutures for these septa, thus delimitating the end of the ammonitella shell (red arrow). (<b>J</b>). Photograph of the specimen drawn at I clearly showing the first septum (red arrow) delimitation the shape of the ammonitella stage. Scale bars: (<b>E</b>). 5 mm; (<b>F</b>–<b>H</b>). 10 mm; (<b>J</b>). 1 mm. See also <a href="#fossstud-02-00011-f008" class="html-fig">Figure 8</a> as a complement.</p>
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<p>Main taxa represented in the reef community preserved in siderite and phosphate nodules of the San Gregorio Formation, Devonian of Uruguay. (<b>A</b>). JMM-IMT 0006, Actinopterygii (skull and anterior region of the trunk). (<b>B</b>). JMM-IMT 0056. Ammonoidea (goniatite ammonoid adult associated to corallites of cf. Rugosa (arrow). (<b>C</b>). JMM-IMT 0331. Trilobita (characteristic red color and badly preserved; could be a molten exhubia). (<b>D</b>). JMM-IMT 0313. Brachiopoda (clusters of <span class="html-italic">Orbiculoidea baini</span>). (<b>E</b>). JMM-IMT 0234. Rugosa (colonial corallites). (<b>F</b>). JMMO-IMT 0380. Well-preserved coral specimen tentatively assigned to Tabulata, showing internal morphology of the wall (w) and trabeculate septal region (st). (<b>G</b>). JMM-IMT 0422 cf. Lycopsida stem or root. (<b>H</b>,<b>I</b>). JMM-IMT165. Cephalopoda Orthoceridae. Fragmentary cone-shaped shell of a nautiloid cephalopod. Scale bars: (<b>A</b>–<b>C</b>,<b>E</b>–<b>G</b>): 10 mm; (<b>D</b>): 3 mm; and (<b>H</b>–<b>I</b>): 20 mm.</p>
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15 pages, 764 KiB  
Article
Demand and Nutrient Elasticities of Egg Consumption: Evidence from Saudi Arabia
by Dana Alsaad and Mohammed Al-Mahish
Economies 2024, 12(9), 245; https://doi.org/10.3390/economies12090245 - 11 Sep 2024
Viewed by 322
Abstract
Chicken eggs are a staple food widely consumed directly or indirectly (as an ingredient in other food items) in Saudi Arabia. Due to the importance of eggs in Saudi consumers’ diet, this study aimed to determine the sociodemographic factors affecting Saudi consumer demand [...] Read more.
Chicken eggs are a staple food widely consumed directly or indirectly (as an ingredient in other food items) in Saudi Arabia. Due to the importance of eggs in Saudi consumers’ diet, this study aimed to determine the sociodemographic factors affecting Saudi consumer demand for eggs and how changes in egg prices and consumer income may influence dietary intake derived from eggs. This study analyzes survey data from 466 respondents. We used a double-log demand model to obtain the price and income elasticities of egg demand. The results show that the price elasticity of egg demand is negative and inelastic, indicating that changes in egg price have a small influence on egg demand. Additionally, income elasticity is positive and <1, implying that eggs are a necessary food item. Cross-price elasticities show that cheese and milk complement egg consumption. Meanwhile, cornflakes (a breakfast cereal) were found to be a good substitute for eggs. Furthermore, the results show that consumer demand for eggs is expected to increase as household size increases. Women exhibited a higher demand for eggs, compared with men. Nutrient income elasticity shows that an increase in consumer income increases protein, fat, and calorie intake from eggs. Nutrient price elasticity shows that an increase in egg price has the greatest negative influence on protein intake, resulting from a decrease in egg consumption. This study recommends that decision makers actively monitor egg prices and take all necessary actions to prevent spikes in egg prices, as this will improve nutritional and food security and help consumers maintain a stable protein intake from egg consumption. Full article
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<p>Average real price of a 30-egg tray (local eggs) in SAR (2012–2022). Source: Authors’ calculation based on GASTAT data.</p>
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32 pages, 2904 KiB  
Article
Per- and Polyfluoroalkyl Substances in the Duluth, Minnesota Area: Exposure to and Biomarker Responses in Tree Swallows Relative to Known Fire-Fighting Foam Sources
by Christine M. Custer, Paul M. Dummer, Matthew A. Etterson, Jonathan T. Haselman, Sandra Schultz, Natalie Karouna-Renier and Cole Matson
Toxics 2024, 12(9), 660; https://doi.org/10.3390/toxics12090660 - 10 Sep 2024
Viewed by 329
Abstract
Tree swallow nest boxes were deployed at sites proximal to two putative aqueous film forming foam (AFFF) sources in the Duluth, MN area, as well as along the St. Louis River and a reference lake for comparative purposes in 2019, 2020 and 2021. [...] Read more.
Tree swallow nest boxes were deployed at sites proximal to two putative aqueous film forming foam (AFFF) sources in the Duluth, MN area, as well as along the St. Louis River and a reference lake for comparative purposes in 2019, 2020 and 2021. The two AFFF sites were the current Duluth Air National Guard Base (ANG) and the Lake Superior College Emergency Response Training Center. Between 13 and 40 per- and polyfluoroalkyl substances (PFAS), depending on year, were detected and quantified in tree swallow egg, nestling carcasses, and stomach contents. Assessments were made of oxidative stress and ethoxyresorufin-O-dealkylase activity in liver tissue, thyroid hormone levels in plasma and thyroid glands, DNA damage in red blood cells, and two measures of immune response (haptoglobin-like activity and immunoglobulin) in plasma of the nestlings. Additionally, other contaminants, such as polychlorinated biphenyls, legacy organochlorine pesticides, and trace elements, were assessed at sites with no previous data. Total egg PFAS concentrations at the ANG site and north of that site were 30–40 times higher than at the reference lake, while nestling PFAS concentrations were 10–15 times higher. In contrast, the St. Louis River sites had slightly, but non-statistically significant, elevated egg and nestling PFAS concentrations relative to the reference lake (2–5 times higher). One PFAS, perfluorohexane sulfonate (PFHxS), was higher, as a proportion of total PFAS, at sites with a known AFFF source compared to the reference lake, as well as compared to sites along the St. Louis River with mainly urban and industrial sources of PFAS. The ratio of total carboxylates to total sulfonates also distinguished between PFAS sources. There were few to no differences in biomarker responses among sites, and no association with PFAS exposure. Full article
(This article belongs to the Special Issue Ecotoxicology and Ecological Risks of PFAS)
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<p>Map of per- and polyfluorinated substances (PFAS) study sites in the Duluth, MN area 2019–2021. Two underlined site names are known as locations at which aqueous film forming foams (AFFFs) were used. Note: ANG = Duluth Air National Guard Base and UMD = farm at University of Minnesota, Duluth.</p>
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<p>Correlation between original 13 (PFAS<sub>13</sub>) and current 40 (PFAS<sub>40</sub>) per- and polyfluoroalkyl substances (PFAS) in tree swallow eggs from the Duluth, MN area in 2021. Lower panel is an enlargement of the lower end of the distribution.</p>
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<p>Total mass (ng) of perfluorooctane sulfonate (PFOS) in pairs of eggs and sibling 12- to 15-day old nestlings at Rice Lake North and the reference site (Boulder Lake, upper panel) in the Duluth, MN area in 2020–2021. Lower panel is expanded to provide more detail at the reference lake. Average accumulation rates (ng/day) are provided for the two sites.</p>
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<p>Correlation between the sum of 40 per- and polyfluoroalkyl substances (PFAS<sub>40</sub>) in diet and nestling carcasses (<b>upper</b>) and diet and eggs (<b>lower</b>) from the Duluth, MN area in 2020–2021.</p>
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<p>Nonmetric multidimensional scaling (NMDS) plot of per- and polyfluoroalkyl substances (PFAS) in tree swallow nestlings (2010 and 2021) by region in the Duluth, MN area for n = 25 PFAS detected in ≥2 samples. Note: NMDS plots are unitless, AFFF = aqueous film forming foam, SLR = St. Louis River.</p>
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<p>Nonmetric multidimensional scaling (NMDS) plot of per- and polyfluoroalkyl substances (PFAS) in tree swallow egg, nestling, and diet samples (2010 and 2021) in the Duluth, MN area for n = 27 PFAS detected in ≥2 samples in at least one of the three matrices. Note that NMDS plots are unitless.</p>
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<p>Average percentage of 40 per- and polyfluoroalkyl substances (PFAS<sub>40</sub>) that individual PFAS comprised in tree swallow nestling carcasses in the three regions near Duluth, MN 2020–2021. St. Louis River includes Boy Scout Landing which has an aqueous film forming foam (AFFF) source and has been pulled out for visualization. Percentages inside circles are for perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS). Concentrations or range of total PFAS<sub>40</sub> concentrations are provided for sites within each region in parentheses. Note: AFFF = aqueous film forming foam, PFOA = perfluorooctanoate, PFNA = perfluorononanoate, PFDA = perfluorodecanoate, PFUnA = perfluoroundecanoate, PFDoA = perfluorododecanoate, PFTrDA = perfluorotridecanoate.</p>
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<p>Average percentage of 40 per- and polyfluoroalkyl substances (PFAS<sub>40</sub>) that individual PFAS comprised in tree swallow eggs in the three regions near Duluth, MN 2020–2021. St. Louis River includes Boy Scout Landing, which has been pulled out (lower right) for visualization. Percentages inside circles are for perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS). Concentrations or range of total PFAS<sub>40</sub> concentrations are provided for sites within each region in parentheses. Note: AFFF = aqueous film forming foam, PFOA = perfluorooctanoate, PFNA = perfluorononanoate, PFDA = perfluorodecanoate, PFUnA = perfluoroundecanoate, PFDoA = perfluorododecanoate, PFTrDA = perfluorotridecanoate.</p>
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<p>Ratio of total carboxylates to total sulfonates in tree swallow eggs (<b>upper</b>) and in nestlings (<b>lower</b>) at sites in the three regions near Duluth, MN, 2020–2021.</p>
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17 pages, 3344 KiB  
Article
A High-Throughput Method for Quantifying Drosophila Fecundity
by Andreana Gomez, Sergio Gonzalez, Ashwini Oke, Jiayu Luo, Johnny B. Duong, Raymond M. Esquerra, Thomas Zimmerman, Sara Capponi, Jennifer C. Fung and Todd G. Nystul
Toxics 2024, 12(9), 658; https://doi.org/10.3390/toxics12090658 - 9 Sep 2024
Viewed by 342
Abstract
The fruit fly, Drosophila melanogaster, is an experimentally tractable model system that has recently emerged as a powerful “new approach methodology” (NAM) for chemical safety testing. As oogenesis is well conserved at the molecular and cellular level, measurements of Drosophila fecundity can [...] Read more.
The fruit fly, Drosophila melanogaster, is an experimentally tractable model system that has recently emerged as a powerful “new approach methodology” (NAM) for chemical safety testing. As oogenesis is well conserved at the molecular and cellular level, measurements of Drosophila fecundity can be useful for identifying chemicals that affect reproductive health across species. However, standard Drosophila fecundity assays have been difficult to perform in a high-throughput manner because experimental factors such as the physiological state of the flies and environmental cues must be carefully controlled to achieve consistent results. In addition, exposing flies to a large number of different experimental conditions (such as chemical additives in the diet) and manually counting the number of eggs laid to determine the impact on fecundity is time-consuming. We have overcome these challenges by combining a new multiwell fly culture strategy with a novel 3D-printed fly transfer device to rapidly and accurately transfer flies from one plate to another, the RoboCam, a low-cost, custom-built robotic camera to capture images of the wells automatically, and an image segmentation pipeline to automatically identify and quantify eggs. We show that this method is compatible with robust and consistent egg laying throughout the assay period and demonstrate that the automated pipeline for quantifying fecundity is very accurate (r2 = 0.98 for the correlation between the automated egg counts and the ground truth). In addition, we show that this method can be used to efficiently detect the effects on fecundity induced by dietary exposure to chemicals. Taken together, this strategy substantially increases the efficiency and reproducibility of high-throughput egg-laying assays that require exposing flies to multiple different media conditions. Full article
(This article belongs to the Special Issue Feature Papers in the Novel Methods in Toxicology Research)
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<p>Quantification of fecundity over three days on different types of media. (<b>A</b>) Diagram showing the workflow for this assay. Flies were fed wet yeast paste for two consecutive days and then 2 females and 1 male were transferred into each well of a 48-well plate with molasses-based, BDSC-based (tan), grape juice<tt>–</tt>agarose-based media (16 wells for each condition, as indicated in gold, tan, and pink colors, respectively) and allowed to lay eggs for 23 h. (<b>B</b>) Graph showing the number of eggs laid in each condition, as determined by manual counts. Sample sizes per condition range from 29 to 61 wells and the data were collected from 2–4 independent replicates. Asterisks indicate statistical significance using Bonferroni-corrected pairwise <span class="html-italic">t</span>-tests between D1 and D2 or D3 for each condition. *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Development of protocol for assaying fecundity over 7 days. (<b>A</b>) Diagram showing a workflow in which flies are maintained on media with yeast mixed in on the days in which images will be acquired to assess fecundity and on media with yeast on top during the remaining days. In the “alternating” conditions shown in panels (<b>C</b>–<b>E</b>), flies were put on media with yeast mixed in on days 1, 3, and 7 and on media with yeast on top on days 2, 4, 5, and 6. (<b>B</b>–<b>D</b>) Representative examples of wells from a 3 day time course in which flies were maintained in wells with the yeast mixed into the media at all time points (<b>B</b>) or alternating between wells with yeast mixed into the media on days 1 and 3 and yeast on top on day 2 (<b>C</b>) and a graph showing the number of eggs laid in each regime (<b>D</b>). (<b>E</b>) Graph showing the number of eggs laid in the alternating regime over a 7-day time course. Sample sizes in (<b>D</b>,<b>E</b>) per condition range from 78 to 144 wells and the data were collected from 3–7 independent replicates. Asterisks indicate statistical significance using Bonferroni-corrected pairwise <span class="html-italic">t</span>-tests between D1 and D3 for each condition in (<b>D</b>) and D1 and D3 or D7 in (<b>E</b>). * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Fly transfer device. (<b>A</b>–<b>G</b>) A fly transfer lid (<b>A</b>) is constructed from a layer of nylon mesh bonded between an array of cups (<b>B</b>) and holes (<b>C</b>). Studs in the hole layer (<b>C</b>) pass through corresponding holes in the cup (<b>B</b>) and nylon layers, creating individual gas exchange tops for each well. A hot soldering iron, guided by holes in a metal template, creates holes in the nylon mesh (<b>D</b>). The final assembly (<b>E</b>) studs are bonded with cyanoacrylate glue. (<b>F</b>) When placed on a CO<sub>2</sub> plate, anesthetized flies fall into their corresponding cup, enabling the plate to be replaced and flipped, simultaneously transferring all flies into new wells. (<b>G</b>) Top and side view of the fly transfer device. The cup layer is shown in blue and the hole layer is shown in red.</p>
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<p>RoboCam device for automated image capture. A 3D printer (<b>A</b>) is modified by adding a camera (<b>B)</b> and light plate (<b>C</b>), where the 48-well plate (<b>D</b>) is located. The light ensures good contrast between the eggs and the media while minimizing shadows and reflections. A single-board computer (<b>E</b>) controls the x, y, and z movement of the camera (<b>B</b>) and saves captured images on a hard drive (<b>F</b>). The user programs the RoboCam using a graphical user interface with a keyboard, mouse, and monitor (<b>G</b>). (<b>H</b>) Camera system moves in a snake path, centering over individual wells.</p>
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<p>Automated image analysis pipeline. (<b>A</b>–<b>C</b>) Graphs showing comparisons of manual egg counts to automated egg counts using Quantifly (<b>A</b>), Stardist with the Versatile_fluo model (<b>B</b>), or Stardist using a custom-build model, flyModel2 (<b>C</b>). Each dot shows the manual and automated counts from a single well. (<b>D</b>) Graph showing the error between manual and automated egg counts. Each dot is the difference between the automated count and the manual count (ground truth). (<b>E</b>–<b>G</b>) Images showing individual steps in the automated image analysis pipeline. Starting from the raw image (<b>E</b>), the Stardist model identifies the eggs and the elipse detection tool identifies the well edge (boxed regions and white circle, respectively in Panel <b>F</b>). Then, the number of boxed regions (eggs) within the well are counted (<b>G</b>). Eggs reflected in the well walls are indicated with yellow arrows.</p>
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<p>Detection of the impact of dietary exposure to rapamycin and bendiocarb. (<b>A</b>) Diagram showing workflow for fecundity assays in which chemicals are added to the diet. Chemicals are dissolved in 100% DMSO to a 1000× concentration. Then, they are diluted to 10× concentration in 1% DMSO with either water or water plus yeast. Finally, 30 µL of the 10× solution is pipetted into each well, which contains 300 µL of media, producing a final concentration of 1x chemical in 0.1% DMSO. Using this protocol, flies were exposed to 0.1% DMSO or indicated concentrations of rapamycin or bendiocarb in 0.1% DMSO for 7 days. Egg counts were quantified on days 1, 3, and 7. (<b>B</b>–<b>D</b>) Images of wells at 1, 3 or 7 days after exposure to 0.1% DMSO (<b>B</b>), 25 µM rapamycin (<b>C</b>), or 10 µM bendiocarb (<b>D</b>). (<b>E</b>,<b>F</b>) Graphs showing the number of eggs laid in the indicated conditions over the 7-day time course. Flies did not survive for 7 days on 25 µM bendiocarb. Asterisks indicate a statistically significant difference compared to the DMSO condition on the same day ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 using pairwise t-tests with a Bonferroni multiple hypothesis test correction.</p>
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<p>Downsampling of data from dietary exposure to rapamycin and bendiocarb. The <span class="html-italic">p</span>-values from 100 iterations of downsampling the data by randomly selecting 4, 8, 16, or 24 wells from the day 1, day 3, and day 7 datasets are displayed on the graphs. <span class="html-italic">p</span>-values to the left of the red dotted line are below the conventional 0.05 threshold for statistical significance.</p>
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